Numerous procedures have been developed to detect antigen by means of the agglutination (clumping) of an artificial carrier particle, such as a latex bead, with antibody bound to the surface.

Latex Agglutination

Antibody molecules can be bound in random alignment to the surface of latex (polystyrene) beads (Figure 1). The number of antibody molecules bound to each latex particle is large, resulting in a high number of exposed potential antigen binding sites. Antigen present in a specimen binds to the combining sites of the antibody exposed on the surfaces of the latex beads, forming cross-linked aggregates of latex beads and antigen. The size of the latex bead (0.8 µm or larger) enhances the ease with which the agglutination reaction is visualized. Levels of bacterial polysaccharides detected by latex agglutination have been shown to be as low as 0.1 ng/mL.

Fig1. Alignment of antibody molecules bound to the surface  of a latex particle and latex agglutination reaction. 

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, some agglutination methods require specimens to be pretreated by heating at 56°C or with ethylenediaminetetraacetic acid (EDTA) before testing. Commercial test systems are usually per formed on cardboard cards or glass slides; manufacturer’s recommendations should be followed precisely to ensure accurate results.

Depending on the procedure, some reactions are reported as positive or negative and other reactions are graded on a 1+ to 4+ scale, with 2+ usually the minimum amount of agglutination visible in a positive sample without the aid of a microscope. Control latex (coated with antibody from the same animal species from which the specific antibody was made) is tested alongside the test latex. If the patient specimen or the culture isolate reacts with both the test and control latex, the test is considered nonspecific and the results therefore are invalid.

Latex tests are very popular in clinical laboratories for detecting antigen to Cryptococcus neoformans in cerebrospinal fluid or serum (Figure 2) and to confirm the presence of beta-hemolytic Streptococcus from culture plates (Figure 3). Latex tests are continually being developed for a variety of organisms. Some examples of additional latex tests are available for the detection of Clostridium difficile toxins A and B, rotavirus, and Escherichia coli 0157:H7 from suspect colonies of E coli.

Fig2. Cryptococcal Antigen Latex Agglutination System  (CALAS) (Meridian Diagnostics, Inc., Cincinnati, Ohio.) Patient 1  shows positive agglutination; patient 2 is negative. 

Fig3. Streptex (Remel, Inc., Lenexa, Kan.) Colony of beta hemolytic Streptococcus agglutinates with group B Streptococcus (Streptococcus agalactiae) latex suspension. 

Coagglutination

Similar to latex agglutination, coagglutination uses anti body bound to a particle to enhance the visibility of the agglutination reaction between antigen and antibody. In this case the particles are killed and treated S. aureus organisms (Cowan I strain), which contain a large amount of an antibody-binding protein, protein A, in their cell walls. In contrast to latex particles, these staphylococci bind only the base of the heavy chain portion of the antibody, leaving both antigen-binding ends free to form complexes with specific antigen (Figure 4). Several commercial suppliers have prepared coagglutination reagents for identification of streptococci, including Lancefield groups A, B, C, D, F, G, and N; Streptococcus pneumoniae; Neisseria meningitidis; and Haemophilus influenzae types A to F grown in culture. The coagglutination reaction is highly specific and demonstrates reduced sensitivity in comparison to commercially prepared latex agglutination systems. Therefore, coagglutination is not usually used for direct antigen detection.

Fig4. Coagglutination. 

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