COMPARISON OF FOUR DIFFERENT METHODS FOR DEVELOPMENT OF PROGESTERONE FLUORESCENT IMMUNOASSAY
TSVETOMIRA IVANOVA, NEDYALKA DIMOVA, GALINA GRIGOROVA, TZONKA GODJEVARGOVA*
University “Prof. Dr. A. Zlatarov”, Department of Biotechnology, Prof. Y. Yakimov Str. 1, 8010 Bourgas, Bulgaria
*Corresponding author: godjevargova@yahoo.com
Keywords: fluorescent immunoassay, magnetic nanoparticles, progesterone, milk
Abstract: A reliable fluorescent immunoassay with magnetic nanoparticle (Method A) for the quantitative determination of progesterone in cow milk was developed. This immunoassay was based on the immobilization of monoclonal anti-progesterone-antibody (mAb) on the amino modified magnetic nanoparticles (MNPs-NH2). Both progesterone in the sample and Progesterone-3-(O-carboxymethyl) oxime-bovine serum albumin-fluorescein isothiocyanate (Prog-3-CMO-BSA-FITC) conjugate competed for the immobilized antibody. The proposed Method A was compared with other three methods (B, C, D) with different configuration of immunoreactions. The Method B is similar to Method A, but sample and conjugate were added sequentially. The Method C was based on the immobilization of Prog-3-CMO-BSA conjugate on the MNPs-NH2, which competed with the progesterone in the sample for binding to the added mAb. The binding of mAb to the Prog-3-CMO-BSA-MNPs-NH2 was detected using a target secondary IgG-FITC antibody. The Method D was based on immobilization of secondary antibody on the MNPs-NH2. The mAb was immunoadsorbed to the immobilized secondary antibody. The progesterone in the sample and Prog-3-CMO-BSA-FITC conjugate competed for binding to mAb. It was found that Method A provided better analytical characteristics. Real milk samples (UHT and raw milk) were investigated for the presence of progesterone by using Method A.