کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8418254 | 1545716 | 2013 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses
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کلمات کلیدی
PBSB-cell ElispotR848Too Numerous To CountTNTCFHACD40LPRNCMIPABTLRFCSTTDPBMCmAbPWMASCSACMonoclonal antibody - آنتی بادی مونوکلونالPolyclonal antibody - آنتی بادی های پلی کلونالCell-mediated immunity - ایمنی سلولیinterleukin - اینترلوکینOptimization - بهينه سازيover night - بیش از شبBiotinylation - بیوتینیل شدنdiphtheria toxin - توکسین دیفتریToll-like receptor - تیالآرRoom temperature - دمای اتاقfetal calf serum - سرم گوساله جنینAntibody-secreting cells - سلول های ترشحی آنتی بادیperipheral blood mononuclear cells - سلول های تک هسته ای خون محیطیpertussis toxin - سموم سورافنیCD40 ligand - لیگاند CD40pokeweed mitogen - ماتوژن pokeweedPhosphate-buffered saline - محلول نمک فسفات با خاصیت بافریFilamentous hemagglutinin - هموگلوتینین رشته ایPertactin - پرتاکتین
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
B-cell responses after infection or vaccination are often measured as serum titers of antigen-specific antibodies. Since this does not address the aspect of memory B-cell activity, it may not give a complete picture of the B-cell response. Analysis of memory B cells by ELISpot is therefore an important complement to conventional serology. B-cell ELISpot was developed more than 25Â years ago and many assay protocols/reagents would benefit from optimization. We therefore aimed at developing an optimized B-cell ELISpot for the analysis of vaccine-induced human IgG-secreting memory B cells. A protocol was developed based on new monoclonal antibodies to human IgG and biotin-avidin amplification to increase the sensitivity. After comparison of various compounds commonly used to in vitro-activate memory B cells for ELISpot analysis, the TLR agonist R848 plus Interleukin (IL)-2 was selected as the most efficient activator combination. The new protocol was subsequently compared to an established protocol, previously used in vaccine studies, based on polyclonal antibodies without biotin avidin amplification and activation of memory B-cells using a mix of antigen, CpG, IL-2 and IL-10. The new protocol displayed significantly better detection sensitivity, shortened the incubation time needed for the activation of memory B cells and reduced the amount of antigen required for the assay. The functionality of the new protocol was confirmed by analyzing specific memory B cells to five different antigens, induced in a limited number of subjects vaccinated against tetanus, diphtheria and pertussis. The limited number of subjects did not allow for a direct comparison with other vaccine studies. Optimization of the B-cell ELISpot will facilitate an improved analysis of IgG-secreting B cells in vaccine studies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 391, Issues 1â2, 31 May 2013, Pages 50-59
Journal: Journal of Immunological Methods - Volume 391, Issues 1â2, 31 May 2013, Pages 50-59
نویسندگان
Maja Jahnmatz, Gun Kesa, Eva Netterlid, Anne-Marie Buisman, Rigmor Thorstensson, Niklas Ahlborg,