2004 Fiscal Year Final Research Report Summary
Physiological role of human specific seminal CD10-CD13 enzyme complex
| Project/Area Number |
15590583
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Legal medicine
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| Research Institution | Fukushima Medical University |
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Principal Investigator |
ABE Sumiko Fukushima Medical University School of Medicine, Department of Legal Medicine, Lecture, 医学部, 助手 (50136975)
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| Project Period (FY) |
2003 – 2004
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| Keywords | CD 10-CD 13 enzyme complex / inducible nitric oxide synthase / sandwich ELISA / human semen / anti-peptide antibod |
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| Research Abstract |
We developed a sandwich ELISA for detection of inducible nitric oxide synthase (iNOS) using a combination of two kinds of anti-iNOS antibodies, anti-peptides and anti- recombinant iNOS antibodies that we produced. An antibodies against the synthetic peptide based on the rat iNOS amino acid sequence and the recombinant mouse iNOS were shown by Western blotting to be positive against the 130-kDa subunit mouse recombinant NOS. The detection limit of the ELISA assay was estimated to be 0.1-2 ng of mouse recombinant iNOS as a standard. Human recombinant iNOS was not detected by this method. The intra-assay and inter-assay coefficients of variation were approximately 5% and 10%, respectively. The recoveries of mouse iNOS added to various biological samples were also examined. The recovery of iNOS added to human serum was insufficient because of a high background. On the other hand, iNOS added to 100,000 x g supernatant of rat liver homogenate and 2% bovine serum albumin solution as well as that added to PBS were detectable.
We also examined rat livers treated with lipopolysaccharide (LPS), which strongly induces iNOS expression. Using this method, 0.59 and 1.4 ng of iNOS, equivalent to 1 mg protein, were detected in control and LPS-treated rat livers. These results suggest that this sandwich ELISA is sensitive and specific for mouse (also rat) iNOS and that it enables detection of iNOS in tissue homogenate.
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