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.
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.
Nakamura
Masahiro
M
Diagnostic Department, Shionogi & Co. Ltd., Settu, Osaka, Japan.
Ohta
Hideki
H
Kume
Noriaki
N
Hayashida
Kazutaka
K
Tanaka
Masaru
M
Mitsuoka
Hirokazu
H
Kaneshige
Toshihiko
T
Misaki
Shintaro
S
Imagawa
Keiichi
K
Shimosako
Ken'ichi
K
Ogawa
Naoko
N
Kita
Toru
T
Kominami
Goro
G
D054058:Acute Coronary Syndrome / Q000175:diagnosis; D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D019540:Area Under Curve; D016466:CHO Cells; D002459:Cell Fusion; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D051379:Mice; D008875:Middle Aged; D009101:Multiple Myeloma / Q000276:immunology; D012372:ROC Curve; D051127:Scavenger Receptors, Class E / Q000097:blood / Q000276:immunology*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; C495137:OLR1 protein, human; D051127:Scavenger Receptors, Class E