LOW-LEVEL QUANTIFICATION OF CHOLESTERYL ESTER TRANSFER PROTEIN IN PLASMA SUBFRACTIONS AND CELL-CULTURE MEDIA BY MONOCLONAL ANTIBODY-BASED IMMUNOASSAY

Sensitive immunoradiometric (IRMA) and ELISA assays for cholesteryl es ter transfer protein (CETP) have been developed using two different mo noclonal antibodies (MAbs). The MAbs were prepared against human plasm a CETP and demonstrated specificity by their inhibition of cholesteryl ester transfer activity and by immunoblots of crude plasma fractions and whole media from transfected CHO cells. For these sandwich-type as says, one MAb, 2F8, is used for capture, and the second MAb, 2E7, is i odinated (IRMA) or conjugated with alkaline phosphatase (ELISA) and us ed for detection. Both assays are linear and provide sensitivities muc h greater than previously reported. The IRMA allows for the accurate q uantification of CETP in the range of 0.5-20 ng/assay (5-200 ng/ml), t he ELISA 0.05-5 ng/assay (0.5-50 ng/ml). Using the IRMA, the mean plas ma CETP concentration in 44 normolipidemic individuals was determined to be 2.10 +/- 0.36 mu g/ml; 2.05 +/- 0.33 for males (n = 25) and 2.16 +/- 0.40 for females (n = 19). Values ranged from 1.28 to 2.97 mu g/m l and CETP mass correlated well with cholesteryl ester transfer activi ty (r = 0.913, n = 23). The distribution of CETP in human plasma was e xamined both by gel permeation fast protein liquid chromatography (FPL C) and by native gel electrophoresis. For FPLC using agarose resins, a low ionic strength, isotonic buffer system resulted in near total rec overies of CETP, and demonstrated a peak for CETP mass centered at mol ecular masses of 150 to 180 kilodaltons, larger than that for free mon omeric CETP. Native acrylamide gel electrophoresis of plasma from six individuals, followed by 2F8/2E7 sandwich immunoblotting, showed CETP migrating within a size range of 170-220 kilodaltons. This result is c onsistent with suggestions that plasma CETP is associated with small-s ized HDL. Agarose gel electrophoresis showed plasma CETP, as well as p urified recombinant CETP, to be prebeta migrating. For determining the concentration of CETP in the media of cultured HepG2 cells, advantage was taken of the high sensitivity of the ELISA. CETP levels were foun d to increase linearly over the 100-h culture period, reaching 8.0 +/- 0.4 ng/ml (18.0 +/- 1.3 ng/mg cell protein). These sensitive, direct immunoassays for CETP mass should be valuable aids for examining the b ehavior of CETP in plasma and other complex systems, as well as for st udying the synthesis and secretion of CETP by different cells and tiss ues.