Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Published: July 31, 2023

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)…

Açıklamalar

The authors have nothing to disclose.

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.

Etiketler

Play Video

Bu Makaleden Alıntı Yapın
Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay. J. Vis. Exp. (Pending Publication), e21525, doi: (2023).

View Video