Development of in-house RIA kit for progesterone in cow's skim milk

A radioimmunoassay (RIA) method for progesterone has been developed. It has been validated for the determination of progesterone in cow skim milk. The main reagents used in the development work were prepared and characterized in-house. The assay uses a radioiodinated tracer purified by gel filtration on Sephadex G-25, standards prepared in skim milk and coated tubes with specific antibodies as the solid phase (separation system). The radiochemical purity of the tracer was greater than 95%, the maximum binding using solid phase reached 43% and the nonspecific binding didn't exceed 5%. Series of progesterone standards using milk matrix with concentration ranging from 0 to 40ng/mL were prepared. Detection limit of the assay was 0.13ng/mL and the precision evaluation gives an intra and inter-assay coefficient variations between 4.94% and 12.66%. The recovery obtained with skim milk samples was 84% to 119% and the parallelism test indicated good linearity (R² > 0.99). The clinical tests give high correlation coefficient (r = 0.998) between progesterone concentrations of cow's skim milk assayed using developed progesterone RIA kit and commercial progesterone RIA kit.