ha &HIT.ppt
- 1. Agglutination tests HA & HI
- 2. 2 Agglutination tests • Antibodies can agglutinate multivalent particulate antigens, such as Red Blood Cells (RBCs) or bacteria • Some viruses also have the ability to agglutinate with RBCs. • This behavior is called agglutination. • Serological tests based on agglutination are usually more sensitive than those based on precipitation
- 3. 3 Examples • Slide Agglutination Test • Plate Agglutination Test • Tube Agglutination Test • Passive Agglutination Test • Microscopic Agglutination Test • Haemagglutination test (HAT)
- 4. 4 Slide Agglutination Test • Used for serotyping (e.g. Salmonella) • Antigen: isolated Salmonella in suspension • Antibody: specific antisera against Salmonella • Place test Salmonella in a drop of saline on a slide • Add a drop of antiserum, mix and rock slide for approx 1 minute • Examine for agglutination
- 6. 6 Tube Agglutination Test • Also known as the standard agglutination test or serum agglutination test (SAT) • Test serum is diluted in a series of tubes (doubling dilutions) • Constant defined amount of antigen is then added to each tube and tubes incubated for ~20h @37°C • Particular antigen clumps at the bottom of the test tube • Test is read at 50% agglutination • Quantitative • Confirmatory test for ELISA reactors • Example: Brucellosis screening
- 8. No agglutination Agglutination 1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl In this case, the titre is 1/40 Tube Agglutination Test
- 9. 9 Passive Agglutination Test • Converting a precipitating test to an agglutinating test • Chemically link soluble antigen to inert particles such as LATEX or RBC • Addition of specific antibody will cause the particles to agglutinate • Reverse PAT: antibody linked to LATEX e.g. Lancefield grouping in Streptococci.
- 10. 10 Quantitative Micro Hemagglutination Test (HA) Haemagglutination Test (HA)
- 13. 13 Viral Haemagglutination • Some viruses and microbes contain proteins which bind to erythrocytes (red blood cells) causing them to clump together • NDV • Adenovirus III • AIV • IBV • Mycoplasma
- 14. 14 Viral Hemagglutination • the attachment of viral particles by their receptor sites to more than 1 cell. • As more and more cells become attached in this manner agglutination becomes visible
- 15. 15 Equivalence point: (suitable proportion between the virus particles and RBCs)
- 16. Negative control well (only RBCs+ buffer) (no haemagglutinin) Positive control well (contains haemagglutinin)
- 17. 17 Readings The results • Titer: The maximum dilution that gives visible agglutination. • The end point: is the well with the lowest concentration of the virus where there is haemagglutination 2 4 8 16 32 64 128 256 512 1024 2048 4096 The HA titer of this virus in this row is 256 or 28 (1:256 dilution contains (1 HA unit) (one haemagglutinating unit)
- 19. Titer = 32 HA units/ml Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 8 16 32 64 128 256 virus serial dilution mix with red blood cells side view top view One HA unit :minimum amount of virus that causes complete agglutination of RBCs
- 20. 20 CALCULATIONS • For HI systems with different HA units: (4 HA units for NDV ,8 HA unit for AIV, 4 or 8 for adenovirus group III). • divide the virus titer on the needed HA unit.
- 21. 21 CALCULATIONS • If the titer is 32, How can you get 4 HA units? • 1/32 contain 1 HA • 1/32 * 4 = 4/32 = 1/8. • 1/8 dilution contain 4 HA units. • So 1/8 = (1/ 1+7) – 10 wells * 50μl/well = 500 μl total volume. • 500/8= 62.5 μl virus + 437.5 HI buffer.
- 22. 22 WHAT DO WE NEED?
- 23. 23 PROCEDURE (CONTROLs) • Always run four control rows: _ Positive: Contains antibodies against the specific virus _ Negative: Contains no antibodies against the specific virus _ Antigen _ RBCs
- 24. 24 WASHING RBCs
- 25. 25 Why do we have to wash RBCs? • To obtain pure RBCs and to get rid from any other blood components such as WBCs, immune complexes, and Abs
- 26. 26 Washing process • Take place 4-5 time . • Until get clear solution above the RBCs after centrifugation . • Using PBS or normal saline . Note :(avoid using water to wash RBCs because it will definitely lead to RBCs lyses)
- 27. 27 Procedure • Obtain blood samples in tubes, spin at 1500 RPM for 5 minutes. • Draw off the supernatant using Pasteur pipette. • Add 2ml PBS to each tube and move to a clean test tube. • Centrifuge again. Each time draw off the washing solution and add 10 ml PBS until the solution above the RBCs layer becomes clear.
- 28. 28 Well – done !!
- 30. 30 In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes
- 31. 31 In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes
- 32. 32
- 33. 33 • Purpose: To quantitate serum antibody to a specific avian antigen Procedure: 1. A constant amount of haemagglutinating (HA) antigen is added to each well in a microtiter plate. 2. The test serum is then placed in the first well and serially diluted. 3. The plates are incubated for one hour and then chicken RBCs are added to each well. If antibody is present in the test serum the RBCs will not agglutinate with the HA antigen. 1. HI NEGATIVE wells will have a diffuse sheet of agglutinated RBCs covering the bottom . 2. HI POSITIVE wells will have a well circumscribed button of unagglutinated RBCs
- 34. 34 Antibody Titer • Is the lowest concentration of antibodies against a particular antigen. Figure 18.6
- 35. 35
- 36. 36 Readings • The end point is the well with the lowest concentration of the serum where a clear button is seen. 2 4 8 16 32 64 128 256 512 1024 2048 4096 The antibody titer in this row will be 512 (29). (the lowest concentration of Abs which inhibit HA caused by the virus )