One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to the ligands, CCR5 activates downstream signaling, which plays critical roles in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of pro-inflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C₅Mab-4 (rat IgG_2a, kappa) and C₅Mab-8 (rat IgG₁, kappa), recognized mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (K_D) values of C₅Mab-4 and C₅Mab-8 for CHO/mCCR5 were determined as 3.5 × 10⁻⁸ M and 7.3 × 10⁻⁹ M, respectively. Furthermore, both C₅Mab-4 and C₅Mab-8 could detect mCCR5 by western blotting. These results indicated that C₅Mab-4 and C₅Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.

C-C motif chemokine receptor-8 (CCR8) belongs to class A of G protein-coupled receptors (GPCRs). CCR8 interacts with the specific chemokine ligand CCL1/I-309 in humans, which is produced by various cells, including tumor-associated macrophages and regulatory T cells (Treg). CCR8 is highly expressed on Treg and T-helper 2 (T_H2) cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer progression. The CCR8⁺Treg has been suggested to be an important regulator in the immunosuppressive tumor microenvironment (TME); therefore, it has been desired to develop sensitive monoclonal antibodies (mAbs) for CCR8. This study developed a specific mAb for human CCR8 (hCCR8), which is useful for flow cytometry by employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-hCCR8 mAb, C₈Mab-21, (mouse IgM, kappa) reacted with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8) cells, TALL-1 (acute T lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and natural killer cells, which express endogenous hCCR8 by flow cytometry. Furthermore, C₈Mab-21 demonstrated a moderate binding affinity for CHO/hCCR8 and TALL-1 with a dissociation constant of 6.5×10^-8 M and 2.0×10^-8 M, respectively. C₈Mab-21, which was established by the CBIS method, could be a useful tool for analyzing the hCCR8-related biological response using flow cytometry.

CD44 is a type I transmembrane glycoprotein, which is associated with poor prognosis in various solid tumors. Since CD44 plays critical roles in tumor development by regulating cell adhesion, survival, proliferation, and stemness, it has been considered a target for tumor therapy. Anti-CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody-drug conjugates and chimeric antigen receptor-T cell therapy. We previously developed anti‐pan-CD44 mAbs, C₄₄Mab-5 and C₄₄Mab-46, which can recognize both CD44 standard (CD44s) and variant isoforms. In the present study, a defucosylated mouse IgG_2a version of the anti-pan-CD44 mAbs (5-mG_2a-f and C₄₄Mab-46-mG_2a-f) was generated to evaluate the antitumor activities against CD44-positive cells. Both 5-mG_2a-f and C₄₄Mab-46-mG_2a-f recognized CD44s-overexpressed CHO-K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5-mG_2a-f and C₄₄Mab-46-mG_2a-f could activate effector cells in the presence of CHO/CD44s cells and exhibited the complement‐dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5-mG_2a-f and C₄₄Mab-46-mG_2a-f significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control defucosylated mouse IgG_2a. These results indicate that 5-mG_2a-f and C₄₄Mab-46-mG_2a-f could exert antitumor activities against CD44-positive cancers and could be a promising therapeutic regimen for tumors.

Vascular cell adhesion molecule-1 (VCAM-1) is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung, and high VCAM-1 expression correlated with poor survival of lung cancer patients. VCAM-1 knockdown reduced invasion in A549 human lung cancer cells, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6) demonstrated that the VCAM-1-D6 domain was critical for VCAM-1-mediated A549 cell invasion. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab), which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed invasion in A549 and NCI-H1299 lung cancer cell lines. Taken together, these results suggest that VCAM-1-D6 is a novel therapeutic target in VCAM-1-mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.

Objective: Spontaneous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after mRNA vaccination causes a marked increase in antibody titer because of the combined effect of vaccine and infection (“hybrid immunity”). In this study, we discuss the significance of the mRNA vaccine booster inoculation that has been repeatedly performed in Japan.Methods: We describe the temporal trends of antibody titers in cases in which antibody titers were markedly increased by hybrid immunization.Results: The antibody titer increased with hybrid immunization and tended to decrease with time. However, several cases maintained high antibody titers for approximately one year after coronavirus disease 2019 (COVID-19 diagnosis), even without booster vaccination.Conclusions: Most Japanese patients naturally infected with COVID-19 were infected after mRNA vaccination, and many maintained high antibody titers due to hybrid immunity. The significance of additional vaccination in hybrid-immunized cases is highly questionable regarding cost-effectiveness and risk-benefit.

This study evaluated batch fermentation modes, namely, separate hydrolysis and fermentation (SHF), Quasi-simultaneous saccharification and fermentation (Q-SSF), and simultaneous saccharification and fermentation (SSF), and fermentation conditions, i.e., enzyme and yeast loadings, nutrient supplementation and sterilization, on high titer bioethanol production from SPORL-pretreated Douglas-fir forest residue without detoxification. The result indicated Q-SSF and SSF were obviously superior to SHF operation in terms of ethanol yield. The enzyme loading showed a strong positive correlation between enzyme loading and the ethanol yield. The nutrient supplementation and sterility was not necessary for ethanol production from SPORL-pretreated Douglas-fir. The yeast loading showed no significant influence on the ethanol yield for typical SSF conditions. The terminal ethanol titer of 43.2 g/L, or 75.1% theoretical based on glucose, mannose, and xylose theoretical was achieved when SSF was conducted at the condition of following: whole slurry solids loading of 15%, enzyme loading of 20 FPU/g glucan, 1.8 g/kg (wet) yeast loading, without nutrition supplementation and sterilization, at 38°C, on shake flask at 150 rpm for 96h. It is believed that with mechanical mixing, enzyme loading can be substantially reduced with affect ethanol yield by using a long fermentation time.

Determination of antibody titre of dogs vaccinated against canine distemper in Jos North and South local Government Areas of Plateau State was carried out by collection of sera of vaccinated dogs and administration of well-structured questionnaires to dog owners. The samples collected were analyzed using the immune-blot ELISA Kit to determining the antibody titre (immunoglobulin G). It indicated that dogs vaccinated against the disease mounted adequate protective immunity. The result revealed that 54 (90.0%) of the sampled dogs have protective immunity, with those given more than one dose having higher level of protective antibody. Statistically, the result showed that the antibody titre did not differ significantly in relation to immunity and sex, breed, age and location but significant difference was seen in relation to number of primary vaccination. The result also revealed that those dogs that received booster doses (secondary vaccination) had more protective antibody. The study was aimed at evaluating the antibody titre of dogs vaccinated against canine distemper in Jos, Plateau State.

Bio-molecular reagents like antibodies required in experimental biology are expensive and their effectiveness, among other things, is critical to the success of the experiment. Although such resources are sometimes donated by one investigator to another through personal communication between the two, there is no previous study to our knowledge on the extent of such donations, nor a central platform that directs resource seekers to donors. In this paper, we describe, to our knowledge, a first attempt at building a web-portal titled Bio-Resource Exchange that attempts to bridge this gap between resource seekers and donors in the domain of experimental biology. Users on this portal can request for or donate antibodies, cell-lines and DNA Constructs. This resource could also serve as a crowd-sourced database of resources for experimental biology. Further, in order to index donations outside of our portal, we mined scientific articles to find instances of donations of antibodies and attempted to extract information about these donations at the finest granularity. Specifically, we extracted the name of the donor, his/her affiliation and the name of the antibody for every donation by parsing the acknowledgements sections of articles. To extract annotations at this level, we propose two approaches – a rule based algorithm and a bootstrapped relation learning algorithm. The algorithms extracted donor names, affiliations and antibody names with average accuracies of 57% and 62% respectively. We also created a dataset of 50 expert-annotated acknowledgements sections that will serve as a gold standard dataset to evaluate extraction algorithms in the future. Contact: [email protected], [email protected] Database URL: http://tonks.dbmi.pitt.edu/brx Supplementary information: Supplementary data are available at Database online.

Our aim was to determine the secondary antibody deficiency profiles of patients in a mesoregion of São Paulo state, Brazil, focusing on infectious diseases. Demographic characteristics, clinical and laboratory data were obtained from electronic files; infections were classified as organ-specific and graded as mild, moderate, life-threatening, and fatal. Non-Hodgkin lymphoma (NHL) accounted for 30% of patients, nephrotic syndrome (NS) 25%; chronic lymphocyte leukemia 20%, and multiple myeloma 15%. Patients with NS were younger than those in other groups, and hypo-γ-globulinemia was detected in 94.1%, IgG <400 mg/dL in 60.0%, IgA <40 mg/dL in 55.0%, and CD19 <20 cells/mm³ in 30.0%. One hundred and one infections were found; 82.1% were classified as mild or moderate, 7.9% as life-threatening, and 1.9% as fatal. Respiratory tract infections were more prevalent (41.5%), and pneumonia accounted for 19.8%. Lower levels of infections were found in patients with NS compared with NHL (P=0.0001). Most patients progressed to hypo-γ-globulinemia and secondary antibody deficiency after treatment with immunosuppressants, and mild and moderate infections were predominant. These therapies are increasing in patients with different diseases, therefore monitoring hypo-γ-globulinemia and infections may help to identify patients at high risk for severe complications, antibiotic prophylaxis or treatment, and immunoglobulin replacement

Patients with predominantly antibody deficiencies (PAD) display hypogammaglobulinemia with a high prevalence of infections, along with autoimmune manifestations, benign and malignant lymphoproliferation and granulomatous disease. It is noteworthy that PAD patients, even those with defects in the same causative genes display a variable clinical phenotype, suggesting that additional genetic polymorphisms, located in either immune-related or non-immune-related genes, may affect their clinical and laboratory phenotype. In this context, we analyzed 80 PAD patients, including 70 with common variable immunodeficiency (CVID) for SERPINA1 defects, in order to investigate their possible contribution to PAD clinical phenotype. Eight CVID patients carried heterozygous pathogenic SERPINA1 defects with normal alpha-1 antitrypsin levels. Interestingly, the presence of the Z allele (rs28929474) which was found in three patients, was significantly associated with liver disease; hepatic complications were also observed in patients carrying the p.Leu23Gln (rs1379209512) and the p.Phe76del (rs775982338) alleles. Conversely, no correlation of SERPINA1 defective variants with respiratory complications was observed, although patients with pathogenic variants exhibit a reduced probability of developing autoimmune diseases. Therefore, we recommend SERPINA1 genetic analysis in PAD, in order to identify patients with a higher risk for liver disease.

