Latex fixation test

Transcription

LATEX AGGLUTINATION TEST The latex agglutination test is a laboratory method to check for certain antibodies or antigens in clinical laboratories for the detection of infectious diseases such as rheumatoid arthritis, hepatitis B, haemophilus influenza, enisseria meningitidism, etc. In 1956 Singer and Plotz first described the Rheumatoid Factor Test, a test based on latex agglutination. In rheumatoid arthritis IgG antibodies produced by lymphocytes in the synovial joint react with the IgM antibodies (RF, rheumatoid factor) to generate immune complexes that activate the complement and cause the tissue destruction. The reaction between a particulate antigen and an antibody results in visible clumping called agglutination. Antibodies that produce such reactions are known as agglutinins. The principle of agglutination reactions are similar to precipitation reactions: they depend on the cross linking of polyvalent antigens. When the antigen is an erythrocyte, it is called hemagglutination. Theoretically, all antibodies can agglutinate particulate antigens but IgM, due to its high specificity, is a particularly good agglutinin. Agglutination tests employ latex particles, gelatin beads, colloidal particles, or preserved mammalian or avian blood cells to facilitate visualization of agglutination. In the latex agglutination test, the sample to be tested is sent to the lab where it mixed with latex beads coated with a specific antigen or antibody. Materials Required: 1.5 ml Vials. Microcentrifuge. Pipette. Micro tips. Laboratory refrigerator. Glycine saline buffer. Blocking buffer. Antigen for coating. Latex beads. Test antiserum. Glass slides. Beaker. Toothpick. Procedure: Coating of Latex To 20 µl of latex beads taken in a 1.5 ml vial, add 40 µl of glycine-saline buffer. Add 60 µl of antigen to the latex and incubate at 37oC for 2 hours. Spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant. Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10 minutes. Repeat the washing once more. Add 90 µl of blocking buffer to the pellet, mix well. Incubate at 40C, overnight. Agglutination Test: To 200 µl of glycine-saline buffer taken in a vial, add 4 µl of test antiserum (50 times diluted). Add 50 µl of antigen to 50 µl of diluted antiserum in a 1.5 ml vial, mix well and incubate at room temperature for 10 minutes. Pipette 10 µl of coated latex on a glass slides. Add 10 µl of diluted test antiserum to slide A. Add 10 µl of antiserum mixed with antigen (from step 8) to B. Add 10 µl of glycine-saline buffer to C. Take a toothpick and mix the contents on each slide. Discard the toothpick after using it for one slide and use a new one for the next slide. After mixing, wait for 2 minutes to observe the result. Result: The clumping of latex beads (agglutination) indicates the presence of suspected particles. Absence of white clumps indicates a negative result, i.e., the suspected particle is not present. ADVANTAGES: Helps in detecting certain antigens or antibodies in a variety of bodily fluids such as blood, saliva, urine or cerebrospinal fluid. Ability to obtain semi-quantitative results. A low individual test cost. Relatively short time to obtain results.