Friday, November 21, 2003

Description
Agglutination Assay


Procedure
Passive agglutination is performed using tanned sheep
red blood cells and P. aeruginosa LPS (heated for 1 hour at 100oC 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 37oC 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, 20g/ml LPS or protein is added, and the mixture is incubated for 1 hour at 37oC 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 4oC. 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 50l of antiserum serially diluted in saline and 50l 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 37oC 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.


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