کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8417173 | 1545676 | 2016 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection
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کلمات کلیدی
PBSB-cell ElispotDENVIFN-γFCSKLHASCFITCmAbDMEMBSA - BSADulbecco's modified Eagle's medium - Medal of Eagle اصلاح شده Dulbeccobovine serum albumin - آلبومین سرم گاوantibody-secreting cell - آنتی بادی ترشح کننده سلولMonoclonal antibody - آنتی بادی مونوکلونالStreptavidin - استرپتاویدینimmunoglobulin - ایمونوگلوبولینinterferon-gamma - اینترفرون گاماinterleukin - اینترلوکینRoom temperature - دمای اتاقfetal calf serum - سرم گوساله جنینfluorescein isothiocyanate - فلوئورسین ایسوتیوسیاناتPhosphate-buffered saline - محلول نمک فسفات با خاصیت بافریDengue virus - ویروس دنگیAntibody - پادتَن یا آنتیبادیAntigen - پادگِن یا آنتیژنkeyhole limpet hemocyanin - کلم هول limpet هموسیانین
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 433, June 2016, Pages 23-30
Journal: Journal of Immunological Methods - Volume 433, June 2016, Pages 23-30
نویسندگان
Peter Jahnmatz, Theresa Bengtsson, Bartek Zuber, Anna Färnert, Niklas Ahlborg,