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

Agglutination Test is a serological test in which the antigen and antibody reaction is seen directly as visible clumping. It is the process where particulate antigen reacts with its homologous antibody and form aggregates called agglutinates. These aggregates can be seen with naked eyes.

This reaction is specific because the antigen combines only with the particular antibody that is produced against it. The antibodies that take part in this process are called agglutinins. The firmness of the antigen-antibody complex depends on the affinity and avidity of the reaction.

It is the reaction between particulate antigen and corresponding antibody. The antigen must be present on the surface of cells like bacteria, RBCs or any inert particle (latex). When the antibody binds with more than one antigen, it forms a lattice-like network and this lattice becomes visible as clumps. This is referred to as the agglutination reaction.

Agglutination reaction is better to take place in IgM as compare to IgG because IgM is larger in size and can bind many antigenic determinants. The reaction can be inhibited if antibody is in excess. This condition is called prozone phenomenon. The prozone phenomenon occurs when either antigen or antibody is present in excess and prevents proper lattice formation.

In 1965, Singer and Plotz first introduced this test for clinical use and it was described as the Rheumatoid Factor Test.

It is the interaction between antigen and antibody that forms clumps. These clumps appear when the antibody attaches on the antigen surface and cross-links them. This reaction is used in diagnostic tests such as blood grouping and Widal test.

What is Prozone phenomenon?

The prozone phenomenon is the condition in which an agglutination test becomes falsely negative due to extreme excess of antibodies in the patient serum. It is the situation where the antigenic particles are completely coated by abundant antibodies, and it is not forming the cross-linked lattice that is required for visible clumping. It is the process where only small soluble complexes is formed and no agglutination can be seen, so the reaction appears negative although antibody level is very high. This is referred to as an antibody excess zone, and it is usually corrected when the serum is diluted to reach the proper antigen–antibody ratio for the reaction.

Purpose of Agglutination Test

• It is used to detect the presence of specific antigen or antibody in a sample.
• It is the process that helps in identifying microorganisms by visible clumping.
• It is used in diagnosis of infectious diseases where antigen–antibody reaction is involved.
• It is applied for blood grouping and cross-matching tests.
• It is used to measure the titre of antibodies in serum during different stages of infection.

Specimen collection

• It is usually collected as patient serum because the antibodies is mainly present in serum.
• Whole blood is taken first, and then the serum is separated after clotting.
• In some tests plasma can also be used, but serum is preferred for accurate reaction.
• The sample should be collected in a clean, dry container to avoid contamination.
• Hemolysed or lipemic samples is avoided because it can interfere with the reaction.
• The specimen is stored at proper temperature if immediate testing is not possible.

Types of Agglutination Test

Agglutination is the process where antigen and antibody react to form a visible clump. It is the reaction which is used for different serological tests and it is divided into three main types.

1. Direct Agglutination

It is the technique where cells or insoluble particles or antigen is directly agglutinated with the corresponding antibody.

This test is again divided into two methods–

A. Slide Agglutination

It is the process where the specimen sample is mixed with specific antiserum on a clean microscopic slide.

In this method the sample drop is taken on the slide. Then antiserum (Anti-A, Anti-B, Anti-D etc.) is added on it. After that it is mixed properly with the help of a sterile toothpick. The mixture is allowed for agglutination. Formation of clumping is then observed.

In slide agglutination test positive result is indicated by formation of clump of simple. The clumping appear within few minutes.

Application–

• It is used for identification of bacteria from clinical specimen.
• It is used for blood grouping.

B. Tube Agglutination

It is the process where a specific antibody is detected from serum sample by using a constant amount of known antigen. In this technique the test serum is serially diluted in different test tubes.

A standardized amount of known antigen is added to each tube and mixed. The tubes are incubated at suitable temperature. After few hours of incubation agglutination is completed and the tubes is examined for clumping.

Result is expressed as titer. It is the highest dilution of test serum at which positive agglutination occur.

