Sensitive Progesterone Determination Using a Magnetic Particle-Based Enzyme-Linked Immunosorbent Assay

Abstract

A highly sensitive magnetic nanoparticle enzyme immunoassay of progesterone was established using horseradish peroxidase as a label. The “enzyme label” was prepared by coupling of progesterone-3-(O-carboxymethyl)oxime to horseradish peroxidase. The antiprogesterone antibody was immobilized on the aminomodified magnetic nanoparticles by glutaraldehydе. The typical standard curve for progesterone in buffer by the magnetic nanoparticles enzyme immunoassay was obtained with a detection limit of 0.04 ng mL−1. These results were compared to the data obtained for progesterone in milk. It has been shown that the progesterone magnetic particle-based enzyme-linked immunosorbent assay in milk had a minor depression in sensitivity (detection limit of 0.09 ng mL−1). The progesterone calibration curves obtained by the random antibody immobilization were compared with results by protein A oriented immobilization of the antibodies. The sensitivity of progesterone magnetic particle-based enzyme-linked immunosorbent assay in milk using random antibody immobilization method was lower than sensitivity of the progesterone magnetic particle-based enzyme-linked immunosorbent assay using protein A oriented immobilization method (detection limit of 0.08 ng mL−1). The influences of milk type and the fat content in milk on the immunoassay were investigated. With an improved sensitivity and simple operation, the magnetic particle-linked antibody for immunoassay of progesterone has great potential to supersede the traditional enzyme-linked immunosorbent assay for progesterone determination.