Predominantly antibody deficiencies (PAD) represent the most common type of primary immu-nodeficiencies in humans, characterized by a wide variation in disease onset, clinical manifesta-tions, complications and outcome. Considering that the prevalence of PAD in Greece is unknown and there is limited knowledge on clinical and laboratory characteristics of affected patients, we conducted a nationwide study, including 153 patients (male/female: 66/87; median age: 43.0 years; range: 7.0-77.0) diagnosed and/or followed-up between April 2007 to September 2023. Furthermore, we classified our cohort into five Groups according to their medical history, im-munoglobulin levels, and CTLA4-mutational status: 123 had common variable immunodeficien-cy (CVID), 12 with CVID-like disease (with a history of a previous B-cell depletion immunother-apy for autoimmune or malignant disease), seven with combined IgA and IgG subclass deficien-cies, five with immune dysregulation syndrome and CTLA4 defects, and six with unclassified hypogammaglobulinemia. We demonstrated a remarkable delay of PAD diagnosis, several years after the onset of related symptoms (median: 9.0 years, range: 0-43.0). Family history of PAD was only 11.8%, with the majority of patients considered sporadic cases. Most patients were diag-nosed in the context of a diagnostic work-up for recurrent infections, or recurrent/resistant auto-immune cytopenias. Interestingly, ten patients (5.6%) had no history of infection, diagnosed due to either recurrent/resistant autoimmunity, or during a work-up of their medical/family history. Remarkable findings included an increased prevalence of lymphoproliferation (60.1%) and atopy (24.2%), while 39 patients (25.5%) developed bronchiectasis and 16 (10.5%) granulomatous dis-ease. Patients with CTLA4 defects displayed a variable clinical phenotype at diagnosis, with four patients diagnosed during a work-up of re-current infections, while one also displayed a massive splenomegaly. Cancer was a common complication in our cohort (25 patients, 16.3%), with B-cell malignancies representing the most common neoplasms (56.7%). Our findings indicate the necessity of awareness about PAD and their complications, aiming for early diagnosis and appropriate management of affected patients

Viral diseases represent a major public health concern and an ever-present risk for developing into a future pandemic. Antiviral antibody therapeutics, either alone or in combination with other therapies, have emerged as valuable preventative and treatment options, including during a global emergency. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.

Tilapia lake virus (TiLV), a major pathogen of farmed tilapia, is known to be vertically transmitted. Here, we hypothesize that Nile tilapia (Oreochromis niloticus) broodstock immunized with a TiLV inactivated vaccine can mount a protective antibody response and passively transfer maternal antibodies to their fertilized eggs and larvae. To test this hypothesis, three groups of tilapia broodstock, each containing 4 males and 8 females, were immunized with either a heat-killed TiLV vaccine (HKV), a formalin-killed TiLV vaccine (FKV) (both administered at 3.6 ×106 TCID50 per fish), or with L15 medium. Booster vaccination with the same vaccines was given 3-weeks later, and mating took place 1 week thereafter. Broodstock blood sera, fertilized eggs and larvae were collected from 6-14 weeks post-primary vaccination for measurement of TiLV-specific antibody (anti-TiLV IgM) levels. In parallel, passive immunization using sera from the immunized female broodstock was administered to naïve tilapia juveniles to assess if antibodies induced in immunized broodstock were protective. The results showed that anti-TiLV IgM was produced in the majority of both male and female broodstock vaccinated with either the HKV or FKV and that and that these antibodies could be detected in the fertilized eggs and larvae from vaccinated broodstock. Higher levels of maternal antibody were observed in fertilized eggs from broodstock vaccinated with HKV than those vaccinated with FKV. Low levels of TiLV-IgM were detected in some of the 1-3-day old larvae but were undetectable in 7-14-day old larvae from the vaccinated broodstock, indicating a short persistence of TiLV-IgM in larvae. Moreover, passive immunization proved that antibodies elicited by TiLV vaccination were able to confer 85% to 90% protection against TiLV challenge in naïve juvenile tilapia. In conclusion, immunization of tilapia broodstock with TiLV vaccines could be a potential strategy for the prevention of TiLV in tilapia fertilized eggs and larvae, with HKV appearing to be more promising than FKV for maternal vaccination.

The importance of antibodies in health care and the biotechnology research and development demands not only knowledge of their experimental structures at high resolution, but also practical implementation of this knowledge for both effective and efficient design and production of antibody for its use in both medical and research applications. While the experimental wet-lab approach is usually costly, laborious and time-consuming, computational (dry-lab) approaches, in spite of their intrinsic limitations in comparison with its experimental (wet-lab) counterpart, provide a cheaper and faster alternative option. For the first time, this article reports a comprehensive set of structural electrostatic features extracted from experimentally determined antigen-antibody-related structures, including especially those structural electrostatic features at the interfaces of all experimentally determined antigen-antibody complex structures as of February 29, 2020, to facilitate effective and efficient machine learning-based computational antibody design using currently available experimental structures inside Protein Data Bank.

Antibody-Drug Conjugates (ADCs) are a powerful therapeutic modality for cancer treatment. ADCs are multi-functional biologics in which a disease-targeting antibody is conjugated to an effector payload molecule through a linker. The success of currently used ADCs has been largely attributed to the development of linker systems which allow targeted release of cytocidal payload drugs inside cancer cells. Many lysosomal proteases are over-expressed in human cancers. They can effectively cleave a variety of peptide sequences, which can be exploited in the design of ADC linker systems. As a well-established linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC) is used in many ADCs that are already approved or under preclinical and clinical development. Although ValCitPABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma, many studies have shown that they are susceptible to carboxylesterase 1C (Ces1C) in mouse and rat plasma, which hinders preclinical evaluation of the ADCs. Furthermore, neutropenia and thrombocytopenia, two of the most commonly observed dose-limiting adverse effects of ADCs, are believed to result from the premature hydrolysis of ValCitPABC by human neutrophil elastase. In addition to cathepsin-cleavable linkers, there is also growing interest in legumain-sensitive linkers for ADC development. Increasing plasma stability while maintaining lysosomal cleavability of ADC linkers is an objective of intensive current research. This review reports recent advances in the design and structure-activity relationship studies of various peptide/peptidomimetic linkers in this field.

We recently reported the isolation and characterization of an anti-SARS-CoV-2 antibody, called IgG-A7, that protected transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from the infection with SARS-CoV-2 Wuhan. We show here that IgG-A7 protected 100% of the transgenic mice infected with Delta (B.1.617.2) and Omicron (B.1.1.529) at doses of 0.5 and 5 mg/kg, respectively. In addition, we studied the pharmacokinetic (PK) profile and Toxicology (Tox) of IgG-A7 in CD-1 mice at single doses of 100 and 200 mg/kg. PK parameters at those high doses were proportional to the dose, with the half-life in serum of ~10.5 days. IgG-A7 was well tolerated with no signs of toxicity in urine and blood samples, nor in histopathology analyses. Tissue Cross-reactivity (TCR) with a panel of mouse and human tissues showed no evidence of IgG-A7 interaction with tissues of these species, supporting the PK/Tox results in vivo and suggesting that while IgG-A7 has a broad efficacy profile it is not toxic in humans. The information generated in CD-1 mouse as PK/Tox model, complemented with the mouse and human TCR, could be of relevance as alternatives to NHPs in rapidly emerging viral diseases and/or quickly evolving viruses such as SARS-CoV-2.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people and caused tremendous morbidity and mortality worldwide. Effective treatment for coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 infection is lacking and different therapeutic strategies are under testing. Host humoral and cellular immunity to SARS-CoV-2 infection is a critical determinant for patients’ outcome. SARS-CoV-2 infection results in seroconversion and production of anti-SARS-CoV-2 antibodies. The antibodies may suppress viral replication through neutralization but also might also participate in COVID-19 pathogenesis through a process termed antibody-dependent enhancement. Rapid progress has been made in the research of antibody response and therapy in COVID-19 patients including characterization of the clinical features of antibody responses in different populations infected by SARS-CoV-2, treatment of COVID-19 patients with convalescent plasma and intravenous immunoglobin products, isolation and characterization of a large panel of monoclonal neutralizing antibodies, as well as preliminary clinical results from several COVID-19 vaccine candidates. In this review, we summarize the recent progress and discuss the implications of these findings in vaccine development.

Monoclonal antibody (mAb) therapy has revolutionized the treatment of various diseases, including cancer and autoimmune disorders. However, the emergence of SARS-CoV-2 variants, as well as other pathogenic variants, poses challenges in maintaining the therapeutic efficacy of mAbs. In this mini review article, we aim to explore the strategies to overcome variants resistance in mAb therapy against viral infections. Firstly, we discuss the mechanisms through which variants can evade the neutralizing effects of mAbs. Understanding these mechanisms is crucial in developing targeted approaches to combat resistance. Next, we delve into the strategies being pursued to address variants resistance. These include developing new mAbs or antibody cocktails that target multiple epitopes, engineering mAbs with improved binding affinities or enhanced neutralizing capabilities, and exploring alternative therapeutic modalities such as bispecific antibodies or antibody-drug conjugates. Furthermore, we highlight the role of computational modeling and artificial intelligence in predicting variant escape mutations and aiding in the design of more effective mAbs. Additionally, we examine the importance of continuous surveillance and monitoring of emerging variants to inform treatment strategies. This article emphasizes the urgent need for proactive approaches to tackle variants resistance in mAb therapy. By combining innovative design strategies, computational modeling, and vigilant surveillance, we can maintain the therapeutic effectiveness of mAbs and stay ahead in the battle against evolving pathogens and viral infections.

Acute ischemic strokes are the third leading cause of death and the leading cause of neurological disability worldwide. The oxygen and glucose deprivation associated with ischemic strokes not only leads to neuronal cell death, but also increases the inflammatory response and decreases functional output of the brain. The only intervention approved by US Federal Drug and Food Administration for treatment of ischemic strokes is tissue plasminogen activator (tPA), however, such treatment can only be given within 4.5 hours of the onset of stroke-like symptoms. This narrow time-range limits its application, and it also might induce detrimental rather than beneficial effects to stroke patients by treatment of the tPA. In order to reduce the infarct volume of an acute ischemic stroke while increasing the time period for treatment, emerging therapies reveal great potential by targeting inflammation, growth factors, ion channels, and neurotransmitter receptors with monoclonal antibody (MAB). With successfully application in the treatment of cancer patient by MAB, in this review, we will focus on recent advances on stroke therapy by using MAB on the treatment of stroke by targeting inflammation, growth factors, ion channels, and neurotransmitter receptors. Therefore, developing specific MAB targeting the signaling pathway of stroke will contribute to stroke therapy.

Currently, hepatitis B virus (HBV) core antibody (anti-HBc antibody) and HBV core-related antigen (HBcrAg) are widely used as serum markers for diagnosis based on the HBV core region. While anti-HBc is very significant for clinical diagnosis, the clinical significance of quantitative assay of anti-HBc antibody has been reevaluated with improvements in diagnostic performance, including its association with clinical stage and prediction of carcinogenesis and reactivation. In addition, concerning the new HBcrAg, a high-sensitivity assay method has recently been established, and its diagnostic significance, including the prediction of reactivation, is being reevaluated. On the other hand, the quantitative level of anti-HBc antibody expressed in different units among assay systems, complicating the interpretation of the results. However, it is difficult to standardize assay systems as they vary in advantages, and caution is needed in interpreting the assay results. In this review, the clinical usefulness and remaining problems of anti-HBc antibody and HBcrAg were summarized.

Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.

Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers with different charges through different linking strategies; “Click” conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and “Lysine” conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 antibody, named H₂Mab-250/H₂CasMab-2 (IgG₁, kappa). H₂Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H₂Mab-250 did not react with non-transformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs including H₂Mab-119 (IgG₁, kappa) reacted with both cancer and normal epithelial cells. Furthermore, a core-fucose deleted IgG_2a-type H₂Mab-250 (H₂Mab-250-mG_2a-f) could trigger the antibody-dependent cellular cytotoxicity activity to BT-474, but not to HaCaT cells. Furthermore, H₂Mab-250-mG_2a-f exhibited an in vivo antitumor effect against BT-474 xenograft. Immunohistochemical analysis demonstrated that H₂Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared to H₂Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H₂Mab-250. H₂Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel anti-giraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method. PMab-301 (mouse IgG₁, kappa) detected girPDPN in various applications, such as flow cytometry, western blot, and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffin-embedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues.

