title: Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N-acetyl-seryl-aspartyl-lysyl-proline.
abstract: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N-terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP-like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β-D-galactosidase-labeled Gly-γAbu-SDKP as a marker antigen, an anti-rabbit IgG-coated immunoplate as a bound/free separator and 4-methylumbelliferyl-β-D-galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP-IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter-assay and 13.3 and 7.8% for intra-assay comparisons. Plasma AcSDKP-IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients.
authors.affiliation: Department of Clinical Pharmacy, Oita University Hospital, Hasama-machi, Oita 879-5593, Japan. y-suzuki@oita-u.ac.jp
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Hiroki
authors.initials: H
authors.lastname: Itoh
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Katagiri
authors.forename: Fuminori
authors.initials: F
authors.lastname: Sato
authors.forename: Kanako
authors.initials: K
authors.lastname: Kawasaki
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Sato
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Mimata
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takeyama
mesh_terms: D000328:Adult; D000368:Aged; D000918:Antibody Specificity; D000925:Anticoagulants / Q000737:chemistry; D015415:Biomarkers / Q000097:blood; D002138:Calibration; D016022:Case-Control Studies; D002940:Circadian Rhythm; D004492:Edetic Acid / Q000737:chemistry; D005260:Female; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007106:Immune Sera; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards; D008297:Male; D008875:Middle Aged; D009842:Oligopeptides / Q000097:blood* / Q000276:immunology; D055550:Protein Stability; D012015:Reference Standards; D051436:Renal Insufficiency, Chronic / Q000097:blood*; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase
chemical_list: D000925:Anticoagulants; D015415:Biomarkers; D007106:Immune Sera; D009842:Oligopeptides; D006493:Heparin; D004492:Edetic Acid; D001616:beta-Galactosidase; C058504:goralatide