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.