Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay
Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay
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DOI：
10.1016/s0022-1759(96)00146-9
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发表时间：
1996-11-29
影响因子：
2.2
通讯作者：
Ahmed, R
中科院分区：
文献类型：
--
作者：
Slifka, MK;Ahmed, R
许多急性病毒感染会引起长期的、有时甚至是终身的体液免疫。为了表征这种免疫反应，记忆B细胞和浆细胞的准确定量是必不可少的。浆细胞可以通过自发分泌抗体的能力在体外直接定量。另一方面，记忆B细胞不会自发分泌抗体，但需要抗原刺激才能增殖和分化为抗体分泌细胞(ASC)。在这项研究中，建立了一种基于ELISPOT的极限稀释试验(LDA)来定量检测成年小鼠感染急性淋巴细胞性脉络膜脑膜炎病毒(LCMV)后的病毒特异性B细胞记忆。从体外刺激的培养中计算抗病毒记忆B细胞前体(MBCp)的频率，使用传统的基于ELISA法的LDA来测量培养上清液中积累的病毒特异性抗体，或者使用一种新的基于ELTSPOT的LDA来直接识别分泌抗体的子细胞。在敏感性方面，基于ELISPOT的LDA和基于ELISA的LDA都计算出LCMV特异性MBCp频率约为1/2×10(4)个脾细胞。然而，与基于ELISPOT的LDA估计MBCp频率所需的12天体外刺激相比，基于ELISPOT的LDA只需要3-6天的刺激来量化MBCp频率。如果细胞分裂被伽玛射线或丝裂霉素C阻断，MBCp频率下降到低于检测的水平(
Many acute viral infections induce long-term, sometimes even life-long humoral immunity. To characterize this immune response, accurate quantitation of memory B cells and plasma cells is essential. Plasma cells can be quantitated directly ex vivo by virtue of their ability to spontaneously secrete antibody. Memory B cells on the other hand, do not spontaneously secrete antibody but require antigenic stimulation in order to proliferate and differentiate into antibody secreting cells (ASC). In this study, an ELISPOT-based limiting dilution assay (LDA) was developed for quantitating virus-specific B cell memory following acute lymphocytic choriomeningitis virus (LCMV) infection of adult mice. Antiviral memory B cell precursor (MBCp) frequencies were calculated from in vitro stimulated cultures using either a conventional ELISA-based LDA to measure accumulated virus-specific antibody in the culture medium or a new ELTSPOT-based LDA that identifies the antibody-secreting daughter cells directly. In terms of sensitivity, the ELISPOT-based LDA and the ELISA-based LDA both calculated LCMV-specific MBCp frequencies to be approximately 1/2 X 10(4) spleen cells. However, compared to the 12 days of in vitro stimulation required to estimate MBCp frequencies by the ELISA-based LDA, the ELISPOT-based LDA required only 3-6 days of stimulation to quantitate MBCp frequencies. If cell division was blocked by gamma-irradiation or treatment with mitomycin C, the MBCp frequency dropped below detection (