title: The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.
abstract: Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Clargo
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Hudson
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Welcome
authors.initials: W
authors.lastname: Ndlovu
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Wootton
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Louise A
authors.initials: LA
authors.lastname: Cremin
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Victoria L
authors.initials: VL
authors.lastname: O'Dowd
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Carla R
authors.initials: CR
authors.lastname: Nowosad
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Dale O
authors.initials: DO
authors.lastname: Starkie
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Sophie P
authors.initials: SP
authors.lastname: Shaw
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Compson
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Dominic P
authors.initials: DP
authors.lastname: White
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Brendon
authors.initials: B
authors.lastname: MacKenzie
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: James R
authors.initials: JR
authors.lastname: Snowden
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Newnham
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Wright
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Stephens
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Meryn R
authors.initials: MR
authors.lastname: Griffiths
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Alastair D G
authors.initials: AD
authors.lastname: Lawson
authors.affiliation: UCB Pharma; Berkshire, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lightwood
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001854:Bone Marrow Cells / Q000276:immunology*; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D008297:Male; D010950:Plasma Cells / Q000166:cytology / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D047889:Receptors, Tumor Necrosis Factor, Type II; D057127:Single-Chain Antibodies
keywords: IgG; PCR; TAP; antibody; bone marrow; fluorescent foci; monoclonal; plasma cell