Document Detail

Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells.
MedLine Citation:
PMID:  17027850     Owner:  NLM     Status:  MEDLINE    
We describe a method for the generation of high-affinity monoclonal antibodies, which combines the power of natural immune responses with in vitro panning, B cell culture, RT-PCR and expression of the recombinant product. B cells from immunised rabbits were incubated at approximately 1000-10,000 cells per well with solid phase antigen coated on the surface of 96-well ELISA plates. Extensive washing removed non-binding cells as well as those B cells, which bound with low affinity. Retained B cells were cultured for 7 days in the presence of activated rabbit splenocyte supernatant and irradiated EL-4-B5 mouse thymoma cells, to induce proliferation and secretion of immunoglobulin. Supernatants were screened to confirm the presence of specific antibody, before the cells were harvested en masse from individual positive wells. Single heavy- and light-chain variable region genes were recovered from individual wells by RT-PCR, critically without the need for isolation of single B cells. Paired VH and VL genes were subsequently expressed as recombinant antibodies and shown to retain the original activity and specificity of the B cell culture supernatants. The method has also been successfully applied to the generation of high-affinity antibodies to antigen expressed on the surface of target cells.
Daniel J Lightwood; Bruce Carrington; Alistair J Henry; Andrew J McKnight; Kenneth Crook; Karen Cromie; Alastair D G Lawson
Publication Detail:
Type:  Journal Article     Date:  2006-09-18
Journal Detail:
Title:  Journal of immunological methods     Volume:  316     ISSN:  0022-1759     ISO Abbreviation:  J. Immunol. Methods     Publication Date:  2006 Oct 
Date Detail:
Created Date:  2006-10-23     Completed Date:  2006-12-19     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  133-43     Citation Subset:  IM    
UCB Celltech, Antibody Centre of Excellence, UK.
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MeSH Terms
Amino Acid Sequence
Antibodies, Monoclonal / biosynthesis*,  genetics
B-Lymphocytes / immunology*
CHO Cells
Clone Cells / immunology
Cloning, Molecular
Enzyme-Linked Immunosorbent Assay
Immunoglobulin Heavy Chains / genetics,  immunology
Immunoglobulin Light Chains / genetics,  immunology
Immunoglobulin Variable Region / genetics,  immunology
Molecular Sequence Data
OX40 Ligand / immunology*
RNA / chemistry,  genetics
Recombinant Proteins / genetics,  immunology
Reverse Transcriptase Polymerase Chain Reaction
Surface Plasmon Resonance
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/Immunoglobulin Heavy Chains; 0/Immunoglobulin Light Chains; 0/Immunoglobulin Variable Region; 0/OX40 Ligand; 0/Recombinant Proteins; 63231-63-0/RNA

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