Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1223556 | Journal of Pharmaceutical and Biomedical Analysis | 2010 | 6 Pages |
Abstract
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 (Kd) 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 (r2 = 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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Masahiro Nakamura, Hideki Ohta, Noriaki Kume, Kazutaka Hayashida, Masaru Tanaka, Hirokazu Mitsuoka, Toshihiko Kaneshige, Shintaro Misaki, Keiichi Imagawa, Ken'ichi Shimosako, Naoko Ogawa, Toru Kita, Goro Kominami,