Mouse anti human igg2

Maxisorp^TM plates (Nunc, Waltham, MA, United States) were coated with rabbit anti-human IgG (Sigma, Darmstadt, Germany) or mouse anti-human IgG2 (Sigma) overnight at +4°C. Plates were washed in wash buffer (PBS pH 7.4, 0.05% Tween20), blocked with blocking buffer (PBS, 1% skimmed milk, 0.05% Tween20) for 1 h at room temperature (RT) followed by an additional wash. Patient and calibration sera for IgG (Dako, Glostrup, Denmark) and pure IgG2 (The Binding site, San Diego, CA, United States) were diluted in a blocking buffer, titrated on plates and incubated for 1 h at RT. After one final wash, all wells were incubated for 20 min with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (Dako P0214). The optical density was measured at 405 nm, and absorbance between calibration sera and patient sera was compared.

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).