title: An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.
abstract: The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs.
authors.affiliation: Karolinska Institutet, Department of Medicine Solna, SE-171 76 Stockholm, Sweden; Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden. Electronic address: peter.jahnmatz@ki.se.
authors.forename: Peter
authors.initials: P
authors.lastname: Jahnmatz
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden.
authors.forename: Theresa
authors.initials: T
authors.lastname: Bengtsson
authors.affiliation: Swedish Orphan Biovitrum AB, Tomtebodavägen 23A, SE-112 76 Stockholm, Sweden.
authors.forename: Bartek
authors.initials: B
authors.lastname: Zuber
authors.affiliation: Karolinska Institutet, Department of Medicine Solna, SE-171 76 Stockholm, Sweden; Karolinska University Hospital, Department of Infectious Diseases, SE-17176 Stockholm, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Färnert
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden; Stockholm University, Department of Immunology, SE-106 91 Stockholm, Sweden.
authors.forename: Niklas
authors.initials: N
authors.lastname: Ahlborg
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis*; D055910:Antibodies, Immobilized / Q000276:immunology; D000918:Antibody Specificity; D000921:Antibody-Producing Cells / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D003429:Cross Reactions; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000888:Antibodies, Anti-Idiotypic; D055910:Antibodies, Immobilized; D000941:Antigens; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; C048844:anti-IgG
keywords: Antibody; Antigen; B-cell ELISpot; FluoroSpot