| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1987: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Numerous enzyme immunoassays (EIAs) have been developed using various enzymes as labels. In general, however, development of a specific and sensitive EIA for haptens, such as steroids and drugs, is not always easy owing to various factors. In order to obtain a practical basis for selecting the enzyme, enzyme labeling method, steroid/enzyme molar ratio in the label, method of determining enzyme activity, and bridging phenomenon were investigated in a testosterone or 11-deoxycortisol EIA system; alkaline phosphatase (AP), -galactosidase ( -GAL), horseradish peroxidase (HRP), glucose oxidase (GOD) were compared with regard to the sensitivity. There is the possibility that the heterogeneity of polyclonal antibodies influencing the results on assay sensitivity. The use of monoclonal antibody eliminates this problem, simplifying the analysis of immune reactions. Thus, the production of monoclonal antibodies to steroid hormones was also carried out.
The following results were obtained: 1) The
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N-succinimidyl ester method is useful for these four enzymes. The labeled antigens prepared at molar ratios of 4-8, 20-40, 10-60, and 10-20 in the GOD, AP, HRP, and -GAL labelings gave high sensitivities, respectively.
2) The use of 3,3',5,5'-tetramethylbenzidine, a safe substrate for HRP, as a chromogen in an EIA system with a GOD label gave satisfactory results.
3) In the testosterone EIA, the highest sensitivity was obtained by the use of HRP, with GOD next, AP third and -gal fourth. The amount of testosterone needed to displace 50% of the bound label in the colorimetric EIAs ranged from 9 to 90 pg.
4) Previously, using -GAL as a label in bridge heterologous assay systems for cortisol, we showed that the bridge length is an important factor influencing the sensitivity, and the use of a shorter bridge for enzyme labeling results in an increase in sensitivity. The present findings suggest that the bridge length effect may depend on the label enzyme.
5) EIA systems using a monoclonal antibody were also developed for the determination of 11-deoxycortisol or 17 -hydroxyprogesterone. Less
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