Application–

• Used for Brucellosis test.
• Used in Widal test for diagnosis of enteric fever.
• Used in Weil-Felix reaction.
• Used for Paul-Bunnel test.

2. Indirect Agglutination

It is the process where soluble antigen is first coated on a carrier particle and agglutination take place on the surface of these carrier molecules. In this technique RBC, latex, bentonite etc. is used as carrier particles. This is referred to as passive agglutination.

3. Reverse Passive Agglutination

In this process antibody is coated on the surface of a carrier molecule. It is then used for the detection of antigen present in the patient serum. This process occur when the antigen binds on the coated antibody which form a visible clumping.

Uses of Agglutination Test

• It is used for ABO and Rh blood grouping.
• It is used for cross matching of donor and recipient blood.
• It is used in Direct and Indirect Coombs test.
• It is used for diagnosis of typhoid fever by Widal test.
• It is used for serodiagnosis of Brucellosis.
• It is used for Weil-Felix reaction in typhus fever.
• It is used for identification and serotyping of bacteria.
• It is used for rapid detection of soluble antigen by reverse passive agglutination.
• It is used in coagglutination test for grouping of Streptococcus and detection of cryptococcal antigen.
• It is used in viral hemagglutination inhibition test.
• It is used for detection of rheumatoid factor by latex agglutination method.
• It is used for detection of C-reactive protein (CRP).
• It is used for detection of different autoantibodies.
• It is used in hormone detection test like early pregnancy test based on agglutination inhibition.
• It is used in immunological research for studying antigen-antibody interaction.
• It is used in special ring test for food analysis and for diagnosis of anthrax.

Advantages of Agglutination

• It is simple to perform and give quick result.
• It can be observed directly by formation of visible clumping.
• It need very less laboratory equipment.
• It is low cost and suitable for field investigation.
• It can detect both antigen and antibody.
• It can be used for quantitative measurement by titer value.
• It can detect soluble antigen by passive agglutination.
• It provide good uniformity and stability when carrier particles is used.
• Latex agglutination increase sensitivity due to many binding sites.
• Coagglutination particles show better stability in different conditions.
• It is important for blood grouping and cross matching.
• It is useful for rapid screening of infectious diseases.

Limitations of Agglutination

• It may give false negative result due to prozone and postzone effect.
• It need an optimum ratio of antigen and antibody for proper clumping.
• It show low sensitivity in chronic or low level infection.
• It cannot detect IgG directly because IgG is small and only sensitization occur without visible clumping.
• It may produce cross reaction when crude antigen is used.
• It may give false positive due to heterophile antibody.
• It depend on suitable temperature, pH and electrolytes for proper reaction.
• It need correlation with clinical condition for correct interpretation.
• It may show delay in detection because antibody need time for seroconversion.
• It may have subjective error because result is observed by naked eye.

Examples of Agglutination test

Agglutination is the process in which particulate antigens reacts with their specific antibodies and visible clumping is formed. These are commonly used in immunology for detection of antigen or antibody. Some of the main examples are–

1. Latex Agglutination Test
   It is performed by using latex beads coated with specific antibody and the reaction is seen as clumping of these beads.
2. Slide Agglutination Test
   In this test the antigen is mixed with antiserum on a slide. It is used for quick identification of bacterial isolates.
3. Tube Agglutination Test
   The reaction is performed in a series of tubes. Widal test for typhoid diagnosis is one of the common examples.
4. Indirect (Passive) Agglutination Test
   Here soluble antigens are attached to carrier particles like RBCs or latex beads. The reaction is used for detection of various antibodies.
5. Reverse Passive Agglutination Test
   In this test the antibody is bound to carrier particles instead of antigen. It is helpful when antigens are detected in patient sample.
6. Coagglutination Test
   It uses Staphylococcus aureus cells as carrier particles because Protein A binds Fc region of antibodies. These cells form visible clumps in presence of antigen.
7. Hemagglutination Test
   It is the reaction where red blood cells act as antigens. Blood grouping test is the common example where RBCs agglutinates with specific antisera.