Detection of virus in sample is an actual problem in biotechnology, virology, medicine using biosensor. History of problem dates back to middle of last century and is associated with development of an ion-sensitive solid-state device for neurophysiological measurements. Aim of work is theoretical investigation process of detection antibody+antigen (AB+AG) complexes using SOI-FET biosensor. On example of crystal with ten biosensors, estimates of probability detection AB+AG complexes influenza virus are made. In detection process, following four results are possible: – target complex is present in test sample, and it was correctly detected; – target complex is present in sample, but it was not detected; – target complex is absent in sample, but background particles are mistakenly detected as target complex; – target complex is absent in sample, background particles present in sample and are correctly detected as noise.

Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID.

This is a SARS-CoV-2 seroepidemiological study in pediatric population(0-16 years) during BA.5 Omicron predominance period in Athens metropolitan area. Serum samples were tested for SARS-CoV-2 nucleocapsid antibodies(Abs-N), representing natural infection during three sub-periods of BA.5 predominance: 01/05/2022-31/08/2022(period A), 01/09/2022-31/12/2022(period B) and July 2023(period C) and epidemiological data were collected. Additionally in period C Abs-N seronegative samples were tested for SARS-CoV-2 spike antibodies(Abs-S). A total of 878 children were tested (males:51.3%) with median age(IQR): 108(36-156) months and seropositivity during the 3 subperiods was: A:292/417(70%), B:288/356(80.9%), C:89/105(84.8%), P-value<0.001. SARS-CoV-2 seropositivity increased for from period A to C for 0-1-year (P-value: 0.044), 1-4 years (P-value: 0.028) and 6-12 years children (P-value: 0.003). Children 6-12y had the highest seroposi-tivity rates in all subperiods (A: 77.3%, B: 91.4% and C: 95.8%). A significant correlation of monthly SARS-CoV-2 median antibody titers with monthly seropositivity rates was detected (rs:0.812, P-value:0.008). During period C, an additional 12/105 (11.4%) Abs-S seropositive and Abs-N ser-onegative samples were detected and total seropositivity was estimated at 96.2% (101/105). The increased SARS-CoV-2 seropositivity detected in current seroepidemiology study illustrates a high exposure rate during BA.5 predominance period. These data could guide public health decisions regarding immunization strategies and protection measures.

LR004 is a novel chimeric (human/mouse) monoclonal antibody developed for the treatment of advanced colorectal carcinoma with detectable epidermal growth factor receptor (EGFR) expression. We aimed to investigate the preclinical pharmacokinetics (PK) and in vivo biodistribution of LR004. The PK profiles of LR004 were initially established in rhesus monkeys. Subsequently, 125I radionuclide-labeled LR004 was developed and its binding activity to the EGFR antigen were evaluated. Furthermore, the biodistribution, autoradiography, and NanoSPECT/CT of 125I-LR004 in xenograft mice bearing A431 tumors were examined. The PK data revealed a prolonged half-life and nonlinear PK characteristics of LR004 within the dose range of 6–54 mg/kg. The radiochemical purity of 125I-LR004 was approximately 98.54%, and iodination of LR004 did not affect its specific binding activity to the EGFR antigen. In a classical biodistribution study, 125I-LR004 exhibited higher uptake in highly perfused organs than in poorly perfused organs. Prolonged retention properties of 125I-LR004 in tumors were observed at 4 and 10 days. Autoradiography and NanoSPECT/CT confirmed the significant accumulation and sustained retention of 125I-LR004 at the tumor site in xenograft mice. These findings demonstrated the adequate tumor targeting capabilities of 125I-LR004 in EGFR-positive tumors, which may improve dosing strategies and future drug development.

New coronavirus (SARS-CoV-2) treatments and vaccines are under development to combat the COVID-19 disease. Several approaches are being used by scientists for investigation including 1) various small molecule approaches targeting RNA polymerase, 3C-like protease, and RNA endonuclease and 2) exploration of antibodies obtained from convalescent plasma from patients who have recovered from COVID-19. The coronavirus genome is highly prone to mutations that lead to genetic drift and escape from immune recognition; thus, it is imperative that sub-strains with different mutations are also accounted for during vaccine development. As the disease has grown to become a pandemic, new B-cell and T-cell epitopes predicted from SARS coronavirus have been reported. Using the epitope information along with variants of the virus, we have found several variants which might cause drifts. Among such variants, 23403A>G variant (p.D614G) in spike protein B-cell epitope is observed frequently in European countries such as the Netherlands, Switzerland and France.

Background: In 80% of patients, COVID-19 presents as mild disease^1,2. 20% of cases develop severe (13%) or critical (6%) illness. More severe forms of COVID-19 present as clinical severe acute respiratory syndrome, but include a T-predominant lymphopenia³, high circulating levels of proinflammatory cytokines and chemokines, accumulation of neutrophils and macrophages in lungs, and immune dysregulation including immunosuppression⁴. Methods: All major SARS-CoV-2 proteins were characterized using an amino acid residue variation analysis method. Results predict that most SARS-CoV-2 proteins are evolutionary constrained, with the exception of the spike (S) protein extended outer surface. Results were interpreted based on known SARS-like coronavirus virology and pathophysiology, with a focus on medical countermeasure development implications. Findings: Non-neutralizing antibodies to variable S domains may enable an alternative infection pathway via Fc receptor-mediated uptake. This may be a gating event for the immune response dysregulation observed in more severe COVID-19 disease. Prior studies involving vaccine candidates for FCoV^5,6 SARS-CoV-1^7-10 and Middle East Respiratory Syndrome coronavirus (MERS-CoV) ¹¹ demonstrate vaccination-induced antibody-dependent enhancement of disease (ADE), including infection of phagocytic antigen presenting cells (APC). T effector cells are believed to play an important role in controlling coronavirus infection; pan-T depletion is present in severe COVID-19 disease³ and may be accelerated by APC infection. Sequence and structural conservation of S motifs suggests that SARS and MERS vaccine ADE risks may foreshadow SARS-CoV-2 S-based vaccine risks. Autophagy inhibitors may reduce APC infection and T-cell depletion^{12 13}. Amino acid residue variation analysis identifies multiple constrained domains suitable as T cell vaccine targets. Evolutionary constraints on proven antiviral drug targets present in SARS-CoV-1 and SARS-CoV-2 may reduce risk of developing antiviral drug escape mutants. Interpretation: Safety testing of COVID-19 S protein-based B cell vaccines in animal models is strongly encouraged prior to clinical trials to reduce risk of ADE upon virus exposure.

The target of an antibody plays a significant role in the success of antibody-based therapeutics and diagnostics, and to an extent, that of vaccine development. This importance is focussed on the target binding site – epitope, where epitope selection as a part of design thinking beyond traditional antigen selection using whole cell or whole protein immunisation can positively impact success. With purified recombinant protein production and peptide synthesis to display limited/selected epitopes, intrinsic factors that can affect the functioning of resulting antibodies can be more easily selected for. Many of these factors stem from the location of the epitope that can affect accessibility of the antibody to the epitope at a cellular or molecular level, direct inhibition of target antigen activity, conservation of function despite escape mutations, and even non-competitive inhibition sites. Through the incorporation of novel computational methods for predicting antigen changes to model-informed drug discovery and development, superior vaccines and antibody-based therapeutics or diagnostics can now be more easily designed to mitigate failures. With detailed examples, this review highlights the new opportunities, factors and methods of predicting antigenic changes for consideration in sagacious epitope selection.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors, and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, anti-CCR1 therapeutic agents have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C₁Mab-6 (rat IgG_2b, kappa), using the Cell-Based Immunization and Screening (CIBS) method. Flow cytometry and Western blot analyses showed that C₁Mab-6 recognizes mCCR1 specifically. The dissociation constant of C₁Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9×10^-9 M, indicating that C₁Mab-6 possesses a high affinity to mCCR1. These results suggest that C₁Mab-6 could be a useful tool for targeting CCR1 in preclinical mouse models.

Antibody-drug conjugates (ADCs) have provided new therapeutic options and significant promise for patients with cancers, particularly where existing treatments are limited. Substantial effort in ADC development is underway globally, with 13 ADCs currently approved and many more in development [1]. Therapeutic benefits of ADCs leverage the ability to selectively target cancer cells through antibody binding, resultant relative sparing of non-malignant tissues, and the targeted delivery of a cytotoxic payload. Consequently, this drug class has demonstrated activity in multiple malignancies refractory to standard therapeutic options [1-4]. Despite this, limitations exist, including narrow therapeutic windows, unique toxicity profiles, development of therapeutic resistance, and appropriate biomarker selection [5-7]. This review will describe the development of ADCs, their mechanisms of action, pivotal trials, and approved indications and identify common themes. Current challenges and opportunities will be discussed for this drug class in cancer therapeutics at a time when significant developments in antibody therapies, immunotherapy and targeted agents are occurring.

Regulatory T cells (Tregs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. Tregs are characterized by high expression of CD25, which is a potentially valuable target for Treg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity, due to its capacity to clear Tregs from circulation and lymph nodes but not those that reside in the tumor. Optimization of the Fc domain of this antibody clone has been shown to enhance intratumoral Treg depletion capacity. Here, we generated a stable cell line that produces optimized recombinant Treg depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from the original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model the resulting αCD25-m2a antibody mediated effective depletion of tumor resident Tregs leading to a high effector T cell (Teff) to Treg ratio. Moreover, combination of the αCD25-m2a with αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10-8 M and 3.2 × 10-8 M, respectively. In western blot analysis, recEpMab-37 detected EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by CBIS method, is useful for detecting EpCAM in various applications.

Since the discovery of the human epidermal growth factor receptor 2 (HER2) as an oncogenic driver in a subset of breast cancers and the development of HER2 directed therapies, the prognosis of HER2 amplified breast cancers has increased meaningfully. Next to monoclonal anti-HER2 antibodies and tyrosine kinase inhibitors, the antibody-drug conjugate T-DM1 is a pillar of targeted treatment of advanced HER2-positive breast cancers. Currently, several HER2 directed antibody-drug conjugates are under clinical investigation for HER2 amplified but also HER2 expressing but not amplified breast tumors. In this article, we review the current preclinical and clinical evidence of the investigational drugs A166, ALT-P7, ARX788, DHES0815A, DS-8201a, RC48, SYD985, MEDI4276 and XMT-1522.

Viruses can infect a cell via one or both routes viz. cell-to-cell (c-c) or cell-free (c-f) . Pathogenesis studies of various viruses, including HIV, have shown that c-c transmission yields a significantly higher infection magnitude than the c-f route. Expectedly, potent antibodies inhibited c-f infection more efficiently than with c-cell transmission. To achieve a one-step, synchronous infection cycle that provides amplified infection, we have studied a consistent and efficient c-c HIV infection model since 1992. H9 cells persistently infected with HTLV-IIIB (H3B cells) and uninfected target CD4+ lymphocyte line (HuT78) were mixed in a ratio of 1:4 respectively. We have recently used this model to produce HIV designer antigens that have been shown to elicit monoclonal as well as polyclonal specific antibodies against novel epitopes that are formed post virus-cell engagement, but prior to fusion. The model can be extended for HIV neutralizing antibody assays or drug inhibitors against high multiplicity of infection.

