Agglutination reactions are used to assess the presence of antibodies in a specimen by mixing

it with particulate antigens.

Learning Objectives

 Describe how agglutination reactions can be used to assess the presence of antibodies
in a specimen

Key Points

 Agglutination reactions produce visible aggregates of antibody – antigen complexes

when antibodies or antigens are conjugated to a carrier.

 Carriers used in agglutination methods could be artificial (e.g., latex or charcoal) or

biological (e.g., erythrocytes).

 There are various methods of agglutination reactions that follow the same principle, but
they differ in the elements they employ based on the desired endpoint and the main
purpose of the test.

Key Terms

 Avidity: The measure of the synergism of the strength of individual interactions

between proteins.

 Erythrocytes: Red blood cells.

 Agglutination: the clumping together of red blood cells or bacteria, usually in response
to a particular antibody

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).
*Hemagglutination Assay- Principle, Types, Method, Uses

The antigen and antibody reaction in which the antigen-antibody complex formed is visible in
the form of clumps is called agglutination.

It occurs on the surface of the cells or components involved as antigens are expressed on their
surface. It occurs between insoluble antigens and soluble antibodies.

Agglutination reaction is of two types:

 Direct agglutination: It includes slide agglutination, tube agglutination, coombs’ test,

and heterophile agglutination test.

 Passive agglutination: It includes latex agglutination, hemagglutination test and,

coagglutination test.

Hemagglutination is a type of passive agglutination reaction.

Hemagglutination assay (HA) is a type of immunoassay in which erythrocytes are used as

carrier particles and are commonly preferred for serological diagnosis of various infections.

George Hirst was the person to discover hemagglutination tests. He was an American virologist.

*Requirements

 RBC suspension: It is coated with antigens specific to the antibody to be detected or

antibodies specific to antigens to be detected; hence called a carrier particle. RBCs can
be of humans, sheep, chicks, etc.

 Serum or blood sample

 Microtitre plates such as 96 well-microtitre (V-bottomed) plate

 Diluent: Phosphate Buffered Saline (PBS)

 Negative and Positive Control Samples

 RDT kits (generally with slides, reagents, and control) are available in case of Rapid
Hemagglutination assay.
 *Principle of Hemagglutination Assay

The primary theme of the hemagglutination test is that when any antigens present on
the surface of Red Blood Cells come in contact with any complementary antibody and
vice-versa, they combine to agglutinate and form noticeable clumps, which can be
observed clearly distinguishing the positive test from the negative one.

*Types of Hemagglutination Assay

It is of mainly two types based on the methodology:

A. Rapid Hemagglutination Assay

As the name suggests, in around one minute, this test can determine the presence of a
haemagglutinating agent. Hence, it can also be called Rapid Diagnosis Test (RDT). The
negative and positive control samples must be tested only once when testing multiple
samples.

Whenever a haemagglutination test is performed, the settling pattern of the red blood
cell suspension must be tested. This is done by combining diluent with red blood cells and
allowing them to settle.

i. The diluent should be dispensed.

ii. Add red blood cells and gently shake them to combine.

iii. Allow the red blood cells to settle before examining the pattern

iv. Examine the cells to see if they are setting in a normal pattern and have no auto-
agglutination. In the micro-agglutination assay, there will be a distinct button of cells
and an even suspension with no signs of clumping in the rapid assay.

Procedure of Rapid Hemagglutination Assay

1. Place four separate drops of 10% chicken red blood cells on a glass slide or the provided
one in the kit.

2. Add one drop of each control and test sample to each drop of blood along with PBS. To
dispense each sample, use separate tips, pipettes, or a flamed loop.

3. At first, PBS is dropped, followed by control and unidentified samples.

4. It should be mixed for one minute by rotating the slide or tile.

5. Observe the result and compare it with the positive and negative control provided in the
kit to analyze the result.

B. Micro-hemagglutination Assay

This method is useful for testing the presence or absence of haemagglutinin in allantoic
fluid from many embryonated eggs. It is a more time-consuming method than RDT. Red
blood cells are dissolved in a 1% solution. Cells settle faster in V-bottom plates, and the
slight difference between positive and negative results is greater than in U-bottom
plates.

*Procedure of Micro-hemagglutination Assay

1. Fill out a recording sheet with information about the samples being tested. The samples
and controls will be placed in the wells indicated on this sheet.

2. Take the sample of about 50 ml with a micropipette and dispense it into a well of the
microwell plate. Use a different tip for each sample to prevent contamination of
samples.

