A NEW RELIABLE CHEMILUMINESCENCE IMMUNOASSAY (CLIA) FOR PROSTAGLANDINE(2) USING ENHANCED LUMINOL AS SUBSTRATE

A sensitive and reliable chemiluminescence immunoassay suitable for th e quantitative determination of prostaglandin E(2) (PGE(2)) has been d eveloped using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE(2 ) antibody (mouse), free antigen and solid phase bound antigen. The MT P was first coated with a bovine serum albumin (BSA)-PGE(2) conjugate. Then, after preincubating, the anti-PGE(2) antibody (Ab) and the anal yte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE(2) conjugate. The monoclonal antibody ca ptured 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, en hanced by p-iodophenol. Photons emitted during the reaction were measu red using a photomultiplier tube. The assay has been validated with as say buffer and human plasma over a concentration range of 20-50,000 pg /ml. The lower limit of quantification is 200 pg/ml (2 pg/well) and 15 0 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 quantifi cation of PGE(2) in biological fluids like plasma and suction blister fluid.