3 3 5 5 tetramethylbenzidine (tmb)

To evaluate the presence of IgG anti-spike antibodies in plasma samples, ELISA was performed by coating high-binding 96-well plates (Thermo scientific, Ca t#44-2404-21) with 50 ul per well of a 1μg/ml of the S1 subunit in sodium bicarbonate solution overnight at 4°C. Plates were washed 6 times with washing buffer (1xPBS with 0.05% Tween-20) and incubated with 200 μl/well blocking buffer (1xPBS with 2% skim milk) for 2 hours at room temperature. Immediately after blocking, plasma samples were assayed at a 1:100 starting dilution and 4 additional serial dilutions in blocking buffer. Plates were incubated again for 2 hours at room temperature. After washing 6 times with washing buffer, plates were incubated with anti-human IgG secondary antibody conjugated to horseradish peroxidase (HRP) diluted in blocking buffer at 1:10,000 dilution. Plates were developed by the addition of the HRP substrate, TMB (Life technologies, Cat #002023) for 8 minutes. After that, the absorbance was immediately measured at 650 nm with an ELISA microplate reader (Infinite M200 PRO). Data were analyzed using Prism V8.0. Plasma dilutions were done using a blocking buffer.

IL-6, IL-12p70 and TNFα levels in culture supernatants were assessed using enzyme-linked immunosorbent assay (ELISA) sets from BD Biosciences. IL-1β was assessed using an ELISA set from BioLegend. ELISAs were completed following the manufacturer’s recommendations. Briefly, capture antibodies were diluted as suggested in the correct coating buffer and 100 μL/well were added to Maxisorp ELISA plates (Nunc, Roskilde, Denmark) overnight at 4 °C. Following washing with PBS/0.05% Tween 20, the plates were blocked with 200 µL/well PBS/10% FBS and undiluted supernatant samples and standards (100 μL/well) were added for 2 h. Biotinylated detection antibodies combined with the avidin–horseradish peroxidase (HRP) conjugate were added (100 μL/well) at the suggested dilutions for 1 h. Lastly, 3,3’,5,5’-Tetramethylbenzidine (TMB, Life Technologies, Carlsbad, CA, USA) was added (100 μL/well) to detect HRP for 15–30 min, the reaction was stopped with 1 M HCl (50 μL/well) and the absorbance was measured at 450 nm. For tumour necrosis factor (TNF) and IL1β, the detection step was separated from the avidin step with the solutions added for 1 h and 30 min, respectively, prior to TMB addition.

A sandwich-ELISA used for quantification of Hp-HbF was developed and displayed a high preference for Hp-HbF compared to uncomplexed HbF (>10x higher recovery of a Hp-HbF calibrator series compared to a HbF calibrator series with the same molar content of HbF). No cross-reactivity was observed with Hp or HbA. Ninety six-well microtiter plates were coated with anti-HbF antibodies (HbF-affinity purified rabbit polyclonal (“Bonita”); 4μg/ml in PBS) overnight at RT. In the second step, wells were blocked for 2 hours using blocking buffer (1% BSA in PBS), followed by an incubation with Hp-HbF calibrator or the patient samples for 2 hours at RT. In the third step, HRP-conjugated anti-Hb antibodies (HbA-affinity purified rabbit polyclonal; DAKO; diluted 1:5000), were added and incubated for 2 hours at RT. Finally, a ready-to-use TMB (Life Technologies) substrate solution was added. The reaction was stopped after 30 minutes using 1.0 M HCl and the absorbance was read at 450nm using a Wallac 1420 Multilabel Counter (Perkin Elmer Life Sciences).

Recombinant human VWF A1A2A3 or recombinant human individual A1, A2, or A3 domains were incubated overnight with purified human C3, C3b, or C4 at 4°C to allow complex formation. The complexes were captured with anti-His antibody and detected by anti-C3 or anti-C4, HRP-conjugated secondary anti-IgG antibody, and the chromogenic substrate, 3,3′,5,5′-tetramethylbenzidine (TMB, Life technologies, Massachusetts, United States) was used for detection and quantification. The color reaction was read at 450 nM in a spectrophotometer.
Specifically, 100 μL of the mixtures of VWF domains and C3, C3b, or C4 were pipetted in duplicate and then incubated in High Bind Stripwells (Greiner Bio-One, Germany) with immobilized mouse monoclonal anti-His tag antibody (160 ng/mL) (Genscript, NJ) for 2 hours at 37°C. Either VWF A1A2A3 or any of the individual A domains with bound C3, C3b, or C4 was captured by the anti-His antibody immobilized onto the wells. Bound C3, C3b, or C4 (10 μg/mL) to 100 μg/mL of either the VWF A1A2A3 or individual A1, A2, or A3 domain was detected by either goat anti-human C3 or anti-C4 plus rabbit anti-goat antibody linked to HRP (Pierce Antibody, Illinois, United States). Detection and quantification in the spectrophotometer was with TMB, as above. Bovine serum albumin in phosphate-buffered saline (PBS; 1%) was used as negative control.

iPSC-MGLCs were seeded at 4 × 10⁴ cells/well in 96-well plates (Corning, USA) in macrophage end-differentiation medium. The following day, the medium was changed to fresh medium, and supernatants were collected after 24 hr. Quantification of sTREM2 from cell culture supernatants was performed using an in-house-generated ELISA using MaxiSORP 96-well plates (Nunc, Thermo Fisher Scientific, USA) coated with 1 μg/mL of a rat anti-mouse/human TREM2 monoclonal antibody (clone 237920, R&D Systems, USA) overnight at 4°C. After a blocking step, cell culture supernatant samples and standards (recombinant human TREM2-His; Life Technologies) were incubated for 2 hr at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/mL; AF1828, R&D Systems). After incubation with streptavidin-horseradish peroxidase (HRP) (0.1 μg/mL; Invitrogen), followed by the addition of a chromogenic substrate solution (TMB, Life Technologies), the reaction was terminated with the addition of stop solution (0.16 M H₂SO₄) and absorbance was read at 450 nm (GENios, Tecan, USA). In addition, a second method using a mesoscale assay validated the results (see Supplemental Experimental Procedures).

Purified RRV (50 µL, 10 μg/mL) was coated onto maxi-sorb ELISA plates (Thermo Fisher) overnight in PBS, pH 7.4. Plates were blocked with PBS supplemented with 1X KPL (SeraCare) for 1 h at room temperature. Human MXRA8-Fc (residues 20-337, UniProtKB: Q9BRK3) and negative human PD1-Fc controls were serially diluted in PBS supplemented with 1X KPL, 50 μL/well was added for 1 h at 37 °C. Plates were washed four times with PBS containing 0.05% Tween-20. Plates were then incubated with 50 μL/well horseradish peroxide conjugated goat anti-human IgG (H+L) (Goat anti-Human IgG Secondary Antibody, HRP (Invitrogen, Thermo Fisher, Cat number: A18829, RRID: AB_2535606)) at 5 μg/mL in PBS supplemented with 1X KPL for 1 h at 37 °C. After five washes with PBS containing 0.05% Tween-20, plates were developed with TMB (Life Technologies) and 2 M H₂SO_4. Absorbance was read at 450 nm.