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An enzyme immunoassay (EIA) was applied for determination of progesterone in swine serum. Procedures for EIA are summarized as follows; 0, 25, 50, 100, 200, 500 and 1, 000 pg of progesterone (standard) in 0.1-0.2 ml of methanol was dried and added with 0.1 m<I>l</I> of 0.1 M phosphate buffer (pH 7.0). Serum (0.05-0.1 m<I>l</I>) was extracted with 20 volume of petroleum ether in a test tube and the ether layer was transferred to another tube, dried and added with 0.1 ml of 0.1 M phosphate buffer. Then 0.1 m<I>l</I> of progesterone antiserum raised in rabbits against 11α-hydroxyprogesterone-hemisuccinate-BSA was added at the dilution of 7, 500 and the mixture was incubated at room temperature for 3 hours. Thereafter 0.1 m<I>l</I> of 11α-hydroxyprogesterone-β-galactosidase conjugate solution (×100) was added and incubated at room temperature for 3 hours again. Free and antibody-bound progesterone were separated by addition of 0.1 ml of anti-rabbit gamma-globulin goat serum (second antibody) ( × 40), incubated at 4°C over night and centrifugated at 3, 500 rpm for 20 minutes. The precipitate was washed once with 2 ml of 0.1 M phosphate buffer and resuspended in 2 ml of the same buffer and measured for enzyme activity by a spectrophotometer (absorbance at 420 nm).<BR>The average recovery rate of 25-1.000 pg of progesterone added to 0.05 ml of serum was 89.5%±9.8 (S.D.). Intra-assay and inter-assay coefficient of variation were below 10%. The serum progesterone levels obtained by EIA and RIA were highly correlated (r=0.96, p ?? 0.01).<BR>Thus, this EIA technic can be used for practical and routine analysis of swine serum progesterone.
- 日本繁殖生物学会の論文
日本繁殖生物学会 | 論文
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