Agglutination

Introduction

Agglutination is the clumping together of particles, a reaction that can be used as an immunological test by exploiting the interaction between antibody and a particulate antigen. The antibodies that produce such a reaction are known as agglutinins.

Method

The agglutination test is often carried out in round-bottomed test tubes, with doubling dilutions of the antiserum (i.e. 1:2, 1:4, 1:8...). The particulate antigen is then added and the mixture incubated at 37 degrees C. The last tube showing clear agglutination constitutes the end of the test. The dilution at which the test ends is known as the titre- the number of antibodies per unit volume of the serum.

• An excess of antibody can inhibit agglutination reactions, again in much the same way as a precipitation reaction- this is known as the prozone effect. At high antibody concentrations, the number of epitopes is outnumbered by antibody binding sites. This means the antibodies bind to the antigen univalently, so are unable to cross-link antigen. This often happens in the first few tubes of the test, so the agglutination often only happens in those containing more dilute antiserum.

Applications

Coombs test. A Rad, WikiMedia Commons 2006

• Widal test: used to test for antibodies to certain bacteria in patient serum, e.g. salmonella
• Blood typing: also known as haemagglutination, the reaction can be used to determine blood type, i.e. an anti-A serum will agglutinate with type A red blood cells but not with B or O.
• Virus diagnosis: some viruses, such as the myxoviruses, cause agglutination of the red blood cells. Antibodies inhibit this process and can be detected in a patient's serum for diagnosis.
• Coombs test: agglutination of red blood cells is an indication that certain antibodies are present on the cell surface, e.g. testing for Rh disease in newborn babies.
• Hormone assay by agglutination inhibition- red blood cells coated with a specific hormone produce agglutination with a hormone-specific antibody. The addition of free hormone (the test sample) will block the antigen-binding sites, preventing agglutination. By comparing the activity of the test sample against that of a known standard hormone, a quantitative result can be obtained.

Originally funded by the RVC Jim Bee Award 2007