Abstract
Food and feed contamination with Aflatoxins pose a serious threat to human health and animal husbandry development and has caused widespread concern, among them, G-group Aflatoxins as the main pollutant has attracted more and more attention. In order to establish a rapid, sensitive, specific and efficient immunoassay method for G-group aflatoxins, this study aimed to designed to synthesize 3 immunogens and coating antigens and identified by UV and SDS-PAGE. Then used to immunize Balb/c mice with prepared of three immunogen the titers were determined by indirect ELISA and the sensitivity was determined by competitive indirect ELISA (icELISA), the specificity was assessed by the cross-reaction test (CR). The results of UV and SDS-PAGE showed that the three immunogens and the corresponding coated antigens were successfully synthesized and the best one was SA method among three synthesis methods of G-group AF artificial antigen and its conjugation ratio of AFG1 to BSA was about 5.64∶1. The immune efficacy of SA method was the best, its AFG1 pAb had high titers of 1∶(6.4×103) by indirect ELISA, a good sensitivity with the 50 % inhibition concentration(IC50) of 13.6 μg/kg－1 to AFG1 by blocking ELISA and a high CR to AFG2 of 82.19 %, it showed high specificity for other aflatoxins. The experimental results not only obtained the ideal G group aflatoxin antibody, but also established a substance and technology foundation for G group aflatoxin immunization methods, and can be referenced in the similar tests.
Highlights
Aflatoxins (AFs) has acute, chronic, carcinogenic and immunosuppressive toxic effects on human health
A certain amount of AFG1, Bovine serum albumin (BSA) and AFG1-BSA, and AFG1were weighed and dissolved with methanol the methanol as a blank control; BSA and AFG1-BSA were dissolved in methanol phosphate buffer solution (PBS) solution [V ∶ V (PBS) = 4∶6)], which was used as blank control
The concentration of concentrated glue is 50 g /L-1, voltage 90 V; separation glue mass concentration is 120 g /L-1, voltage 45 V; 10 μL per hole per hole, 10 μg of protein, Coomassie blue staining, the molecular binding ratio of AFB1 and BSA was calculated by UV gel imaging system analysis software
Summary
Aflatoxins (AFs) has acute, chronic, carcinogenic and immunosuppressive toxic effects on human health. Most countries have made clear provisions on the maximum residue limit (MRL) of AFB1 in food (Wu et al, 2013; Rushing & Selim, 2019). The characteristics of contaminated food are that a variety of toxins almost exist at the same time and have toxic additive effects. It has become a development trend to formulate MRL and corresponding detection methods for total aflatoxins (TAFs) (AFB1 + AFB2 + AFG1 +AFG2) (Aiko & Mehta, 2015; Xie et al, 2016). China's existing standard has not yet involved TAFS limited requirements, but the detection method of TAF MRL in grain was prescribed in “LS/T 6128-2017: Determination of aflatoxin B1, B2, G1, and G2 in grain: ultra-high performance liquid chromatograph (China, 2017)
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