Development of Rapid and Sensitive One‐Step Direct Enzyme Linked Immunosorbent Assay for 17‐α‐OH‐Progesterone in Serum

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Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17‐α‐hydroxy‐progesterone (17‐α‐OH‐P) in human serum. The antiserum was raised against 17‐α‐hydroxy‐progesterone‐3‐O‐carboxymethyloxime bovine serum albumin (17‐α‐OH‐P‐3‐O‐CMO‐BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17‐α‐hydroxy‐progesterone‐3‐O‐carboxymethyloxime with horseradish peroxidase (HRP). Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose‐response studies were carried out by incubating 25 µL enzyme conjugate along with 50 µL of standards on the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100 µL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The detection limit of the assay was 180 pg/mL. The assay was more specific as compared to most other reported immunoassays for 17‐α‐OH‐P. Cross reaction with analogous C18, C19, and C21 steroids was less than 0.1% except for progesterone which showed 2.1% cross reaction. The intra‐ and inter‐assay coefficients of variation ranges from 3.7–7.5% and 6.9–11.7%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.9 (n=30).