Low level quantification of c &wyl ester transfer protein in plas,ma subfractions and cell culture media by monoclonal antibody-based immunoassay

Sensitive immunoradiometric (IRMA) and ELISA Supplementary key words sandwich blotting immunoblotting assays for cholesteryl ester transfer protein (CETP) have been native gel electrophoresis prebeta migrating Superose Superdex developed using two different monoclonal antibodies (MAbs). HepG2 cells The MAbs were prepared against human plasma 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 assays, one MAb, 2F8, is used for capture, and the second MAb, 2E7, is iodinated (IRMA) or conjugated with alkaline phosphatase (ELISA) and used for detection. Both as- says are linear and provide sensitivities much greater than previ- ously reported. The IRMA allows for the accurate quantifica- tion of CETP in the range of 0.5-20 ng/assay (5-200 ng/ml), the ELISA 0.05-5 ng/assay (0.5-50 ng/ml). Using the IRMA, the mean plasma CETP concentration in 44 normolipidemic in- dividuals was determined to be 2.10 k 0.36 pglml; 2.05 k 0.33 for males (n = 25) and 2.16 + 0.40 for females (n = 19). Values ranged from 1.28 to 2.97 pg/ml and CETP mass correlated well with cholesteryl ester transfer activity (r = 0.913, n = 23). The distribution of CETP in human plasma was examined both by gel permeation fast protein liquid chromatography (FPLC) and by native gel electrophoresis. For FPLC using agarose resins, a low ionic strength, isotonic buffer system resulted in near total recoveries of CETP, and demonstrated a peak for CETP mass centered at molecular masses of 150 to 180 kilodaltons, larger than that for free monomeric CETP. Native acrylamide gel elec- trophoresis 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 consistent with suggestions that plasma CETP is associated with small-sized HDL. Agarose gel electrophoresis showed plasma CETP, as well as purified recombinant CETP, to be prebeta migrating. For de- termining the concentration of CETP in the media of cultured HepG2 cells, advantage was taken of the high sensitivity of the ELISA. CETP levels were found to increase linearly over the 100-h culture period, reaching 8.0 k 0.4 ng/ml (18.0 k 1.3 ng/mg cell protein). 811 These sensitive, direct immunoassays for CETP mass should be valuable aids for examining the be- havior of CETP in plasma and other complex systems, as well as for studying the synthesis and secretion of CETP by different cells and tissues.-Clark, R. W., J. B. Moberly, and M. J. Bamberger. Low level quantification of cholesteryl ester transfer protein of plasma subfractions and cell culture media by monoclonal antibody-based immunoassay. J. Lipid Res. 1995. 36: 876-889. Cholesteryl ester transfer protein