Agglutination test ,antigen antibody reaction

Direct
Passive
Reverse Passive
Agglutination Inhibition
Coagglutination
Agglutination tests can be done :
On slides
In tubes
In microtritation plates
-Difference between precipitation and agglutination reaction.

1. Agglutination test Ajay Subedi Janamaitri foundation institute of health sciences,Balaju,Kathmandu,Nepal

2. 2 In 1896, Gruber and Durham published the first report about the ability of antibody to clumps cells, based on observation of agglutination of bacterial cells by serum ABO blood group Typhoid fever Brucellosis

3. Agglutination test It is one of important laboratory method to detect antigen antibody reaction. It provides flexible and useful method for semi quantitating of either antigen or antibody concentration. The reaction occurs between insoluble antigen and appropriate antibody. The reaction will results in forming aggregate or agglutinate. Antibodies that produce such reactions are called agglutinins .

4. Stages of agglutination reaction Primary phage- Sensitization Antibody reacts with single antigenic determinants on or close to particle surface. It is a rapid and reversible reaction. Based on Law of Mass Action

5. Secondary phase- Lattice formation A single antibody molecule binds to antigenic determinants on adjacent particles. The visible reaction occur under appropriate conditions and over time, particles remain connected and interconnected by antibody bridge. IgM is 700 times more efficient in agglutination than IgG (Restricted hinge region of IgG than IgM )

6. Enhancement of lattice formation Erythrocytes and bacterial cells have slightly negative charge in their surface Like charges tend to repel Use of low ionic strength saline (Use of 5 to 30% albumin neutralize the surface charge) Increase the viscosity (red cells)- add dextran or polyethylene glycol (PEG) Agitation and centrifugation Temperature- (IgG- 30-37/ IgM-4-27); ABO-IgM pH- best 6.5-7.5; IgM- best at low pH 6

7. An excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition is called the prozone effect.

8. 8 Agglutination tests- type of particles involved Antibodies can agglutinate multivalent particulate antigens, such as Red Blood Cells (RBCs) or bacteria or latex particles 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

9. Types of agglutination Rxn Direct Passive Reverse Passive Agglutination Inhibition Coagglutination 9

10. Active/Direct agglutination test combination of an insoluble particulate antigen with its soluble antibody – forms antigen-antibody complex – particles clump/agglutinate • used for antigen detection . Examples The particle antigen may be a bacterium. e.g.: Serotyping of E. coli, Salmonella using a specific antiserum. The particle antigen may be a parasite. e.g.: Serodiagnosis of Toxoplasmosis. The particle antigen may be a red blood cell. e.g.: Determination of blood groups.

11. 11 Passive Agglutination Test Converting a precipitating test to an agglutinating test Chemically link soluble antigen to inert particles such as LATEX carbon, gelatin, silicates or RBCs. Particle size- 7um to 0.8um Addition of specific antibody will cause the particles to agglutinate Examples: RA factor , antinuclear antibodies (ANA) –detects autoimmnity , ASO, Spirochete antibody(TPHA). Y Y Y + =

12. Hemeagglutination E.g. Rose waaler test –detects rheumatoid arthritis RA factor in rheumatoid arthritis patients acts as antibody to human gamma globulin. It can agglutinate red cells coated with globulins Antigen is sheep RBCs suspension sensitized with rabbit anti sheep erythrocytes antibody ( amboreceptor ).

13. Examples Microhemagglutination test for Syphilis (MHA-TP) Hemagglutination treponemal test for Syphilis (HATTS) Passive hemagglutination tests for antibody to extracellular antigen of Streptococci Rubella indirect hemagglutination test Hemagglutination Inhibition Test (HAI)  for Avian Influenza Quantitative Micro Hemagglutination Test (HA)

14. 2.Latex agglutination : latex particles used as carrier molecules Eg : antigen coated latex particles are used in detection of ASO , S.agalactiae , Clostridium difficile toxins A and B , rotavirus Antibody coated latex particles are used in detection of CRP, RA factor, HCG. Reaction is dependent on Amount and avidity of antigen bound to carrier Time of the incubation with specimen Environment of interaction (pH, Protein concentration etc )

15. 3.RPR or rapid plasma reagin : Used for syphillis Carbon is the carrier particle Cardiolipin antigen is coated with charcoal (carbon) Detect reagin – an antibody like substance in the plasma of syphillis

16. Reverse Passive Agglutination test Antibody is attached to the particulate carrier. One of the most commonly used laboratory tests that involves latex particle agglutination is the procedure for C-Reactive Protein (CRP). Reverse PAT: antibody linked to LATEX e.g. Lancefield grouping in Streptococci. 16

17. Agglutination Inhibition Antibodies against the viral protein can prevent haemagglutination so this phenomenon is called the haemagglutination -inhibition (HAI).

18. Positive Negative Hemagglutination inhibition for detection of antibodies

19. C o-agglutination: The test is based on the presence of protein A on the outer surface of Staphylococcus aureus produced by most strains(Heat killed Cowan 1 strain). The antibody( IgG ) binds by Fc portion to protein A and the antigen combining Fab portion is free. Coagglutination test can be done for sterptococcus ( lancefield groups), Spneumoniae , N.gonorrhoea , N.meningitidis

20. Co-agglutination reaction

21. Agglutination reaction When a particulate antigen is mixed with its antibody in the presence of electolytes at a suitable temperature and pH, the particles are clumped or agglutinated Agglutination tests can be done : On slides In tubes In microtritation plates

22. 22 Slide Agglutination Test Used for serotyping (e.g. Salmonella ) Antigen: isolated Salmonella in suspension Antibody: specific antisera against Salmonella Procedure : 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

23. 23 Slide Agglutination Test

24. 25 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

25. 26 Tube Agglutination Test

26. No agglutination Agglutination 1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl In this case, the titre is 40 Tube Agglutination Test

27. Negative Positive Positive Negative Slide Microtitration plate

28. Coombs ( Antiglobulin )Tests Direct Coombs Test Detects antibodies on erythrocytes + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin)

29. Coombs ( Antiglobulin )Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum Y Y Y Y Y Patient’s Serum Target RBCs + ↔ Step 1 + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin) Step 2

30. Heterophile Agglutination Tests • Weil – Felix Test or Reaction in Serodiagnosis of typhus fevers is heterophile agglutination test and sharing of common antigen between typhus, Rickettsiae and some strains of Proteus bacilli.

31. Cold Agglutination test • Positive in Mycoplasma ( Primary Atypical ) Pneumonia • The patients sera agglutinated human O group erythrocytes at 4 o c the agglutination being reversible at 37 0 c.

32. Properties Precipitation reaction Agglutination reaction Definition It is the antigen antibody reaction where the antibody reacts with the soluble antigen to form precipitin It is the antigen antibody reaction where the antibody reacts with the soluble antigen to form agglutinin Size of an antigen larger Comparatively smaller Solubility of antigen Soluble form Sedimented form Sensitivity Less sensitive More sensitive Media used Either liquid or gel matrix Does not require Types It is of three types: Precipitation in solution, precipitation in agar by diffusion and electrophoresis It is of two types: Active and passive agglutination Matrix Precipitation reaction can be performed on glass slides, petri plates and test tubes Agglutination reaction can be performed on microtitre plate, glass slides and test tubes Resulted compound Precipitins Agglutinins Formation of resulted compound Either found as suspension or sink to the bottom The end product sinks to the bottom Appearance of end product Appear as large, insoluble mass of visible precipitate Appear as large visible aggregates

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