Development of Dot-ELISA Technique for Estimation of Milk Progesterone and Pregnancy Diagnosis using PVDF Membrane

An analytical hormone separation method and dot enzyme linked immunosorbant assay (dot-ELISA) for quantitation of hormone are described. For extraction of progesterone from milk chloroform methanol mixture was used. For Dot ELISA, conjugate was prepared by immunoglobulin isolated from whole serum by ammonium sulphate precipitation method and conjugated with Horse radish Peroxidase by modified gluteraldehyde procedure. For immobilization of progesterone to PVDF membrane a number of solvents were tried viz., Methanol, Methanol + Carbonate-bicarbonate buffer, Methanol + PBS, Ethanol and 70 per cent isopropanol. On observation following Dot ELISA procedure, 70 per cent isopropanol was selected as best. Dot ELISA of serially diluted progesterone was performed to get the best titre of conjugate selected for further studies. For standard colour of dot with different progesterone concentration, Dot ELISA with varying concentration of progesterone was performed. The new technique was thus developed for estimation of progesterone using PVDF membrane and 70 per cent isopropanol as solvent. The developed Dot ELISA was also used for estimation of progesterone and pregnancy diagnosis from cattle milk sample and technique was validated. This techniques may be useful for estimation of other steroid hormones.