Goat anti human igm antibody

Leptospiral LPS extracted as aforementioned was used to generate the antigen tested line (T-line), whereas protein A served as the controlled line (C-line). Both lines were immobilized on a nitrocellulose (NC) membrane (AE99, Whatman Schleicher & Schuell; Dassel, Germany) using XYZ 3060 (BioDot; Irvine, CA). The membrane was dried and kept in a dehumidifier cabinet. Immunogold nanoparticles (40-nm, Diagnostic Consulting Network; Carlsbad, CA) conjugated with goat anti-human IgM antibody (Jackson ImmunoResearch; West Grove, PA) (adjusted to 14 µg/ml in 2 mM borate buffer (pH 9.0) containing 1% BSA) were then impregnated (GF33, Whatman Schleicher & Schuell). The conjugated pad (GF33 membrane), NC membrane, sample pad and wicking pad (Whatman) were cut into a 5-mm-wide strip by a Guillotine Cutter (Arista Biologicals Inc.; Allentown, PA) and finally assembled on a plastic back plate. This in-house IgM/LPS-based rapid detection kit (LEPkit) was then sealed and kept at room temperature until used.

Isolated CD19⁺ B cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin/streptomycin (100 U/ml), and 2 mM Glutamax in 96-well round bottom plates at a density of 50 × 10⁴ cells/well. T cell-independent mimicking stimulations were performed in 96-well round bottom plates in the presence of CpG-ODN2006 (1 µg/ml TLR9 agonist, InvivoGen). T cell-dependent mimicking stimulations were performed in 96-well round bottom plates with 0.5 µg/ml mouse anti-human-CD40 antibody (Ebioscience #16-0409-85), 5 µg/ml goat anti-human-IgM antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) with or without the presence of IL-10 (Invitrogen, 50 ng/ml). Assessment of the induced transitional CD24^hiCD38^hi phenotype and Bregs was done on B cells harvested after 48 H CpG (1 µg/ml) stimulation. They were further blocked with brefeldin A for 6 h and re-stimulated with PMA and ionomycin for 4 h for the assessment of cytokine producing B cells by intracellular flow cytometry. For the assessment of TLR9 responsiveness of B cells they were cultured further and assessed for surface expression of antibody isotypes and production of immunoglobulins after 7 days. Supernatants were harvested and cells stained for flow cytometric analysis.