- The reaction is specific; an antigen combines only with its homologous antibody and vice versa. The specificity however is not absolute and cross reactions may occur due to antigenic similarity or relatedness.
- Entire molecules react and not fragment.
- There is no denaturation of the antigen or the antibody during the reaction.
- The combination occurs at the surface, therefore it is the surface antigens that are immunologically relevant.
- The combination is firm and irreversible. The firmness of the union is influenced by the affinity and avidity of the reaction.
- Affinity refers to the intensity of attraction between the antigen and antibody molecules. It is a function of the closeness of fit between an epitope and the paratope (antigen combining region of its antibody).
- Avidity is the strength of the bond after the formation of the antigen antibody complexes. It reflects the overall combining property of the various antibody molecules in an antiserum, possessing different affinity constants with the multiple epitopes of the antigen.
- Antigens and antibodies can combine in varying proportions, unlike chemicals with fixed valencies. Both antigens and antibody are multivalent, antibodies are generally bivalent, though IgM molecules may have five or ten combining sites. Antigens may have valencies up to hundreds.
At high antibody concentrations, the number of antibody binding sites may greatly exceed the number of epitopes present in the antigens. As a result, most antibodies bind antigen only univalently instead of multivalently. Antibodies that bind univalently can not cross-link one antigen to another.
Prozone effects are readily diagnosed by performing the assay at a variety of antibody ( or antigen) concentration. As one dilutes to an optimum antibody concentration, one sees higher levels of agglutination. When using polyclonal antibodies incomplete antibodies also causes prozone effect.
- Agglutination is more sensitive than precipitation for the detection of antibodies.
- Agglutination occurs optimally when antigens and antibodies react in equivalent proportions.
Types of agglutination
- Slide agglutination: Serotyping.
- Tube agglutination: e.g. Widal test.
- Indirect (passive agglutination): where soluble antigens are coated on vehicle particle e.g. latex particle, RBCs.
- When a drop of the appropriate antiserum is added to a smooth, uniform suspension of a particulate antigen in a drop of saline on a slide or a tile, agglutination takes place.
- A positive result is indicated by the clumping together of the particles and the clearing of the drop. Depending up on the titre of the serum, agglutination may occur instantly or with in seconds.
- Clumping occurring after a minute may be due to drying of the fluid and should be disregarded.
- It is essential to have on the same slide a control consisting of the antigen suspension in saline, without the antiserum, to ensure that the antigen is not autoagglutinable.
- Slide agglutination is a routine procedure for the identification of many bacterial isolates from clinical specimens. It is also the method used for blood grouping and cross matching.
This is the standard quantitative method for the measurement of antibodies.
- When a fixed volume of a particulate antigen suspension is added to an equal volume of serial dilutions of an antiserum in test tubes, the agglutination titre of the serum can be estimated.
- Tube agglutination is routinely employed for the serological diagnosis of typhoid, brucellosis and typhus fever ( weil- felix reaction).
The procedure involves adding a suspension of dead typhoid bacterial cells to a series of tubes containing the patient’s serum, which has been diluted out to various concentrations. After the tubes have been incubated for 30 minutes at 37° C, they are centrifuged and examined to note the amount of agglutination that has occurred. The reciprocal of the highest dilution at which agglutination is seen is designated as the antibody titer of the patient’s serum. For example, if the highest dilution at which agglutination occurs is 1:320, the titer is 320 antibody units per milliliter of serum. Naturally, the higher the titer, the greater is the antibody response of the individual to the disease.
- The size of the latex bead (0.8µm or larger) enhances the ease with which the agglutination reaction is recognized.
- Levels of bacterial polysaccharides detected by latex agglutination have been shown to be as low as 1.0 ng /ml because the pH, osmolarity and ionic concentration of the solution influence the amount of binding that occurs, conditions under which latex agglutination procedures are carried out must be carefully standardized.
- Additionally, some constituents of body fluids such as rheumatoid factor, have been found to cause false- positive reactions in the latex agglutination systems available. To counteract this problem. It is recommended that all specimens be treated by boiling or with ethylenediaminetetraacetic acid (EDTA) before testing.
- Commercial test systems are usually performed on cardboard cards or glass slides; manufacturers recommendations should be followed precisely to ensure accurate results.
- Reactions are graded on a 1+ to 4+ scale, with 2+ usually the minimum amount of agglutination seen in a positive sample.
- Control latex (coated with antibody form the same animal species from which the specific antibody was made) is tested alongside the latex. If the patient specimen or the culture isolate reacts with both the test and control latex, the test is considered non specific and therefore uninterpretable.
- Latex tests are very popular in clinical laboratories to detect antigen to Cryptococcus neoformans in CSF or serum and to confirm the presence of beta- hemolytic streptococcus form the culture plates. Latex tests are also available to detect Streptococcus agalactiae, Clostridium difficile toxins A and B and rotavirus.
- Direct coombs test: Detection of incomplete antibodies on patients RBCs. Antibodies attached on the surface of the RBCs ( patient RBCs) + Antihuman globulin = agglutination.
- Indirect coombs test:Detection of antibodies in patients sera. Rhesus positive RBC + Patient serum ( if contains incomplete circulating Abs coats the surface of the RBC)+ Antihuman globulin which makes the bridge = agglutination