Agglutination Reaction Definition, Types, Applications

In 1896, two bacteriologists, Herbert Edward Durham and Max von Gruber, independently discovered specific agglutination. In honour of the discoverers, this reaction was given the name GruberDurham reaction. Later, Gruber designated as “agglutinin” any chemical that induced an agglutination reaction (from the Latin). Fernand Widal (1862–1929) employed agglutination to diagnose typhoid disease in the same year. Blood serum from a typhoid carrier clumped a culture of typhoid bacteria, whereas serum from a typhoid-free individual did not. The Widal test is the first sero-diagnostic test for an infectious disease. In 1900, Karl Landsteiner discovered another significant practical application of the agglutination reaction: blood group (ABO) typing. This marked the beginning of the science of transfusion medicine and safe blood transfusion.

What is Agglutination Reaction?

In the antigen-antibody interaction known as agglutination, particles of antigen combine with their respective antibodies under the influence of electrolytes at a predetermined temperature and pH, causing the particles to clump together visibly.
When there is a 1:1 ratio of antigen to antibody, agglutination is most efficient.

There are many similarities between agglutination reactions and precipitation processes, which are comparable in their basic properties. Antibodies and the cells that harbour the antigen on their surface form a lattice network, much like what happens in a precipitation reaction. When cells clump together, the outcome is more noticeable than when a soluble antigen is present alone.
Since the agglutination process occurs on the particle’s surface, the antigen must be accessible so that it can bind with the antibody and form visible clumps, which is not the case with the precipitation reaction. In agglutination procedures, the antibody solution is diluted serially and a constant amount of particle-based antigen is added to each dilution.
Clumping is observed visually after incubation at 37 degrees Celsius for several hours. Titer is defined as the inverse of the greatest dilution at which clumping occurs with the antiserum.

Because of the abundance of antigenic determinants on the cell surface, the phenomenon of antibody excess is quite uncommon.
Some antibody responses involve recognition of antigenic markers on a cell surface but no agglutination of the target cells.
They prevent the whole antibodies from fully agglutinating. Antibodies with this property are known as blocking antibodies.

Some examples of these blocking antibodies are those that target the Rh factor and the brucella virus.
The detection of antigens and antibodies in serum and other body fluids can be accomplished by a number of different agglutination reactions. Extremely sensitive, and the test result is readily apparent to the naked eye, they are an excellent choice.

Process Of Agglutination

By suspending cells containing the antigen (epitopes), antigen-bearing bacteria, or particles in a solution containing particular antibodies termed “agglutinins,” agglutination can be induced.

To visualise the specificity of an agglutination reaction, think of a lock and key. In molecular terms, an antibody has a characteristic “Y” shape.
The “Y” shaped Fab section is composed of the hypervariable regions of the heavy and light chains and contains the combining site.
As seen in the Figure, the antigenic determinant fits snugly into a cleft formed by the antibody’s combining site.

So the cleft generated by the Fab is the lock, and the antigenic determinant is the “Key” that opens the lock. Agglutination occurs if and only if the conditions are right for it to do so. This idea applies to any response between an antigen and an antibody.
There are two phases of agglutination. Sensitization comes first, followed by lattice creation.

Sensitization

This phenomenon occurs when an antibody binds to its respective antigen. The reaction is affected by the pH level, the incubation temperature, and the incubation period.

While IgG antibodies are most active at 37 degrees C, IgM antibodies are most active between 4 and 22 degrees C. The incubation period is flexible, lasting anywhere from 15 to 60 minutes.

Lattice formation

Like a Jaal, lattice looks like a grid. Sensitized particles bond with one another by cross-linking to produce it. Time is needed, however the result may be visible to the naked eye, unlike with sensitization.
In general, IgM is superior for this type of reaction due to its larger size, however IgG antibodies may require improvement.

Types of agglutination reactions

Direct agglutination describes responses in which the antigens are already present on the particle. In contrast, passive agglutination makes use of particles that have been artificially modified to bear antigens.

