Homologous ELISA for measurement of medroxyprogesterone acetate in serum

In order to develop ELISA for medroxyprogesterone acetate, medroxyprogesterone acetate-3-carboxymethyloxime (MPA-3-CMO) was coupled to bovine serum albumin (BSA) for immunogen preparation and to horseradish peroxidase (HRP) for enzyme conjugate preparation by N-hydroxysuccinimide mediated carbodiimide reaction. The immunogen was used to raise the antiserum in New Zealand white rabbit. The immunoreactivity of MPA-3-CMO-BSA-antibody and MPA-3-CMO-HRP enzyme conjugate was checked by checkerboard assay. The MPA-3-CMO-HRP enzyme conjugate and MPA-3-CMO-BSA-antibody were used for further development, standardization and validation of the assay. Sensitivity, ED₅₀ and affinity of the assay were found to be 0.114 ng/mL, 2.75 ng/mL and 9.9 × 10⁻⁸ L/mol respectively. The % cross-reaction of analogous steroids with MPA-3-CMO-BSA-antibody was less than 0.025%. The recovery of the exogenously spiked MPA serum pools were in the range of 96.83-105.47%. The intra- and inter-assay coefficients of variation was less than 7.02%. The correlation coefficient of the serum level of MPA measured by the developed assay with the commercially available kit was found to be 0.95 (n = 37). This developed ELISA was further validated by measuring serum level of MPA in rat after administering them different doses of MPA intramuscularly.