Immunoassay of tryptase from human mast cells

Abstract A sandwich ELISA was developed for the measurement of tryptase. The assay utilizes the mouse monoclonal anti-tryptase antibody, termed G5 (IgG2b kappa ) in the solid phase and monospecific goat IgG anti-tryptase antibody together with tryptase in the fluid phase. The immunoassay will quantify 0.1 ng−5.6 ng of tryptase per 100 μl of sample solution to within 0.1 ng. Intra-assay coefficients of variation were determined at 0.3 ng, 1.0 ng and 3.0 ng of tryptase per assay, respectively, to be 19%, 7% and 4% with buffer and 10%, 4%, and 4% in the presence of 20% plas,a. Inter-assay coeffients of variation at the same respective levels of tryptase were 22%, 18% and 15% with buffer and 18%, 11% and 14% with 20% plasma. Net absorbance values obtained with a standard amount of tryptase in buffer alone and up to 50% (v/v) normal human citrate-treated plasma were within 10% of one another, indicating nearly complete detection of tryptase added to plasma. This represents the first sensitive immunoassay for a preformed mediator specific for human mast cells.