Active/Direct agglutination
Passive agglutination

1. Direct agglutination

As a category, direct agglutination reactions can be broken down into four subcategories: (a) slide agglutination, (b) tube agglutination, (c) heterophile agglutination, and (d) antiglobulin (Coombs’) test.

a. Slide agglutination test

A simple agglutination process is carried out on a glass slide. One common use of direct slide agglutination is the classification of bacteria.
A drop of standard antiserum is mixed with a bacterial solution for this test.

The formation of bacterial clumps and the disappearance of the background solution indicate a successful response.
In a positive test, clumping happens immediately, usually within a few seconds. On the same slide, you’ll find a “control” made up of a suspension of antigen in saline without any antiserum.
It helps verify the findings and identify potential false positives caused by antigen autoagglutination.

Uses of Slide agglutination test

Common practise for determining the species of bacteria recovered from clinical samples, such as Salmonella, Shigella, Vibrio, etc.
To determine blood types and for use in transfusions.

b. Tube agglutination test

The tube agglutination test is carried out in glass tubes, as the name suggests. Serum from the patient is diluted in a succession of tubes, and then bacterial antigens relevant to the condition under suspicion are added. Physical clusters of agglutination are evidence of a response between an antigen and an antibody. It is the gold standard for determining the concentration of antibodies in blood samples. Regularly used for the serodiagnosis of both enteric fever and brucellosis, tube agglutination tests demonstrate the presence of antibodies in the serum by demonstrating the presence of:

Widal test

The presence of antibodies against Salmonella typhi, S. paratyphi A, and S. paratyphi B in patient serum is used to diagnose enteric fever using the Widal test.

The standard agglutination test

For the purpose of serodiagnosis, the standard agglutination test is frequently employed. However, the tube agglutination test for brucellosis is made more difficult by the prozone phenomenon.
This is because there is a lot of brucella antibody in the patient’s serum, leading to false negative results.

Multiple serum dilutions eliminate the potential for false positive results.
There is also the issue of serum antibodies that are either blocking or incomplete.
Antiglobulin (Coombs’ test) is used to detect these antibodies and prevent this from happening.

c. Heterophile agglutination test

This test relies on the presence of heterophilic antibodies in specific bacterial illnesses’ serum:

Weil–Felix test: Weil–Felix test is an example of heterophile agglutination response for rickettsial infection serodiagnosis. In this test, cross-reacting antibodies against a rickettsial infection are discovered using cross-reacting antigens (e.g., Proteus strains OXK, OX19, and OX2). Antibodies against rickettsial organisms cross-react with antigens from Proteus strains OXK, OX19, and OX2.
Paul–Bunnell test: The Paul–Bunnell test is another heterophile agglutination test that uses sheep erythrocytes as antigens to detect antibodies in infectious mononucleosis.

Test for Streptococcus MG agglutination: Similar to the Streptococcus MG agglutination test, which detects antibodies to Mycoplasma pneumoniae causing primary atypical pneumonia, is the Streptococcus MG agglutination test.

d. Antiglobulin (Coombs’) test

Coombs’, Mourant, and Race originally developed the Coombs’ test to detect anti-Rh antibodies that do not agglutinate Rh erythrocytes in saline.
When serum containing incomplete anti-Rh antibodies is combined with Rh erythrocytes in saline, incomplete antibody antiglobulin coats the surface of the erythrocytes without causing agglutination.

These erythrocytes are agglutinated when treated with antiglobulin or Coombs’ serum (rabbit antiserum against human globulin).
There are two forms of Coombs’ test: (a) the direct Coombs’ test and (b) the indirect Coombs’ test.

i. Direct Coombs’ test

This test involves the in vivo sensitization of red blood cells (RBCs) using incomplete antibodies.

By agglutinating the patient’s red blood cells with antiserum against human immunoglobulin, this test can identify cell-bound antibodies.

ii. Indirect Coombs’ test

This test involves the in vitro sensitization of RBCs with partial antibodies. In this test, normal red cells and antiserum to human immunoglobulin are combined with the patient’s serum.
Agglutination happens if the patient’s serum has antibodies.

