EVALUATION OF THE EFFICIENCY OF THREE DIFFERENT FLUORESCENT CONJUGATES USED AS TRACERSIN PENICILLIN MAGNETIC NANOPARTICLE IMMUNOASSAY
SVETLA IVANOVA, KATYA GABROVSKA, GALINA GRIGOROVA, TZONKA GODJEVARGOVA*
Department of Biotechnology, University “Prof. Dr. Asen Zlatarov”, Burgas, Bulgaria
*Corresponding author: godjevargova@yahoo.com
Keywords: immunoassay; Penicillin G; fluorescent conjugates; protein A; magnetic nanoparticles
Abstract: Three conjugates between the investigated antibiotic Penicillin G (PEN) and fluorescent dyes (ATTO derivatives) were prepared and used as tracers. The first step in all conjugation reactions was activation of PEN by carbodiimide method. Conjugate 1 was synthesized in two steps: activated PEN was conjugated to bovine serum albumin (BSA), and the purified and lyophilized PEN-BSA conjugate was labeled with ATTO 647 NHS dye (PEN–BSA-ATTO 647 NHS conjugate). Conjugate 2 was prepared by using ethylenediamine as a spacer between activated PEN and ATTO 647 NHS (PEN–EDA-ATTO 647 NHS conjugate). Conjugate 3 was synthesized by direct coupling of activated PEN to ATTO 488 amine (PEN–ATTO 488 amine conjugate). The fluorescent conjugates were purified by gel chromatography. A fluorescent immunoassay for PEN determination on the basis of the obtained three conjugates and immobilized anti-penicillin antibody on magnetic nanoparticles (MNPs) was developed. An antibody against PEN was immobilized on MNPs by the random and oriented method. The measuring protocol was based on the principles of a non-competitive indirect fluorescent immunoassay. It was found that the assay sensitivity was the highest when using oriented antibody immobilization and Conjugate 3 (PEN-ATTO 488) as a tracer.