EFB Dengue Literature Review

ELISpot

7 min read

ELISpot

Overview

The Enzyme-Linked Immunospot (ELISpot) assay quantifies individual antibody-secreting cells (ASCs) by capturing antibodies secreted by single cells onto a membrane coated with antigen (for antigen-specific counting) or anti-Ig antibody (for total ASC counting). Each secreting cell produces a discrete visible “spot” detected by a secondary enzyme-conjugated antibody. Results are expressed as spot-forming units (SFUs) per input cell number, or as a frequency of antigen-specific cells among total IgG⁺ (or IgA⁺/IgM⁺) spots.

ELISpot provides functional single-cell resolution that flow cytometry cannot — it identifies ASCs not by surface phenotype but by what they actually secrete. It is particularly useful for quantifying rare antigen-specific responses within a large polyclonal ASC background.

Key Points from Literature

  • Polyclonal vs. antigen-specific frequencies in SLE: ELISPOT with 9G4 idiotype capture, influenza antigen, tetanus toxoid, and SLE autoantigens (anti-dsDNA, anti-Ro, anti-Sm) in SLE patients with large ASC expansions showed that: (1) influenza- and tetanus-specific ASCs were detectable in SLE patients despite no recent immunization or exposure (range 0–1.2% of IgG⁺ ASCs for influenza; 0–0.65% for tetanus) but were undetectable in healthy subjects; (2) even in patients with large ASC expansions, anti-dsDNA + anti-Ro + anti-Sm together accounted for less than 3% of IgG⁺ ASCs; (3) 9G4⁺ (VH4-34-encoded) ASCs had a median frequency of 2.4% and could account for up to 20% of IgG⁺ ASCs (see Tipton2015 - ASC Diversity and Origin in SLE, n=46 SLE patients, n=7 healthy controls for ELISpot panels).

  • Biological implication of ELISpot data: The detection of influenza/tetanus memory ASCs in SLE without recent immunization — but absent in healthy controls — demonstrates polyclonal bystander activation of pre-existing memory ASC pools during disease flares. This supports a model of generalised B cell hyperactivation in SLE rather than purely autoantigen-driven expansion.

  • Technical protocol: Wells coated with anti-IgG (5 µg/ml), anti-IgA, or antigen (trivalent influenza vaccine 6 µg/ml, monoclonal 9G4 antibody). After overnight incubation, spots detected with alkaline phosphatase–conjugated secondary antibody and Blue Alkaline Phosphatase Substrate (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Referenced in COVID-19 EF context: Woodruff2020 references ELISpot as part of the Sanz lab’s standard ASC characterisation toolkit but does not present new ELISpot data in this paper; primary ASC quantification was performed by flow cytometry and BCR sequencing (see Woodruff2020 - EF B Cell Responses in COVID-19).

  • DN2 cell IgG ASC output by ELISPOT: Sorted DN2 cells cultured with TLR7 (R848) + IFN-γ + IL-21 generate IgG ASC frequencies comparable to SWM by ELISPOT, despite their lower surface IgG levels. DN2 per-cell IgG secretion is higher than DN1 or SWM. This confirms functional ASC differentiation capacity of DN2 cells and validates ELISPOT as a readout for EF pathway output (see Jenks2018 - DN2 B Cells and EF Pathway in SLE, sorted population cultures).

  • First DENV-specific ELISpot in acute dengue (Wrammert2012): ELISPOT plates coated with purified formalin-inactivated DENV-2 (strain 16681, Microbix Biosystems; 10 µg/ml) or polyvalent anti-human Ig (10 µg/ml, Jackson ImmunoResearch). PBMCs plated in 3–5-fold dilution series from 10 × 10⁵. Detection with biotinylated anti-human IgG, IgA, or IgM (Invitrogen) + avidin-HRP (Vector Labs) + AEC substrate. Automated counting (CTL). Results: ≥70% of IgG spots were DENV-specific; IgA ~100-fold lower; IgM near-absent in secondary infections (42/46). DENV-2 antigen detected cross-serotype responses equivalently in DENV-1 and DENV-3 patients, confirming shared epitope targeting. Non-dengue febrile illness controls (hepatitis A, unidentified) showed substantial total PB responses but zero DENV-specific spots — validating assay specificity (see Wrammert2012 - Plasmablast Responses in Acute Dengue, n=46 dengue + controls, adapted from Crotty et al. 2004).

