Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N-acetyl-seryl-aspartyl-lysyl-proline.
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| 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.
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| Authors | Yosuke Suzuki, Hiroki Itoh, Fumihiko Katagiri, Fuminori Sato, Kanako Kawasaki, Yuhki Sato, Hiromitsu Mimata, Masaharu Takeyama |
| Journal | Journal of peptide science : an official publication of the European Peptide Society (J Pept Sci) Vol. 18 Issue 4 Pg. 276-81 (Apr 2012) ISSN: 1099-1387 [Electronic] England |
| PMID | 22396066 (Publication Type: Journal Article) |
| Copyright | Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. |
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