Mouse anti human igg3

For IgG subclass assay, each plasma sample was tested in quadruplicates. After initial incubation of FV2-coupled microspheres with plasma and washing as described above, microspheres were incubated with 100 µL mouse anti-human Ab to detect specific IgG subclasses: (a) mouse anti-human IgG1 (Molecular Probes, Ref. A10630) 1:1000 dilution, (b) mouse anti-human IgG2 (Sigma, ascites HP6014 I5635) 1:1000 dilution, (c) mouse anti-human IgG3 (Sigma, clone HP6050 I7260) 1:2000 dilution, and, (d) mouse anti-human IgG4 (Sigma HP6023 I9888) 1:1000 dilution. Plates were incubated for 1 h, at 25 °C on the shaker. After washing as described above, microspheres were resuspended in 100 µl of R-phycoerythrin-conjugated donkey anti-mouse IgG H + L (Jackson 715-116-150) at 1:250 dilution. Plates were incubated for 1 h, at 25 °C on a shaker, washed as before and resuspended in 100 µl PBS-1 % BSA; 85 µl of the microsphere suspension was analysed using a Liquichip M100 reader (Qiagen, Valencia, CA, USA). To test the quality of mouse-anti-human Ab, beads coupled to human monoclonal IgG1k, human monoclonal IgG2k, human monoclonal IgG3λ and human monoclonal IgG4λ were used. These beads were assayed as described above. Each sub-class was highly specific with no significant cross-reactivity with other IgG sub-classes (Additional file 2).

To test if there is a dose-dependent cytotoxicity-blocking effect with anti-human IgG antibodies, cells were treated with 5 µl of MS or HC A-FT, 5% NHS, plus various amounts of anti-human IgG antibodies. The following blocking antibodies were used: mouse anti-human IgG1 (Sigma #I2513), mouse anti-human IgG3 (Sigma #SAB4200759), goat anti-human IgG (H + L) (Vector Lab, #AI-3000), goat anti-human IgG-Fc (Rockland # 609-1103), and mouse anti-CD20 (R&D system #MAB4225). In addition, we tested MS drug Mitoxantrone (Cayman Chemical #14842) and pan-caspase inhibitor Z-VAD-FMK (Promega #G7231).