1996 Fiscal Year Final Research Report Summary
Development of immuno PCR method and its biomedical application
| Project/Area Number |
07807211
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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 |
Laboratory medicine
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| Research Institution | Okayama University |
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Principal Investigator |
ENDO Hiroshi Okayama University School of Health Sciences Professor, 医療技術短期大学部, 教授 (70033090)
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| Co-Investigator(Kenkyū-buntansha) |
MORI Shuji Okayama University School of Health Sciences Assistant, 医療技術短期大学部, 助手 (50220009)
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| Project Period (FY) |
1995 – 1996
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| Keywords | immuno PCR / immunoassay / C- reactive protein |
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| Research Abstract |
A high sensitive assay, which was termed immuno PCR,for C- reactive protein as model molecule was developed. PCR (polymerase chain reaction) was employed in detection system of this assay. C- reactive protein was immobilized on microplate through APPC (p-aminophenylphosphorylcholine) - BSA,based upon its Ca^<2+>-dependent binding property to phosphorylcholine. After blocking, 1st Ab, biotinylated 2nd Ab, avidin and biotinylated template DNA were added in turn. In final, PCR was performed with SP6 promoter primer and T7 promoter primer. As assessed by image analyzer after electrophoresis followed by EtBr staining, the amounts of PCR product to be amplified (497 bp) was shown to be dependent on the amounts of initially added C- reactive protein. In addition, it varied with some parameters, including PCR cycles, temperature and period for annealing. C- reactive protein as little as 2.5 ng/ml could be detected by his immono PCR,while not by sandwich- type conventional enzyme immunoassay.
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