A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E2 using enhanced luminol as substrate

Abstract

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E2 (PGE2) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE2 antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE2 conjugate. Then, after preincubating, the anti-PGE2 antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE2 conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated anti-mouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10-50,000 pg/ml. The lower limit of quantification is 100 pg/ml (2 pg/well) and 150 pg/ml (3 pg/well) for buffer and plasma, respectively. The intra-day coefficients of variation (CV) for the range of 100-50,000 pg/ml are 3.2-8.9% (buffer) and 4.2-17.7% (plasma) and inter-day CV are 2.9-19.8% (buffer) and 3.6-21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.