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

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Analytical Letters 2015, 48, pp page number

  • Tsvetomira Ivanova and Tzonka Godjevargova
  • University “Prof. Dr. A. Zlatarov”, Department of Biotechnology, Bourgas, Bulgaria

This publication describes the synthesis, silane functionalization and use of magnetic Iron nanoparticles in combination with enzyme-linked immunosorbent assay (ELISA) to facilitate the hybrid technique of ‘magnetic nanoparticles – ELISA’ for use in the capture of progesterone in numerous types of milk. The synthesis and functionalization of the magnetic nanoparticles were carried out in an automated Globe system (now replaced by the Syrris Orb Jacketed Reactor) to furnish nanoparticles with a mean size of 182.4nm and 292.8nm after functionalization.

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 anti-progesterone antibody was immobilized on the amino modified magnetic nanoparticles by glutaraldehyde?. The typical standard curve for progesterone in the 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 the 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.

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This paper used the Syrris Globe system, which has now been replaced by the Syrris Orb Jacketed Reactor.

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