Research Of Screening And Identification Of Monoclonal Antibody And Single Chain Variable Fragments Against Citrinin

Citrinin is a secondary metabolites generated by Penicillium, Aspergillus and other fungal strains, widespread in a variety of agricultural and food products. Early years, citrinin has been the use of antibiotics because it has good antibacterial effect, but later study found that it also has a strong renal toxicity and carcinogenicity, which is serious harmful to human and animal health. Therefore, establishing the detection method of a set of residual citrinin is very necessary, and the preparation of anti citrinin specific antibody is of great practical significance for the establishment of clinical detection methods. In this study, artificial antigen CIT-BSA and CIT-OVA were prepared by chemical coupling method; one anti-citrinin hybridoma cell line named 3B11 and its monoclonal antibodies was obtained using cell fusion technology; one large capacity, natural murine ribosome display single chain antibody library was constructed according to the principles of genetic engineering; the anti-citrinin scFv gene was screened using ribosome display technology. This research will lay the foundation of detection of citrinin residues.1. Synthesis and characterization of the artificial antigens of citrininAs a hapten, without immunogenic, antibody of anti-citrinin could not be directly made from immunizing animals, it must be conjugated with a protein carrier to make the artificial antigens, then antibody could be made from immunizing animals. In this study, EDC and NHS were used as a coupling agent,the carbodiimide method was used to prepare artificial antigen CIT-BSA and CIT-OVA, BSA and OVA as the protein carrier, and UV spectroscopy, SDS-PAGE and MALDI-TOF mass spectrometry were used to identify the products before and after the coupling reaction. Results showed that the artificial antigen CIT-BSA was made, and the conjugation ratio was about 5:1. The antigen can be used for animals immunization to prepare the antibody.2. Preparation and identification of the monoclonal antibodies of anti-citrininThe artificial antigen CIT-BSA was used for animal immunization, spleen cells of the immunized Balb/C mice were fused with SP2/0 cell, indirect ELISA and competitive ELISA were used to screen,5 secreting anti-citrinin hybridoma cell lines were screened after 3 subclone, named 2A9,3B5,3B11,3C5,6C1. After identification, the antibody types were IgM, IgGl and IgG2a; IC50 value of monoclonal antibody 3B11 was 150.9 ng/mL, the lowest detection line IC10 was 5.42 ng/mL; and the cross-reactivity rate of patulin, aflatoxin B1, fumonisin B1 and ochratoxin A were less than 0.01%; antibody affinity constant was 7.75×107 L/moL, which was a high-affinity antibodies. Monoclonal antibody prepared in this experiment has a good inhibitory effect, sensitivity, and specificity, it would provide a technical basis for the establishment and development of enzyme-linked immunosorbent assay kit detection method for rapid detection of citrinin.3. Construction and identification of high-capacity, naive mouse ribosome display single chain Fv libraryTo construction of high-capacity, naive mouse ribosome display single chain Fv library. Total RNA was extracted from the spleen cell of the specific pathogen free mouse followed RT-PCR using random primer. The heavy chain variable region gene (VH), light chain variable region gene (VL) and the kappa chain constant region gene (Cκ) of the immunoglobulin were amplified respectively by PCR. They were linked by a linker encoding (Gly4Ser) 3 by splicing by overlap extension PCR (SOE-PCR) to construct single chain Fv library and ribosome display library. The positive clones were identified by PCR and sequencing after linked and transformed to competent cells. The constructed ribosome display library showed a good integrity and diversity, with a capacity of 5.6×1013 and a gene length of about 1100bp. This study has constructed a high-capacity, naive mouse ribosome display single chain Fv library successfully, which would play roles in screening scFv.4. Selection of anti-CIT Specific scFvs by ribosome displaySingle chain variable fragments (scFvs) against citrinin (CIT) were selected from a naive scFv library constructed from the splenocytes of non-immunized mice by a improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/CIT-BSA and ovalbumin (OVA)/CIT-OVA were used as the antigen to select the specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, the expression vector pTIG-TRX containing the specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for expression. Thirteen positive clones were selected and three clones (named 23,68 and 109) showed higher binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with the carrier proteins. IC50 value of scEv 68 was 1242.2 ng/mL, the lowest detection line IC10 was 3.1 ng/mL; and the cross-reactivity rate of patulin, aflatoxin B1, fumonisin B1 and ochratoxin A were less than 0.01%. The specific scFvs could be used to establish a novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for selection of scFvs specific to other small molecules using ribosome display.In summary, monoclonal antibodies and single chain variable fragments against citrinin has been prepared in this study, based on the artificial antigens, which would laid the foundation for the establishment of citrinin residue detection methods, and also for single chain variable fragments against other haptens.