X0939

Western blotting was performed to determine the immunogenic region of Pap31 and SCS-α. Both the purified complete proteins and their respective 3 truncated constructs were electrophoresed on 15% SDS-PAGE gels and electrotransferred onto a PVDF membrane (GE Healthcare, Buckinghamshire, UK) using a Bio-Rad Trans-Blot SD Cell apparatus. A blocking step for 1 h at room temperature using 5% skim milk in PBS was performed. The membranes were immunoblotted during 90 min with sera (1:50 dilution in Tris-buffered saline [TBS]-Tween with 1% bovine serum albumin [BSA]). Western blottings with at least 10 sera from exposed people were performed for both Pap31 and SCS-α. A commercial negative control (DAKO X0939) consisting of a pool of at least 30 sera from healthy blood donors was used. After washing 3 times with TBS-0.05% Tween20) for10 min, the membranes were incubated for 1 h with a polyclonal rabbit anti-human IgM 1:1000 dilution in TBS-T with 1% BSA conjugated with peroxidase (Dako, Glostrup, Denmark) using o-phenylenediamine tablets as substrate (Sigma, St. Louis, MO, USA).

IgM and IgG antibody levels were measured by ELISA as described by Matsuoka et al. [17 (link)] using sonicated B. bacilliformis whole cells as antigens. Total protein concentration was quantified by the Pierce assay (Thermo Scientific, Rockford, USA). IgM were detected with rabbit anti-human IgM (1:1000) conjugated with peroxidase and using o-Phenylenediamine (Dako, Glostrup, Denmark) as substrate (Sigma, St. Louis, MO). IgG were detected with rabbit anti-human IgG (1:1000) conjugated with alkaline phosphatase and using Sigma P104 phosphatase substrate. Optical densities were measured as absorbance at 450 nm and 405/655 nm for IgM and IgG, respectively. Each sample was analyzed in triplicate intra-plate and results were reported as three independent ELISA experiments. The negative control, included a pool of sera from 30 healthy donors (X0939, Dako, Glostrup, Denmark). In the absence of an established cut off, evidence of infection was considered in the samples above the Finite Mixture Model (FMM) cut off that provides a statistical framework for the analysis of immunoglobulin values [18 (link)].