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 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 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 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 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 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.-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 (CETP) catalyzes the transfer of cholesteryl esters (CE), triglyceride, and phospholipid between lipoproteins in the plasma of many animal species, including humans (1). Most plasma CE is generated on high density lipoproteins (HDL) through the action of 1ecithin:cholesterol acyltransferase (LCAT). CETP acts to redistribute a portion of this CE to triglyceride-rich lipoproteins, which are transformed via lipolysis to remnants and low density lipoprotein (LDL), or to LDL directly. Because this exchange of CE for triglyceride causes a net loss of CE from HDL, CETP has been proposed to play an important role in determining the balance between LDLand HDL-cholesterol levels. That CETP can play such a role in vivo is suggested by the very high HDL levels observed for subjects genetically defective for CETP expression (2), and by the reduced HDL levels displayed by CETP transgenic mice (3). These contrasting states represent potentially antiatherogenic and atherogenic lipoprotein profiles, respectively, and call for more detailed studies on the role of CETP in intravascular lipoprotein metabolism, as well as its function in different cells and tissues. Abbreviations: CETP, cholesteryl ester transfer protein; CE, cholesteryl ester; E, total cholesterol; CO, cholesteryl oleate; CMC, critical micelle concentration; VLDL/IDL, very low density lipoprotein/intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; IRMA, immunoradiometric assay; MAb, monoclonal antibody; FPLC, fast protein liquid chromatography. *To whom correspondence should be addressed. 2Present address: Baxter Healthcare Corporation, Renal Division Research MPR-Dl, McGaw Park, IL 60085-6730. 876 Journal of Lipid Research Volume 36, 1995 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom For CETP to transfer neutral lipid it must associate with the lipoproteins involved and bind the lipid to be transferred. Much effort has been expended to determine the degree to which CETP is associated with lipoprotein subclasses in the plasma and how factors such as surface charge, apolipoprotein content, plasma proteins, and ionic strength may be involved in such interactions. CETP interactions in whole plasma or in defined assays with isolated lipoproteins or synthetic vesicles have frequently been examined using gel filtration techniques to separate components followed by assessment of CETP content by CE-transfer activity (4-6). Other studies have examined CETP interaction with lipoproteins immobilized on Sepharose (7) or used both gel filtration and immobilized lipoproteins in separate experiments (8). In one investigation, plasma lipoprotein subclasses were specifically removed by apolipoprotein A-I and A-I1 immunoaffinity and the amount of CETP activity remaining in the nonbinding plasma fraction was determined (9). Single dimension native polyacrylamide gel electrophoresis (PAGE) (10, 11) and two-dimensional agarose/PAGE (12) have also been used to characterize the CETP-associated species in human plasma with regard to size classes and surface charge. In none of these studies, however, was an attempt made to determine CETP mass. In studying the effects of anti-CETP antibodies on CETP binding to lipoproteins or vesicles in artificial systems, Swenson et al. (13) determined relative mass levels in chromatography fractions by sodium dodecyl sulfate (SDS)/PAGE followed by scanning of immunoblots, but did not determine actual concentrations. Likewise, demonstrations of the secretion of CETP by cells in culture have either relied solely on CEtransfer activity and inhibition of such by specific antibodies (14-16) or combined this evidence with that derived by immunoprecipitation and immunoblotting (17). The lack of quantitative information on CETP mass in the above studies appears due in large part to lack of a sensitive and reliable assay for measuring CETP at the low levels found in chromatographic fractions and cell culture media. We have developed a series of monoclonal antibodies (MAb) to human CETP. Two MAbs, identified as 2F8 and 2E7, have been found to function well in double-MAb sandwich immunoassays. Immunoradiometric (IRMA) and ELISA assays have been standardized using 2F8 as the capture antibody and either iodinated or alkaline phosphatase-conjugated 2E7 as the detection MAb. Conditions for optimizing the detection of CETP under different conditions, including the selection and concentration of appropriate detergents, are described, as well as the use of these assays to measure CETP in gel filtration fractions and cell conditioned media. The IRMA and ELISA for CETP described in this report are direct noncompetitive assays that take advantage of the high specificity and low background of monoclonal antibodies, and are unaffected by widely varying lipid and lipoprotein levels. For quantification of CETP at low levels, the ELISA provides a sensitivity 40-50 times greater than that previously reported for both indirect competitive (10) and immunoradiometric (18, 19)