Anti human horseradish peroxidase conjugated secondary antibody

Nunc MaxiSorp ELISA plates (ThermoFisher Scientific) were coated with 0.02 mg/mL of Pfs230D1, Pfs230D1-E2p, and Pfs25 in Carbonate buffer, pH 9.6 overnight at 4 °C. The plates were then washed three times with PBS and 0.05% Tween 20 (PBST). The plates were then blocked with 2% Bovine Serum Albumin (BSA) (Sigma) in PBST for 1 h at room temperature. The plates were then washed three times with PBST. The plates were then incubated with 250 ng/mL of LMIV-01 in 2%BSA, PBST for 1 h at room temperature. The plates were then washed three times with PBST. The plates were then incubated with a 1:5000 dilution of anti-human horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The plates were then washed three times with PBST. The plates were then incubated with 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Millipore Sigma Aldrich). The reaction was stopped by the addition of 2 M sulfuric acid to a final concentration of 1 M. The plates were read with a Synergy H1 plate reader and 5gen v3.08 software. The data were graphed and analyzed using GraphPad Prism 9 for MacOS.

Protein lysates were obtained by extracting liver tissue with T-Per Tissue Protein Extraction Reagent (Pierce, Rockford, United States) supplemented with NaF (10 mM), EDTA (2 mM), benzamidine (10 mM), PMSF (1 mM), leupeptin (1 μg/ml), Na3VO4 (2 mM), and aprotinin (1,5 μg/ml). Protein content was measured by Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, United States) following the manufacturer’s instructions. For immunoblotting, 20 μg of protein extracts were denaturated at 95°C, separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (NuView Precast gels, Peqlab, Erlangen, Germany) and blotted onto a nitrocellulose membrane (Hybond ECL Nitrocellulose Membrane, GE Healthcare, Buckinghamshire, United Kingdom). Small and large hepatitis delta antigens (S-HDAg, L-HDAg) were detected using a highly diluted human anti-Delta antibody (anti-HDAg-positive human serum (1:2,000). An anti-human horseradish-peroxidase conjugated secondary antibody (Jackson Immunoresearch, Suffolk, United Kingdom) was used in a dilution of 1:10,000. Signals were visualized with Super Signal West Dura Chemiluminescent Substrate (Pierce) and the Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, United States).