Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins

Abstract

To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G1 and G2. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G1 and G2, and did not cross-react with aflatoxins B1, B2, or M1. Its IC50 values for aflatoxins G1 and G2 were 17.18 ng·mL−1 and 19.75 ng·mL−1, respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL−1. To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G1 and G2 and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B1-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.

Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis

“…This allowed us to expose the carboxyl group that differentiates it from PZA and enabled us to generate a specific immunoassay. Nevertheless, despite these limitations, the IC50 achieved by our assay was high (1.16 mg/mL) compared to other competitive immunoassay studies using small molecules such as aflatoxin g1 (278.321 g/mol, IC50: 17.18 ng/mL) [ 24 ].…”

Design of Hapten Synthesis and Antibody Characterization of G-group Aflatoxins

“…AFG1 is converted to AFG2a under the action of H2SO4, the aldehyde group of afg2a and the amino group of BSA form unstable Schiff base. Under the reduction action of NaBH4, which formed a stable AFG2A-BSA (Chu et al, 1985;Li et al, 2015). The synthesis route is shown in Figure 2.…”

Advances in Antibody Preparation Techniques for Immunoassays of Total Aflatoxin in Food

“…Chu et al [ 30 ] established this method in 1985. Peiwu Li et al [ 31 ] prepared AFG1 mAb with this method, and the IC50 of AFG1 and AFG2 was 17.18 and 19.75 μg/kg, respectively. The route of synthesis of the AFB2a-BSA antigen via the SA method is shown in Figure 9 .…”