**7. A method for quantifying serum levels of autoantibodies against β2GPI/HLA-DR complexes**

We developed and modified a method to measure serum levels of autoantibodies against β2GPI/HLA-DR complexes (anti-β2GPI/HLA-DR) [4, 19].

Green fluorescent protein (GFP)-labeled β2GPI/HLA-DR complex-expressing 293 T cells and DsRed-labeled HLA-DR-expressing 293 T cells were generated by transient transfection [19]. A serum sample from a patient in whom anti-β2GPI/ HLA-DR were detectable after a 106 -fold dilution was used as a standard serum. The anti-β2GPI/HLA-DR level of a standard serum was defined as 1,000 units. The mean fluorescence intensity (MFI) of IgG binding to transfected cells in the sample sera was analyzed by flow cytometry. Specific IgG binding to the β2GPI/HLA-DR complex was calculated by subtracting the MFI of IgG binding to HLA-DR-expressing cells from β2GPI/HLA-DR complex-expressing cells. Serum levels of anti-β2GPI/HLA-DR in each sample were calculated from the standard curve generated by measuring specific IgG binding to the β2GPI/HLA-DR complex in serially diluted standard serum.
