Design of antigen synthesis and preparation and characterization of specific and eurytopic antibodies against B-group aflatoxins
The aim of this study was to prepare B-group aflatoxins(BGAFs) antibody with strong specificity and good eurytopicity. According to the molecular structure and active site of aflatoxin B1 (AFB1), the BGAFs artificial antigen AFB1-BSA was prepared by 6 methods such as oxime active ester(OAE)，methylation of ammonia(MOA)，mixed anhydride(MA)，semi acetal(SA)，epoxide(EP) and enol ether derivative(EED) and identified by UV and SDS-PAGE. Polyclonal antibodies against AFB1(AFB1 pAb) were prepared by immunizing New Zealand rabbits with AFB1-BSA, and the titers of AFB1 pAb was detected by indirect ELISA, the sensitivity of AFB1 pAb was analyzed by indirect competitive ELISA(icELISA) and the specificity and eurytopicity of AFB1 pAb was analyzed by cross-reactivity(CR) test. The results showed that AFB1-BSA was synthesized successfully and the best one was OAE method among 6 synthesis methods of BGAFs artificial antigen and its conjugation ratio of AFB1 to BSA was about 8.46∶1. The immune efficacy of OAE method was the best, its AFB1 pAb had high titers of 1∶（1.28×104） by indirect ELISA, a good sensitivity with the 50% inhibition concentration(IC50) of 10.32 μg/L to AFB1 by icELISA and a high CR to AFB2 of 75.21%, AFG1 of 44.13%, AFG2 of 14.72%, AFM1 of 16.36% and AFM2 of 1.44%, respectively. In this study, AFB1 pAbs with high titer, sensitivity, specificity and eurytopicity were prepared, which laid a matter and technical foundation for the establishment of BGAFs immunoassay.
2. Luo, X.; Li, K.; Xing, J.; Qi, L.; Yang, M.; Wang, R.; Wang, L.; Li, Y.; Chen, Z. (2018). In vivo toxicity assessment of aflatoxin B(1)-contaminated corn after ozone degradation. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 35, 341-350, doi:10.1080/19440049.2017.1395518.
3. CHINA. National Food Safety Standard Limit of mycotoxins in food. (2017). Vol. GB 2761 —2017.
4. Gefen, T.; Vaya, J.; Khatib, S.; Rapoport, I.; Lupo, M.; Barnea, E.; Admon, A.; Heller, E.D.; Aizenshtein, E.; Pitcovski, J. (2015). The effect of haptens on protein-carrier immunogenicity. Immunology 144, 116-126, doi:10.1111/imm.12356.
5. Mongkon, W.; Sugita-Konishi, Y.; Chaisri, W.; Suriyasathaporn, W. (2017). Aflatoxin B1 Contamination of Dairy Feeds after Storage in Farm Practice in Tropical Environmen. Biocontrol Sci 22, 41-45, doi:10.4265/bio.22.41.
6. Xiao LW, Xu X, Zhao W, et al. (2017). Study on the development and performance of a paper strip for rapid quantitative detection of aflatoxin B1 based on time-resolved fluorescent nano-spheres. Grain Processing. 42, 41-43.
7. Kolosova, A.Y.; Shim, W.B.; Yang, Z.Y.; Eremin, S.A.; Chung, D.H. (2006). Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples. Anal Bioanal Chem 384, 286-294, doi:10.1007/s00216-005-0103-9.
8. Yu, Y.Y.; Chen, Y.Y.; Gao, X.; Liu, Y.Y.; Zhang, H.Y.; Wang, T.Y. (2018). Nanoparticle based bio-bar code technology for trace analysis of aflatoxin B1 in Chinese herbs. Journal of food and drug analysis. 26, 815-822, doi:10.1016/j.jfda.2017.11.003.
9. Wang YN, Wang XF, Niu LL, et al. (2016). Research progress in hapten molecule design and immunogen synthesis and antibody characteristics of aflatoxin B1. Science and Technology of Food Industry. 23, 367-376.
10. Zhou, Y.; Wu, J.; Yu, W.; Xu, Y.; Wang, P.; Xie, B.; Chen, F. (2007). Preparation for aflatoxin B(1)-cationized bovine serum albumin based on Mannich-type reaction. J Immunol Methods. 328, 79-88, doi:10.1016/j.jim.2007.08.009.
11. Rushing, B.R.; Selim, M.I. (2017). Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B(2a) and Biological Amines. Chem Res Toxicol, 30, 1275-1285, doi:10.1021/acs.chemrestox.7b00002.
