Agglutination

its about agglutination and various clinical test based on agglutination

1. agglutination By Monisha Jayabalan I MSc Medical Biochemistry

2. The interaction between antigen and antibody is called antigen-antibody interaction The antibody binds with the antigen to form a complex molecule called immune complex This reaction is specific in nature ( ex: lens antigen ) The part of the antibody which combines with antigen is called paratope or antigen binding site The part of the antigen which combines with the antibody is called epitope or antigenic determinant

3. Agglutination happens when there is a reaction between antigen and antibody It is defined as the interaction between antibody and a particulate antigen which results in clumping The antibodies that produces such reactions are called as agglutinins The particulate antigens aggregated are called agglutinogens

4. PROZONE EFFECT Excess antibody has got the capacity to inhibit the agglutination reaction As the antibody concentration is lowered below the prozone , the reaction occurs.

5. MECHANISM Primary phenomenon (SENSITIZATION) First reaction involving Ag- Ab combination Single antigenic determinant on the surface particle Initial reaction: rapid and reversible Cross link formation  visible aggregates (stabilization)

6. Secondary phenomenon : LATTICE FORMATION Ab + multivalent Ag  stable network (visible reaction) conc. of Ag and Ab Governed by physiochemical factors: Ionic strength of milieu pH temperature

7. Lattice Formation The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination If both antigen and antibody are SOLUBLE reaction will become visible over time, ie , p recipitation

8. AGGLUTINATION TEST Agglutination test refers to the examination of clump formation when particulate antigen and its antibody are combined It has wide application in clinical field

9. APPLICATIONS OF AGGLUTINATION TEST ABO blood grouping Rh blood grouping Widal test for typhoid Weil felix test for typhus Coomb’s test for the identification of anti Rh antibodies Brucella agglutination test for brucellosis Leptospira agglutionarion for leptospirosis Cold agglutination test for pneumonia, malaria and trypanosomiasis Haemagglutination inhibition test for the diagnosis of certain viral and parasitic diseases

10. ABO blood grouping and Rh typing To the drop of blood sample anti-serum A and to another drop anti-serum B is added. If the blood sample is clumped with antiserum A it is GROUP A If clump formation occur with anti-serum B then its GROUP B Clumping with both anti- sera then it is GROUP AB No clumping its GROUP O

11. For Rh typing, a drop of blood sample is mixed with a drop of anti-Rh serum if clumping occurs its Rh+ve If there is no clumping then its Rh- ve

12. WIDAL TEST It is a serological test for enteric fever It based on measurement of agglutinating antibodies against O and H antigens bacteria causing typhoid fever is mixed with serum containing specific antibodies obtained from an infected individual When the antiserum of the person is added to the antigen then there is clump formation

13. The result of the tests are scored from 0 to 4+, ie , 0 (no agglutination) , 1+ (25% agglutination), 2+ (50% agglutination), 3+ (75% agglutination) or 4+ (100% agglutination) . The smallest quantity of serum that exhibits a 2+ or 50% agglutination is considered the end-point of serum activity or titre . O > 1 in 80 H > 1in 160

14. TITRE AND END POINT Titre : The maximum dilution that gives visible agglutination. The end point: is the well with the lowest concentration of the virus where there is agglutination

15. COOMB’S TEST This test was devised by coomb in 1945 There are two types of coomb test namely direct coomb test and indirect coomb test The two Coombs tests are based on the fact that anti-human antibodies, which are produced by immunizing non-human species with human serum, will bind to human antibodies, commonly IgG or IgM .

16. DIRECT COOMB TEST The patient's red blood cells (RBCs) are washed (removing the patient's own serum ) and then centrifuged with antihuman globulin (also known as Coombs reagent). If immunoglobulin or complement factors have been fixed on to the RBC surface in-vivo , the antihuman globulin will agglutinate the RBCs and the direct Coombs test will be positive.

17. INDIRECT COOMB TEST The IAT is a two-stage test. First stage Washed test red blood cells (RBCs) are incubated with a non human serum. If the serum contains antibodies to antigens on the RBC surface, the antibodies will bind onto the surface of the RBCs. Second stage The RBCs are washed three or four times with isotonic saline and then incubated with antihuman globulin. If antibodies have bound to RBC surface antigens in the first stage, RBCs will agglutinate when incubated with the antihuman globulin (also known Coombs reagent) in this stage, and the indirect Coombs test will be positive .

18. AGGLUTINATION INHIBITION TEST a serologic technique useful in testing for certain unknown soluble antigens. The unknown antigen is mixed with a known agglutinin. If a reaction occurs, the agglutinin can no longer adhere to the cells or particles that carry its corresponding antigen, and the unknown antigen is thus identified . A fine example is pregnancy test

19. The latex agglutination inhibition test was also developed in the early 1960s. It used polystyrene latex particles coated with hCG . A mixture of centrifuged human urine and rabbit hCG anti-serum was prepared and added to the hCG-coated latex particle suspension. If the urine was free of hCG , the latex particles would agglutinate in response to the rabbit hCG anti-serum . If hCG was present in the urine , the hCG would neutralise the rabbit hCG anti-serum and agglutination of the latex particles would not occur.