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Production of Single-chain Variable Fragment Antibody Against Fumonisin B1and Its Preliminary Application in Immunoassay

Author: ZouLong
Tutor: XuYang
School: Nanchang University
Course: Fermentation Engineering
Keywords: fumonisin monoclonal antibody single-chain variable fragment prokaryotic expression enzyme-linked immunosorbent assay stability corn
CLC: TS207.5
Type: Master's thesis
Year: 2013
Downloads: 61
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Abstract


Fumonisins are a group of secondary fungal metabolites primarily produced by Fusarium moniliforme, one of the fungi that frequently colonize corn and other ceresls. Fumonisins are hepatotoxic, nephrotoxic and neurotoxic, in addition, they may be correlated to carcinogenicity. Fumonisin B1(FB1) is the most prevalent and toxic fumonisin. Effective detection and control of fumonisin contamination in food are the important measures to prevent the food safety issues. Currently, a number of analysis methods have been used for quantification of FB1, such as high performance liquid chromatography (HPLC), liquid chromatography with tandem mass detection, immunoassay and electrochemical analysis, and immunoassay shows its unique advantages in simplicity, sensitivity and high throughput. Single-chain variable fragment (scFv) is the smallest functional unit with complete antigen combination properties of monoclonal antibodies (McAb). Compared with McAb, scFv is easy to genetic modification and production, moreover, beneficial to biological synthesis of enzyme labeled antibody and development of a variety of immunological detection mode. This study successfully produced McAb against FB1and its recombinant scFv, and the scFv-based enzyme-linked immunosorbent assay (ELISA) was established for determination of FB1in corn samples. The main research results are as follows:1Two different artificial immunogens, namely FB1-GA-KLH and FB1-EDC-KLH, were synthetized using the glutaraldehyde and carbodiimide crosslinking method, respectively. Both groups of mice immunized with each of the above immunogen secreted anti-FB1antibodies in their serum, and the sensitivities and selectivity of the two groups of antiserum were not singnificantly different, and non-high cross-reactivities with fumonisin B2were showed. The fusion of the splenocytes immunized with FB1-GA-KLH conjugate with myeloma cells yielded two monoclonal antibodies against FB1, namely clone1D11and clone3F11.2The specific heavy-and light-chain variable regions were amplified from hybridoma cell total RNA by using mouse anticody variable regions degenerate primers, and then constructed into scFv genes (VH-linker-VL) by splice overlap extension PCR with linker sequences. Then scFv antibodies were displayed on the surface of filamentous phage M13K07by means of fusion protein for identification of the scFv antibody activities. After sequencing, the amino acid esquences were used to comparative analysis, physical and chemical property analysis, and homologous modeling. As results, the scFv displayed on phage surface show antibody activity, the sequences have the high homology with variable regions of mouse antibody and can form correct space conformation. In addition, the scFv are hydrophilic proteins with about25-26kDa of molecular weight and neutral or slightly alkaline isoelectric point.3Taking clone1D11as research object, the prokaryotic expression vectors of scFv and its fusion protein with alkaline phosphatase (scFv-AP) were constructed and transformed into E. coli. By optimizing expression conditions, the scFv and its fusion protein were mainly in the formation of inclusion bodies. After denaturation, purification and refolding, the scFv and its fusion protein were characterized by ELISA. The calibration curve of scFv-based ELISA on FB1was linear from2.10μg L-1to76.45μg L-1(R2=0.9922), with an IC50value of12.67μg L-1and a cross-reactivity with FB2of5.23%, which were similar with parent McAb. The scFv-AP fusion protein with antibody activity and enzyme activity simultaneously could not be used for development of detection system for relatively low enzyme activity.4The scFv in this study was susceptible to heat treatment, which could result in irreversible inactivation, and it could be completely disabled with treatment at65℃for10min. The circular dichroism spectrum analysis results demonstrated that the irreversible inactivation of scFv was caused by the destruction of the secondary structure. Methanol was harmful to the sensitivity of scFv-based ELISA, but20%of final concentration would be accepted. On the basis of scFv, an indirect competitive ELISA for determination of FB1in corn samples was established. The detection capacity (CCβ) and limit of detection (LOD) of this assay were15.00μg kg-1and8.32μg kg-1, respectively. The recovery values and coefficient of variation values were86.74%to105.41%and9.72%to11.62%, respectively. In addition, the determined results of30naturally contaminated corn samples by the scFv-based ELISA are in agreement with the findings of HPLC (R2=0.969).

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CLC: > Industrial Technology > Light industry,handicrafts > Food Industry > General issues > Food standards and testing > Determination of food contamination
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