Development of a sensitive enzyme immunoassay (EIA) for determination of progesterone in milk by a second antibody technique

A sensitive enzyme immunoassay (EIA) for determination of progesterone in whole milk using a second antibody technique was developed. The microtitration plates used in the assay were firstly coated with sheep anti-rabbit IgG and incubated at 4 degree C overnight. The immunoreaction was performed by incubating a mixture of 2 μl of antibody against progesterone. The standard curve was sensitive in the range of 0.2 pg/well ∼12.5 pg/well, corresponding to 0.1 ng/ml ∼6.25 ng/ml. The detecting limit of the assay is 0.15 pg/well and the mild-point relative binding (50% relative binding) is at 0.92 pg/well. The intra-and inter-assay coefficients of variation were 4.68% and 11.10% respectively. Recovery of the assay is 92.67 +- 10.79%. Milk samples from dairy cows were tested by running parallel RIA and EIA. A good correlation (r = 0.97, n = 4.8, P<0.001) was obtained and the estimated values were similar in both techniques. The results show the EIA developed in the study not only provides very reliable assay, with a sensitivity 80 times higher than the RIA previously validated in the laboratory, but also reduces the amount of valuable antibody and enzyme label in comparison to direct coating