A canine population’s immune resistance to canine distemper virus (CDV), canine parvovirus (CPV), and infectious hepatitis virus (CAV-1) was evaluated. In this study, a total of 112 sera were analyzed. Animals were considered as vaccinated if, in the last two years, they had re-ceived at least one dose of a vaccine that provides joint protection against CDV, CPV, and CAV-1. Animals that had never received any dose of these vaccines were designated as non-vaccinated. CDV, CPV, and CAV-1 antibodies were detected via a modified solid-phase enzyme-linked im-munosorbent assay (ELISA), which detects IgG antibody levels in sera and provides semi-quantitative results in less than 30 min. In total, 41.1% of the dogs had been vaccinated, and 58.9% of dogs were designated as non-vaccinated. Overall, 90.2%, 92.0%, and 78.6% of the tested dogs had positive results for the presence of IgG antibodies against CPV, CDV, and CAV-1, re-spectively. CPV antibodies were present in 93.5% (43/46) of the vaccinated and 87.9% (58/66) of the non-vaccinated dogs, while CDV antibodies were present in 95.5% (63/66) of the vaccinated and 87.0% (40/46) of the non-vaccinated dogs. Finally, CAV-1 antibodies were present in 84.8% (56/66) of the vaccinated and of 69.6% (32/46) the non-vaccinated dogs.

Antibody-drug conjugates (ADCs), a groundbreaking advancement in cancer therapy, synergistically combine the precision of monoclonal antibodies with the potent destruction capabilities of cytotoxic drugs, signifying a new era in the targeted oncological treatments. This comprehensive paper delves into the evolving field of antibody-drug conjugates (ADCs) and their pivotal role in cancer therapy. We have conducted an in-depth exploration of the structural components of ADCs, including target antigens, monoclonal antibodies, chemical linkers, and cytotoxic payloads, each playing a crucial role in determining the efficacy and safety profile of these novel therapies. A detailed market analysis reveals the leading pharmaceutical companies shaping the ADC landscape, reflecting the growing interest and potential in this therapeutic approach. We further dissect the engineering intricacies of ADCs, focusing on their ability to target specific cancer cell antigens while minimizing off-target effects, thus offering a more focused treatment with reduced systemic side effects compared to traditional chemotherapy. This approach holds promise in a heightened therapeutic index and decreased toxicity. The mechanisms and effector functions of ADCs are explored, highlight- ing their multifaceted strategy in combating cancer. We include a comprehensive look at how ADCs operate from initial blood circulation to the induction of apoptosis in cancer cells. We also review several key ADCs currently in use or under investigation, offering insights into their unique mechanisms of action, pharmacokinetics, and clinical implications. The discussion extends to drug creation modalities, focusing on innovative conjugation techniques that enhance ADC homogeneity and therapeutic efficacy. In conclusion, the paper underscores the transformative potential of ADCs in the realm of cancer treatment, marking a shift towards more precise and targeted therapeutic approaches. By offering a thorough analysis of ADC technology, this paper sheds light on the prospects of these groundbreaking therapies in the ongoing fight against cancer.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular ATP into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment (TME) attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (mCD39) mAb, C₃₉Mab-1 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of C₃₉Mab-1 using flow cytometry. We performed the PA tag (12 amino acids)-substituted analysis (named PA scanning) and RIEDL tag (5 amino acids)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C₃₉Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275^th to 279^th, 282^nd to 291^st, and 306^th to 323^rd amino acids of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

To increase the prediction accuracy of positive oral food challenge (OFC) outcome during stepwise slow oral immunotherapy (SS-OIT) in children with hen’s egg (HE) allergy, we evaluated the predictive value of the combination of antigen-specific IgE (sIgE) with antigen binding-avidity and sIgG4 values. Sixty-three children with HE allergy undergoing SS-OIT were subjected to repeated OFC with HE. We measured the ovomucoid (OVM)-sIgE by ImmunoCAP or densely carboxylated protein (DCP) microarray, sIgG4 by DCP microarray, and the binding-avidity of OVM-sIgE defined as the level of 1/ IC50 (nM) measured by competitive binding inhibition assays. The OFC was positive in 37 (59%) patients undergoing SS-OIT. Significant differences in DCP-OVM-sIgE, CAP-OVM-sIgE, I/ IC50, DCP-OVM-sIgG4, the multiplication products of DCP-OVM-sIgE and binding-avidity of DCP-OVM-sIgE (DCP-OVM-sIgE/IC50) and DCP-OVM-sIgE/sIgG4 were compared between the negative and positive groups (p<0.01). Among them, the variable with greatest area under the receiver operating characteristic curve was DCP-OVM-sIgE/IC50 (0.84), followed by DCP-OVM-sIgE/sIgG4 (0.81). DCP-OVM-sIgE/IC50 and DCP-OVM-sIgE/sIgG4 are potentially useful markers for the prediction of positive OFC during HE-SS-OIT and may allow proper evaluation of the current allergic status in the healing process during HE-SS-OIT

Advances in the treatment of cancer with immunotherapy necessitate a locally administered sustained-delivery system to improve treatment efficacy and reduce toxicity. Towards this end, the encapsulation and release characteristics of human antibody immunoglobulin-G (IgG-FITC) were investigated with the coaxial electrospray technique. The antibody solution was exchanged for a solution at the antibody’s isoelectric pH. The shell solution consisted of PLGA in ethyl acetate. The impact of shell flow rates was investigated in the fabrication of IgG-FITC-loaded PLGA microparticles and the release-profile of IgG-FITC from the PLGA microparticles was characterized. Encapsulation efficiency ranged from 0.04-1.52%. Fluorescence images suggest core localization in the formulation with a lower shell flow rate and shell localization in the formulation with the higher shell flow rate. SEM image analysis revealed that lower shell flow rate resulted in the smaller average particle size with minimal difference in zeta potential between formulations. The lower shell flow rate exhibited a release profile with a t1/2 of 36 hours whereas the higher shell flow rate had a t1/2 &lt; 3 hours. Antibody-fluorescence signals were measured for 28 days and indicated a biphasic release profile with a burst-release followed by a sustained-release in both formulations.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human rotavirus-specific immunity, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 has been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb; Cx₁Mab-1 (rat IgG₁, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Cx₁Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx₁Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6×10^-9 M and 2.1×10^-8 M, respectively. Furthermore, Cx₁Mab-1 could detect mCXCR1 by western blot analysis. These results indicated that Cx₁Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 signaling induces chemotaxis of immune cells to inflammation sites and promotes inflammation in inflammatory diseases. Various mouse models to mimic the pathogenesis of each disease have been developed to understand mechanisms and evaluate therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx₃Mab-4 (rat IgG₁, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx₃Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx₃Mab-4 was determined as 1.3 × 10^-9 M, indicating that Cx₃Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx₃Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

CD44 has been known as a marker of tumor initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in malignant progression of cancers by promoting the stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and establishment of the therapy. However, the function of the variant 4-encoded region has not to be elucidated. Therefore, specific monoclonal antibodies (mAbs) against the variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) monoclonal antibodies (mAbs) by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterized them. One of the established clones (C₄₄Mab-108; IgG₁, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The K_D of C₄₄Mab-108 for CHO/CD44 v3-10 was 3.4 × 10⁻⁷ M. In western blot analysis, C₄₄Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C₄₄Mab-108 stained formalin-fixed paraffin-embedded oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C₄₄Mab-108 is useful to detect CD44v4 in various applications.

The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.

Although there are extensive literature reports on the use of gold nanoparticle (AuNP) based homogeneous assays for detection of biomolecules, very few experimental description and procedures involving their preparation are described. In this study, AuNPs conjugated to Bovine Serum Albumin or Envelope protein from Dengue II were developed as a homogeneous immunoassay for antibody detection. We report here optimization of key parameters to prepare an immunoassay like conjugation protein concentration, centrifugation time, electrolyte addition and assay temperature. We determined that saturating protein concentrations improved AuNPs surface coverage and uniformity of the assay and addition of sodium chloride improved sensitivity of the antibody detection method and assay stability. Furthermore, we showed that dynamic light scattering can be used to monitor changes in gold nanoparticles in the preparation and detection steps. Additionally, numerical simulations of the plasmonic optical response of AuNPs were carried out to scan for size-dependent response of the AuNPs. The AuNPs homogeneous immunoassay developed was further used in the detection of antibodies in vitro to detect Dengue virus infection.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but severe adverse event that was first observed during the global vaccination campaign against SARS-CoV-2 infection, specifically in those receiving adenoviral vector-based vaccines for Coronavirus disease 2019 (COVID-19). VITT develops 4 to 42 days post-vaccination and is characterized by the development of platelet-activating anti-platelet factor 4 (PF4) antibodies, leading to thrombocytopenia and thrombosis at unusual sites. The rise in awareness and subsequent prompt recognition of VITT was paramount in reducing mortality. Moreover, as vaccination campaigns around the world continue, a better understanding of VITT not only has important clinical implications but is also crucial for future vaccine development. In this review, we summarize the clinical features, pathophysiology, and incidence rates of VITT as well as highlight other anti-PF4 antibody-mediated disorders of growing clinical significance.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wildtype subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers by a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as target, yielding synthetic multienzyme-complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was achieved also on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable, TVCVPA-assisted electrochemical biosensing through modularly IgG-assembled sensor enzymes.

Data on immunogenicity, immune response persistency, and safety of COVID-19 boosters in patients with comorbidities are limited. Therefore, we aimed to evaluate and compare three different boosters in individuals who received two doses of the BBIBP-CorV vaccine including underlying diseases and healthy cases as control. One hundred forty subjects including 63 ones with at least one of three underlying diseases (UD) (including obesity, hypertension, and diabetes mellitus) and 77 healthy ones (HC) who had received a booster dose (either PastoCovac Plus or PastoCovac or BBIBP-CorV) were enrolled. The presence of SARS-CoV-2 antibodies was assessed before the booster injection and 28, 60, 90, and 180 days after it. Moreover, the adverse events (AEs) were recorded on days 7 and 21 post-booster shot for evaluating safety outcomes. Significantly increased titers of anti-spike, anti-RBD, and neutralizing antibodies were observed in both UD and HC groups 28 days after the booster dose, although the titer rise of anti-spike IgG and anti-RBD IgG was insignificantly inferior in the UD group compared to the HC group. All antibodies’ titers declined, with no significant differences between the two groups over time. Notably, all specific antibodies persisted up to 180 days; particularly the neutralizing antibody in both groups. Furthermore, no significant difference in antibody levels was observed between each UD subgroup and the HC group, except for neutralizing antibodies in the hypertension sub-group. PastoCovac Plus and PastoCovac boosters induced higher antibodies’ fold rise in UD individuals than BBIBP-CorV booster recipients. Safety outcomes did not show any serious AEs after the booster injection. The overall incidence of AEs post the booster injection was higher in the UD group than the HC group. Furthermore, the highest systemic AEs rate was reported in the UD group receiving the BBIBP-CorV booster. In conclusion, administration of COVID-19 boosters can equally induce robust and persistent humoral immune responses in individuals with or without UD primarily vaccinated with 2-doses of the BBIBP-CorV. Protein-based boosters with higher antibodies' fold rise and lower AEs in individuals with comorbidities might be considered a better choice for these individuals.

