An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

Abstract

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products.

A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenicAspergillusspecies

“…This is nearly identical with that of the competitive indirect STC‐EIA of Li et al . (), who reported IC 50 values of 0·38–0·47 ng ml −1 ; however, the test of these authors suffered from significant matrix effects, requiring matrix calibrant standard curves for each specific matrix and IAC clean‐up of extracts. In contrast, the STC‐EIA reported here tolerated fungal mycelium extracts very well, allowing a simple extract dilution instead of extract purification.…”

“…() and Li et al . () under modified reaction conditions and with a simplified clean‐up procedure using liquid–liquid‐extraction. In brief, 500 mg glycolic acid was dissolved in 2 ml of trifluoroacetic acid.…”

“…More recently, Li et al . () reported monoclonal antibodies against STC, employing a method originally described for AFB 2 by Cervinio et al . (), and their use in competitive indirect EIA.…”

“…However, the report of Li et al . () lacked details about the preparation of the STC derivative, the competitive indirect EIA format was relatively laborious, and its application to the analysis of spiked cereal samples required time‐consuming immunoaffinity chromatography (IAC) clean‐up. IAC clean‐up of STC using commercial columns was also described by other authors (Sasaki et al .…”

“…Approaches to establish enzyme immunoassays (EIA) based on antibodies against STC-hemiacetal as the antigen were reported some decades ago, but these either required tedious chemical conversion of STC into its hemiacetal before detection (Li and Chu 1984;Morgan et al 1986;Lou et al 1996) or lacked sensitivity (Li et al 1996), and were obviously not further studied. More recently, Li et al (2014) reported monoclonal antibodies against STC, employing a method originally described for AFB 2 by Cervinio et al (2007), and their use in competitive indirect EIA. However, the report of Li et al (2014) lacked details about the preparation of the STC derivative, the competitive indirect EIA format was relatively laborious, and its application to the analysis of spiked cereal samples required time-consuming immunoaffinity chromatography (IAC) clean-up.…”