Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution |
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Authors: | Senthilkumar Krishnaswamy Masahiko Miyamoto Tadazumi Komiyama |
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Institution: | a Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan b GeneCare Research Institute, Kamakura 247-0063, Japan |
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Abstract: | Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1 + HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC50 values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40 μM and 2.20 μM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a Kd value of 3.09 × 10−11 M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs. |
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Keywords: | Phage display Competitive panning elution Candida albicans membrane fraction HM-1 killer toxin HM-1-neutralizing monoclonal antibody High-affinity antibody |
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