An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.

Hui Wu,Qi Zhang,Fei Ma,Chenyu Du,Min Li,Hengling Wang,Peiwu Li,Zhaowei Zhang,Xiaoxia Ding

doi:10.1371/journal.pone.0106415

Hui Wu, Qi Zhang + Show 7 more

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https://doi.org/10.1371/journal.pone.0106415

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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.

Highlights

Sterigmatocystin (STG) is toxic, mutagenic, and carcinogenic secondary metabolites primarily produced by Aspergillus species such as Aspergillus versicolor, Aspergillus flavus, Aspergillus nidulans, and Aspergillus rugulosus [1,2]
The toxicity of sterigmatocystin is only second to aflatoxin B1, which seriously threatens human and animal health [1,3,4,5]
We present a simple, fast and reliable synthesis for novel STG derivatives and STG bovine serum albumin conjugates

Summary

Introduction

Sterigmatocystin (STG) is toxic, mutagenic, and carcinogenic secondary metabolites primarily produced by Aspergillus species such as Aspergillus versicolor, Aspergillus flavus, Aspergillus nidulans, and Aspergillus rugulosus [1,2]. Sterigmatocystin can contaminate many types of food and feed, especially wheat, maize, peanuts, and forage [3]. The toxicity of sterigmatocystin is only second to aflatoxin B1, which seriously threatens human and animal health [1,3,4,5]. To protect the agricultural environment, assess quality of commercial agroproducts and foods, and safeguard health and lives of consumers, a few countries have established maximum limits of sterigmatocystin in agro-products. The regulatory sterigmatocystin level of below 25 mg?kg is accepted in China [6].

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