title: 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.
abstract: OBJECTIVE
  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.
  
  
  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.
authors: Department of Vascular Physiology, National Cerebral and Cardiovascular Center, Osasa, Japan.
authors: Shin
authors: S
authors: Iwamoto
authors: Yoshiko
authors: Y
authors: Fujita
authors: Akemi
authors: A
authors: Kakino
authors: Kazuki
authors: K
authors: Yanagida
authors: Haruo
authors: H
authors: Matsuda
authors: Ryo
authors: R
authors: Yoshimoto
authors: Tatsuya
authors: T
authors: Sawamura
mesh_terms: D000595:Amino Acid Sequence; D053283:Apolipoprotein B-48 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D051127:Scavenger Receptors, Class E / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D053283:Apolipoprotein B-48; D017931:DNA Primers; D008024:Ligands; C495137:OLR1 protein, human; D011993:Recombinant Fusion Proteins; D051127:Scavenger Receptors, Class E