Monoclonal antibodies to progesterone: characterization and selection for enzyme immunoassay in bovine milk.

Thirty-one stabile murine monoclonal antibody (MAb) producing cell lines to progesterone were generated by using a short and a long immunization protocol. Long-term immunization with high doses of 11alpha-hydroxyprogesterone-hemisuccinate-bovine serum albumin (11alpha-OH-P-HS-BSA) antigen led to very good antibody response in Balb/c mice. The donor mouse produced antiserum with a high titre of 1/250,000. Eleven MAbs were selected for further characterization since they showed high sensitivities (<35 pg/well to inhibit 50% of the tracer) in bridge homologous enzyme immunoassay (EIA). The results were compared to the donor mouse polyclonal antiserum. The MAbs and the donor mouse antiserum were generally found to be highly specific, when tested with 30 different steroids. Employing MAb 9C11, with affinity constant, K(alpha), to 11alpha-OH-P-HS of 1.1 x 10(10) M(-1), a bridge heterologous microtitre plate EIA for milk progesterone was developed, using the second-antibody coating technique and horseradish peroxidase (HRP) as an enzyme label. The assay is simple and convenient to use, as it permits direct addition of undiluted milk samples, at the same time maintaining high sensitivity, high precision, and a wide range of optical density (OD) values. The major advantage of the assay developed, compared to previously published direct addition milk progesterone immunoassays, is that progesterone concentrations, measured by the EIA, were not influenced by changing milk fat concentrations, even when milk samples containing up to 10% of milk fat were used for analysis.