3. Place negative and positive controls on one of the plates.

4. Pour 50 mL of PBS into each well. These wells will serve as auto-agglutination controls
for red blood cells.

5. Fill each well with 25 mL of 1% red blood cells.

6. Gently tap the plate’s sides to mix. Cover the plate with a plate cover.

7. Let the plate stand for about 40 minutes, and observe/record the data.

*Result Interpretation of Hemagglutination Assay

The appearance of clumps in the case of agglutinated suspension can indicate a positive
test in all tests. It can be compared with the positive control set to analyze properly. A
positive test suggests that the respective sample is contaminated with antibodies or
antigens related to a pathogen.

Note: The clumps can be observed at the bottom of the well in the case of Micro-well
and on the surface in the case of RDT.
*Applications of Hemagglutination Assay

 It can be used to detect the humoral immune response of the body against any disease
or infective agents.

 It can be used for determining different blood cell types or groups.

 It can detect and quantify viral infections such as paramyxovirus, influenza, etc.

 Different rapid diagnosis test kits based on hemagglutination are designed. For e.g. an
RDT kit for detecting HbsAg in case of Hepatitis B infection.

 It can also detect various bacterial infections, such as syphilis.

 *Limitations of Hemagglutination Assay

 Faults in incubation time and concentration of RBC may lead to wrong results.

 Different factors considered in the reaction must be specific. Their non-specificity may
result in an incorrect result.

 Determination of quantitative values and result interpretation require qualified

individuals.

 Result interpretation is made manually without any digital data, so the different
observers might have errors or fluctuations in analysis.

 Latex Agglutination Test

 Direct Agglutination Test refers to the assays in which the antigen directly
agglutinates with the antibody.
 Indirect or passive agglutination involves coating of antigen on the surface of a
carrier molecule (e.g. RBC, latex or bentonite), such that the antibody binds to the
coated antigen and agglutination takes place on the surface of the carrier molecule.
They are also referred to as ‘particle agglutination test’.

 Reverse passive agglutination test is a special type of particle agglutination test

in which the antibody is coated on a carrier molecule which detects antigen in the
patient’s serum.

Latex Agglutination Test

A group of passive agglutination tests carried out by coating either antigen or

antibody on an artificial carrier particle, called latex bead are called as latex
agglutination test. They may alternatively referred to as latex fixation test and
may be:

Latex Agglutination Test (LAT) for Antibody Detection: A Passive

Agglutination Test with Antigen bound to the surface of latex beads (polystyrene
latex particles; 0.8- 1 μm in diameter).

Latex Agglutination Test (LAT) for Antigen Detection: A Reverse Passive

Agglutination Test with Antibody bound to the surface of latex beads.
Objectives of Latex Agglutination Test

To detect microbial and viral infections, autoimmune diseases, hormones, drugs or

serum proteins by agglutination reaction of antigen-antibody.

*Principle of Latex Agglutination Test

Antibody or antigen molecules can be bound in random alignment to the surface of

latex (polystyrene) beads. The number of antibody or antigen molecules bound to
each latex particle is large, resulting in a high number of exposed potential binding
sites. Antigen or antibody present in a specimen binds to the combining sites of the
corresponding antigen/antibody exposed on the surfaces of the latex beads,
forming cross-linked aggregates of latex beads and antigen/antibody. Large
particle size of latex facilitates the visualization of the antigen-antibody reaction.

Requirements for Latex Agglutination Test

1.5 ml Vials, Microcentrifuge, Pipette, Microtips, Laboratory refrigerator, Glycine

saline buffer, Blocking buffer, Antigen for coating, Latex beads, Test antiserum,
Glass slides, Beaker, Tooth pick.
Procedure of Latex Agglutination Test

The common procedure for both types of latex test involves

coating microbeads of latex with pathogen-specific antigens or antibodies.
Patient’s cerebrospinal fluid, serum or urine is mixed with the coated latex
particles in serial dilutions with normal saline and observed
for agglutination (clumping).

Coating of Latex (For detection of antibodies)

1. To 20 μl of latex beads taken in a 1.5 ml vial add 40 μl of glycine-saline buffer.

2. Add 60 μl of antigen to the latex and incubate at 37oC for 2 hours.
3. Spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant.
4. Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10
minutes.
5. Repeat the washing once more.
6. Add 90 μl of blocking buffer to the pellet, mix well.
7. Incubate at 4oC, overnight.

