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A direct enzyme immunoassay of progesterone in skim milk was established. 1. 11α-hydroxyprogesterone-hemisuccinate was conjugated with β-D-galactosidase from E. coli by a mixed anhidride method. After purification, the conjugate was used as tracer of immunoassay. 2. Anti-11α-hydroxyprogesterone-hemisuccinate-bovine serum albumin rabbit serum and anti-rabbit γ-globulin goat serum were used as the first and the second antibodies, respectively. 3. Milk sampled from one of any udder quarter regardless of time of milking was defatted by centrifugation (3, 000 rpm, 10 minutes) and subjected to progesterone measurement. 4. Skim milk (0.05 ml) was diluted 4 times with 0.1M phosphate buffer pH 7.0, added with 0.1 ml of the first antibody and incubated at 4C for 1h. To this mixture 0.1 ml of diluted normal rab-bit serum and 0.1ml of the diluted second antibody solution was added. After incubation at 4 C overnight, it was centrifuged at 3, 000 rpm for 20 minutes to separate the bound from free form. The precipitate was washed once and resuspended to the buffer. Then the enzyme activity was deter-mined. Skim milk from cows in estrus treated with dextran and charcoal, which were diluted 4 times with the buffer containing standard progesterone (0-1, 000 pg), were also assayed. 5. The sensitivity of this enzyme immunoassay was 4 pg/tube. Inter- and intra-assay coefficients of variation were 10.7% and 8.6% each. Milk progesterone values obtained by the present direct enzyme immunoassay and the previous technique with extraction of progesterone from milk were highly correlated (r=0.95, P<O.01). It is concluded that the direct enzyme immunoassay of skim milk progesterone is highly sensitive, reliable, and practical, and therefore applicable for the assessment of luteal activity of cows.
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日本繁殖生物学会 | 論文

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