Development of a Highly Sensitive Biotin-Streptavidin Amplified Enzyme-Linked Immunosorbent Assay for Determination of Progesterone in Milk Samples

Agricultural product contamination by endocrine-disrupting compounds (EDCs) residues is an international public health issue and requires continuously stringent administration. A high-throughput biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for detection of progesterone (P4) has been established to be applied in determining milk samples. Under experimental optimization, BA-ELISA based on anti-P4-specific antibody (P4-pAb-based BA-ELISA) showed a linear range from 0.44 to 31.06 ng/mL. The IC50 was 3.68 ng/mL, and the limit of detection (LOD) was 1.25 × 10−1 ng/mL, with negligible cross-reactivities on structural P4 analogues. The signal amplification system enhanced sensitivity on account of strongly supramolecular affinity between biotin and streptavidin. Good recoveries ranged from 87.81 to 113.11%, and low coefficient of variation (below 9.75%) of the immunoassay validated the feasibility and applicability to monitor P4 in milk samples. Milk samples with different spiked levels were tested by both the established P4-pAb-based BA-ELISA and liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Our results, combined with the advantages of specific antibodies, elucidated that P4-pAb-based BA-ELISA is an applicable method for routine screening analysis of P4.