Development and Characterization of Monoclonal Antibodies Against Nitro-166Tyrosine of High-Density Lipoprotein: Apolipoprotein A1.

Abstract

Apolipoprotein A1 (ApoA1) of the high-density lipoprotein (HDL) plays a cardinal role in alleviating atherosclerosis in various ways. Its role in reverse cholesterol transport is preeminent. However, the ApoA1 undergoes oxidation under chronic inflammatory conditions and these oxidations are mediated by myeloperoxidase. It has been reported that the oxidation of the amino acids such as methionine, tyrosine, and tryptophan residues at specific sites of ApoA1 renders it not only dysfunctional but also proinflammatory and proatherogenic. Thus, assessing the quality of ApoA1 and, in turn, that of HDL in circulating blood can serve as an early diagnostic tool for cardiovascular diseases (CVDs). In this study, we developed monoclonal antibodies (mAbs) specific to modified ApoA1 with its tyrosine residue at the 166th position nitrated to 3-nitrotyrosine. A 20 amino acid peptide around the modification of interest was designed using an antigenicity prediction tool. The peptide was custom synthesized with ovalbumin as conjugate and used as an antigen to immunize BALB/c mice. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the immunized mouse. A hybridoma clone 2E5B7, thus developed and characterized, was found to secrete mAb of the desired specificity and sensitivity against nitrated 166Tyrosine. The lowest concentration of the antigen that could be detected by the mAb with confidence was 15 ng. The mAb was able to detect nitrated 166Tyrosine peptide ovalbumin conjugate antigen spiked in human plasma with high specificity. The generated mAb could be potentially used in immuno-based diagnostic systems to screen the quality of HDL and in turn assess CVD risks in humans.