Biotin anti human igg

For the ELISpot assay of PS-specific IgG-secreting cells in humans and mice, a 96-well filtration plate (Millipore, Billerica, MA, USA) was coated with 1 mg/ml phosphatidylserine-BSA antigen (Cloud-Clone) (100 μl/well) at 4 degrees centigrade and incubated overnight. Then, purified B-cell subsets (2000 cells per well) were cultured in antigen coating filtration plates for 48 h. Then, biotin anti-human IgG (Clone: G18-145, BD Pharmingen) and biotin anti-mouse IgG (Clone: Poly4053, BioLegend) were added as detection antibodies. AKP streptavidin (BD Biosciences) and NBT/BCIP substrate (Millipore) were used. Finally, PS-specific IgG-secreting cells were quantified. To measure and compare the antibody-secreting capacity of various B-cell subsets, we performed separate experiments using sorting-purified B1a, B1b and B2 cells from the peritoneal cavity of 10-week-old female MRL/Lpr mice in the ELISPOT assay and measured the parameters, including the spot number as spot forming units (SFU) and the spot size as relative spot volume (RSV), using an IRIS reader (Mabtech, Sweden) [24 (link)].

Human serum samples (1:100 dilution) and mouse serum samples (1:200 dilution) were collected to measure the anti-dsDNA and anti-phosphatidylserine (anti-PS) IgG levels accordingly. Briefly, 1 mg/ml BSA-PS antigen (Cloud-Clone) or fetal calf dsDNA antigen (Sigma, US) was coated in 96-well ELISA plates (Thermo Fisher Scientific) (100 μl/well) at 4 degrees centigrade overnight. After blocking with 0.5% gelatin and 0.5% BSA buffer, human serum samples (1:100 dilution) and mouse serum samples (1:200 dilution) were then added. Biotin anti-human IgG (Clone: G18-145, BD Pharmingen, San Diego, CA, USA) or biotin anti-mouse IgG (Clone: Poly4053, BioLegend) were then added as detection antibodies. The HRP-streptavidin (BioLegend) and TMB substrate set (BioLegend) were then added accordingly. All data were collected in a microplate absorbance reader (Tecan, Austria) at OD450 nm absorption [7 (link)].