Heat shock proteins (Hsps) are highly conserved molecules that are constitutively expressed and upregulated in response to physiological stress conditions. These immunogenic chaperones can have essential functions in fungi, particularly in dimorphic pathogens. Histoplasma capsulatum and Paracoccidioides species are dimorphic fungi that are the causative agents of histoplasmosis and paracoccidioidomycosis, respectively, which are systemic mycoses with significant rates of morbidity and mortality. Current treatment consists of long-term antifungal agents, and there is an urgent need for new therapeutic approaches with higher efficacy, lower toxicity, better biodistribution and improved selectivity. We engineered an immunoglobulin G1 (IgG1) isotype chimeric mouse-human monoclonal antibody, titled ch-MAb 4E12, from the parental IgG2a MAb 4E12, a monoclonal antibody to H. capsulatum Hsp60 that is protective in experimental histoplasmosis and paracoccidioidomycosis models elicited by H. capsulatum var. capsulatum and Paracoccidioides lutzii, respectively. The ch-MAb 4E12 increased phagolysosomal fusion and enhanced the yeasts uptake by PMA differentiated human THP1 macrophage cells in vitro. At low concentrations, the chimeric antibody significantly reduced the pulmonary and splenic fungal burden compared to an irrelevant antibody or no treatment. These results are the first to show that a chimeric mouse-human antibody can modify infection caused by dimorphic fungi.

Corona viruses hijack human enzymes to assembly sugar coat on Spike glycoproteins. The mechanism that human antibodies may uncover the antigenic viral peptide epitopes hidden by sugar coat are unknown. In this study, we analyzed recombinant SARS-CoV-2 Spike protein secreted from BTI-Tn-5B1-4 cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired MS/MS spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of Spike proteins according to Cryo-EM and homolog-modeled structures, and available antibodies that bind to SARS-CoV-1. The results showed that all 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose type of N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the Spike receptor binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains included those located on Central Helix, between amino acids 967 and 1016 of SARS-CoV-1, and 985 to 1034 of SARS-CoV-2 Spike protein. As the majority of antibody paratopes bind to peptide portion with or without sugar modification, we propose a snake-catcher model that a minimal length of peptide is first clamped by a paratope, and the binding is either strengthened by sugars close to peptide, or not interfered by sugar modification.

The first exposure to influenza is thought to impact subsequent immune responses later in life. The consequences of this can be seen during influenza epidemics and pandemics with differences in morbidity and mortality for different birth cohorts. There is a need for better understanding of how vaccine responses are affected by early exposures to influenza viruses. In this analysis of hemagglutination inhibition (HI) antibody responses in two cohorts of military personnel we noticed differences related to age, sex, prior vaccination, deployment and birth year. These data suggest that HI antibody production, in response to influenza vaccination, is affected by these factors. The magnitude of this antibody response is associated with, among other factors, the influenza strain that circulated following birth.

Rocky Mountain or Brazilian spotted fever, caused by Ricketsia ricketssi, is a fulminant, seasonal, and neglected disease that occurs in focal points of North America and South America. Rapid detection are essential for a better prognosis and survival rate of the infected individual. However, disease diagnosis still faces challenges as the accuracy of many of the available laboratory tests fluctuates. Therefore, this review aimed to identify methods for antibody or antigen detection, its gaps, and evolution over time. A search was conducted to find all studies in the Pubmed database that described antibody or antigen detection for Ricketsia ricketsii infection. Initially, a total of 403 articles were screened. Of these articles, only 19 fulfilled the pre-established inclusion criteria and were selected. Among the different methods applied, the IFA technique was the one most frequently found in the studies. However, it presented varied results such as low specificity using the indirect method. Other techniques, such as ELISA and immunohistochemistry, were also found, although in smaller numbers and with their own limitations. Although some studies showed promising results, there is a pressing need to find new techniques to develop a rapid and effective Ricketsia ricketssi infection diagnosis.

We previously developed a highly sensitive and specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa). In this study, we produced a humanized IgG1 version (humLpMab-23) and its defucosylated form (humLpMab-23-f) of an anti‑PDPN mAb to potentiate the ADCC activity. The humLpMab-23 could recognize PDPN‑overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN) and PDPN-positive PC-10 and LN319 cells by flow cytometry. Furthermore, we found that humLpMab-23-f exerted ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10 and LN319 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that humLpMab-23-f could be useful for an antibody treatment regimen for PDPN-positive human cancers.

Background and Objectives: Treatment for antineutrophil cytoplasmic antibody-associated vasculitis (AAV) must deal with immunosuppression as well as infections associated with compromised immune system, such as tuberculosis (TB). Our aim was to overcome the gap in the literature concerning the risk of incidental TB after diagnosis of AAV. Materials and Methods: This retrospective population-based cohort study was based on the data from the National Health Insurance Research Database in Taiwan. We used a novel algorithm to identify patients with newly diagnosed granulomatous polyangiitis (GPA) or microscopic polyangiitis (MPA) between January 1, 2000 and December 31, 2012. The primary outcome was risk of incidental TB. Cox proportional hazard models were used to evaluate the association between AAV and incidental TB. Results: A total of 2,257 patients with AAV and a propensity-score matched cohort of 9,028 patients were studied. Overall, patients with AAV were at a 1.48x higher risk of contracting incidental TB than were patients in the matched cohort (adjusted HR 1.48; 95% confidence interval [CI], 1.02-2.15). Note that the highest risk of contracting incidental TB was in the first two years following a diagnosis of AAV, with a nearly 1-fold increase in risk (adjusted HR, 1.91; 95% CI, 1.01-3.60). Female AAV patients were 3.24x more likely than females without AAV to develop TB (adjusted HR 3.24; 95%CI, 1.85-5.67). Conclusion: Patients with AAV face an elevated risk of contracting incidental TB, particularly within the first two years after AAV diagnosis. The risk of contracting TB is higher among female AAV patients than among females without AAV.

Background: Vaccination is one of the most effective life-saving medical interventions, and the introduction of SARS-CoV-2 vaccines was intended to prevent the serious implications of COVID-19. The objectives of the study were: i) to observe the humoral immune response to BNT162b2 vaccine and SARS-CoV-2 infection (mainly breakthrough infections), ii) to demonstrate the persistence of anti-SARS-CoV-2 antibodies over time in relation to the number of received vaccine doses and the course of infection, and iii) to determine adverse effects after primary vaccine doses. Methods: To assess the humoral response, IgG and IgA anti-S1 antibodies were quantified by ELISA assays. In total, the tests were carried out seven times in almost two years. Results: We demonstrated strong immunogenicity (compared to levels before primary vaccination, 150- and 20-fold increases in IgG and IgA, respectively) of the BNT162b2 vaccine. Over time, we observed a systematic decline in antibody levels, which may have contributed to breakthrough infections. Although they caused seroconversion similar to the booster, antibody levels in such patients fell more rapidly than after re-vaccination. On the other hand, in individuals who did not receive booster(s) and who did not present breakthrough infection, anti-SARS-CoV-2 antibodies returned to pre-vaccination levels after 20 months. The most commonly recognized adverse effects were injection site redness and swelling. Conclusion: Vaccination is highly effective in preventing the most severe outcomes of COVID-19 and should be performed regardless of prior infection. Booster doses significantly enhance anti-SARS-CoV-2 antibody levels and, in contrast to those obtained by breakthrough infection, they remain longer.

Treatment with the anti-CGRP antibody fremanezumab is successful in the prevention of chronic migraine. In preclinical rat experiments, fremanezumab has been shown to reduce calcitonin gene-related peptide (CGRP) release from trigeminal tissues and the aversive behaviour to noxious facial stimuli, which are characteristic pathophysiological changes accompanying severe primary headaches. To further decipher the effects of fremanezumab underlying these antinociceptive effects in rats, immunohistochemistry and ELISA techniques were used to analyse the content and concentration of CGRP in the trigeminal ganglion as well as the ratio of trigeminal ganglion neurons immunoreactive to CGRP and CGRP receptor components 1-10 days after subcutaneous injection of fremanezumab (30 mg/kg) compared to an isotype control antibody. After fremanezumab treatment, the fraction of trigeminal ganglion neurons immunoreactive to CGRP and the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) was significantly lowered compared to the control. The content and concentration of CGRP in trigeminal ganglia were not significantly changed. A long-lasting reduction of CGRP receptors expressed in trigeminal afferents may contribute to the attenuation of CGRP signalling and antinociceptive effects of monoclonal anti-CGRP antibodies in rats.

Mathematical modeling is crucial in investigating the pandemic of the ongoing coronavirus disease (COVID-19). The primary target area of the SARS-CoV-2 virus is epithelial cells in the human lower repertory track. During this viral infection, infected cells can initiate innate and adaptive immune responses to viral infection. Immune response in COVID -19 infection can lead to longer recovery time and more severe secondary complications. We formulate a target cell limited mathematical model by incorporating a saturation term for SARS-CoV-2 infected epithelial cell loss reliant on infected cell levels. Forward and backward bifurcation between disease-free and endemic equilibrium points has been analyzed. Global stability of both disease-free and endemic equilibrium is provided. We have seen that the disease-free equilibrium is globally stable for $R_0<1$, and endemic equilibrium exists and is globally stable for $R_0>1$. Impulsive application of drug dosing has been applied for the treatment of covid-19 patients. Also, the dynamics of the impulsive system are discussed when a patient takes drug holidays. The numerical simulations are performed in support of our analytical findings and for the qualitative analysis of the system's dynamics with and without impulse drug dosing.

Influenza virus imprinting is now understood to significantly the influence immune responses and clinical outcome of influenza virus infections that occur later in life. Due to the yearly cycling of influenza viruses, humans are imprinted with the circulating virus of their birth year to subsequently build a complex influenza virus immune history but very little is known about how the imprinting strain influences vaccine responses. To investigate the imprinted host immune responses to split-virion vaccination, we imprinted ferrets with a sublethal dose of the historical seasonal H1N1 strain A/USSR/90/1977. After a +60 day recovery period was given to build immune memory, ferrets were immunized and the challenge at Day 123. Samples were collected throughout the time course and immunological assays were performed to investigate recall mechanisms. The preimmune-vaccinated ferrets did not experience significant disease during challenge while naïve-vaccinated ferrets had severe disease. Hemagglutination inhibition assays showed preimmune ferrets had a more robust antibody response post vaccination, increased virus neutralization activity. Virus specific immunoglobulins were of predominantly the IgG isotype suggesting B cell maturity and plasticity at vaccination. These results are important and should be considered for vaccine design.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinases family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, sensitive monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop highly sensitive and specific anti-EphB4 mAbs for several applications using the Cell-Based Immunization and Screening (CBIS) method. EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, western blotting, and immunohistochemistry. B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell lines, MCF-7 and MDA-MB-468, but did not react with EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (KD) values were determined to be 2.9 × 10‑9 M, 1.3 × 10‑9 M, and 3.3 × 10‑9 M by flow cytometric analysis for CHO/EphB4, MCF-7, and MDA-MB-468 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in western blotting, and stained breast cancer tissues immunohistochemistry. Altogether, B4Mab-7 demonstrated high sensitivity and specificity against EphB4 in various applications.

Bisphenol A diglycidyl ether (BADGE) is widely existed in the inner coating of canned foods. It migrates into food and generate various derivatives in the process of storage, such as Bisphenol A (2, 3-dihydroxypropyl) glycidyl ether (BADGE·H2O), Bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE·HCl) and Bisphenol A (3-chloro-2-hydroxypropyl) (2, 3-dihydroxypropyl) glycidyl ether (BADGE·HCl·H2O), which have negative effects on human health. A gold nanoparticle-based immu-nochromatographic assay for simultaneous detection of BADGE and its derivatives was developed by using a broad-spectrum polyclonal antibody, and the detection can be finished in 15 min. The visuali-zation of results was processed by Adobe Photoshop CC software to achieve quantitative analysis and the detection limit (IC15) is 0.97ng/mL. The recoveries of BADGE and its derivatives at various spiking levels in canned food samples ranged from 79.86% to 93.81%. The detection results of the proposed immu-nochromatographic assay were also validated by HPLC analysis, and got good consistency (R2=0.9580).