12. Kononenko, G.P.; Burkin, A.A.; Soboleva, N.A. (2002). [Comparative characteristics of immunoreagents based on aflatoxin B1 hemiacetals and sterigmatocystine]. Prikl Biokhim Mikrobiol 38, 571-577.
13. Wang YN, Wang XF, Niu LL, et al. (2017). Advance in immunoassay of total aflatoxins in food. Science and Technology of Food Industry. 13, 344-351.
14. Wang YN, Wang XF, Wang ZL. (2018). Study and application of detection methods of total aflatoxins in food. Food and Fermentation Industries. 44, 1 , 285-290.
15. Cervino, C.; Knopp, D.; Weller, M.G.; Niessner, R. (2007). Novel aflatoxin derivatives and protein conjugates. Molecules 12, 641-653, doi:10.3390/12030641.
16. Wang YN, Wang SY, Zhang HT, et al. (2014). Establishment of Hybridoma Cell Lines Secreting Anti-Cadmium Ion Monoclonal Antibody and Identificat of Their Immunological Properties. Acta Agriculturae Boreali-occidentalis Sinica., 11, 24-29.
17. Ju RH, Wen K, Duan LL, et al. (2015). Preparation and Characterization of Polyclonal Antibodies Against Acrylamide. Journal of Nuclear Agricultural Sciences. 29, 10, 1979-1984.
18. Zhao HH, Zhang ZR, Li XJ, et al. (2016). Prokaryotic Expression of Tartary Buckwheat Flavonol SynthaseFtFLS2 and Preparation of Its Polyclonal Antibody. Journal of Nuclear Agricultural Sciences. 30, 2, 240-245.
19. Chen T, Wang CQ, Li XF, et al. (2014). Determination of aflatoxin B1 in peanut under different storage conditions by indirect ELISA. China Oils and Fats. 9, 88-91.
20. Zhang C, Pan JR, Shuai RQ, et al. (2016). Research on Enzyme Linked Immunosorbent Assay for Multi-residues of Nitroimidazoles in Foods of Animal Origin. Journal of Nuclear Agricultural Sciences. 30, 2, 323-331.
21. Liu, H.X.; Yang, Y.X.; Ma, M.G.; Wang, X.M.; Du, X.Z. (2015). Self-assembled Gold Nanoparticles Coating for Solid-Phase Microextraction of Ultraviolet Filters in Environmental Water. Chinese Journal of Analytical Chemistry 43, 207-211.
22. Zeng, H.; Chen, J.; Zhang, C.; Huang, X.A.; Sun, Y.; Xu, Z.; Lei, H. (2016). Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition. Anal Chem 88, 3909-3916, doi:10.1021/acs.analchem.6b00082..
23. Shi HY, Wang MH. (2008). Effect of Hapten Space Arm Length on Immune Recognition. Chinese Journal of Pesticide Science. 2, 172-177.
24. Guo NF, Yu JC, Ma FM, et al. (2014). Study of Comparison the Commonly Used Artificial Antigen Identification Methods. Journal of Food Science and Biotechnology. 33 5,517-521.
25. Morita, I.; Oyama, H.; Yasuo, M.; Matsuda, K.; Katagi, K.; Ito, A.; Tatsuda, H.; Tanaka, H.; Morimoto, S.; Kobayashi, N. (2017). Antibody Fragments for On-Site Testing of Cannabinoids Generated via in Vitro Affinity Maturation. Biological & pharmaceutical bulletin, 40, 174-181, doi:10.1248/bpb.b16-00669.
26. Hayashi, N.; Saegusa, J.; Uto, K.; Oyabu, C.; Saito, T.; Sato, I.; Kawano, S.; Kumagai, S. (2016). Evaluation of a Computer-Aided Microscope System and Its Anti-Nuclear Antibody Test Kit for Indirect Immunofluorescence Assay. Rinsho Byori, 64, 142-151.
27. Wang YN. (2020). Preparation of Broad-Specificity Antibodies and Development of Immuno-chromatographic Strip for Detection of Total Aflatoxins in Animal Food. Henan Institute of Science and Technology.
28. Xie H, Zhang X, Wang X, et al. (2015). Preparation of anti-aflatoxin B1 monoclonal antibodies and its use in an indirect competitive ELISA for aflatoxin B1. Microbiology China. 10, 2033-2040.
29. Xiao Z, Li PW, Zhuang Q, et al. (2011). Production and characteristics of specialised monoclonal antibodies against aflatoxin B1. Chinese Journal of Oil Crop Sciences. 33, 001, 66-70.
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