Agglutination Assays

Reference:

Lanyi, B., and T. Bergan. Methods in Microbiology, Vol 10: 93-168.

Bacterial Agglutination

Bacterial agglutination is performed using either P. aeruginosa cells autoclaved at 120^oC for 1 hour, then centrifuged and resuspended in saline or live P. aeruginosa cells grown to mid log phase, then centrifuged and resuspended in saline. (For either method resuspend the bacteria so the concentration is 5 X 10⁸ bacteria/ml.) In both methods the cell preparations are incubated with serial dilutions of antiserum for 1 hour at 37^oC. The agglutination of live cells is characterized by a coarse granular bacterial clumping, as opposed to the finer granular clumping of heat-killed P. aeruginosa with homologous LPS O-antigen-specific serum. Bacterial agglutination is scored on a scale of +1 (weak agglutination) to +4 (strong reaction). The agglutination can also be performed on glass slides using a drop each of antiserum and bacterial preparation, and the results observed after 3 to 5 minutes.

Passive Agglutination

Passive agglutination is performed using tanned sheep red blood cells and P. aeruginosa LPS (heated for 1 hour at 100^oC prior to use). Sheep erythrocytes are washed three times in saline (0.9% NaCl), adjusted to 4% erythrocytes (v/v) in PBS, pH 7.5 and 2.5mg of tannic acid in 50ml PBS is added to 50ml of the cell suspension. After a 15 minutes incubation at 37^oC with occasional mixing, the cells are centrifuged (100 x g for 20 minutes) and washed with 100ml PBS. One half of the cells are kept as a control in PBS containing 1mM NaN₃. To the other half of the cells, 20µg/ml LPS or protein is added, and the mixture is incubated for 1 hour at 37^oC with very gentle agitation at regular intervals. The antigen-coated cells are washed three times in saline and 1mM NaN₃ is added for storage at 4^oC. The cells are made up to 1% (v/v) in saline before use.

Passive agglutination is performed in 96 well conical bottom plates (Linbro, Flow Labs) using 50µl of antiserum serially diluted in saline and 50µl of 1% (v/v) antigen-coated sheep erythrocytes. Control wells contain the antiserum and tanned, non-antigen-coated sheep erythrocytes. The plates are incubated at 37^oC for 1 hour. The titre is the inverse of the highest serum dilution showing agglutination. Non-agglutinated cells give a tight button of cells at the bottom of the well, while the agglutinated cells form a mat at the well bottom.