Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Abstract

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity. J. Vis. Exp. (2016)

This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody's isotype, which correlates with the B cells' secretory activity.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 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 review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cyt…

Representative Results

Figure 1. Illustration of the Gating Strategy to Separate the Naïve B cells, Memory B cells, and PBs in the PBMCs. (A) Cells (2 x 106) were stained with 2 µg each of CD19-APC, CD27-eFluor450, and CD38-PE mAbs (step 3.4). The lymphocyte compartment was gated (G1) on the dot plot for forward scattering (FSC) versus side scattering (SSC). (B)…

Materials

BD Vacutainer K2E	BD Biosciences	367525	10 ml tube
Ficoll-Paque Plus	GE Healthcare	17-1440-02	Endotoxin-free
Trypan blue 0.5% solution	Biological Industries	03-102-1B
IMag Human B lymphocyte enrichment set	BD Biosciences	558007
Biotinylated CD27 mAb	Biolegend	302804	Clone O323
Streptavidin magnetic microbeads	BD Biosciences	9000810
15 ml Falcon tubes	BD Falcon	352196
Blue nylon mesh cell strainer, 40 μm	BD Falcon	352340
Anti-human CD19-APC	Biolegend	302212	Clone HIB19
Anti-human CD27-eFluor 450	eBioscience	48-0279-42	Clone O323
Anti-human CD38-PE-Cy7	Biolegend	303516	Clone HIT2
Anti-human CD38-PE-Cy7	BD Biosciences	560677	Clone HIT2
Anti-human CD45-FITC	Biolegend	304006	Clone HI30
Anti-human CD45-FITC	BD Biosciences	555482	Clone HI30
Anti-mouse/rat/human CD27-PerCP Cy5.5	Biolegend	124213	Clone LG.3A10
Anti-human CD27-PerCP Cy5.5	BD Biosciences	65429	Clone L128
Anti-human CD19-FITC	Miltenyi Biotec	130-098-064	Clone LT19
Anti-human CD19-FITC	GeneTex	GTX75599	Clone LT19
Anti-human CD20-FITC	BD Biosciences	555622	Clone 2H7
Biotinylated anti-human CD27	Biolegend	302804	Clone O323
Biotinylated anti-human CD27	eBioscience	13-0279-80	Clone O323
7-aminoactinomycin D (7-AAD)	BD Biosciences	559925
CpG (ODN 2006)	InvivoGen	tlrl-2006	Type B CpG
Recombinant human IL-2	PeproTech	200-02
Recombinant human IL-10	PeproTech	200-10
Recombinant human IL-21	PeproTech	200-21
Recombinant human sCD40L	PeproTech	310-02
Protein A of S. aureus Cowan (SAC)	Sigma-Aldrich	82526
Pokeweed mitogen (PWM)	Sigma-Aldrich	L9379
MultiScreen filter plates, 0.45 µm pore size	Merck Millipore	MSIPS4510	Sterile, clear 96-well filter plate with hydrophobic PVDF membrane
BCIP/NBT solution	Sigma-Aldrich	B6404
BCIP/NBT single reagent, alkaline phosphatase substrate	Merck Millipore	ES006
Human IgG	Jackson ImmunoResearch	009-000-003
Human IgG, Fc fragment	Jackson ImmunoResearch	009-000-008
F(ab')2 fragment of goat anti-human Ig (IgG+IgM+IgA)	Jackson ImmunoResearch	109-006-127
Goat anti-human IgG-alkaline phosphatase, Fcγ fragment specific	Jackson ImmunoResearch	109-055-008
Goat anti-human IgM-alkaline phosphatase, Fcµ fragment specific	Jackson ImmunoResearch	109-055-095
Goat anti-human IgG-peroxidase, Fcγ fragment specific	Jackson ImmunoResearch	109-035-008
Goat anti-human IgM-peroxidase, Fcµ fragment specific	Jackson ImmunoResearch	109-035-095
BD ELISPOT AEC substrate kit	BD Biosciences	551951
C.T.L. ImmunoSpot analyzer	C.T.L.