Generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone-modified low density lipoprotein

Eric Ferguson,Sampath Parthasarathy,Joy Joseph,B Kalyanaraman

doi:10.1016/s0022-2275(20)32578-5

Eric Ferguson, Sampath Parthasarathy + Show 2 more

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https://doi.org/10.1016/s0022-2275(20)32578-5

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Abstract

Elevated plasma homocysteine (homocysteinemia) are presumed to be responsible for the development of coronary artery disease, however, the precise etiology is unclear. We examined the possibility that the adduct formed from the reaction between homocysteine thiolactone, a metabolic product of homocysteine, and apolipoprotein B-100 lysyl residues of low density lipoprotein (LDL) was immunogenic. New Zealand White rabbits were immunized with this adduct at 6-week intervals. Antisera collected following the 3rd immunization was assayed for antibody titers using solid phase ELISA techniques. Titers (defined as the inverse of the greatest serum dilution in which there was a significant difference (P < 0.05) between the percentage antibody bound from the antiserum and the pre-immune serum) were approximately 105. In competition-based ELISAs, homocysteine thiolactone-treated LDL competed for binding with the antiserum, as the 50% inhibitory concentration was approximately 10 μg/ml. Neither homocysteine, homocystine (homocysteine disulfide), nor Cu2+-oxidized LDL competed for binding. LDL in which lysyl residues were derivatized by acetylation or methylation were not recognized by the antiserum. Homocysteine thiolactone-treated plasma competed for binding to the antiserum, whereas native plasma did not. All lipoprotein fractions from the homocysteine thiolactone-treated plasma competed for binding to the antiserum. We conclude that homocysteine thiolactone-modified LDL is highly immunogenic and specific for homocysteine thiolactone-modified lysines. The potential for using this antibody as a diagnostic tool for measuring plasma homocysteine concentrations and the implications for understanding diseases induced by homocysteinemia are discussed.—Ferguson, E., S. Parthasarathy, J. Joseph, and B. Kalyanaraman. Generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone-modified low density lipoprotein. J. Lipid Res. 1998. 39: 925–933.

Highlights

IntroductionElevated plasma homocysteine (homocysteinemia) are presumed to be responsible for the development of coronary artery disease, the precise etiology is unclear
Elevated plasma homocysteine are presumed to be responsible for the development of coronary artery disease, the precise etiology is unclear
Dithio-bis-2-nitrobenzoic acid (DTNB), potassium bromide, Folin and Ciocalteu’s Phenol Reagent, guanidine hydrochloride, sodium dodecyl sulfate (SDS), Sephadex G-25, polyoxyethylenesorbitan monolaurate (Tween-20), ethylenediaminetetraacetic acid (EDTA), butylated hydroxytoluene (BHT), sodium cyanoborohydride, materials for all phosphate-buffered saline (PBS, sodium phosphate (25 mm), sodium chloride (125 mm), pH 7.4), homocysteine thiolactone hydrochloride, and homocysteine were obtained from Sigma Chemical Co

Summary

Introduction

Elevated plasma homocysteine (homocysteinemia) are presumed to be responsible for the development of coronary artery disease, the precise etiology is unclear. We examined the possibility that the adduct formed from the reaction between homocysteine thiolactone, a metabolic product of homocysteine, and apolipoprotein B-100 lysyl residues of low density lipoprotein (LDL) was immunogenic. In competition-based ELISAs, homocysteine thiolactone-treated LDL competed for binding with the antiserum, as the 50% inhibitory concentration was approximately 10 ␮g/ml. Homocystine (homocysteine disulfide), nor Cu2ϩ-oxidized LDL competed for binding. Homocysteine thiolactone-treated plasma competed for binding to the antiserum, whereas native plasma did not. All lipoprotein fractions from the homocysteine thiolactone-treated plasma competed for binding to the antiserum. The potential for using this antibody as a diagnostic tool for measuring plasma homocysteine concentrations and the implications for understanding diseases induced by homocysteinemia are discussed.—Ferguson, E., S. Generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone-modified low density lipoprotein. While there is evidence that indicates homocysteine thiolactone may be involved in atherosclerosis and thromboembolic disorders, a method for the detection of the homocystamide adduct has been lacking

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