The world is currently going through a serious pandemic of viral infection with SARS-CoV-2, a new isolate of coronavirus, resembling and surpassing the crisis that occurred in 2002 and 2013 with SARS and MERS, respectively. SARS-CoV-2 has currently infected more than 142,000 people, causing 5,000 deaths and reaching more than 130 countries worldwide. The very large spreading capacity of the virus clearly demonstrates the potential threat of respiratory viruses to human health, alarming governments around the world that preventive health policies and scientific research are pivotal to overcoming the crisis. Coronavirus disease 2019 (COVID-19) causes flu-like symptoms in most cases. However, approximately 15% of patients will need hospitalization, and 5% require assisted ventilation, depending on the cohorts studied. What is intriguing, however, is the higher susceptibility of elderly individuals, especially those who are more than 60 years old and have comorbidities, including hypertension, diabetes and heart disease. In fact, the death rate in this group may be up to 10-12%. Interestingly, children are somehow protected and not included as a risk group.Thus, here, we discuss some possibilities of molecular and cellular mechanisms by which elderly subjects may be more susceptible to severe COVID-19. In this sense, we raise two main points: i) increased ACE-2 expression in pulmonary and heart tissue of chronic angiotensin 1 receptor (AT1R) blocker users and hypertensive individuals and ii) antibody-dependent enhancement (ADE) after previous exposure to other circulating coronaviruses. We believe these are pivotal points for a better understanding of the pathogenesis of severe COVID-19 and must be addressed with attention by physicians and scientists in the field.

Therapeutics antibodies have increasingly shifted the paradigm of disease treatments, from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. In fact, the constant region can govern effects that might be useful in reducing the unwanted consequences resulted from systemic circulation. For this reason, apart from the commonly used IgG isotypes, IgA antibodies are promising therapeutics drugs, given its localized mucosal effects. While the antibody Fc effector cell activity has been well explored, recent research has shown evidences that the constant region of the antibody can also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To further investigate this, we analyzed the IgA antibody constant and its allosteric effects onto the antigen binding regions, using recombinant Pertuzumab IgA1 and IgA2 variants. We found mutations in the C-region to reduce Her2 binding, and our computational structural analysis showed that such allosteric communications were highly dependent on the antibody hinge, providing the evidence to consider antibodies as a whole protein rather than a sum of functional regions.

The details of antigen-antibody interactions and the identification of epitopes are critical for the development of monoclonal antibody drugs. Ab42 is a native human-derived anti-CFH monoclonal antibody. In this study, the interaction between antigen pCFH and antibody (Ab42) was theoretically demonstrated by molecular docking and MD simulation, combined with free energy calculation and computational alanine scanning (CAS), and key amino acids and epitopes were identified. Experimental alanine scanning (EAS) was then carried out to verify the results of the calculation, and our results indicated that Ab42 antibody forms hydrogen bonds and interacts hydrophobically with pCFH through the Tyr315, Ser100, Gly33, and Tyr53 residues on its CDR, while the main pCFH epitopes are located at the six sites of Pro441, Ile442, Asp443, Asn444, Ile447, and Thr448. In conclusion, this study has explored the mechanism of antigen-antibody interaction from both theoretical and experimental aspects, and our results have important theoretical significance for the design and development of relevant antibody drugs.

EphB2 is a member of the Eph family tyrosine kinase receptors. EphB2 binds to ephrin-B1, ephrin-B2, and ephrin-B3, which are critical regulators of vascular and nervous development through controlling cell migration and axon guidance. EphB2 is overexpressed in tumors, including glioma, breast cancer, hepatocellular carcinoma, and malignant mesothelioma, and it functions as a tumor promoter. Therefore, the development of monoclonal antibodies (mAbs) against EphB2 is essential for tumor diagnosis and therapy for EphB2-positive tumors. In this study, we developed novel mAbs for human EphB2 using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-EphB2 mAbs, Eb2Mab-3 (mouse IgG1, kappa), reacted with EphB2-overexpressed Chinese hamster ovary-K1 (CHO/EphB2) and an endogenously EphB2-expressing cancer cell line (LS174T) by flow cytometry. Using flow cytometry, the dissociation constant (KD) values of Eb2Mab-3 for CHO/EphB2 and LS174T were determined as 1.1 × 10−9 M and 3.6 × 10−10 M, respectively. These results indicated that Eb2Mab-3 possesses a high affinity for detecting EphB2 and could apply to tumor therapy.

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8⁺ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C₈Mab-2, using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of C₈Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C₈Mab-2 recognizes the N-terminal region (1–33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the _17-DFFTAP_-22 sequence is important for the recognition by C₈Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C₈Mab-2.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL has been considered as a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells such as vascular and lymphatic endothelial cells.Therefore, cancer-specificity or selectivity is required for the reduction of adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG₁, kappa), by immunizing mice with soluble PODXL ectodomain derived from a glioblastoma LN229 cell.PcMab-6 reacted with the PODXL-positive LN229 cells, but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H), but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47 showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG_2a type (PcMab-6-mG_2a) and a humanized IgG₁-type (humPcMab-6) mAbs, and further produced the core fucose-deficient types (PcMab-6-mG_2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG_2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG_2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

Despite successful vaccination efforts, the emergence of new SARS-CoV-2 variants poses ongoing challenges to control COVID-19. Understanding humoral responses concerning SARS-CoV-2 infections and their impact is crucial for developing future vaccines that are effective worldwide. Here, we identified 41 immunodominant linear IgG B-cell epitopes in its Spike glycoprotein with a SPOT synthesis peptide array probed with a pool of serum from hospitalized COVID-19 patients. Bioinformatics showed a restricted set of epitopes unique to SARS-CoV-2 compared to other coronavirus family members. Potential crosstalk was also detected with Dengue virus (DENV), confirmed by screening individuals infected with DENV before the COVID-19 pandemic in a commercial ELISA assay for anti-SARS-CoV-2 antibodies. Higher reactivity indices were often measured in individuals with a pre-pandemic dengue infection than those with COVID-19. A high-resolution evaluation of antibody reactivity against peptides representing epitopes in the spike protein identified ten sequences in the NTD, RBD, and S2 domains. Functionally, antibody-dependent enhancement (ADE) in SARS-CoV-2 infection of monocytes was observed with pre-pandemic dengue-positive sera. A significant increase in viral load was measured compared to controls, with no detectable neutralization or considerable cell death, suggesting its role in viral entry. This study highlights the importance of identifying the epitopes generated during the humoral response to a pathogenic infection to understand the potential interplay of previous and future conditions on disease. Vaccine development and optimization strategies should be mindful of the potential for A.D.E. that could differ geographically due to endemic biological risks.

We report on an immunofluorescent paper-based assay for the detection of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper-based device was fabricated by using lamination technique for easy and optimized handling. Our approach utilises a two-step strategy that involves (i) initial coating of the paper-electrode with recombinant SARS-CoV-2 nucleocapsid antigen to capture the target SARS-CoV-2 specific antibodies, and (ii) subsequent detection of SARS-CoV-2 antibodies using fluorophore-conjugated IgG antibody. The fluorescence readout was observed with fluorescence microscopy. The images were processed and quantified using a MATLAB program. The assay can selectively detect SARS-CoV-2 humanized antibodies spiked in PBS and healthy human serum samples with the relative standard deviation of approximately 6.4% (for n = 3). It has broad dynamic ranges (1 ng to 50 ng/µL in PBS and 5 to 100 ng/µL in human serum samples) for SARS-CoV-2 humanized antibodies with the detection limits of 2 ng/µL (0.025 IU/mL) and 10 ng/µL (0.125 IU/mL) in PBS and human serum samples, respectively. We believe that our assay has the potential to be used as a simple, rapid, and inexpensive paper-based diagnostic device with a portable fluorescent reader to provide point-of-care diagnosis. This assay can be used for rapid examination of a large batch of samples toward clinical screening of SARS-CoV-2 specific antibodies as a confirmed infected active case or to evaluate the immune response to a SARS-CoV-2 vaccine.

After the influenza H1N1 pandemic of 2009, the seasonal A/Brisbane/59/2007 strain was replaced by the A/California/07/2009 strain for the influenza virus vaccine composition. After several seasons with no indications on the occurrence of antigenic drift, A/Michigan/45/2015 was chosen as the H1N1 vaccine strain for season 2017/2018. Since the immune response to influenza is shaped by the history of exposure to antigenically similar strains, the potential cross-protection between seasonal human influenza vaccine strains and the emerging pandemic strains was investigated. Human serum samples were tested by haemagglutination inhibition and single radial haemolysis assays against A/Brisbane/59/2007, A/California/07/2009, and A/Michigan/45/2015 strains. Strong cross-reactions between A/California/07/2009 and A/Michigan/45/2015 strains were observed in 2009/2010, most likely induced by the start of the 2009 pandemic, and the subsequent post-pandemic seasons from 2010/2011 onwards when A/California/07/2009 becomes the predominant strain. In 2014/2015 season, population immunity against A/California/07/2009 and A/Michigan/45/2015 strains increased again, associated with strong cross-reactions. While haemagglutination inhibition assay has a higher sensitivity for detection of new seasonal drift, the single radial haemolysis assay is an excellent tool to determine the presence of pre-existing immunity, allowing a potential prediction on the booster potential of influenza vaccines against newly emerging drifted strains.

The ongoing pandemic of COVID-19 has not only commenced a global health emergency but agitated various aspects of humanity. During this period of crisis researchers over the world have ramped their efforts to constrain the disease in all possible ways whether it is vaccination, therapy, or diagnosis. Since the spread of the disease has not yet elapsed sharing the ongoing research findings could be the key to disease control and management. An early and efficient diagnosis could leverage the outcome until a successful vaccine is developed. Molecular tests both in-house and commercial kits are preferably being used worldwide in the COVID-19 diagnosis. However, the limitation of high prices and lengthy procedures impede their use for mass testing. Keeping the constant rise of infection in mind search for an alternative test that should be cost-effective, simple, and suitable for large scale testing and surveillance is a need of an hour. One such alternative could be the immunological tests. Therefore, in the last few months deluge of immunological rapid tests has been developed and validated across the globe. The objective of the present review is to share the diagnostic performance of various immunological assays reported so far in SARS-CoV-2 case detection. The article consolidated the studies (published and preprints) related to the serological tests such as chemiluminescence, enzyme-linked and lateral flow-based point-of-care tests in COVID-19 diagnosis and updated the current scenario. This review will hopefully be an add-on in COVID-19 research and will contribute to congregate the evidence for decision-making.

Porcine circovirus type 2 (PCV2) is a small non-enveloped DNA virus that causes swine immunosuppression by inducing apoptosis in lymphocytes. The ORF3 protein plays a major role in PCV2-induced apoptosis in porcine kidney cells, but there is little information regarding this protein in PCV2-infected lymphocytes. In this study, hybridoma screening and epitope mapping were determined by using an indirect ELISA. The mAb 7D3 against ORF3 peptide (residues 35–65) of PCV2 were generated in this study. In vivo situation, the mAb 7D3 recognized ORF3 protein existed in PCV2-infected apoptotic porcine PBMCs. It is noteworthy that thimerosal interfered with the binding of mAb 7D3 to epitope and it was diminished by adding cysteine. Additionally, thimerosal interacting with cysteine-containing peptide was demonstrated by the PTI assay. Furthermore, thimerosal specifically interacted with the antigen-binding sites of mAb 7D3. This study suggested that thimerosal blockade the occlusion of the antigen-binding sites of mAb 7D3 to bind ORF3 peptide (residues 35–65) via thimerosal interacting with cysteine residues which should be located within the antigen-binding sites of mAb 7D3. Overall, the mAb 7D3 has been characterized and it will be a valuables tool in future studies of ORF3 function and the wider mechanism of cell apoptosis caused by PCV2 infection. Similarly, these techniques will be useful for applications in detecting thimerosal too.

