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