Peroxidase conjugated goat anti human igg

To detect antibodies against polyomaviruses, virus-like particle-based enzyme immunoassays similar to the assays developed for MCPyV, LPyV and HPyV9 were used [24] (link), [28] (link). Briefly, ELISAs were performed in microplates coated with 100 ng of extract enriched for VLPs. VLP concentrations were determined using the Qubit Protein Assay Kit (Invitrogen). Sera were tested at 1∶100 dilution and peroxidase-conjugated goat anti-human IgG (Southern Biotech, Clinisciences, Nanterre, France) diluted 1: 20,000 was used to detect binding of human and great ape IgGs. The cut-off values were set at 0.200, determined as previously [24] (link), [28] (link).

A codon-optimized nucleotide fragment encoding full-length nucleoprotein was synthesized and cloned into pETM11 expression vector (European Molecular Biology Laboratory). The His-tagged SARS-CoV-2 N protein was bacterially expressed in E. coli BL21 (DE3) and purified as a soluble dimeric protein by affinity purification using a Ni-NTA Protino column (Macherey Nagel) and gel filtration using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare). Ninety-six–well ELISA plates were coated overnight with N in phosphate-buffered saline (PBS) (50 ng per well in 50 μl). After washing four times with PBS–0.1% Tween 20 (PBST), 100 μl of diluted sera (1:200) in PBST–3% milk was added and incubated for 1 hour at 37°C. After washing three times with PBST, plates were incubated with 8000-fold diluted peroxidase-conjugated goat anti-human IgG (Southern Biotech) for 1 hour. Plates were revealed by adding 100 μl of horseradish peroxidase (HRP) chromogenic substrate [3,3′,5,5′-tetramethylbenzidine (TMB), Eurobio Scientific] after three washing steps in PBST. After 30-min incubation, ODs were measured at 405 nm (OD_405nm). OD measured at 620 nm was subtracted from values at 405 nm for each sample.

The anti‐NeuGc Abs in the sera of patients with HCC and healthy volunteers were measured by ELISA, according to previously reported methods.¹⁴, ¹⁵ ELISA microplates (Costar 3591; Corning, New York, NY, USA) were coated with NeuGc‐ and NeuAc‐conjugated polyacrylamide (GlycoTech, Gaithersburg, MD, USA) overnight. We added peroxidase‐conjugated goat anti‐human IgG (Southern Biotech, Birmingham, AL, USA) or peroxidase‐conjugated mouse anti‐human IgG subclasses (Southern Biotech). The optical density was measured at 490 nm using the MTP 310 microplate reader (Corona Electric Co., Ltd., Ibaraki, Japan). We decided to analyze x100 and x500 dilution data based on the preliminary data (n = 16, Figure S1) in which the serum was diluted in 4 steps.