Agglutination Test

1. To 200 μl of glycine-saline buffer taken in a vial, add 4 μl of test antisera. (50 times
diluted).
2. Add 50 μl of antigen to 50 μl of diluted antiserum in a 1.5 ml vial, mix well and incubate
at room temperature for 10 minutes.
3. Pipette 10 μl of coated latex onto a glass slides.
4. Add 10 μl of diluted test antiserum to slide A.
5. Add 10 μl of antiserum mixed with antigen (from step 1) to B.
6. Add 10 μl of glycine-saline buffer to C.
7. Take a tooth pick and mix the content in each slide. Discard the tooth pick after using in
one slide (take a new one for the next slide).
8. After mixing, wait for 2 minutes to observe the result.
Results and Interpretation of Latex Agglutination Test

Depending on the procedure, some reactions are reported as positive or negative

and other reactions are graded on a 1+ to 4+ scale, with 2+ usually the minimum
amount of agglutination visible in a positive sample without the aid of a
microscope.

Positive: Agglutination of the beads evident by clumps in any of the dilutions is

considered a positive result.

It confirms either that the patient’s body has produced the pathogen-specific
antibody (if the test supplied the antigen) or that the specimen contains the
pathogen’s antigen (if the test supplied the antibody).

Negative: No agglutination or formation of clumps.

Absence of pathogen-specific antigen or antibody.

Applications of Latex Agglutination Test

 Latex tests are very popular in clinical laboratories for detecting antigen to Cryptococcus
neoformans in cerebrospinal fluid or serum. It is also used for detection of capsular
antigens of Pneumococcus, Haemophilus influenzae and Meningococcus.
 To confirm the presence of beta-hemolytic Streptococcus from culture plates.
  Latex tests are continually being developed for a variety of organisms such as for the
detection of Clostridium difficile toxins A and B, rotavirus, and Escherichia coli 0157:H7
from suspect colonies of E coli.
 Latex agglutination test using latex particles coated with anti-CRP antibodies is the most
widely used method employed worldwide for detection of C-reactive protein. Detection
limit of CRP by latex agglutination test is 0.6 mg/dl.
 Latex Agglutination Test (LAT) for Antibody Detection is used for detection of ASO
(antistreptolysin O antibody).

*Coagglutination test:

Coagglutination is a type of agglutination reaction in which Cowan I strain of S. aureus is used as

carrier particle to coat antibodies. Cowan I strain of S. aureus contains protein A, an anti-
antibody, that combines with the Fc portion of immunoglobulin, IgG, leaving the Fab region free
to react with the antigen present in the specimens. In a positive test, protein A bearing S.
aureus coated with antibodies will be agglutinated if mixed with specific antigen. The advantage
of the test is that these particles show greater stability than latex particles and are more
refractory to changes in ionic strength.

Uses of Coagglutination test

1. Detection of cryptococcal antigen in the CSF for diagnosis of cryptococcal meningitis;

2. Detection of amoebic and hydatid antigens in the serum for diagnosis of amoebiasis and
cystic echinococcosis,
3. Grouping of streptococci and mycobacteria and for typing of Neisseria gonorrhoeae.

*Agglutination inhibition:

Agglutination inhibition or hemagglutination inhibition refers to the inhibition of these

reactions by soluble antigen which reacts with the combining sites of the antibodies and
thereby prevents their binding to and agglutination of the particles.

The hemagglutination inhibition (HI) assay is used to titrate the antibody response to a viral
infection. The HI assay takes advantage of some viruses' ability to hemagglutinate (bind) red
blood cells, therefore forming a “lattice” and preventing the red blood cells from clumping.

In hemagglutination (or hemagglutination inhibition assay, HIA, or passive hemagglutination

assay, PHA), RBCs (generally sheep), which have been coated or coupled with antigen (thus
called sensitized RBCs), are incubated with antibody and sample. After incubation, the degree of
agglutination is determined. In this assay, antigen in the sample competes with the antigen
coated on the RBCs for their interaction with antibody. Another variation of the HIA is reverse
passive hemagglutination assay, in which the purified antibody is coupled to the RBCs.
Agglutination is observed when antigen or sample extract is added to the system. Although HIA
is relatively simple, food extracts often interfere with the assay and a large amount of antiserum
is needed. Only semiquantitative information can be obtained.

Hemagglutination inhibition tests are simple, sensitive, inexpensive, and rapid and therefore
are often the method of choice for assaying antibodies to influenza A virus.

The test relies on the hemagglutination activity of the influenza HA and the ability of HA-specific
antibodies to inhibit the virus from agglutinating erythrocytes (Fig. 13-5). Briefly, dilutions of
serum are incubated with virus, and erythrocytes are added. After incubation, the HI titer is read
as the highest dilution of serum that inhibits hemagglutination.