| Aflatoxins are a group of structurally related toxic metabolites produced by fungi, which are highly toxic and carcinogenic compounds. Aflatoxins have been classified as Group 1 carcinogens by International Agency for Research on Cancer. Aflatoxin M1 (AFM1) is the hydroxylated metabolite of Aflatoxin B1 (AFB1). Lactating animals that ingest feedstuffs contaminated with AFB1 excrete AFM1 into milk, and subsequently it can be found in a large variety of milk products. With the adjustment of diet, AFM1 in milk and milk products is a health danger for human, especially for infants and children who are more susceptible to adverse effect of mycotoxins, which represents a worldwide concern. Limits of AFM1 and technical barriers to trade have been setted by many countries. The traditional methods consume a lot of standard materials and organic solvents, which are harmful to operators and cause secondary pollution to the environment. Therefore, the safe and environmentally friendly assay is needed to protect the safety of consumer and break technical barriers. The main conclusions of this paper are as follow.1. Balb/c mice were immunized by AFM1-BSA, which was emulsified by antigen emulsified device. After cell fusion and screening by semi-solid HAT medium, a monoclonal cell line, excreting anti- AFM1 antibody, was obtained. It exhibited high affinity for AFM1 of 1.74×109 L/mol and no cross-reactivity to aflatoxin B1, B2, G1 and G2, which was the most specific monoclonal antibody for AFM1. The subtype of antibody was IgG2a. High-quality monoclonal antibodies provide a good foundation for further research.2. Based on the monoclonal antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM1 in milk and infant milk products. Assays were performed in the AFM1-BSA coated (0.0625μg/mL) ELISA format in which the antibody was diluted 1:10000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimized. Finally, the limits of detection (LOD) were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91 to 110%. Thirty samples were analyzed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method. The ultra-sensitive test method for AFM1 was developed, which provided a powerful security for milk and milk products safety.3. Due to the harm of AFM1 standard to operators and environment, anti-idiotype antibody was used as surrogate calibrator for green analysis of AFM1. Polyclonal anti-idiotype antibody, used as an AFM1 surrogate, was generated by immunizing rabbits with F(ab')2 fragments, which were digested from the anti-AFM1 monoclonal antibody by pepsin. Polyclonal anti-idiotype antibody exhibited high specificity to the anti-AFM1 monoclonal antibody, and no cross-reactivity to either of the other anti-aflatoxin monoclonal antibodies or the isotype matched monoclonal antibody was observed. The concentrations at the same inhibition value were compared. And the results showed that a perfect correlation was obtained. Polyclonal anti-idiotype antibodies were used for ELISA as surrogate calibrators. The inter-assay and intra-assay variation was less than 10.8%, recovery ranged from 85.2 to 110.9%. A reference nationl standard method, immunoaffinity column coupled to high-performance liquid chromatography method, was used to validate the developed method, and a good correlation was obtained with R = 0.9922.4. The non-labeled secondary antibody could compete with enzyme-labeled secondary antibody for binding to the primary antibody. The method utilized a non-labeled secondary antibody as competitor in ELISA, obtaining the same performance as the reference standard. The non-labeled secondary antibody showed excellent correlations with aflatoxin standards in competitive ELISA at the same inhibition value. Therefore, a novel format of general surrogate calibrator for aflatoxins in ELISA was reported in this study. After the researches of the recognizing site and recovery tests, the non-labeled secondary antibodies can successfully replace the aflatoxin standards as the general surrogate calibrators in ELISA.5. A screening method for AFM1 in milk, based on immunoaffinity column clean-up, ASPEC XL SPE sampler automated processing and laser induced fluorescence detection, was developed in this paper. The sensitivity and selectivity of the method are enhanced by a preconcentration of immunoaffinity column and intense excitation of diode laser resource. The LOD is 2.3 pg/mL, the limit of quantitation (LOQ) is 24.7 pg/mL. The recoveries of AFM1 at 50, 100 and 500 pg/mL ranged from 87 to 106%, relative standard deviation was below 8%. The developed method was compared to nationl standard method for analysis of 25 milk samples, the results showed that a good correlation was obtained by the two methods. Overall, this study provides an environmentally friendly, high sensitive, fast, and reliable analytical method for detecting AFM1 in milk.
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