title: High-resolution analysis of the B cell repertoire before and after polyethylene glycol fusion reveals preferential fusion of rare antigen-specific B cells.
abstract: OBJECTIVE
  The hybridoma technology is one of the most important advances in clinical immunology. Little is known about the differences between the antibodies produced during the in vivo immune response and those recovered in hybridoma libraries. Here, we investigate a potential fusion bias inherent to the hybridoma production process.
  
  
  METHODS
  Transgenic rats carrying human Ig heavy and light chain loci were immunized with measles virus (MV) to generate human mAbs. Usin g high-throughput sequencing of IgH mRNA, we compared the IgH repertoire of lymph nodes and the derived hybridoma library using the sequences of the MV-specific hybridoma clones as a reference set with known specificity.
  
  
  RESULTS
  We observed that large clonotypes from the lymph nodes were not represented in the hybridoma library, but low-frequency B cell populations became highly enriched and most hybridoma clones were derived from these. Our data also showed that identical CDR3s evolved from diverse VDJ recombinations, indicating convergence of different B cells subpopulations towards expression of antibodies with similar paratopes.
  
  
  CONCLUSIONS
  The efficient generation of mAbs results from a fusion process highly selective for rare antigen-specific B cells rather than in vivo expanded populations. Antibodies of particular interest may therefore be missed during classical hybridoma production.
authors.forename: Axel R S X
authors.initials: AR
authors.lastname: Dubois
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Buerckert
authors.forename: Regina
authors.initials: R
authors.lastname: Sinner
authors.forename: William J
authors.initials: WJ
authors.lastname: Faison
authors.forename: Anne M
authors.initials: AM
authors.lastname: Molitor
authors.forename: Claude P
authors.initials: CP
authors.lastname: Muller
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D000956:Antigens, Viral / Q000008:administration & dosage; D001402:B-Lymphocytes / Q000145:classification / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology; D002459:Cell Fusion / Q000379:methods; D002999:Clone Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D006825:Hybridomas / Q000737:chemistry / Q000276:immunology*; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000737:chemistry; D008198:Lymph Nodes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008459:Measles virus / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D055647:Rats, Transgenic; D060152:V(D)J Recombination / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011092:Polyethylene Glycols
keywords: B cell repertoire; Hybridoma; immunoglobulin; lymph nodes; measles virus; next-generation sequencing; polyethylene glycol; transgenic animals