Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire.
The ability to isolate antibodies against any antigen of interest has become increasingly important as antibodies have proved their utility both in antigen detection, quantification and as specific in vivo targeting agents. To this end, we have constructed a large antibody phage library in the single chain Fv (scFv) phagemid format based on the naive human variable (V) gene repertoire dictated by IgD and IgM. Optimizing each step of the library construction has resulted in a highly diverse and functional library, as assessed by sequencing analysis, large-scale automated expression analysis and antigen screening. Furthermore, the versatile format of the library, which comprises 14 separate sub-libraries, adds considerably flexibility with respect to which part of the antibody repertoire that is to be probed. This versatility has been further exploited to generate a refined antibody library, which exhibits one of the highest prokaryotic expression levels reported to date for a naive repertoire. The construction of the refined library was based on the functional purification of expressed V genes in the context of the protein L interaction with correctly folded V genes of the kappa light chain family. Antigen screening of this library indicated that the functional purification improved the ability to retrieve antigen specific antibodies, but at the cost of potential loss of diversity in the isolated repertoire.
Løset
Geir Åge
GÅ
Affitech AS, Oslo Research Park, Gaustadalléen 21, 0349 Oslo, Norway. g.a.loset@bio.uio.no
Løbersli
Inger
I
Kavlie
Anita
A
Stacy
John Eric
JE
Borgen
Tine
T
Kausmally
Louise
L
Hvattum
Else
E
Simonsen
Bjørg
B
Hovda
Maria Befring
MB
Brekke
Ole Henrik
OH
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification*; D000941:Antigens / Q000276:immunology; D006801:Humans; D007072:Immunoglobulin D / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library
D000906:Antibodies; D000941:Antigens; D007072:Immunoglobulin D; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D019151:Peptide Library