In brucellosis and other disorders, Coombs’ tests are performed to detect (a) anti-Rh antibodies and (b) incomplete antibodies.

2. Passive agglutination

Carrier particles coated with soluble antigens are utilised in passive agglutination.
This is typically done to transform precipitation reactions into agglutination reactions, as the latter are simpler to run and understand and more sensitive for detecting antibodies than precipitation reactions.

Reverse passive agglutination refers to the detection of antigens by adsorption of the antibody rather than antigens on the carrier particle.
Prior to the 1970s, erythrocytes were the predominant particles employed to coat antigens.
In recent years, however, a range of different particles, such as polystyrene latex, bentonite, and charcoal, have been utilised for this purpose.

The size of particles ranges from 7 for RBCs to 0.05 for extremely small latex particles. The application of synthetic beads or particles offers consistency, uniformity, and stability.
Visually, reactions are similarly simple to interpret. Depending on the carrier particles utilised, passive agglutination reactions can be of the following types: I latex agglutination test, (ii) hemagglutination test, and (iii) coagglutination test.

a. Latex agglutination test

It is a test that uses latex particles as antigen or antibody carriers.

Singer and Plotz discovered by accident in 1955 that IgG spontaneously adsorbs to the surface of polystyrene latex particles.
Latex particles are affordable, generally stable, and immune to antibody cross-reactivity.
These particles may be coated with antibodies for antigen detection in serum and other bodily fluids. The use of monoclonal antibodies has decreased cross-results, hence decreasing the number of false-positive reactions.

In addition, the high particle size of latex permits enhanced naked-eye detection of antigen–antibody interactions.
Typically, the tests are conducted on cardboard cards or glass slides, and favourable reactions are rated on a scale from 1 to 4.

Limitations of Latex Agglutination Test

Due to the fact that the pH, osmolarity, and ionic concentration of the solution determine the quantity of binding that happens, latex agglutination processes must be conducted under precisely defined circumstances.

Some components of bodily fluids, such as rheumatoid factor, have been identified as causing false positive results in the existing latex agglutination systems.
Some agglutination procedures require specimens to be processed at 56°C or with ethylenediaminetetraacetic acid (EDTA) prior to testing, which makes it a laborious procedure.

Advantages of Latex Agglutination Test

The size of the latex bead (at least 0.8 m) facilitates the visualisation of the agglutination response.

LAT is currently one of the most popular examinations because to its simplicity and speed. Many previous serological tests, such as co agglutination assays, are believed to have been supplanted by it.
They are affordable, relatively stable, and do not react with any other antibodies.
The lowest levels of bacterial polysaccharides identified by latex agglutination were 0.1 ng/mL.

Uses of Latex agglutination test

The tests are utilised to quickly identify antigens of group B Streptococcus, Staphylococcus aureus, Neisseria meningitidis, Cryptococcus neoformans, etc.
In addition to detecting soluble microbial antigens in urine, spinal fluid, and serum for the diagnosis of a range of infectious disorders, these assays have shown beneficial for detecting soluble antigens in urine, spinal fluid, and serum.
In serum samples, these assays are used to detect RA factor, ASLO, CRP, etc.

b. Hemagglutination test

In hemagglutination tests, RBCs serve as carrier particles. RBCs from sheep, humans, and chickens, among others, are routinely employed in tests.
This test is known as an indirect hemagglutination (IHA) test when RBCs are coated with antigen to detect antibodies in the blood.
The IHA is a widely utilised test for the serodiagnosis of numerous parasite disorders, such as amoebiasis, hydatid disease, and toxoplasma.

