Titermax

A portion of the gene that encoded the major outer membrane protein (OmpA)of CaLas (YP_003065185) designated Omp3f was subcloned into the pET102/D-TOPO vector system (Life Technologies, Carlsbad, CA). The expressed protein fragment was purified by His/Ni chromatography (Life Technologies, Carlsbad, CA) under denaturing conditions and used for the preparation of polyclonal antibodies (Ding et al., submitted). A New Zealand white rabbit was immunized 4 times over the course of 56 days with 0.3 mg purified antigen mixed with an equal volume of TiterMax (Sigma Aldrich, St. Louis, MO) adjuvant per immunization (Cocalico Biologicals, Reamstown, PA). A commercial goat anti-rabbit polyclonal antibody (#12–448, EMD-Millipore, USA) conjugated with alkaline phosphatase was used as secondary antibody.

Immunization of C56BL/6 mice (four groups of three individuals) was carried out by injecting twice 5 µg Ovalbumin (OVA) (Hyglos GmbH, Bernried am Starnberger See, Germany) together with 2.5 mg β-sulfoquinovoside mixture (6) according to published protocols^{60 (link)}. OVA-specific Ig production was measured by ELISA according to Leung S et al.^{61 (link)}. Titermax and Complete Freund’s Adjuvant (Sigma Aldrich, St. Louis, MO, USA) were used according to manufacturer instructions. Statistical analyses were performed by ANOVA using the GraphPad Prism 4.0 Software (GraphPad Software, Inc, La Jolla, CA).

Recombinant mouse (rm)Ear11 was expressed as a 6xHis fusion protein in Sf9 cells using Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific) and purified from cell culture supernatants on a Nickel NTA resin column (GE Healthcare) followed by gel filtration on HiLoad 16/60 Superdex 75 column (GE Healthcare) and then HisTrap HP column (GE Healthcare). Purified protein was dialysed into sterile, endotoxin-free PBS. Endotoxin concentration was quantified at 32 EU/mg using the Limulus/Amebocyte Lysate (LAL) assay according to manufacturer’s instructions (Pierce, ThermoFisher).
A male rat was immunised by sub-cutaneous injection of 100 μg of rmEar11 combined in a 1:1 ratio with TiterMax (Sigma-Aldrich) on day 0, 28, and 32. A single intravenous injection of 50 μg of Ear11 was given on day 84. Monoclonal antibodies were generated by standard protocols.

All subcutaneous (s.c.) immunizations were carried out with 100 μg mDsg3 or a combination of 30 μg hDsg1 and 70 μg hDsg3 emulsified in the adjuvant TiterMax^™ (Sigma-Aldrich, St. Louis, Missouri, USA). Immunizations occurred at day 0, 14, 35, and 56, adapted from a mouse model of immunization-induced EBA [6 (link)]. Subsequent injections were given (in this order): in both foot pads, in the tail base, in the right lateral abdominal region and in the left lateral abdominal region. The follow-up was 84 days. Mice were weighed weekly and clinically examined for evidence of cutaneous/mucosal lesions. Serum samples were obtained every second week. A high-resolution mouse endoscope (HOPKINS Optik 64019BA; Karl StorzAidaVet, Tuttlingen, Germany) was used to determine the extent of oral lesions as described [10 (link)]. Biopsies of the skin, buccal mucosa, and esophagus were obtained at the end of the observation period.

Naive and infected mice (7 dpi) were subcutaneously immunized with 100 mg of ovalbumin (OVA; Sigma-Aldrich) emulsified in an equal volume of TiterMax adjuvant (Sigma-Aldrich). After 14 days, the animals were sacrificed and sera were collected to assess OVA-specific antibodies using the ELISA method [27 (link)].

The recombinant PfCSP was shipped to Green Mountain Antibodies, Inc. (Burlington, VT, USA) for the immunization of mice, followed by the fusion to generate monoclonal antibodies. Briefly, mice were primed with 50 μg of PfCSP emulsified with complete Freund’s adjuvant, followed by weekly immunization of 50 μg of PfCSP emulsified with TiterMax^® (Sigma-Aldrich, St. Louis, MO, USA) and SAS^® (Sigma-Aldrich) (alternate week). One week after administering seven doses of immunization, the lymph node was isolated. B cells were purified from using anti-B220 magnetic-activated cell sorting (MACS), and then fused with a mouse myeloma cell line. Cloning was achieved by limiting dilution. After re-cloning, positive clones that secrete immunoglobulin G (IgG) against the full-length PfCSP were selected by enzyme-linked immunosorbent assay (ELISA) (Table 1).