Definition of Agglutination
When a particulate antigen (agglutinogen) combines with its antibody (agglutinin) in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. Agglutination is the aggregation of already insoluble particles or cells into larger clumps. Interaction between the antibody (Ab) and particulate antigen results in visible clumping called agglutination.
Types of agglutination
- Slide agglutination e.g. Serotyping, blood grouping ( AB0 and Rh typing), and cross match
- Tube agglutination (Bacterial Agglutination) test e.g. Widal test, Weil-Felix reaction, Paul-Bunnel test
- Haemagglutination: It is further divided into sub-types active and passive haemagglutination test. The active haemagglutination test is again divided into direct and indirect active haemagglutination, similarly passive haemagglutination into direct and indirect passive haemagglutination. e.g. TPHA test, Coomb’s test
- Miscellaneous types- It contains Passive agglutination with latex and other particles; and co-agglutination test.
Slide Agglutination
- Smooth and uniform suspension of particulate Ag is prepared in a drop of saline on a glass slide or tile.
- A drop of appropriate antiserum is added and mixed.
- Positive result – indicated by the clumping together of the particles and clearing of drop
- Clumping is visible to the naked eye sometimes requires confirmation under the microscope.
- Control should be run in each test.
Uses
- Identification of bacterial isolates from clinical specimens
- Blood grouping and cross-matching
Advantages
- Rapid
- Requires smaller quantities of reagents
Tube agglutination (Bacterial Agglutination) test
- A fixed volume of particulate Ag suspension is added to the equal volume of serially diluted serum in test tubes and incubated.
- The highest dilution of serum that gives a positive agglutination reaction is recorded as an antibody titer.
Applications
For the diagnosis of-
- Typhoid fever (Widal test)
- Brucellosis (Brucella agglutination test)
- Typhus fever (Weil -Felix Reaction)
Advantage
- More quantitative than slide agglutination test
Haemagglutination
- Agglutination tests in which red blood cells are used.
- Different types of haemagglutination tests- A) Active haemagglutination test and B) Passive haemagglutination Test.
a) Direct active haemagglutination test
In this test, RBCs are used as an antigen (Ag). Examples are as follows-
- Paul-Bunnel test for infectious mononucleosis in which sheep RBCs are used as antigen.
- Cold agglutination test for primary atypical pneumonia in which human ‘O’ group RBCs are used as an antigen Mycoplasma -atypical pneumonia
Indirect active haemagglutination test
Red blood cells (RBCs) are coated with immunoglobulin G ( IgG) antibodies (Abs) and the rabbit anti-IgG Abs are added which cause agglutination of RBCs. e.g. Coomb’s test
Direct Coomb’s test (DCT)
- Used to detect monovalent maternal Ab already present on RBCs
- Sensitization of RBCs with incomplete Abs takes place in vivo
- When such RBCs are treated with Coomb’s serum (rabbit antiserum against human g-globulin), agglutination occurs.
Use
In the hemolytic diseases of newborns (HDN) due to Rh incompatibility.
Indirect Coomb’s test (ICT)
- Sensitization of RBC with Ab is performed in vitro by incubating
- the patient’s serum with Rh-positive RBC( group ‘O’ or same group RBCs) and then Coomb’s serum is added.
- Results into agglutination
Uses
- Used for detection of anti-Rh Ab (free) in the patient’s serum.
- For a demonstration of any type of incomplete or non-agglutinating antibodies. e.g. non-agglutinating antibodies in brucellosis
Direct Passive haemagglutination test
- The antigen is attached or absorbed on the surface of RBC
- RBC acts as inert carrier or Ag – are agglutinated by antibodies. e.g. Treponema pallidum haemagglutination (TPHA) test used for the serodiagnosis of syphilis
Indirect Passive haemagglutination test:
Sheep RBCs are sensitized with rabbit anti-sheep erythrocyte Ab (Amboceptor) and used as antigen. e.g. Rose –Waller test for rheumatoid arthritis-autoantibody (RA- factor) acts as an Ab and agglutinates Sheep RBC sensitized with amboceptor.
Passive agglutination with latex and other particles
- Inert particles are used as carriers of Ag
- Ag molecules are nonspecifically adsorbed to the surface of latex particles, which have a uniform diameter of 0.8-1m
- The addition of a specific antibody transforms the latex (milk) from a milky white liquid to a coarse suspension of visible granules
- The test can be performed in the test tube but more commonly on slides and examples are as follow-
√RA factor test
√Pregnancy test
√CRP test (c-reactive protein)
√Antisterptolysin O test (ASO)
√the test can also be used for the diagnosis of
- Cryptococcal meningitis
- Amoebiasis
- Meningococcal infections
- Pneumococcal infections
- Hepatitis B virus infections and others
Co-agglutination test
The ability of protein A of Staphylococcus aureus (Cowan – 1 strain) to react non specifically with the Fc portion of IgG leaving Fab portion free to react with its homologous Ag is used for detection of Ag. IgG on protein A of Staphylococcus reacts with specific Ag causes clumping of staphylococci. This clumping or agglutination of staphylococci is called co-agglutination.
Uses
- Streptococcal grouping
- Typing of N. gonorrhoeae
- Grouping of mycobacteria
- For detection of bacterial Ags in blood, urine, and CSF in pneumococcal, gonococcal, meningococcal, streptococcal, Salmonella, and Haemophilus infections.
Key Notes
- Agglutination is more sensitive than precipitation for antibody detection.
- Antigen-antibody reaction in which antibody combines with a particulate antigen.
Further Readings
- Ivan Roitt, Jonathan Brostoff and Davis Male-Immunology, 3rd Ed, 1993
- Jenis Kuby, Barbara A. Osborne: Textbook of Immunology, (3rd Ed.)1992.
- Basic Immunology by Abdul K. Abbas and Andrew H. Lichtman
- ROITT’S ESSENTIAL IMMUNOLOGY, Ivan M Roitt, and Peter J. Delves 3rd,8th,10th Edition.
- A Textbook of Microbiology by P Chakraborty
- Textbook of Microbiology by Ananthanarayan and Paniker
- Review of Medical Microbiology and Immunology by Warren Levinson
- Diagnostic Microbiology by Connie R. Mahon