Preparation Of Monoclonal Antibody And Single-chain Antibody Fragment To The Hapten Aflatoxin B1

The aflatoxin Bl is one of the most toxic mycotoxin in the natural world.The international organizations take it as 10,000 times as basic toxin unit comparing to other materials.The major harm to human lies in its strong performance of the toxic and carcinogenic.It is produced by the fungus Aspergillus flavus,a parasite widely existing in peanuts,corn,and so on.In the modern society,the people have to put more and more concern on the issues of the potential risk of food with the economic development,which has attracted the attention of government world wide.Among all the governments,the EU has been carrying out the most stringent standard of the safety and test on the food since 2000;in which the AFB1 is limited in 2μg/kg and the total aflatoxin is no more than 4μg/kg.It is because of such restriction which has created new barriers to trade among countries,which also caused a great economic loss in the agricultural goods exporting in China.Immunization of Balb/C mice with the conjugant of AFB1-BSA is carried out by hypodermic injection.Antisera of mice are detected by indirect enzyme-link immunosorbent assay(ELISA),7～10 d after 3rd immunization.The titer of serum is up to 16000.Three days after 4th booster immnization,spleen cells of the mice with the highest titer of serum antiobodies are fused with SP2/0 murine myeloma cells by 50% PEG4000,and hybridoma cells are prepared.The hybridoma cell line of OG9 are screened for specificity to AFB1 using indirect and competive ELISA,and cloned by limited dilution method.Then the total RNA of cells is extracted after the specific one was selected.We have got the targeted genes of V_H V_L and Linker which is used to connect V_H with V_L in scFv antibody,though the RT-PCR according to the corresponding primers.Then the gene of scFv is amplified though the special PCR according to the designed primers by overlap extension.Then the scFv gene is connected with the phagemid vector pCANTAB-SE, purchased from Pharmcia company,after they were digested by the specific enzymes SfiI and NotI,then the ligated product is electro-transformed into Escherichia coli TG1. Finally,we have got a scFv antibody library.Our results provided a basis for further work on antibody engineering to neutralize the toxin.