Molecular Cloning and Expression of Recombinant Phage Antibody against Fumonisin B1
| Autor: | Hui Ren Zhou, L. Patrick Hart, James J. Pestka |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
medicine.diagnostic_test
medicine.drug_class chemical and pharmacologic phenomena Biology Molecular cloning biology.organism_classification Monoclonal antibody Microbiology Molecular biology law.invention Filamentous bacteriophage law Polyclonal antibodies Immunoassay Recombinant DNA medicine biology.protein Avidity Antibody Food Science |
| Zdroj: | Journal of Food Protection. 59:1208-1212 |
| ISSN: | 0362-028X |
| DOI: | 10.4315/0362-028x-59.11.1208 |
| Popis: | Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B 1 (FB 1 )-specific antibodies, composed of a single chain containing a variable heavy (V H ) and light (V L ) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB 1 -BSA conjugate or from an existing hybridoma cell line that produces anti-FB 1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of V H and V L cDNAs, (iii) ligation of V L and V H chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB 1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB 1 , the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB 2 or FB 3 . The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other foodborne disease hazards. |
| Databáze: | OpenAIRE |
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