Details

Autor(en) / Beteiligte

• 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

Titel

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

Ist Teil von

• Journal of pharmaceutical and biomedical analysis, 2010-01, Vol.51 (1), p.158-163

Ort / Verlag

England: Elsevier B.V

Erscheinungsjahr

2010

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.