The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

摘要

人外周血通常用于评估体液免疫反应。本文介绍了从外周血中分离人 B 细胞、在培养物中将人 B 细胞分化为分泌抗体 （Ab） 的 B 细胞 （ASCs） 以及通过 ELISpot 测定计数总 IgM 和 IgG-ASCs 的方法。

摘要

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified naïve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

引言

B cells play a central role in the development of humoral immunity. They initially develop in the bone marrow and enter the blood stream as naïve B cells, which can migrate into the lymphoid tissues, such as the spleen, lymph nodes, and tonsils, for further development. Upon Ag encounter, some naïve B cells migrate into lymphoid follicles, where germinal center B cells can differentiate into memory B cells and plasmablasts (PBs)/plasma cells (PCs). While most PBs/PCs egress into the blood stream, a few eventually reside in the bone marrow to undergo terminal differentiation into long-lived PCs¹. B cells in circulation are heterogeneous, and at ste....

方案

Human peripheral blood must be obtained from healthy donors under informed consent, and the use of blood samples must conform to the approved guidelines established by individual institutional review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cytometry (Figure 1) and ELISpot assays (Figure 3) was approved by the Internal Review Board of National Taiwan University Hospital (protocol number 201307019RINB).

1. Isolation and Purification of Human Peripheral Blood B Cells

1. Draw ~ 10 mL of blood from the median cubital vein (in the cubital fossa anterior to the elbow) i....

结果

PBMCs were depleted of RBCs and adherent cells (steps 1.2 to 1.7). An aliquot (2 x 10⁶) of cells were subjected to a flow cytometric analysis to illustrate the populations of naïve B cells, memory B cells, and PBs/PCs in peripheral blood (Figure 1). In this donor's PBMCs, about 10% of the lymphocytes were CD19⁺ B cells. In the B-cell compartment, the percentage of CD19⁺CD27^- naïve B cells was around 50%. On the other hand, about.......

讨论

Isolation and Purification of Human Peripheral Blood B Cells

Normally, RBCs can be efficiently ruptured and cleared by lysis buffer (step 1.2). It is important not to incubate PBMCs with the RBC lysis buffer longer than 5 min, as cell viability might be affected by the ammonium chloride. Alternatively, RBCs and platelets can be simultaneously removed by the following protocol.

Mix fresh whole blood with acid-citrate-dextrose (ACD) buffer (39 mM citric acid, 75 mM sodiu.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
BD Vacutainer K2E	BD Biosciences 367525		10 mL 管
Ficoll-Paque Plus	GE Healthcare	17-1440-02	无内毒素
台盼蓝 0.5% 溶液	生物工业	03-102-1B
IMag 人 B 淋巴细胞富集套装	BD Biosciences 558007
生物素化 CD27 mAb	Biolegend	302804	克隆 O323
链霉亲和素磁性微珠	BD Biosciences 9000810
15 mL Falcon 管	BD Falcon	352196
蓝色尼龙网状细胞过滤器，40 &μ;m	BD Falcon	352340
抗人 CD19-APC	Biolegend	302212	克隆 HIB19
抗人 CD27-eFluor 450	eBioscience	48-0279-42	克隆 O323
抗人 CD38-PE-Cy7	Biolegend	303516	克隆 HIT2
抗人 CD38-PE-Cy7	BD Biosciences	560677	克隆 HIT2
抗人 CD45-FITC	Biolegend	304006	克隆 HI30
抗人 CD45-FITC	BD Biosciences	555482	克隆 HI30
抗小鼠/大鼠/人 CD27-PerCP Cy5.5	Biolegend	124213	克隆 LG.3A10
抗人 CD27-PerCP Cy5.5	BD Biosciences	65429	克隆 L128
抗人 CD19-FITC	Miltenyi Biotec	130-098-064	克隆 LT19
抗人 CD19-FITC	GeneTex	GTX75599	克隆 LT19
抗人 CD20-FITC	BD Biosciences 555622		克隆 2H7
生物素化的抗人 CD27	Biolegend	302804	克隆 O323
生物素化的抗人 CD27	eBioscience	13-0279-80	克隆 O323
7-氨基放线菌素 D （7-AAD）	BD Biosciences	559925
CpG （ODN 2006）	InvivoGen	tlrl-2006	B 型 CpG
重组人 IL-2	PeproTech	200-02
重组人 IL-10	PeproTech	200-10
重组人 IL-21	PeproTech	200-21
重组人 sCD40L	PeproTech	310-02
S. aureus Cowan （SAC）	蛋白 ASigma-Aldrich	82526
商陆有丝分裂原 （PWM）	Sigma-Aldrich	L9379
MultiScreen 滤板，0.45 µm 孔径	Merck Millipore	MSIPS4510	无菌、透明的 96 孔滤板，带疏水性 PVDF 膜
BCIP/NBT 溶液	Sigma-Aldrich	B6404
BCIP/NBT 单一试剂，碱性磷酸酶底物	Merck Millipore	ES006
人 IgG	Jackson ImmunoResearch	009-000-003
人 IgG，Fc 片段	JacksonImmunoResearch	009-000-008
F（ab'）2 山羊抗人 Ig 片段（IgG + IgM + IgA）	Jackson ImmunoResearch	109-006-127
山羊抗人 IgG-碱性磷酸酶，Fc&γ;片段特异性	Jackson ImmunoResearch	109-055-008
山羊抗人 IgM-碱性磷酸酶，Fcµ 片段特异性	JacksonImmunoResearch	109-055-043
山羊抗人 IgG-过氧化物酶，Fc&γ;片段特异性	Jackson ImmunoResearch	109-035-008
山羊抗人 IgM-过氧化物酶，Fcµ 片段特异性	Jackson ImmunoResearch	109-035-095
BD ELISPOT AEC底物试剂盒	BD Biosciences	551951
C.T.L. ImmunoSpot 分析仪	C.T.L.