title: Generation of a recombinant bacteriophage antibody library to mycobacterium tuberculosis.
abstract: Monoclonal antibodies to Mycobacterium tuberculosis (Mtb) may be useful for laboratory diagnosis, antigenic characterization, and studying the immune response to infection and vaccination. To investigate the potential of the recombinant phage antibody technique for the isolation of single-chain fragments (ScFv) reactive with Mtb culture proteins, we generated a bacteriophage antibody library from splenic tissue of mice immunized with heat-killed Mtb. Heavy- and light-chain immunoglobulin genes were isolated and amplified by the polymerase chain reaction (PCR), and the products were assembled into ScFv gene fragments and cloned into the pCANTAB5-E phagemid. Phagemids were introduced into E. coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Immunoselection of the Mtb phage library against Mtb cell wall extract or culture filtrate proteins selected antigen-specific and cross-reactive phage antibodies, none of which demonstrated reactivity to the immunodominant 65-kDa Mtb antigen. These results suggest that the phage antibody system has the potential to generate a diverse population of the reactive phage that may prove useful for investigating the immune response to Mtb infection and vaccination.
authors.affiliation: Department of Medical and Research Technology, Univerity of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Cummings
authors.forename: N E
authors.initials: NE
authors.lastname: Hooper
authors.forename: S S
authors.initials: SS
authors.lastname: Rowland
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D051379:Mice; D009160:Mycobacteriophages / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D019151:Peptide Library; D013154:Spleen / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library