Goat anti mouse or goat anti human igg conjugated to hrp

The proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Bio-Rad, St-Laurent, QC, Canada), blocked and incubated with primary antibody [Mouse monoclonal anti-βactin (Sigma, Oakville, Canada) or Human polyclonal anti-HDAg [27 (link)]]. Washed membranes were incubated for 1 h at RT with a goat-anti-mouse or goat-anti-human IgG conjugated to HRP (Jackson ImmunoResearch, Baltimore, MD, USA). Protein bands were visualized with the Clarity western ECL (Bio-Rad, St-Laurent, QC, Canada) using a Sapphire Biomolecular Imager (Azure Biosystems, Montréal, QC, Canada).

EIA was performed according to a comprehensive published method (Protocol 3.3.1. in CPM [15 (link)]) with the following modifications (a step-by-step protocol is provided in S1 Text). Briefly, purified recombinant proteins quantified by the Lowry protein assay kit (Thermo Fisher Scientific, Waltham, MA) or whole-cell extract of Leptospira diluted in 1X sodium carbonate coating buffer was used to coat Nunc MaxiSorp flat-bottom EIA plates (eBioscience, San Diego, CA) at 0.5–1 μg/ml (protein) or 10⁵-10⁸cells/well (Leptospira extract) overnight at 4°C. The following day the plates were washed with 1XPBS, blocked with 1% BSA for 2h, washed again, and incubated with human or murine serum diluted at 1:50 or 1:100. Goat anti-mouse or goat anti-human IgG conjugated to HRP diluted at 1:10000 (Jackson ImmunoResearch, West Grove, PA) was used as the secondary antibody. The OD₄₅₀ was read on a SpectraMax Plus EIA reader (Molecular Devices). For determination of Ig class and IgG isotypes the secondary antibody conjugated to HRP was diluted as follows: anti-human -IgG1, -IgG2, -IgG3, -IgG4 (1:1000, Invitrogen), anti-human IgA (1:8000, Southern Biotech), anti-human IgD (1:1200, Southern Biotech) and IgM (1:10,000, Jackson ImmunoResearch Laboratories, Inc.). The EIA cutoff was set at three standard deviations above the average OD₄₅₀ of all healthy control samples.