Mechanisms
New monoclonal-antibody immunoassays quantify CETP mass down to nanogram levels in plasma and cell culture (J Lipid Res 1995)
Original title: Low level quantification of cholesteryl ester transfer protein in plasma subfractions and cell culture media by monoclonal antibody-based immunoassay
Two monoclonal antibodies raised against human plasma CETP, 2F8 for capture and 2E7 for detection, were used to build sandwich-type immunoradiometric and ELISA assays, validated by their ability to inhibit cholesteryl ester transfer activity and by immunoblotting. The IRMA quantified CETP over 0.5-20 ng/assay (5-200 ng/ml) and the ELISA over 0.05-5 ng/assay (0.5-50 ng/ml), both more sensitive than prior methods. Using the IRMA, mean plasma CETP in 44 normolipidemic individuals was 2.10 plus or minus 0.36 micrograms/ml, correlating well with cholesteryl ester transfer activity (r equals 0.913, n equals 23). Gel permeation and native electrophoresis showed plasma CETP migrating at 150-220 kilodaltons, larger than free monomeric CETP and consistent with association with small HDL, while the ELISA tracked CETP secretion by cultured HepG2 cells rising linearly to 8.0 plus or minus 0.4 ng/ml over 100 hours, establishing sensitive new tools for studying CETP mass in plasma and cell systems.
Original abstract
Sensitive immunoradiometric (IRMA) and ELISA assays for cholesteryl ester transfer protein (CETP) have been developed using two different monoclonal antibodies (MAbs). 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 assays are linear and provide sensitivities much greater than previously reported. The IRMA allows for the accurate quantification 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 individuals was determined to be 2.10 +/- 0.36 micrograms/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 micrograms/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 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 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 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 found 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 behavior of CETP in plasma and other complex systems, as well as for studying the synthesis and secretion of CETP by different cells and tissues.
Summary written by cetpinhibition.org from the published abstract; figures as published. Page updated 18 August 2026. Methods.