Extraction and labeling of aflatoxin B1 from Aspergillus flavus as a main constituent in radioimmunoassay technique

In this study among the tested strains, Aspergillus flavus RCMB 002002(3) exhibited the highest Aflatoxin B1 productivity. The optimum biosynthetic conditions were reported to be static growth on yeast-extract sucrose liquid medium (YES) at 25°C with initial ph value of 5.5 and 4% inoculum size for 21 days. Also, addition of Zn²⁺ and Cu²⁺ stimulate AFB1 production using sucrose and yeast extract as best carbon and nitrogen sources, respectively. Purification of Aflatoxin B1 from other relative toxins was carried out by using preparative thin Layer chromatography (TLC) and analyzed these toxins by High Performance Liquid Chromatography (HPLC). For radioimmunoassay technique the basic reagent was (Tracer), so optimization of iodination of tyrosine methyl ester (TME) was carried out, the best ph value was 7.4; amount of chloramine-T (50 µg), amount of TME (25 µg) and the reaction time (2 min). Synthesis of Aflatoxin B1 - Oxime was carried out by reaction of Aflatoxin B1 with carboxy methoxyl amine and purification on silica gel column. Aflatoxin B1 - Oxime was conjugated with ¹²⁵ I -TME by mixed anhydride method and was purified by solvent extraction method for preparation of tracer. The RIA was standardized for liquid phase system using polyethylene glycol (PEG), regarding, the effect of incubation temperature, incubation time and sample volume was 25 °C, 3 h and 100 μl, respectively. Then, standard curve for assess Aflatoxin B1 in sample was constructed