| Project/Area Number |
10670326
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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 |
Hygiene
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| Research Institution | Nippon Medical School |
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Principal Investigator |
INAGAKI Hirofumi Nippon Medical School, Department of Hygiene and Public Health, Assistant Professor, 医学部, 講師 (50213111)
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| Co-Investigator(Kenkyū-buntansha) |
MINAMI Masayasu Nippon Medical School, Department of Hygiene and Public Health, Professor, 医学部, 教授 (00019639)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Cholinesterase / Monoclonal Antibody / Enzyme-linked Immunosorbent Assay (ELISA) / Organophosphates |
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| Research Abstract |
A new sandwich ELISA system for human serum butyrylcholinesterase (BChE; EC 3.1.1.8) was established using novel anti-BChE monoclonal antibody named BCE-01. The detection limit of the ELISA was around 5 ng/ml and the effective assay range was from 5 to 2,000 ng/ml.
BChE concentration in serum samples from eighteen healthy volunteers determined by the ELISA was 56.5±10.7 mg/l (Mean±S.D.) and homospecific activity (enzyme activity (IU/I)/enzyme concentration determined by the ELISA (mg/l)) was 21.2±1.3 IU/mg (Mean±S.D.). The coefficient of variance (C.V.) for the homospecific activity was 6.0%, that was smaller than those for BChE concentration and BChE activity (19.0% and 18.9%, respectively). This indicated that the homospecific activity is constant among serum samples from health donors in spite of large inter-individual variation in BChE concentration and BChE activity, and that more quantitative evaluation of intoxication with BChE inhibitors can be carried out by determination of BChE homospecific activity than simply measuring serum BChE activity.
On the other hand, the binding of BCE-01 monoclonal antibody to BChE was not affected by the modification of BChE with DEP and neostigmine methoylsulfate. In addition, BCE-01 antibody did not inhibit BChE enzyme activity. These results indicated that the epitope of BCE-01 antibody exist on other than BChE enzyme active site and that the binding of the inhibitor on the active site of BChE does not affect recognition and binding of BCE-01 antibody. Therefore, BChE concentrations in the sera from the patients intoxicated with cholinesterase inhibitors were also considered to be accurately determined by the ELISA.
Lastly, the serum samples from four patients intoxicated with organophosphate compunds were analyzed using the ELISA. The serum BChE homospecific activity was shown to increase along with recovery, and the disappearance of the inhibited enzyme from the blood was investigated.
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