title: ELISpot and ELISA analyses of human IL-21-secreting cells: Impact of blocking IL-21 interaction with cellular receptors.
abstract: Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n=24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n=18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden. Electronic address: jenny@mabtech.com.
authors.forename: Jenny
authors.initials: J
authors.lastname: Huang
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden. Electronic address: cecilia@mabtech.com.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Ehrnfelt
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden; Stockholm University, SE-114 18 Stockholm, Sweden. Electronic address: staffan@mabtech.com.
authors.forename: Staffan
authors.initials: S
authors.lastname: Paulie
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden. Electronic address: bartek@mabtech.com.
authors.forename: Bartek
authors.initials: B
authors.lastname: Zuber
authors.affiliation: Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden; Stockholm University, SE-114 18 Stockholm, Sweden. Electronic address: niklas@mabtech.com.
authors.forename: Niklas
authors.initials: N
authors.lastname: Ahlborg
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D002176:Candida albicans / Q000276:immunology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D058501:Enzyme-Linked Immunospot Assay; D057809:HEK293 Cells; D006801:Humans; D020381:Interleukin-17 / Q000276:immunology / Q000378:metabolism; D007378:Interleukins / Q000032:analysis* / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D010835:Phytohemagglutinins / Q000276:immunology; D053655:Receptors, Interleukin-21 / Q000037:antagonists & inhibitors*; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D013745:Tetanus Toxoid / Q000276:immunology; D000098546:Interleukin-21
chemical_list: D000911:Antibodies, Monoclonal; C508717:IL17A protein, human; D020381:Interleukin-17; D007378:Interleukins; D010835:Phytohemagglutinins; D053655:Receptors, Interleukin-21; D013745:Tetanus Toxoid; D000098546:Interleukin-21
keywords: Antigen; ELISA; ELISpot; Interleukin-21; T cell; Th17