Agglutination Test – definition, Types, Uses, Advantages, Disadvantages

Agglutination test is an immunological and serological test which is used to detect specific antigen or antibody in the patient sample.

It is based on the visible clumping reaction between particulate antigen and its specific antibody. In this reaction, antibody binds with more than one antigen particle and form a lattice like network. So the particles become clumped and this clumping is called agglutination.

In agglutination test, the antigen is usually insoluble or particulate in nature. It may be red blood cells, bacterial cells, latex beads or other particles. When these particles react with their specific antibody, visible clumps are formed. These clumps can be seen by naked eye in many cases.

The reaction mainly depends on proper antigen-antibody reaction. The antibody acts as a bridge between antigen particles. This bridging causes cross linking of particles. Then large aggregate is produced.

Agglutination test is mainly of two types. These are direct agglutination test and indirect agglutination test.

In direct agglutination test, the antigen is naturally present on the surface of cells or particles. For example, antigen present on RBC or bacteria directly reacts with antibody and agglutination occur.

In indirect agglutination test, soluble antigen or antibody is artificially attached on carrier particle. These carrier particles may be latex bead or red blood cell. After this, the coated particle reacts with specific antibody or antigen and visible clumping is formed.

Agglutination test is simple, rapid and less expensive test. It is widely used in clinical diagnosis. It is used for blood grouping, diagnosis of bacterial infections like typhoid fever by Widal test, and detection of hormone like hCG in pregnancy test.

So, agglutination test is an important serological test. It helps to identify antigen or antibody by formation of visible clumps. This is a common method in diagnostic microbiology and immunology.

Principle of Agglutination Test

Principle of Agglutination Test is based on the specific reaction between particulate antigen and its specific antibody. The antigen may be present on the surface of bacteria, red blood cells (RBC) or it may be attached artificially on latex beads.

In this test, antibody first binds with the specific antigenic site or epitope present on the particle. This first binding is not visible by naked eye. It is called sensitization phase.

After this, the antibody acts as a bridge between two or more antigen particles. As antibody has more than one binding site, it can join neighbouring particles together. This produces cross linking of antigen particles.

Due to this cross linking, a lattice like network is formed. The particles come together and form visible clumps. This visible clumping is called agglutination.

For proper agglutination, the antigen and antibody must be present in correct proportion. This condition is called zone of equivalence. If antigen or antibody is in excess amount, clumping may not occur properly.

Agglutination also depends on suitable condition like proper pH, temperature and ionic strength. The antibody bridging must also overcome the repulsive force between suspended cells. This repulsive force is known as zeta potential.

So, in agglutination test, specific antigen-antibody reaction occur and then cross linked aggregates are formed. These aggregates are seen as visible clumps.

Prozone Phenomenon

Prozone phenomenon is an immunological phenomenon where very high amount of antibody is present in the patient serum and it gives false negative or weak reaction in the test.

It is also known as hook effect or antibody excess. It is mainly seen in serological tests like agglutination test and precipitation test. In these tests, proper proportion of antigen and antibody is needed for visible reaction.

In normal condition, antibody binds with antigen and form cross linking. This cross linking produces lattice network. So visible clumping or precipitate is formed.

But in prozone phenomenon, antibody is present in excess amount. The excess antibody completely covers all antigenic sites. Due to this, one antibody cannot join two antigen particles properly. So lattice formation does not occur.

As a result, visible clumping is not formed. The test may appear negative although the patient sample contains high amount of antibody. This is called false negative result.

Prozone phenomenon is clinically important. It may occur in diseases where large amount of antibody is produced. It is seen in secondary syphilis, brucellosis, some autoimmune diseases and also in HIV co-infection.

This effect can be removed by doing serial dilution of patient serum. By dilution, antibody concentration becomes reduced to proper level. Then antigen and antibody react in correct proportion and true positive reaction is obtained.

Types of Agglutination Test

Agglutination test are classified into two main ways.

A. Types on the basis of reaction mechanism

1. Direct Agglutination Test

In direct agglutination test, the antigen is naturally present on the surface of particle.

The particle may be bacterial cell or red blood cell (RBC). Here no artificial carrier is used. The antibody directly reacts with antigen and visible clumping is formed.

2. Indirect Agglutination Test

In indirect agglutination test, soluble antigen or antibody is coated artificially on a carrier particle.

The carrier particle may be latex bead, bentonite or treated RBC. After coating, it reacts with specific antibody or antigen. Then visible agglutination occur.

3. Reverse Passive Agglutination Test

In reverse passive agglutination test, specific antibody is coated on carrier particle.

This test is used to detect soluble antigen in patient sample. When antigen binds with coated antibody, cross linking occur and clumping is produced.

4. Agglutination Inhibition Test

In agglutination inhibition test, the reaction is based on inhibition of agglutination.

The soluble antigen in patient sample first binds with antibody and neutralizes it. So antibody cannot bind with antigen coated particles. Here absence of clumping indicates positive result.

5. Coagglutination Test

In coagglutination test, killed Staphylococcus aureus cells are used as carrier particles.

The cell wall contains protein A which binds with antibody. The antibody remains outward and captures specific antigen. Then agglutination occur.

6. Hemagglutination Test

Hemagglutination test is agglutination of red blood cells (RBCs).

It is used in blood grouping, Coombs test and viral hemagglutination inhibition test. The clumping of RBCs is the main reaction here.

B. Types on the basis of test method

1. Slide Agglutination Test

Slide agglutination test is performed on glass slide or card.

Antigen and antibody are mixed on slide. Then immediate visible clumping is observed. It is a rapid qualitative test.

