Ap conjugated donkey anti human igg

Standard ELISA was performed using microtiter plates coated with 5 μg/ml of relevant recombinant protein.
For detection of equine Abs in sera samples, AP-conjugated Rabbit anti-horse IgG (Sigma, A6063; used at 1:4000 working dilution) was applied following detection using PNPP substrate (Sigma, N1891).
For phage-ELISA, anti-M13 Ab (Sino Biological, 11973-MM05T; used at 300 ng/nl) followed by HRP-conjugated sheep anti-mouse (GE, LNA931V/AG; used at 1:2000 working dilution) were used, Detection performed with TMB substrate (Millipore, ES001).
For detection of recombinant chimeric Abs, AP-conjugated Donkey anti-human IgG (Jackson ImmunoResearch, 709-055-149; used at 1:2000 working dilution) was applied following detection using PNPP substrate.
Specificity ELISA of the Abs performed at least 3 times for each Ab in serial concentrations. Representative results are shown at constant Ab concentration (3 μg/ml). Plotted signals represent OD [405 nm] values of each Ab against its cognate specific antigen.
For BoNT native toxins binding evaluation, Indirect ELISA performed. Anti-BoNT/A or anti-BoNT/B rabbit polyclonal Abs (28 (link)) or anti-BoNT-HC murine mAbs (21 (link)) at a concentration of 5 μg/ml used for toxins [10 ng/ml] capturing. Tested mAbs were then reacted with captured toxins and detected as detailed above.

Samples were subjected to SDS-PAGE on 4–20% Tris-glycine gels and electrotransferred to PVDF membrane. PVDF membranes were then clamped into the Mini-Protean II Multiscreen apparatus (Bio-Rad), and individual lanes were blocked and probed with human sera diluted at 1∶100, unless otherwise noted. Secondary antibodies used were either alkaline phosphatase (AP)-conjugated goat anti-human IgG+IgM or AP-conjugated donkey anti-human IgG diluted 1∶10,000 (Jackson ImmunoResearch). Quantification of autoantibody reactivity on immunoblots was performed via computer-assisted densitometric scanning (Epson 8836XL high-resolution scanner and NIH Image J densitometry software). Autoantibody levels were expressed in arbitrary densitometry units. Statistics were performed using GraphPad Prism 5.0. The Mann-Whitney U test (two tailed) was used to evaluate the difference between two groups, with p<0.05 taken as significant. Spearman correlations were used to explore the relationship between fold change in autoantibody levels and outcome measures.

Specific ELISA was performed against SARS-CoV-2 spike glycoprotein ectodomain (expressed and purified as described [28 (link)] for the evaluation of the endogenous humoral response of each tested serum sample. Maxisorp™ 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with 1 µg/mL of spike protein in NaHCO3 buffer (50 mM, pH 9.6), washed, and blocked with PBT buffer (1xPBS, 0.05% Tween 20, 2% BSA). Samples were diluted 1:200 for determining their anti-spike titer (Figure 1). Human or mouse Abs were visualized by AP-conjugated donkey anti-human IgG (Jackson ImmunoResearch, Bar Harbor, ME, USA, Cat# 709-055-149, lot 130049; used at 1:1,000) or donkey anti-mouse IgG (H+L) minimal cross (Jackson ImmunoResearch, USA, Cat# 715-055-150, lot 142717; used at 1:2,000), respectively. PNPP substrate (Sigma, Rehovot, Israel, Cat# N1891) was applied for the reaction development. Washing steps were carried out with 1xPBST (1xPBS, 0.05% Tween 20) and all incubation were performed at room temperature.

Direct ELISA (65 (link)) consisted of coating microtiter plates with 1 × 10⁷ PFU/mL of inactivated VACV IHD-J or ΔH3 or with 2 µg/mL recombinant proteins. For phage ELISA, HRP-conjugated anti-M13 antibody (Sino Biological, USA, Cat# 11973-MM05T-H lot HO13AU601; used at 250 ng/mL) was used following detection with TMB substrate (Millipore, USA). ELISA of both serum and recombinant antibodies was applied with AP-conjugated donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149 lot 130049; used at 1:2,000 working dilution) following detection using PNPP substrate (Sigma, Israel).

Direct ELISA³³ consisted of coating microtiter plates with 2 μg/ml of recombinant SARS-CoV-2 spike, S1 domain, RBD or NTD subunits. For phage ELISA, HRP-conjugated anti-M13 antibody (Sino Biological, USA, Cat# 11973-MM05T-H lot HO13AU601; used at 1:5000 working dilution) was used following detection with TMB substrate (Millipore, USA). ELISA of both sera and recombinant scFv-Fc human antibodies was applied with AP-conjugated Donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149 lot 130049; used at 1:2000 working dilution) following detection using PNPP substrate (Sigma, Israel).