An alternative protein standard to measure activity of LOX-1 ligand containing apoB (LAB) - utilization of anti-LOX-1 single-chain antibody fused to apoB fragment

Aim: we have recently demonstrated that the circulating level of lox-1 ligand containing apob (lab) predicts the risk of cardiovascular events; however,as is the case in other assays measuring oxidized ldl (oxldl),chemical unstability and inter-lot variance of standard oxldl may limit the utility of measuring lab. this study aimed to develop an alternative protein standard that is simultaneously recognized by lox-1 and anti-apob antibody instead of copper-oxidized ldl. methods and results: cdnas encoding the variable regions of anti-lox-1 monoclonal antibody were cloned from hybridomas and reorganized to express anti-lox-1 single-chain variable fragment (fv). cdnas of four regions of human apob (b1 to b4),which were reported to be epitopes of many anti-apob antibodies,were also cloned. after confirming the respective reactivity of fv and apob fragments to lox-1 and anti-apob antibodies,cdnas of fv and apob fragments were connected to express fv-apob chimeric proteins. these fusion proteins were found to be recognized by both lox-1 and anti-apob antibodies. among them,the fusion proteins of fv-b1 and fv-b3 gave saturable binding curves against immobilized lox-1 when detected by anti-apob antibodies. the binding curves of different fv-b1 preparations to lox-1 were almost identical while those of oxldl varied among the preparations,suggesting better quality control of fv-b1 preparations. conclusions: the fusion proteins composed of fv-form anti-lox-1 antibody and apob fragment are useful alternatives to copper-oxidized ldl in determining lab,which would facilitate the application of modified ldl analyses to the clinical diagnosis and risk evaluation of cardiovascular disease.