Human igm elisa quantitation set

Each CD4⁺ T cell subset (1 × 10⁵ cells/well) was cocultured with B cells (1 × 10⁵ cells/well) and T_FH cells (5 × 10⁴ cells/well) in round-bottomed 96-well plates. Recombinant staphylococcal enterotoxin B (SEB) (2 μg/ml; Toxin Technology, Sarasota, FL, USA) was added to stimulate B cells. Blocking antihuman IL-10 mAb (25209; R&D Systems), blocking antihuman Fas ligand mAb (100410; R&D Systems), or mouse immunoglobulin G1κ (IgG1κ) isotype control (eBioscience) was added at a concentration of 10 μg/ml on the first day of the coculture in the same experimental system as above. To assess cell-cell contact, a coculture experiment was performed using a Transwell plate (HTS Transwell-96 System [3381]; Corning Life Sciences, Acton, MA, USA) with the following cells: B cells, 3 × 10⁵ cells/well; T_FH cells, 2 × 10⁵ cells/well; and CD4⁺CD25⁻LAG3⁺CD45RA⁻ T cells, 1 × 10⁵ cells/well. The concentrations of IgG, IgA, and IgM in the 12-day culture supernatant were measured by ELISA (Human IgG ELISA Quantitation Set, Human IgA ELISA Quantitation Set, and Human IgM ELISA Quantitation Set; Bethyl Laboratories, Montgomery, TX, USA).

B cells and PBMCs were cultured at 3x10⁵/well in 96 well plates. ELISA was performed using Human IgG ELISA Quantitation Set, Human IgA ELISA Quantitation Set, and Human IgM ELISA Quantitation Set (Bethyl Laboratories).

Plasma concentrations of IgG, IgM and soluble CD52 were quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocols (Human CD52 ELISA kit, Aviva Systems Biology, San Diego, CA, USA; Human IgG ELISA quantitation set, Bethyl Laboratories, Montgomery, TX, USA; Human IgM ELISA quantitation set, Bethyl Laboratories, Montgomery, TX, USA). For quantitation of soluble CD52, plasma samples were diluted 1:10. Duplicates were measured for each sample. The optical density values were obtained on a SpectraMax M3 (Molecular Devices, San Jose, CA, USA) at wavelength of 450 nm. Quantification of α2,3 sialylation was determined as previously described (20 (link)). Briefly, plates were coated with MAA-II (Vector Labs, Burlingame, CA, USA) at 20 µg/ml overnight at 4°C, washed twice with PBS, blocked for 1 h at RT with 1% BSA in PBS and plated with 20 µg/ml of CD52-Fc or control Fc prior to incubation with anti-IgG Fc HRP antibody (Bethyl Laboratories, 1:1000 dilution). After two washes with PBS, TMB was added and color development stopped by addition of 0.5M H₂SO₄.