Little is known about the long-term durability of the induced immune response in subjects af-fected by obesity, particularly in those with an abdominal distribution of adipose tissue. We evaluated SARS-CoV-2-specific antibody response after BNT162b2 vaccine booster dose, com-paring individuals with abdominal obesity (AO) with those without, discerning between indi-viduals previously infected or not. IgG-TrimericS were measured in 511 subjects at baseline, 21 days after vaccine dose-1 and at one, three, six and nine months after dose-2 and at one and three months after booster dose. Nucleocapsid antibodies were assessed at baseline and at the end of the study to detect SARS-CoV-2 infection. To evaluate the three-months difference in absolute variation of IgG-TrmicericS levels from booster dose we used multivariable linear regression that showed interaction between AO and SARS-CoV-2 infection status (p=0.016). AO is associated with higher absolute IgG-TrimericS variation in prior infected individuals, regardless of possi-ble confounders and IgG-TrimericS levels at booster dose (p=0.0125). No interaction was evinced using BMI in the same regression model (p=0.418). The robust response in the development of antibodies after booster dose, observed in people with OA and previous infection, may support the recommendations to undergo the booster dose also in this population group.

PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by atomistic molecular dynamics simulations. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of mutation and for the neighboring residues. This stabilization affects not only protein-protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Further, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16 we obtained an experimental EC50 value of 3nM for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Their high affinity makes PG16-derived peptides useful building blocks for further optimization to obtain a potent inhibitor that efficiently blocks HIV-1 infection.

Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions can also play essential roles in antigen binding. Here, we describe the humanization of the pan-HLA-DR mAb 44H10, a murine antibody displaying significant involvement of the framework region in antigen binding. Using a structure-guided approach, we identify and restore framework residues that directly interact with the antigen or indirectly modulate antigen binding by shaping the antibody paratope and engineer a humanized antibody with affinity, biophysical profile, and molecular binding basis comparable to that of the parental 44H10 mAb. As a humanized molecule, this antibody holds promise as a scaffold for the development of MHC class II-targeting therapeutics and vaccines.

In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infection, ferrets (Mustela putorius furo) have been used to investigate the pathogenesis. Podoplanin (PDPN) is an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of those tissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry. The N-terminal ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292 epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at the C-terminus of PMab-292 epitope. The epitope sequence (₃₄-VRPEDD-₃₉) also exists between Val26 and Asp31 of ferPDPN, indicating that PMab-292recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.

CD44 is a type I transmembrane glycoprotein, and possesses various isoforms which are largely classified into CD44 standard and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 mAb, C₄₄Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C₄₄Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10), and found that C₄₄Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C₄₄Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C₄₄Mab-108 includes Asp280 and Trp281 of CD44v3-10.

Development of new neurotherapeutics is strongly depending on appropriate animal model chosen in preclinical studies. A cuprizone model is an effective tool for studying demyelination and re-myelination processes in the brain, but BBB integrity in cuprizone model is still a topic for debate. Several publications claim that BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could be explained by the increased BBB permeability. In our work we aim to analyze permeability of BBB for different macromolecules, particularly, antibody-conjugates in cuprizone-intoxicated mice. We compared the traditional approach using Evans blue injection with following dye extraction and detection of antibody-conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in cuprizone model. First, we validated our model of demyelination by detecting changes in gene expression of myelin basic protein and proteolipid protein, and by performing MRI and histological analysis. Our results suggest that the methods with better sensitivity were able to detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that ques-tioned BBB integrity in cuprizone model and demonstrate possibility for delivery of anti-body-conjugates to demyelinated corpus callosum

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of diagnostic biomarkers and effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeting monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells in immunohistochemistry. These results indicate that C44Mab-94 is useful for detecting CD44v8 in various applications and is expected to be useful for the application of GC diagnosis and therapy.

Background: Fabry disease cardiomyopathy (FDCM) has manifested some resistance to enzyme replacement therapy (ERT) particularly in its advanced phase. Recently myocardial inflammation of autoimmune origin has been demonstrated in FDCM. Aims: Aim of the study is the assessment of circulating anti-globotriaosylceramide (GB3) antibody as biomarker of myocardial inflammation in FDCM. Its sensitivity was based on the evidence of overlapping myocarditis at left ventricular endomyocardial biopsy. Methods and Results: From January 1996 to December 2021, 85 patients received in our Department the histological diagnosis of FDCM and 48 (56.5%) of them had an overlapping myocardial inflammation with negative PCR for the common cardiotropic viruses, positive anti-heart and anti-myosin abs. The presence of anti-GB3 antibodies was evaluated with an Elisa assay (BioGeM scarl Medical Investigational Research, MIR - Ariano Irpino, Italy) in the FDCM patients and compared with controls of healthy individuals. Circulating levels of anti-GB3 autoantibodies were compared with the presence or absence of myocardial inflammation at histology and with the severity of FDCM. The assay results showed that 42 out of 48 FDCM subjects with myocarditis were positive for anti-Gb3 antibodies while 30 out of 37 FDCM patients without myocarditis had negative anti-GB3 antibodies, with a sensitivity of 87.5 % and a specificity of 83.5%. Conclusions: The present study suggests a potential positive role of antiGB3 antibodies as marker of overlapping cardiac inflammation in patients with FDCM.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors, and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, the elucidation of each CD44 isoform’s function in tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through immunization of CD44v3–10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3) determined by enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer cell lines (COLO205 and HSC-3) by flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3–10, COLO205, and HSC-3 was 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M, respectively. C44Mab-6 could detect the CD44v3–10 in western blotting, and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments, and expected for the application of tumor diagnosis and therapy.

African swine fever (ASF) is a lethal disease in pigs that has grave socio-economic implications worldwide. For the development of vaccines against African swine fever virus (ASFV), immunogenic antigens that generate protective immune responses need to be identified. There are over 150 viral proteins - many of which are uncharacterized – and humoral immunity to ASFV has not been closely examined. To profile antigen specific antibody responses, we developed luciferase-linked antibody capture assays (LACAs) for a panel of ASFV capsid proteins and screened sera from inbred and outbred animals that were previously immunized with low virulent ASFV before challenge with virulent ASFV. Antibodies to B646L/p72, D117L/p17, M1249L and E120R/p14.5 were detected in this study; however, we were unable to detect B438L specific antibodies. Although OURT88/1 induced viremia was observed in the presence of anti-B646L/p72 antibodies as well as B602L antibodies they were associated with recovery from lethal disease in inbred and outbred animals. However, these antibodies did not correlate with protection against Georgia 2007/1 infection. Antibody responses against M1249L and E120R/p14.5 were observed in animals with reduced clinical signs and viremia. Here we present LACAs as a tool for targeted profiling of antigen specific antibody responses to inform vaccine development.

Since the detection of the first COVID-19 patient, 2 years have passed, during which more than 287,862,000 people fell ill globally, of which about 1.9% died. Implementation of SARS-CoV-2 control programs required efforts from almost all countries. An important direction in the fight against COVID-19 was the formation of herd immunity, the main tool for managing the pandemic. Study goal: to assess the seroprevalence of antibodies (Abs) to SARS-CoV-2 nucleocapsid (Nc) and receptor binding domain (RBD) in the St. Petersburg population during the COVID-19 pandemic. Materials and methods. A longitudinal cohort randomized monitoring study of Ab seroprevalence (SARS-CoV-2 Nc, RBD) was organized and conducted according to a unified methodology developed by Rospotrebnadzor with the participation of the St. Petersburg Pasteur Institute. For this purpose, a cohort of 1000 volunteers was formed who participated in all five stages of seromonitoring. The cohort was divided into 7 age groups: 1-17; 18-29; 30-39; 40-49; 50-59; 60-69; 70; and older (70+) years. Seropositivity levels (Nc, RBD) were assessed by quantitative and qualitative enzyme immunoassays. During the 2nd year of monitoring, some volunteers were vaccinated with the GamCOVIDVac (84%) or EpiVacCorona (11.6%) vaccines approved in Russia. Statistical processing was carried out using the Excel 2010 software package. Confidence intervals for shares and percentages (95% CI) were calculated using the method of A. Wald and J. Wolfowitz with adjustment (A. Agresti, B.A. Coull). The statistical significance of differences was calculated by z-test, using the appropriate online calculator (p<0.05), unless indicated. Results. There was a trend towards: an increase in Nc seropositivity in stages 1-3 of seromonitoring, with a decrease in stages 4-5 among children and adults. The share of RBD seropositive steadily increased during all 5 stages of seromonitoring. The most frequently found were low anti-RBD Abs levels (22.6-220 BAU/ml). High Ab levels were recorded statistically significantly less frequently. Asymptomatic forms were observed in 84-88% of SARS-CoV-2 seropositive volunteers. By the 5th stage of monitoring, this indicator significantly decreased to 69.8% (95% CI: 66.1-73.4). The monitoring revealed a statistically significant increase in anti-RBD Abs, alongside a statistically significant decrease in the proportion of Nc seropositive. This dynamic was especially characteristic of persons vaccinated with GamCOVIDVac. Conclusion. Prior to the use of specific vaccines, a seroprevalence of anti-Nc Abs was noted. After the introduction of the GamCOVIDVac vaccine in adults, a decrease in the level of anti-Nc Abs was noted due to an increase in the proportion of RBD seropositive persons.

Pretilachlor is a chloroacetamide herbicide, mainly used for the control of weeds and broadleaf weeds in rice, and widely used in China. For the detection of residues of pretilachlor in the en-vironment and food, a highly sensitive and specific monoclonal antibody against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47±2.35 μg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 μg/L was developed for the detection of pretilachlor residues in the environment and food crops. The specificity of the antibody was explained by computer simula-tions and experimental validation. No cross-reactivity of this monoclonal antibody to alachlor, acetochlor and metalaxyl, and it cross-reacted less than 3.0% to both butachlor and propisochlor. The limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 μg/L. The recoveries of all samples were 78.3%~91.3%. The ic-ELISA method was validated by high-performance liquid chromatography, thus, it can be used for residue detection of pretilachlor in the environment and grains.

As the coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses. The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Antiviral ADCC requires three components: 1) antibody; 2) effector lymphocytes with the Fc-IgG receptor CD16A; and 3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals’ variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that support ADCC which encompasses afucosylation. This assay utilizes externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a-externalization vs. 51Cr cell death were similar for NK-92 CD16A and blood NK cells. Notably, the %CD107a-positive cells negatively correlated with dead Raji cells and was nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC.

