Development of anti-chloro 192 tyrosine HDL apoA-I antibodies for the immunodiagnosis of cardiovascular diseases.

High density lipoproteins (HDL) are considered cardio protective. Apolipoprotein A-I (apoA-I), a major component of HDL helps in reverse cholesterol transport, whose function is greatly affected during atherosclerosis due to oxidation by myeloperoxidase. Amino acid tyrosine residue of apoA-I at position 192 and 166 are sensitive to oxidation by myeloperoxidase resulting in the generation of chlorinated and nitrated apoA-I and they are believed to be present in atherosclerotic plaques and in circulation. These oxidized apoA-I have been suggested as potential indicator(s) of CVD risks in humans. To detect the levels of oxidized apoA-I there is a need for developing monoclonal antibodies (mAbs) with high specificity and sensitivity that could be utilized routinely in clinical immune based assays for blood plasma or for in vivo imaging. In this study, chemically chlorinated apoA-I (chlorinated 192tyrosine- apoA-I) and a short synthetic peptide, containing the corresponding chlorinated tyrosine residue, conjugated to keyhole limpet hemocyanin (KLH) carrier protein were used for immunization. Stable hybridoma clones F7D5 and G11E3 were found to be highly sensitive and reactive towards chlorinated 192tyrosine- apoA-I. Interestingly, these mAbs also displayed positive reaction with atherosclerotic plaques obtained from mouse and human biopsies. In vitro or in vivo diagnostic tests could be developed either by detecting oxidized apoA-I in human plasma or by directly imaging atheroma plaques as both mAbs were shown to stain human atheroma. The anti-chlorinated 192tyrosine- apoA-I mAbs described in this study may have a high diagnostic potential in predicting CVD risks.

Development of Monoclonal Antibody Against Chlorinated 192tyrosine Containing ApoAI Peptide to Screen Quality of Human High Density Lipoprotein (HDL).

ApoAI, a major protein component of HDL is considered to be a key factor which contributes for HDL's cardio- protective functions. ApoAI is sensitive to myeloperoxidase mediated oxidative modifications under chronic inflammatory conditions such as atherosclerosis. The amino acid tyrosine at position 192 of ApoAI is considered to be one of the vulnerable sites for oxidation which impairs HDL functions and its quality. The presence of oxidized ApoAI in plasma may serve as a useful indicator of CVD risks in humans, but its detection in the clinical settings requires monoclonal antibodies (mAbs) with high specificity. In this study, we have developed mAbs against chloro- tyrosine at position 192 of ApoAI. We designed a small synthetic peptide of 7 amino acids length containing modified tyrosine residue (189-LAE-3-Cl-Y-HAK-195) based on antigenicity prediction. We coupled this peptide to Keyhole Limpet Hemocyanin (KLH) for generating mAbs. Hybridoma clones, KLH-ApoAI-F2 and KLH-ApoAI-H9, were found to be highly specific to chloro-192Tyr containing peptide of ApoAI and not to either nitro-192Tyr containing or to chloro-166Tyr and nitro-166Tyr containing peptides of ApoAI. The utility of these mAbs for screening the quality of HDL as an indicator of CVD risks in humans is discussed.