It is known as reverse passive hemagglutination when antibodies are linked to RBCs to detect microbial antigen (RPHA).
In the past, the RPHA has been widely used to identify viral antigens, such as HBsAg in the serum, for the diagnosis of hepatitis B infection.
Antigens in numerous other viral and parasite illnesses have also been detected with this test.

c. Viral hemagglutination

Numerous viruses, such as influenza, measles, and mumps, can agglutinate RBCs without antigen–antibody interactions.
This method is known as viral hemagglutination. This event is referred to as hemagglutination inhibition and occurs when a virus-specific antibody inhibits the hemagglutination.
This is the basis for the viral hemagglutination inhibition test, which is used to identify neutralising antibodies in the sera of patients against agglutinating viruses.

The serum of the patient is first incubated with a viral preparation in order to perform this test.
Then, RBCs that are known to be agglutinated by the virus are added to the mixture.
Absence of agglutination or a reduction in agglutination suggests the existence of antibody in the patient’s serum.

Coagglutination test

Coagglutination is a sort of agglutination reaction in which antibodies are coated with the Cowan I strain of S. aureus.
Cowan I strain of S. aureus has protein A, an anti-antibody, which binds to the Fc region of immunoglobulin, IgG, freeing the Fab region to react with the antigen contained in the specimens.
Protein A containing S. aureus that has been coated with antibodies will agglutinate when combined with a specific antigen in a positive test.

The advantage of this test is that these particles are more stable and resistant to changes in ionic strength than latex particles.

Uses of Coagglutination test

Detection of cryptococcal antigen in the cerebrospinal fluid (CSF) to diagnose cryptococcal meningitis; Detection of amoebic and hydatid antigens in the serum to diagnose amoebiasis and cystic echinococcosis, respectively.
Streptococci and mycobacteria classification and Neisseria gonorrhoea typing.

Prozone And Post Zone Phenomena

If antigen and antibody are not combined in the appropriate proportions, a false negative antigen antibody reaction, either agglutination or precipitation, can result. This can occur if either the antigen or the antibody is in excess (Prozone) (Post zone).

Prozone phenomenon

When tested undiluted, certain sera do not exhibit agglutination. After dilution, the same sera exhibit a positive agglutination/precipitation reaction. This phenomena, in which agglutination or precipitation occurs at higher serum dilution ranges but not at lower dilutions or when undiluted, is known as the “Prozone phenomenon.” Antibody excess leads to the creation of very tiny complexes that do not cluster together to generate apparent agglutination, hence producing a false negative reaction. Prozone response is likely responsible for false-negative results. Prozone response can also be caused by blocking antibodies or nonspecific serum inhibitors. When distinct antigens are in close proximity to one another, the antibodies corresponding to each antigen may inhibit binding and compete with one another.

Post-zone phenomenon

This refers to the reaction wherein excess of antigen results in no lattice formation and a false negative agglutination reaction. Antigen overload is also a likely source of antigen-antibody agglutination/precipitation reactions that are falsely negative.

Applications Of Agglutination

Typing recipient and donor blood cells for blood transfusion.
To recognise and classify bacterial cultures.
To identify the presence of a specific antibody and quantify its concentration in the serum of a patient.

Cross-matching and blood grouping
Characterization of Bacteria Serotyping of Vibrio cholera, Salmonella Typhi, and Paratyphi, for example.
Diagnosis of numerous diseases using serology. Example: Rapid plasma regains (RPR) and Antistreptolysin O (ASO) tests for syphilis and rheumatic fever, respectively.

Detection of an unidentified antigen in clinical specimens. Detection of the Vi antigen of Salmonella Typhi in the urine, for instance.

References

https://nios.ac.in/media/documents/dmlt/Microbiology/Lesson-60.pdf
https://www.dshs.texas.gov/lab/serology_agg.shtm

https://science.umd.edu/classroom/bsci423/song/Lab6.html
https://patentscope.wipo.int/search/en/detail.jsf?docId=WO1988001374
https://www.biologyonline.com/dictionary/agglutination

https://iscnagpur.ac.in/study_material/dept_zoology/2.2_ANM_agglutination_reaction.pdf
https://biologyreader.com/agglutination-reaction.html
https://microbeonline.com/antigen-antibody-reactions/

https://microbiologynotes.org/agglutination-reaction-definition-uses-and-application/