Smartphone-based point-of-care testing (POCT) is rapidly emerging as an alternative to traditional screening and laboratory testing, particularly in resource-limited settings. In this proof-of-concept study, we present a smartphone- and cloud-based artificial intelligence quantitative analysis system (SCAISY) for relative quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific IgG antibody lateral flow assays that enables rapid and accurate evaluation of test strips. By capturing an image with a smartphone camera, SCAISY quantitatively analyzes antibody levels and provides results to the user. We analyzed changes in antibody levels over time in over 248 individuals, including vaccine type, number of doses, and infection status, with a standard deviation of less than 10%. We also tracked antibody levels in six participants before and after SARS-CoV-2 infection. Finally, we examined the effects of lighting conditions, camera angle, and smartphone type to ensure consistency and reproducibility. This study suggests that SCAISY is a simple and powerful tool for real-time public health surveillance, enabling the acceleration of quantifying SARS-CoV-2-specific antibodies generated by either vaccination or infection and tracking of personal immunity levels.

The successful vaccination implies the induction of effective specific immune responses. We intend to find biomarkers among various immune cell subpopulations, cytokines and antibodies which could be used to predict the levels of specific antibody- and cell-mediated responses after measles-mumps-rubella vaccination. We measured 59 baseline immune status parameters (frequencies of 42 immune cell subsets, levels of 13 cytokines, immunoglobulins) before vaccination and 13 response variables (specific IgA and IgG, antigen-induced IFN-γ production, CD107a expression on CD8+ T lymphocytes, and cellular proliferation levels by CFSE dilution) 6 weeks after vaccination for 19 individuals. Statistically significant Spearman correlations between some baseline parameters and response variables were found for each response variable (p&lt;0.05). Due to the low number of observations relative to the number of baseline parameters and missing data for some observations, we utilized three feature selection strategies to select potential predictors of the post-vaccination responses among baseline variables: (a) screening of the variables based on the correlation analysis, (b) supervised screening based on the information of changes of baseline variables at day 7, (c) implicit feature selection is performed using regularization-based sparse regression. We identified optimal multivariate liner regression models for predicting the effectiveness of vaccination against measles-mumps-rubella using the baseline immune status parameters. It turned out that the sufficient number of predictor variables ranges from one to five depending on the response variable of interest.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a high rate of mortality in kidney transplant recipients (KTRs). The current vaccine strategy for KTRs seems to be unable to provide effective protection against coronavirus disease 2019 (COVID-19) [2] and the occurrence of severe COVID-19 in some vaccinated KTRs suggested a lack of immunity. We analized here the antibody response in a group of 32 kidney transplant recipients (KTR) followed at the Nephrology and Dialysis unit of the Hospital Pio XI of Desio, Italy, compared with a group of 23 healthy workers classified as Late responders (HCW-LR) (10 males age 27-62) and 13 females (age 46-64). We observed that the patients need additional vaccine boosters due to their immunocompromised status and therapy to protect them from infections related to viral variants, in accordance with the literature.

Breast cancer patients with high levels of HER2 (human epidermal growth factor receptor 2) expression had worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa). This was then altered to produce H2Mab-77-mG2a-f, an afucosylated mouse IgG2a. In the present work, we examined the reactivity of H2Mab-77-mG2a-f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressed breast cancer cell line, was identified by H2Mab-77-mG2a-f with a strong binding affinity [a dissociation constant (KD): 5.0 × 10-9 M]. H2Mab-77-mG2a-f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in western blot analysis. Furthermore, H2Mab-77-mG2a-f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2Mab-77-mG2a-f. Additionally, in the BT-474-bearing tumor xenograft model, H2Mab-77-mG2a-f substantially suppressed tumor development when compared to the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2Mab-77-mG2a-f. These findings point to the possibility of H2Mab-77-mG2a-f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

This study assesses the durability of severe acute respiratory coronavirus-2 (SARS-CoV-2) anti-nucleocapsid (anti-N) immunoglobulin G (IgG) after infection, and examines its association with established risk factors among South African healthcare workers (HCWs). Blood samples were obtained from 390 HCWs with diagnosis of coronavirus disease 2019 (COVID-19) for assay of the SARS-CoV-2 anti-N IgG at two time points (Phase 1 &amp; 2) between November 2020 and February 2021. Out of 390 HCWs with COVID-19 diagnosis, 267 (68.5%) had detectable SARS-CoV-2 anti-N IgG antibodies at the end of phase I. These antibodies persisted for 4-5 and 6-7 months in 76.4% and 16.1%, respectively. In the multivariate logistic regression model analysis, black participants were more likely to sustain SARS-CoV-2 anti-N IgG for 4-5 months. However, participants who were HIV positive were less likely to sustain SARS-CoV-2 anti-N IgG antibodies for 4-5 months. In addition, individuals who were &lt;45 years of age were more likely to sustain SARS-CoV-2 anti-N IgG for 6-7 months. Of the 202 HCWs selected for phase 2, 116 participants (57.4%) had persistent SARS-CoV-2 anti-N IgG for an extended mean period of 223 days (7.5 months). Findings support the longevity of vaccine responses against SARS CoV-2 in black Africans.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent KD of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

Background: Asthma is the most common chronic disease affecting children and altering their quality of life. The severity of asthma is often modulated by immunoglobulin E (IgE)-mediated allergen sensitization and is associated with comorbid allergic dis-eases. Omalizumab is a humanized monoclonal antibody anti-IgE, the first biological therapy approved to treat patients aged ≥6 years with severe allergic asthma. The primary objective of our study was to investigate the efficacy and safety of Omali-zumab in Romanian paediatric patients with severe allergic asthma. Methods: In this observational real-life study, 12 children aged 6 to 18 years, (mean age 12.4 years ) with severe allergic asthma received Omalizumab as an add-on treatment. The levels of asthma control, exacerbations, lung function and adverse events were evaluated at baseline and after the first year of treatment. Results: We noticed general improvements in total asthma symptom scores and the rate of exacerbation of severe asthma. Omalizumab increased the initial variables of lung function, and no serious adverse reactions were reported. FEV1 improved statistically significant after one year of treatment with Omalizumab, [ΔFEV1 (% pred.) =18.3, and similarly, ΔMEF50 (%) = 25.8]. The mean severe exacerbation rates due to asthma decreased from 4.1 (2.8 SD) to 1.15 (0.78 SD) during the treatment year (p<0.0001) with Omalizumab. Conclusions: Treatment with Omalizumab can be an effective and safe therapeutic option for Romanian children with severe allergic asthma, providing clinically relevant in-formation on asthma control and exacerbation rate in children and adolescents. The results highlighted the effect of Omalizumab in young patients, starting from the first year of treatment.

MicroRNAs (miRNAs) play important roles in the control of HIV-1 infection. Here, we performed RNAseq profilings of miRNAs and mRNAs expressed in CD4+ T-lymphocytes upon HIV-1 infection. Our results reveal significant alterations of miRNAs and mRNAs expression profiles in infected relative to uninfected cells. One of the miRNAs markedly downregulated in infected cells is miRNA-26a. Among the putative targets of miRNA-26a are CD59 receptor transcripts, which are significantly upregulated in infected CD4+ T-cells. Addition of miRNA-26a mimics to CD4+ T-cells reduces CD59 at both mRNA and surface protein levels, validating CD59 as a miRNA-26a target. Consistent with the reported inhibitory role of CD59 in complement-mediated lysis (CML), knocking-out CD59 in CD4+ T-cells renders both HIV-1 infected cells and progeny virions more prone to antibody-dependent CML (ADCML). Addition of miRNA-26a mimics to infected cells leads to enhanced sensitivity of progeny virions to ADCML, a condition linked to a reduction of CD59 packaging into released virions. Lastly, HIV-1-mediated downregulation of miRNA-26a expression is shown to be dependent on integrated HIV-1 expression but does not involve viral accessory proteins. Overall, these results highlight a novel mechanism by which HIV-1 limits ADCML by upregulating CD59 expression via miRNA-26a downmodulation.

Most studies on vaccines of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused on antibody, but cellular immunities are also critical. We aimed to evaluate the immune reactions of hemodialysis (HD) patients after the administration of the booster dose from the perspective of both humoral and cellular immunities. Hemodialysis patients (HD group) and age- and sex-matched non-dialysis individuals (control group) receiving three doses of BNT162b2 vaccine were measured for anti-SARS-CoV-2 immunoglobulin (IgG) and T-SPOTⓇ.COVID test (T-SPOT) before, 3 weeks, and 3 months after the booster dose. The HD group had significantly higher SARS-CoV-2 IgG levels 3 weeks and 3 months after the booster dose than the control group, although both groups had no difference in SARS-CoV-2 IgG levels before the booster dose. Moreover, the HD group had significantly higher T-SPOT levels before and 3 weeks after the booster dose than the control group, but the difference was not significantly different 3 months after the booster dose. Furthermore, the incidence rates of local and systemic adverse reactions were significantly higher in the HD group than in the control group. HD patients obtained higher SARS-CoV-2 IgG levels and SARS-COV-2-specific T-cell responses after the booster dose than control.

Vaccines trigger a complicated immunological response that includes B and T cells, with B cells producing antibodies. SARS-CoV-2 immunity weakens over time after vaccination. Discovering key changes in antigen-reactive antibodies over time after vaccination could help improve vaccine efficiency. In this study, we collected data on blood antibody levels in a cohort of healthcare workers vaccinated for COVID-19 and obtained 73 antigens in samples from four groups according to the duration after vaccination, including 104 unvaccinated healthcare workers, 534 healthcare workers within 60 days after vaccination, 594 healthcare workers between 60 and 180 days after vaccination, and 141 healthcare workers over 180 days after vaccination. An efficient machine learning based framework containing four feature selection methods (least absolute shrinkage and selection operator, light gradient boosting machine, Monte Carlo feature selection, and maximum relevance minimum redundancy) and four classification algorithms (decision tree, k-nearest neighbor, random forest, and support vector machine) was designed to screen out essential antigens. Several efficient classifiers with weighted F1 value around 0.75 were constructed. This study revealed that S1+S2, S1.mFcTag, S1.HisTag, S1, S2, Spike.RBD.His.Bac, Spike.RBD.rFc, and S1.RBD.mFc were most highly ranked among all features, where S1 and S2 are the subunits of Spike, and the suffixes represent the tagging information of different recombinant proteins. Meanwhile, the classification rules were extracted from the optimal decision tree to explain quantitatively the roles of antigens in the classification. This study identified antibodies associated with decreased clinical immunity based on populations with different time spans after vaccination. These antibodies have important implications for maintaining long-term immunity to SARS-CoV-2.

Vaccines are crucial for controlling the COVID-19 pandemic, and booster doses are becoming increasingly important. This study aimed to assess the efficacy of the ChAdOx1 nCoV-19 vaccine from AstraZeneca as a third dose in healthcare workers at different time intervals (one, three, and six months). Two methods to measure immune response—ELISA (EUROIMMUN Medizinische Labordiagnostika AG, Luebeck, Germany) and ELISpot (Mabtech AB, Macka Strand, Sweden)—were used. A total of 170 participants were included in the study. The results showed that while IgG levels decreased at six months compared to levels at one and three months, they were still significantly higher than the baseline. Furthermore, neutralizing levels at three and six months after the third dose were not significantly different. These findings suggest that the immune response induced by the vaccine is robust and effective for several months. These results have significant implications for public health policymakers, as they provide strong support for booster vaccinations. The ChAdOx1 nCoV-19 vaccine appears to be a reliable option for preventing the spread of COVID-19, and this study provides valuable information for healthcare workers and policymakers in managing the pandemic.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti‐EGFR and human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti‐EGFR mAb (E134Bf) and a mouse-dog chimeric anti‐HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermore, E134Bf-H77scFv exerted antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.