  • DENV-specific plasmablast quantification by ELISpot in acute dengue: UV-inactivated DENV-1 (Hawaii), DENV-2 (16681), and DENV-3 (H87) whole virus particles were used as capture antigens. PBMC plated without prior stimulation (spontaneous secretion). Secondary DFC patients (n=9) had ~40,000 DENV-3-reactive IgG spots per 10⁶ PBMC (72% of all IgG spots); healthy naive had zero DENV-reactive spots. Cross-serotype testing (n=14) showed plasmablasts reacted with all three serotypes but with 3-fold preference for the infecting serotype (DENV-3, p<0.01). Minor influenza virus reactivity (520 spots/10⁶ PBMC, 0.7% of IgG ASCs) was also detected. Cryopreserved PBMC validated for this assay (see GarciaBates2013 - Plasmablast Response and Dengue Severity, adapted from Crotty et al. 2003).

  • ELISpot used to establish murine ABC cytokine output ex vivo. On TLR7/9 activation, ABCs produce higher IFN-γ and exceptionally high IL-10 than follicular B cells, confirmed by transcriptional array plus ELISpot (see Cancro2020 - Age-Associated B Cells, review — no original data; mouse).

  • ELISPOT used as an orthogonal check on flow-defined ASC frequencies. Stone enumerated antibody-secreting cells by both CD138⁺CD93⁺ flow cytometry and ELISPOT on sort-purified subsets, plus a bulk antibody secretory rate (ng/hour/10⁶ cells) from re-cultured cells — three readouts agreeing that the CD138⁺CD93⁺ double-positive fraction contains the secreting cells (see Stone2019 - T-bet Promotes ASC Differentiation by Limiting IFN-gamma Inflammation, mouse, in vitro, 7 independent experimental samples/group). Ex vivo ELISPOT on bone marrow and lymph node quantified antigen-specific IgG, IgG1, IgG2b and IgG2c ASCs after influenza, resolving an isotype-selective defect (IgG2c lost, IgG2b intact) that a total-IgG readout would have missed. Worth noting for panel design: a subclass-resolved ELISPOT detected a phenotype invisible to bulk titre.

  • A deliberately short 6-hour IgG ELISPOT, used to count only actively secreting cells. Serially diluted B cells were transferred directly onto anti-human-IgG-coated plates for 6 hr (not overnight), detected with alkaline-phosphatase-conjugated anti-human IgG and BCIP/NBT substrate, read on a CTL ELISPOT reader with correction for non-specific background (see Zumaquero2019 - IFN-gamma Programs T-bet-hi B Cells for ASC Differentiation, human, n=20 HD + n=40 SLE + in vitro reconstruction). The short incubation matters for interpretation: it enumerates cells already secreting at the moment of transfer rather than cells that differentiate during the assay, which is what makes the 2.5-day DN2-versus-naive comparison a measure of pre-existing secretory capacity.

  • ★ A paired media-only vs R848 design that separates constitutive from TLR-inducible antibody secretion. Plates were coated with sonicated cell lysate from LCMV-infected BHK-21 cells rather than a purified antigen; sorted B cell subsets were plated in media alone overnight, or with R848 (10 ng/mL) for 72 hours, then developed with alkaline-phosphatase-conjugated anti-mouse IgG and Vector Blue and read on an ImmunoSpot analyser or counted blinded (see Song2022 - Tfh Outside Germinal Centers Drive T-bet CD11c B Cells, mouse, LCMV-Armstrong). The two arms are what allowed the authors to place T-bet⁺CD11c⁺ cells between memory B cells (TLR-dependent) and plasma cells (TLR-independent) — a distinction a single-condition ELISpot would have missed entirely. The same paired layout was used for anti-dsDNA and anti-chromatin autoreactivity.

Contradictions & Debates

None documented in current wiki sources.

Plasmablast, DN2 B Cell, Activated Naive B Cell, FACS Sorting, BCR Sequencing, Extrafollicular Response, In Vitro B Cell Stimulation, PRNT

Sources

Graph View

Backlinks