Different buffer effects in selecting HM-1 killer toxin single-chain fragment variable anti-idiotypic antibodies.
We reported previously competitive panning elution with PBS (pH 7.0) that contains HM-1 killer toxin (HM-1) and Candida albicans membrane fraction (CaMF) to release phages bound with CaMF as an antigen. Here, as an alternative strategy, we isolated high-binding affinity recombinant single-chain fragment variables (scFvs) with in vitro anti-fungal activity from an scFv phage library. The competitive panning elution contained acidic, neutral and basic pH buffers with original antigen HM-1 or HM-1 peptide 6 used to release phages bound with HM-1-neutralizing monoclonal antibody (nmAb-KT). For neutral pH eluted conditions, 87.5% of clones showed high-binding affinity against nmAb-KT by using ELISA, but was 16% and 26% for acidic and basic eluted conditions, respectively. After nucleotide sequencing, we obtained seven different anti-idiotypic antibodies from the different selection procedures. The clone expression and purification by using a HisTrap HP column, showed that clones scFv S3, S4 and S7 had in vitro antifungal activity against Saccharomyces cerevisiae, Candida albicans. The purified scFvs showed strong binding affinity with nmAb-KT by using ELISA. These results showed that changing the buffer pH with competing elements plays important role in elution of bound phages to targeted antigen and also in identification of positive scFv phages.
Krishnaswamy
Senthilkumar
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan.
Kabir
M Enamul
ME
Miyamoto
Masahiko
M
Furuichi
Yasuhiro
Y
Komiyama
Tadazumi
T
D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification* / Q000494:pharmacology; D000935:Antifungal Agents / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D000937:Antigen-Antibody Reactions; D002021:Buffers; D002176:Candida albicans / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000737:chemistry* / Q000276:immunology*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000187:drug effects
D000888:Antibodies, Anti-Idiotypic; D000935:Antifungal Agents; D002021:Buffers; D007128:Immunoglobulin Fragments; D009183:Mycotoxins; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins