title: Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining.
abstract: Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of V(H) and V(L) genes obtained from lymphocytes of immunized animals. Recently, a different approach to antibody isolation that circumvents library screening and instead relies on the mining of the V(H) and V(L) gene repertoires obtained by high throughput sequencing of cDNAs from bone marrow antibody-secreting cells was reported. Here we compared the antibodies obtained via phage library screening or via repertoire mining of V gene cDNAs obtained from total splenocytes of mice immunized with the hapten trinitrophenyl (TNP) conjugated to carrier proteins. We show that, despite the large heterogeneity of B lymphocytes in the spleen, the most abundant V genes encoded antigen-specific antibodies, indicating that total splenocytes can be used in place of bone marrow plasma cells for antibody discovery at least in high titer animals. While both phage display and repertoire mining yielded antigen-specific antibodies showing comparable affinities by enzyme-linked immunosorbent assay analysis, clones obtained by the latter approach displayed higher selectivity towards TNP relative to control haptens. Interestingly, the antibody genes isolated by phage display were of low abundance or absent from the V gene repertoire obtained by 454 sequencing. Similarly, the highly abundant V genes identified by repertoire mining, that as soluble antibodies were antigen-specific, were found to be poorly displayed on phage and were not enriched by phage panning. Thus, our results reveal that phage display and repertoire mining of immune repertoires are complementary technologies that can yield different antigen-specific antibody clones.
authors.affiliation: Department of Molecular Microbiology and Biotechnology,  The George S Wise Faculty of Life Sciences, Ramat Aviv 69978,  Israel.
authors.forename: Ido
authors.initials: I
authors.lastname: Saggy
authors.forename: Yariv
authors.initials: Y
authors.lastname: Wine
authors.forename: Leeron
authors.initials: L
authors.lastname: Shefet-Carasso
authors.forename: Limor
authors.initials: L
authors.lastname: Nahary
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
authors.forename: Itai
authors.initials: I
authors.lastname: Benhar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000915:Antibody Affinity; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015723:Gene Library; D006241:Haptens / Q000276:immunology*; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D016415:Sequence Alignment; D013154:Spleen / Q000166:cytology; D014301:Trinitrobenzenes / Q000276:immunology*
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D006241:Haptens; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D014301:Trinitrobenzenes