Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N-acetyl-seryl-aspartyl-lysyl-proline
Article first published online: 7 MAR 2012
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Journal of Peptide Science
Volume 18, Issue 4, pages 276–281, April 2012
How to Cite
Suzuki, Y., Itoh, H., Katagiri, F., Sato, F., Kawasaki, K., Sato, Y., Mimata, H. and Takeyama, M. (2012), Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N-acetyl-seryl-aspartyl-lysyl-proline. J. Peptide Sci., 18: 276–281. doi: 10.1002/psc.2400
- Issue published online: 21 MAR 2012
- Article first published online: 7 MAR 2012
- Manuscript Accepted: 10 JAN 2012
- Manuscript Revised: 26 DEC 2011
- Manuscript Received: 25 OCT 2011
- enzyme immunoassay;
- clinical application;
- chronic renal failure
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. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.