title: Quantifiable analysis of human immunoglobulin heavy chain class-switch recombination to all isotypes.
abstract: Somatic recombinational events, including the immunoglobulin heavy chain class-switch, are a normal feature of B-cell maturation. To enable comprehensive and sensitive class-switch analysis in ex vivo human B cells, we have developed multiple digestion-circularization PCR (DC-PCR) techniques for quantifiable detection of switching to all immunoglobulin isotypes. This technology was validated by extensive sequencing of PCR products, tests with control non-lymphoid cells and B-cell lines of known isotypic specificities, and by demonstrating DC-PCR selectivity in a model system. With tonsillar B-cell DNA, switching to gamma 3, gamma 1, alpha1, gamma 2, gamma 4 and alpha2 isotypes was reproducibly detectable among different individuals. Levels of epsilon switching were relatively low and usually required higher total amounts of template DNAs for detection. Quantitation of alpha1 class switching in a panel of human tonsillar whole B cells was performed by the internal-competitor approach, and showed a pattern consistent with previous studies on IgA+ tonsillar cells. We demonstrate that these assays can rapidly show germline status or specific switch rearrangements in B lymphoid cell lines.
authors.affiliation: Institute for Immunology and Allergy Research, PO Box 145, Westmead, Sydney, Australia.
authors.forename: H A
authors.initials: HA
authors.lastname: Weckert
authors.forename: J A
authors.initials: JA
authors.lastname: Hughes
authors.forename: E M
authors.initials: EM
authors.lastname: Benson
authors.forename: I S
authors.initials: IS
authors.lastname: Dunn
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D015322:Gene Rearrangement, B-Lymphocyte; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D014066:Palatine Tonsil / Q000166:cytology / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic
chemical_list: D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D004247:DNA