IMMUNOLOGICAL STUDIES OF HUMAN CONSTITUTIVE CYCLOOXYGENASE (COX-1) USING ENZYME IMMUNOMETRIC ASSAY

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclo nal antisera and 4 of the mAbs strongly recognized human COX in platel et extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were perf ormed in 96-well microtiter plates coated with one: mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. O ne combination (solid phase CX-101 + CX-105-AChE) exhibited the best s ensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly c ross-reacted with human COX-1 from platelet extracts. Another combinat ion (solid phase CX-111 + CX-110-AChE) exhibited good recognition of h uman COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best comprom ise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both specie s were observed. In parallel, polyclonal antibodies were raised in rab bits against a peptide of 12 amino acids corresponding to the aminoter minal part of human COX-1. These polyclonal antibodies were affinity-p urified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to an alyze the COX-1 content of human platelets and cultured human umbilica l vein cells (HUVEC). The results obtained with each assay were compar ed in terms of sensitivity and specificity. The validity of both assay s was checked by analyzing platelets and HUVEC extracts previously fra ctionated by molecular sieve chromatography.