Characterization And Application Of Aspergillus-specific Single-chain Antibodies And Their Encoding Genes

The Aspergillus flavus is important plant pathogen that infects agricultural crops before harvest and during storage. Usually, A. flavus usually infects maize, peanuts and cotton causing crop yield loss, food mildew and huge economic losses. Additionally, A. flavus is also a human and animal pathogen, which leads to serious aspergillosis disease. A. flavus and A. parasiticus are the predominant producers of aflatoxins, which are secondary metabolites with carcinogenic and mutagenic effects to human and animals. The infection of A. flavus in agricultural products usually leads to high-level aflatoxin contamination, which is a severe threaten to human and animal health. High concentrations of aflatoxins intake will cause acute aflatoxicosis, and daily intake of low dose of aflatoxins will induce liver cancer and other diseases. Thus detection of A. flavus fast and sensitively will play a critial in prevention of disease caused by A. flavus and monitoring the contamination of aflatoxins.Immunoassay has been widely used in the detection of various pathogens. Immunoassay is based on the specific recognition between antigen and antibody, so antibody with high affinity palys an essential role in this system. Traditional immunoassay relies on polyclonal and monoclonal antibody, which are produced by animal immunization and hybridoma cell technology, and the cost of production is usually high. High-affinity single-chain antibody (scFv) can be isolated from immune or naive antibody library through phage display. In addition, phage display selection system can immediately provide gene sequences of single-chain antibodies against the target. The single-chain antibody can be easily expressed in bacteria and fused to other moleculars by subcloning which have advantages in immunoassay. In this study, we aimed to isolate single-chain antibody against A. flavus cell wall target from an immunized chicken antibody library using phage display. Based on scFv fusion proteins, new kinds of Aspergillus pathogens immunoassay methods were developed. The main results of this study are as follows:1. A.flavus cell wall proteins were used as antigen to immunize Balb/c mice. Four A. flavus specific monoclonal antibodies were isolated through hybridoma cell technology and purified from ascitic fluids. Among them mAb2A8showed the strongest binding ability in indirect ELISA, and can be used in immunoassay. 2. A. flavus cell wall proteins were used as antigen to immunize White Leghorn chickens. The genes of variable domians of heavy chain and light chain were cloned and constructed into a recombinant antibody library with a size of1.2×107. The antibody library was screened using phage display, and35positive clones were identified through expression ELISA. There were four types of antibodies named AfSA4, AfSA5, AfSA8and AfSD10according to their amino acid sequences. These scFv antibodies were expressed in bacteria and then purified using affinity chromatography.3. Alignment of amino acid sequences from AfSA4, AfSA5, AfSA8and AfSD10showed that there is variation in the composition of amino acid and length of CDR regions. Indirect ELISA showed that AfSA4exhibited the strongest binding activity towards A. flavus and A. parasiticus. The3D model prediction of scFv antibodies were also performed, and the comparison of structures showed that the interaction (e.g. hydrogen bond) between the loops of L-CDR2and H-CDR3of AfSA4facilitates the forming of a compact motif, which may play a critical role in high binding ability. Immunofluorescence showed that the binding of AfSA4localized to the cell wall of A. flavus and A. parasiticus. Whereas, antibodise AfSA5, AfSA8and AfSD10were not able to bind to the surface of cell wall. Immunoblot analysis showed that the binding properties of different scFv antibodies are varied.4. Phage ELISA was also used to identify positive clones from library after panning. The sequences of positive clones were compared with those obtained by expression ELISA. A high reactive scFv antibody AfPD12was expressed in bacteria and purified consequently. Immunofluorescence showed that AfPD12also binds to the cell wall of A. flavus and A. parasiticus. Immunoblot demonstrated that AfPD12recognizes the cell wall proteins of A. flavus.5. The genes of AfSA4and AfPD12were fused to that of alkaline phosphatase (AP) to generate fusion protein AfSA4-AP and AfPD12-AP, which were expressed and purified in bacteria. ELISA and immunoblot showed that scFv and scFv-AP exhibited the same specificity towards Aspergillus, so AP did not interfere the binding property of scFv in this study. The simultaneous dimerization of AP molecular imparts avidity to scFv-AP, which would improve the binding performance of scFv-AP. Surface plasmon resonance (SPR) analysis showed that the affinity of AfSA4-AP fusion was about6-fold higher than AfSA4, and AfPD12-AP also exhibited14-fold higher affinity compared with AfPD12. Indirect ELISA demonstrated that scFv-AP fusions were more sensitive in immunoassay. 6. The gene of AfPD12was fused to a protein tag that can be biotinylated when expressed in bacteria. The bio-AfPD12fusion protein was expressed in bacteria and purified consequently. Western blot analysis showed that bio-AfPD12was successfully biotinylated, and streptavidin specifically recognized bio-AfPD12. Indirect ELISA showed that bio-AfPD12could be used in the immunoassay.7. Monoclonal antibody mAb2A8was used as capture antibody, and AfSA4-AP fusion was used as detection antibody to set up a sensitive sandwich ELISA (ds-ELISA). This ds-ELISA was able to detect A. flavus and A. parasiticus simultaneously with a detection limit of10-3μg/mL. The sensitivity reached up to1μg/g when the ds-ELISA was used to detect fungi in maize and peanut matrix. The ds-ELISA had no cross reactivity towards health materials and non-Aspergillus fungi, so this immuassay can be used in the detection of aflatoxin producing fungi.8. Capture antibody AfPD12and detection antibody AfSA4-AP fusion were used to set up a sandwich ELISA, which showed a sensitivity of10-2μg/mL. The using of capture antibody AfPD12avoided the need of monoclonal antibody. A double sandwich fluorescence-linked immunosorbent assay (ds-FLISA) was developed based on AfPD12and bio-AfPD12. The detection limits of ds-FLISA against A. flavus and A. parasiticus were10-1μg/mL.In summary, different scFv antibodies were isolated from chicken antibody library using phage display, and their properties were analyzed through structure and immunorecognition in this study. ScFv antibodies with higher affinity were constructed into biofunctional scFv-AP and bio-scFv fusion. The well-matched monoclonal antibody, scFv and its fusion proteins were used to develope ds-ELISA and ds-FLISA method. The immunoassay developed in this study can be used in the detection of aflatoxin producing fungi, and will play an important role in ensuring agricultural production and food safety.