Journal of Lipid Research, Vol 36, 876-889, Copyright © 1995 by Lipid Research, Inc.

ARTICLES

Low level quantification of cholesteryl ester transfer protein in plasma subfractions and cell culture media by monoclonal antibody-based immunoassay

RW Clark, JB Moberly and MJ Bamberger
Department of Cardiovascular and Metabolic Diseases, Pfizer, Inc., Groton, CT 06340, USA.

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.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				R. W. Clark, R. B. Ruggeri, D. Cunningham, and M. J. Bamberger Description of the torcetrapib series of cholesteryl ester transfer protein inhibitors, including mechanism of action J. Lipid Res., March 1, 2006; 47(3): 537 - 552. [Abstract] [Full Text] [PDF]

				R. W. Clark, T. A. Sutfin, R. B. Ruggeri, A. T. Willauer, E. D. Sugarman, G. Magnus-Aryitey, P. G. Cosgrove, T. M. Sand, R. T. Wester, J. A. Williams, et al. Raising High-Density Lipoprotein in Humans Through Inhibition of Cholesteryl Ester Transfer Protein: An Initial Multidose Study of Torcetrapib Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 490 - 497. [Abstract] [Full Text]

				T. Oka, S. Yamashita, T. Kujiraoka, M. Ito, M. Nagano, Y. Sagehashi, T. Egashira, M. N. Nanjee, K.-i. Hirano, N. E. Miller, et al. Distribution of human plasma PLTP mass and activity in hypo- and hyperalphalipoproteinemia J. Lipid Res., August 1, 2002; 43(8): 1236 - 1243. [Abstract] [Full Text] [PDF]

				Z. Guo, C. Yuan, T. Wei-Lavery, Y. Fang, R. A. Garvin, H. I. Nishida, and T. Nishida Secretion of Phospholipid Transfer Protein by Human Hepatoma Cell Line, Hep G2, Is Enhanced by Sodium Butyrate J. Nutr., November 1, 1999; 129(11): 1984 - 1991. [Abstract] [Full Text]

				K. Sasai, K. Okumura-Noji, T. Hibino, R. Ikeuchi, N. Sakuma, T. Fujinami, and S. Yokoyama Human cholesteryl ester transfer protein measured by enzyme-linked immunosorbent assay with two monoclonal antibodies against rabbit cholesteryl ester transfer protein: plasma cholesteryl ester transfer protein and lipoproteins among Japanese hypercholesterolemic patients Clin. Chem., July 1, 1998; 44(7): 1466 - 1473. [Abstract] [Full Text] [PDF]