Agglutination is the clumping together of antigens and antibodies. It occurs when the antibodies bind to particulate antigens. This causes the antigens to crosslink and form visible aggregates. Common applications of agglutination tests include blood typing (ABO and Rh), diagnosis of typhoid (Widal test), and identification of antibodies against Rh antigens (Coombs test). The titer or end point of an agglutination test refers to the highest dilution at which antigen-antibody clumping is still visible.

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.

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

7. 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

8. • 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, precipitation

9. 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

10. 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

11. 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

12. • 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

14. 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

15. • 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

16. 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

17. 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.

19. • 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.

20. • 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.

21. 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

22. • 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.