2. Tube Agglutination Test

Tube agglutination test is performed in test tubes.

Patient serum is serially diluted and mixed with standard antigen. It is used to detect antibody amount or antibody titer.

3. Microscopic Agglutination Test (MAT)

Microscopic agglutination test (MAT) is observed under microscope.

Serum dilution is mixed with live antigen culture. It is mainly used for diagnosis of leptospirosis. The reaction is observed under dark field microscope.

4. Latex Agglutination Test

Latex agglutination test uses latex beads coated with antigen or antibody.

It is rapid and sensitive test. It is used to detect bacterial antigen, viral infection, C-reactive protein (CRP) and other serum substances.

5. Heterophile Agglutination Test

Heterophile agglutination test detects heterophile antibodies.

These antibodies cross react with unrelated antigen of other species. It is used in diagnosis of infectious mononucleosis.

General Procedure of Agglutination Test

1. Patient serum is collected in a clean test tube. The serum may contain specific antibody or sometimes antigen which is to be detected.
2. The serum is diluted serially with normal saline. This is mainly done in tube agglutination test. It helps to reduce excess antibody and also to find the antibody titre.
3. A standard suspension of particulate antigen is added to the diluted serum. The antigen may be bacterial cell, red blood cell (RBC) or antigen coated latex bead.
4. The antigen and serum are mixed properly. In slide test, mixing is done on glass slide or card. In tube test, mixing is done in the test tube.
5. The mixture is allowed to react for some time. In slide test, the slide is gently rocked for few minutes. In tube test, the tube is incubated at suitable temperature for required time.
6. After incubation, the reaction is observed carefully. The formation of visible clumps or granules are checked by naked eye. In some special test, microscope may be used.
7. If visible clumping is formed, the test is positive. It indicates that specific antigen-antibody reaction has occurred and cross linking of particles has taken place.
8. If the mixture remains smooth and uniform, the test is negative. It indicates that specific antigen or antibody is absent or not present in detectable amount.

Advantages of Agglutination Test

• Agglutination test gives rapid result. In slide agglutination test, visible clumping may be seen within few seconds to minutes.
• It is simple to perform. The procedure is easy and does not need highly trained person in many routine tests.
• It is less expensive test. Only small amount of reagent and sample are needed.
• It does not need complex instrument. So it can be used in small laboratory and also in field condition.
• It has good sensitivity. The use of particulate antigen or carrier particle makes the reaction more visible.
• It is more sensitive than precipitation test in many cases. Because large particles form visible clumps more easily.
• It can be used for detection of many biological substances. It is used to detect bacterial cells, viral particles, serum proteins, hormones and autoantibodies.
• It is useful for quick diagnosis of infection. Many agglutination tests are used in routine microbiology and serology laboratory.
• The result can be observed by naked eye in many tests. So reading of result is simple and fast.
• It can be used for both antigen and antibody detection. By changing the coated particle, different types of sample can be tested.

Limitations of Agglutination Test

• Agglutination test is not useful in very early stage of infection. In early infection, detectable amount of antibody may not be formed in patient serum. So false negative result may occur.
• It is mostly semi quantitative test. In tube agglutination test, result is given as titre from serial dilution. It does not give exact concentration of antibody.
• The result depends on visual observation. Weak clumping may be difficult to read. So different technicians may give different interpretation.
• False negative result may occur due to prozone phenomenon. In this condition, excess antibody prevents proper lattice formation and clumping is not seen.
• False negative result may also occur due to postzone phenomenon. Here excess antigen prevents cross linking of particles. So visible agglutination does not occur.
• False positive result may occur due to cross reaction. Some unrelated antibodies or conditions like rheumatoid factor, autoimmune diseases, malaria, previous infection or vaccination may give non-specific clumping.
• The test is affected by quality of sample. Turbid, lipemic or hemolyzed sample may interfere with the reaction. Sometimes auto-agglutination may look like positive result.
• It needs proper test condition. Proper pH, temperature and ionic strength are required for correct antigen-antibody reaction.
• Technical error may affect the result. If slide becomes dry, protein crystallization may occur and it may look like false agglutination.
• It may not identify the exact antibody or exact cause in some cases. So further confirmatory test is needed for proper diagnosis.

Uses of Agglutination Test

• Agglutination test is used for blood grouping. It helps to detect ABO and Rh blood group before blood transfusion.
• It is used for blood cross matching. Donor blood and recipient serum are tested to see any incompatible antigen-antibody reaction.
• It is used for diagnosis of bacterial infection. Widal test is used for diagnosis of typhoid and paratyphoid fever.
• It is used for diagnosis of syphilis. Tests like VDRL and RPR are used for this purpose.
• It is used for diagnosis of brucellosis and leptospirosis. These tests help to detect specific antibody in patient serum.
• It is used for viral disease diagnosis and immunity study. Viral hemagglutination inhibition test is used in influenza, mumps, measles and rubella.
• It is used in Coombs test. This test helps in diagnosis of autoimmune hemolytic anemia, hemolytic disease of newborn and transfusion reaction.
• It is used for pregnancy test. hCG hormone in urine can be detected by agglutination inhibition test.
• It is used to detect clinical markers. Rheumatoid factor (RF) and C-reactive protein (CRP) can be detected by agglutination test.
• It is used for rapid antigen detection from patient sample. Bacterial antigen, fungal antigen and cryptococcal antigen can be detected from serum or CSF.
• It is used to measure antibody titre. This helps to know the level of antibody in infection or after vaccination.
• It is used for bacterial identification. It helps to identify Staphylococcus aureus, streptococcal groups and other bacterial strains.

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