Direct Agglutination Assay
Direct Agglutination Assay
Direct Agglutination Assay
PRINCIPLE
Direct agglutination assay involves clumping of cells or particulate antigen
by specific antibody with 2 or more receptors linking the particles in
suspension. Such agglutination reaction requires a minimum cell surface
antigen density and access of antibodies to their complementary cell
surface antigens. The most common example of a direct agglutination
reaction is red blood cell aggregation through the addition of ABO
isohemagglutinins.
MATERIALS
Reagents
1.Known group A, B, AB and O human erythrocytes, washed and
resuspended in normal saline to make a 3-4% cell suspension
2.Normal saline (NaCI 0.85%)
3.Anti-A, Anti-B, Anti-AB and anti-Rh serum
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[PRACTICAL 5] MTEB 2404
EQUIPMENTS
PROCEDURE
1. Place a drop of Anti-A serum on the left side of the glass slide and
a drop of Anti-B
serum on the right side of the slide.
2.Place a drop of 3-4% cell suspension of known A cells next to each drop
of serum.
3.Mix the 2 drops on each half of the slide with an applicator or
toothpick. DO NOT mix the serum together.
4. Rotate the slide gently while examining it macroscopically.
Record the result observed.
5. Repeat steps 1 to 4 to observe the reaction of known cells of
group B, ANTIBODY
and O in the presence of Anti-A and Anti-B serum.
6.Determine your own blood group. Swab a subject's finger with alcohol.
Using a disposable, sterile lancet, prick the swabbed spot. Allow several
drops of blood to flow directly into a tube containing 1 ml saline.
7.Mix the blood suspension and proceed as in steps 1 to 4. Record the
results.
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Result
Rh Blood
Anti- Anti- Antibo Antibo Anti-
NO Anti-A Contr Grou
B AB dy A dy B D
ol p
A
1 Rh(D)
+ve
B
2 Rh(D)
+ve
AB
3 Rh(D)
+ve
O
4 Rh(D)
+ve
A
Rh(D)
5
- ve
B
Rh(D)
6
- ve
AB
Rh(D)
7
- ve
8 O
Rh(D)
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[PRACTICAL 5] MTEB 2404
- ve
: Agglutination
: No agglutination
Discussion:
The basic technique in identification of
the antigens and antibodies of blood groups is the agglutination
test. Agglutination of red cells results from antibody cross-linkages
established when different specific combining sites of one antibody react
with antigen on two different red cells. By mixing red cells (antigen)
and serum (antibody), either the type of antigen or the type of antibody
can be determined depending on whether a cell of known antigen
composition or a serum with known antibody specificity is used.
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[PRACTICAL 5] MTEB 2404
An antibody that does not clump red cells when they are suspended
in saline solution is called incomplete. Such antibodies block the antigenic
sites of the red cells so that subsequent addition of complete antibody of
the same antigenic specificity does not result in agglutination. Incomplete
antibodies will agglutinate red cells carrying the appropriate antigen,
however, when the cells are suspended in media
containing protein. Serum albumin from the blood of cattle is a substance
that is frequently used for this purpose. Red cells may also be rendered
specifically agglutinable by incomplete antibodies after treatment with
such protease enzymes as trypsin, papain, ficin, or bromelain.
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[PRACTICAL 5] MTEB 2404
Conclusion:
The agglutination reaction can be observed by doing the ABO grouping
test using sera reagent which shown positive by forming clumping within
specific antibody.
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