Igg1 biotin

For class switch recombination to IgG1 resting splenic B cells were isolated from 2.5 to 5 months old Kmt2d^+/+ CD19-Cre- (wt, 2 females and 3 males) and Kmt2d^f/f CD19-Cre⁺ (Kmt2d^−/− 2 females and 3 males) mice by immunomagnetic depletion with anti-CD43 MicroBeads (anti-Ly48, Miltenyi Biotech), and cultured at 0.5×106 cells/ml with LPS (25 µg/ml; Sigma), IL-4 (5 ng/ml; Sigma) and RP105 (Anti-Mouse CD180; 0.5 µg/ml; BD Pharmingen) for 4 days. B cells were infected at 24 and 48 hours in culture with pMX-Cre-IRES-GFP as described³⁸ to enhance Kmt2df/f deletion. Class switching to IgG1 was measured at 96 hours in the GFP⁺ population (>90%) by flow cytometry using the following antibodies: IgG1-biotin (BD Pharmingen, clone A85-1 #553441) following streptavidin-Pacific Bule (Molecular Probes), B220-Alexa700 (Biolegend, clone RA3 #103232). Data acquisition was performed on the BD LSR II Flow Cytometer (BD Biosciences) equipped with CellQuest software (Becton Dickinson). Analysis was performed with FlowJo software (Tree Star).

IgG subclass distribution of gB-specific IgG in sera was measured by BAMA as previously described (44 (link)). For measurement of IgG1, sera were diluted 1:500, and for measurement of IgG2, IgG3, and IgG4, sera were diluted 1:40. Sera were then co-incubated with gB covalently coupled to Luminex beads. In separate assays for each IgG subclass, binding was detected by biotin-conjugated subclass-specific antibodies at a concentration of 4 μg/mL: IgG1-biotin (BD Pharmingen; clone G17–1), IgG2-biotin (BD Pharmingen; G18–21), IgG3-biotin (Calbiochem; clone HP6047), and IgG4-biotin (BD Pharmingen; clone G17–4). The plates were washed, then binding was detected by addition of phycoerythrin (PE)-conjugated streptavidin (2 μg/mL, Southern Biotech). Data acquisition, data analysis, and quality control criteria were as described above for BAMA. The lower limit of positivity was defined as 100.