Horseradish peroxidase hrp conjugated goat anti human igg

Serum levels of SARS-CoV-2 RBD IgG were measured as previously described^{33 (link)}. Briefly, SARS-CoV-2 RBD was produced in Sf9 insect cells infected with recombinant baculoviruses (Invitrogen, CA, USA). Following purification, the protein was concentrated to 5 mg/mL by ultrafiltration. Ninety-six well microplates were coated with RBD at 1 μg/mL. Serum samples were diluted 1:500 in phosphate-buffered saline-Tween (PBS-T) containing 1% bovine serum albumin and run in triplicate (mean values are reported). The plates were incubated with 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Jackson Laboratories). After three washes with PBS-T, the binding was detected using SigmaFast OPD reagent (Sigma) according to the manufacturer’s recommendations. Color development was stopped with 3 M H₂SO₄ and read on a Multiskan FC (ThermoFisher Scientific) plate reader at 492 nm. Serial sera from individual patients were analyzed in the same run. The cut-off discriminating between positive and negative sera was set as the mean absorbance of control sera plus three times the standard deviation.

Human serum anti-Neu5Gc IgGs were detected by ELISA as described previously [65 (link)]. Briefly, microtiter plates were coated in triplicates with Neu5Acα2-polyacrylamide (PAA) or Neu5Gcα2-PAA (GlycoTech) at 500 ng/well in 50 mM of sodium carbonate-bicarbonate buffer (pH 9.5) at 4°C for overnight. Plates were washed with Tris-buffered saline (TBS) and blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 hours at room temperature (RT). Dilutions of human serum (1/50 in TBST) were added in triplicates to the wells and incubated for 4 hours at RT. Wells were washed with TBS, and then horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories) diluted in TBST (1:5000) added to the wells for 1.5 hours at RT. Samples in wells were developed with a buffer containing HRP substrate O-phenylenediamine and measured at OD_{490 nm} on a SpectraMax 250 (Molecular Devices). Anti-Neu5Gcα2-PAA IgG values were obtained after subtraction of the values obtained with Neu5Acα2-PAA, thus negating any non-specific binding to Neu5Ac or PAA. Data normalized OD_{490 nm} levels were quantified into μg/ml using standard dilution curves of purified human IgG (Jackson ImmunoResearch Laboratories) per experimental day.

The ACD-plasma of patients was collected in BD Vacutainer^® and maintained on ice during the procedures of depletion to avoid the activation of the complement. IgG depletion was obtained by passing human plasma-citrate at 10% diluted in TSB (3 mL) on a protein-G Sepharose^TM Fast Flow (GE Healthcare, Uppsala, Sweden) column, as indicated by the manufacturer’s instructions. Bound IgG were eluted with 0.1 M Glycine-HCl pH 2.8 and measured using the Pierce^TM Coomassie (Bradford reagent) protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). The actual depletion of IgGs was evaluated by Western blot analysis after loading 1 µL/lane ACD-plasma on SDS-PAGE (10–12% acrylamide-bis; Bio-Rad Laboratories, Milan, Italy) and the use of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1 µg/mL; Jackson ImmunoResearch, West Grove, PA, USA).