Agglutination test

Precipitation tests
Agglutination Tests:

Definition:
When the specific antibodies (agglutinins) bind to surface
antigens of bacteria/virus or any antigens immobilized in
particulate matter (such as latex particle) and cause the
formation of a visible clumps, such test is called
agglutination test.
Agglutination:

 Agglutination is the visible expression of the aggregation of antigens and antibodies.

Agglutination reactions apply to particulate test antigens that have been conjugated to a
carrier. The carrier could be artificial (such as latex or charcoal particles) or biological
(such as red blood cells). These conjugated particles are reacted with patient serum
presumably containing antibodies.
 The endpoint of the test is the observation of clumps resulting from that antigen-
antibody complex formation.
 The quality of the result is determined by the time of incubation with the
antibody source, amount and avidity of the antigen conjugated to the carrier, and
conditions of the test environment (e.g., pH and protein concentration).
Agglutination test can be performed
in:

1. Surface of glass slides: Rapid reading/evaporation

2. Test tubes: More sensitive because of longer incubation
Types of Agglutination..

 Identification of an organism with known antibodies.

 Identification of serum antibodies with known antigens.
Methods of agglutination:

Various methods of agglutination are used in diagnostic immunology

1. Latex agglutination :
In latex agglutination, many antibody molecules are bound to latex
beads (particles), which increases the number of antigen-binding sites. If an antigen is present
in a test specimen, it will bind to the antibody and form visible, cross-linked aggregates.
Latex agglutination can also be performed with the antigen conjugated to the beads for testing
the presence of antibodies in a serum specimen.
Latex agglutination test
2. Direct bacterial agglutination:
Direct bacterial agglutination uses whole
pathogens as a source of antigen. It measures the antibody level produced
by a host infected with that pathogen. The binding of antibodies to surface
antigens on the bacteria results in visible clumps.
3. Hemagglutination :
It uses erythrocytes as the biological
carriers of bacterial antigens, and purified polysaccharides or
proteins for determining the presence of corresponding antibodies in a
specimen.
Direct coombs test

 Direct Coombs’ test (DAT): Detects nonagglutinating antibodies or complement proteins

on red blood cells in vivo
 Antibody Is used to clump bacterial cells or other large structures
 Some bacteria and viruses cross-link red blood cells and clump them together
Direct coomb’s test:
Agglutination of red blood cells is called hemagglutination. One common assay that uses
hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT),
which generally looks for nonagglutinating antibodies. The test can also detect complement attached to
red blood cells.

The antibodies bound to red blood cells in these conditions are most often IgG, and because of the
orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is
unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood
cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies
(that may be combined with anti-complement) . The Coombs’ reagent links the IgG attached to
neighboring red blood cells and thus promotes agglutination.
 

 
Indirect coomb’s test

 There is also an indirect Coombs’ test known as the indirect

antiglobulin test (IAT). This screens an individual for
antibodies against red blood cell antigens (other than the A and B
antigens) that are unbound in a patient’s serum IAT can be used
to screen pregnant women for antibodies that may cause
hemolytic disease of the newborn. It can also be used prior to
giving blood transfusions.
Precipitation Reactions

 Precipitation reactions are serological assays for the detection of

immunoglobulin levels from the serum of a patient with infection.

 Key Points
Precipitation
assays are performed in semi-solid media such as agar or agarose
where antibodies and antigens can diffuse toward one another and form a
visible line of precipitation.
Precipitation methods:

 There are several precipitation methods applied in the diagnostic laboratory. These
include single, double, and electroimmunodiffusion.
 The most widely used gold standard precipitation methods are Ouchterlony
test /double immunodiffusion test
1. Precipitin ring test:
To perform this test, a set of test tubes is prepared by adding an
antigen solution to the bottom of each tube. Each tube receives
the same volume of solution, and the concentration of antigens
is constant (e.g., 1 mg/mL). Next, glycerol is added to the
antigen solution in each test tube, followed by a serial dilution
of the antiserum. The glycerol prevents mixing of the antiserum
with the antigen solution, allowing antigen-antibody binding to
take place only at the interface of the two solutions. The result
is a visible ring of precipitin in the tubes that have an antigen-
antibody ratio within the equivalence zone. This highest dilution
with a visible ring is used to determine the titer of the
antibodies. The titer is the reciprocal of the highest dilution
showing a positive result, expressed as a whole number. In
Figure the titer is 16.
Precipitin ring test:
2. Double immunodiffusion test/ ouchterlony assay :

When agar is highly purified, it produces a clear, colorless gel. Holes are
punched in the gel to form wells, and antigen and antisera are added to
neighboring wells. Proteins are able to diffuse through the gel, and
precipitin arcs form between the wells at the zone of equivalence. Because
the precipitin lattice is too large to diffuse through the gel, the arcs are
firmly locked in place and easy to see.
Double immunodiffusion test
3. Flocculation assay
It is similar to a precipitin reaction except that it involves insoluble antigens
such as lipids. A flocculant is similar to a precipitin in that there is a visible
lattice of antigen and antibody, but because lipids are insoluble in aqueous
solution, they cannot precipitate. Instead of precipitation, flocculation
(foaming) is observed in the test tube fluid.
4. Radial immunodiffusion
The radial immunodiffusion (RID) assay is similar to the Ouchterlony assay
but is used to precisely quantify antigen concentration rather than to compare
different antigens.
It’s a semi-quantitation of proteins by gel diffusion using antibody
incorporated in agar.

5. Electroimmunodiffusion:
It’s a variation of the double immunodiffusion method reaction that uses an
electric current to enhance the mobility of the reactants toward each other.
Difference b/w agglutination and precipitation
test:

The significant differences between precipitation and agglutination reaction are due to
the following properties like:
 Size: The size of antigen in precipitation reaction is larger in comparison to the
agglutination reaction.
 Solubility: In a precipitation reaction, the antigen is in soluble form whereas, in an
agglutination reaction, the antigen is in sedimented form.
 Sensitivity: Agglutination reaction is more sensitive in comparison to the precipitation
reaction.
 Comparison Chart
Properties Precipitation reaction Agglutination reaction
It is the antigen antibody reaction where It is the antigen antibody reaction where
Definition the antibody reacts with the soluble antigen the antibody reacts with the soluble antigen
to form precipitin 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

It is of three types: Precipitation in solution,
It is of two types: Active and passive
Types precipitation in agar by diffusion and
agglutination
electrophoresis
Precipitation reaction can be performed on Agglutination reaction can be performed on
Matrix
glass slides, petri plates and test tubes microtitre plate, glass slides and test tubes

Resulted compound Precipitins Agglutinins

Either found as suspension or sink to the
Formation of resulted compound The end product sinks to the bottom
bottom
Appear as large, insoluble mass of visible
Appearance of end product Appear as large visible aggregates
precipitate
Similarities:

 Both precipitation and agglutination reaction are the two most common serological tests.
 Precipitation, as well as agglutination reaction, can be used for the quantitative study of
antigen and antibody.
 Agglutination and precipitation reaction requires both the reactants, i.e. antigen and
antibody to form an immunocomplex or lattice