Generation of monoclonal antibodies against a soluble form of lectin-like oxidized low-density lipoprotein receptor-1 and development of a sensitive chemiluminescent enzyme immunoassay

• Published in: Journal of pharmaceutical and biomedical analysis 2010-01, Vol.51 (1), p.158-163
• Main Authors: Nakamura, Masahiro, Ohta, Hideki, Kume, Noriaki, Hayashida, Kazutaka, Tanaka, Masaru, Mitsuoka, Hirokazu, Kaneshige, Toshihiko, Misaki, Shintaro, Imagawa, Keiichi, Shimosako, Ken’ichi, Ogawa, Naoko, Kita, Toru, Kominami, Goro
• Format: Article
• Language: English
• Subjects: Acute coronary syndrome; Acute Coronary Syndrome - diagnosis; Adult; Animals; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - immunology; Area Under Curve; Cell Fusion; Chemiluminescent enzyme immunoassay; CHO Cells; Cricetinae; Cricetulus; Enzyme-Linked Immunosorbent Assay - methods; Female; Human serum; Humans; Immunoassay - methods; Luminescent Measurements - methods; Male; Mice; Middle Aged; Monoclonal antibody; Multiple Myeloma - immunology; ROC Curve; Scavenger Receptors, Class E - blood; Scavenger Receptors, Class E - immunology; Sensitivity and Specificity; Soluble LOX-1
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2009.06.019

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), expressed prominently in atherosclerotic lesions, is cleaved and released as a soluble LOX-1 (sLOX-1), which is a specific biomarker to diagnose acute coronary syndrome (ACS) at an early stage. Although sLOX-1 levels in patient's blood were successfully measured with our previously established enzyme-linked immunosorbent assay (ELISA), the assay was not sensitive enough to detect normal serum levels of sLOX-1 in healthy human subjects. We therefore developed sensitive and specific monoclonal antibodies (mAbs) against sLOX-1 in order to establish a more sensitive immunoassay. Mice were immunized with recombinant human LOX-1 extracellular domain. mAbs were subsequently generated by standard myeloma cell fusion techniques with a novel screening method using time-resolved fluorescence immunoassay. Using two anti-human sLOX-1 mAbs and alkaline phosphatase as a label, a sandwich chemiluminescent enzyme immunoassay (CLEIA) was developed. In total, nine mAbs were obtained. The dissociation constant ( K d) values of these mAbs for sLOX-1 were 0.12–1.32 nM. Characteristics of these mAbs were estimated and the best combination for CLEIA was selected. The newly established CLEIA could determine sLOX-1 levels as low as 8 pg/mL, and thus, was sensitive enough to measure serum sLOX-1 levels in normal human subjects and to evaluate subtle differences. Values for sLOX-1 measured by monoclonal CLEIA and polyclonal ELISA were highly correlated ( r 2 = 0.7594, p < 0.0001). Area under the curve values of the receiver-operating characteristic curves in detecting ACS were 0.948 and 0.978 for monoclonal CLEIA and polyclonal ELISA, respectively. Thus, a more sensitive sLOX-1 CLEIA was established using newly developed mAbs against sLOX-1. In addition to its advantage in early diagnosis of ACS, this assay may also be useful in predicting cardiovascular disease risk in disease-free subjects.