Detection of sCTLA-4 protein in plasma and serum samples. (A) Analysis by sCTLA-4 (left panel) and pan–CTLA-4 (right panel) immunoassays of 2-fold serial dilutions of recombinant sCTLA-4 spiked into 1/20 and 1/40 dilutions of plasma and serum samples. An isotype-matched mouse IgG2a aκ capture Ab is used as a negative control. Serial dilutions of culture supernatant of sCTLA-4 HeLa cell transfectants in standard assay buffer were included in each assay. (B) Detection of recombinant sCTLA-4 spiked into human whole blood. Mouse IgG2a aκ isotype-matched Ab used as capturing reagent served as a negative control. (C) Analysis of sCTLA-4 in serum samples from autoimmune patients and controls using the sCTLA-4 immunoassay (left panel) and the pan–CTLA-4 immunoassay (right panel). Serial dilutions of recombinant sCTLA-4 were spiked into serum diluted 1:20 (dark gray columns) and 1:40 (light gray columns), and representative examples from a total of 61 donors are shown (see Supplemental Fig. 3 for results from all donors), as follows: 4 GD patients and 3 age- and sex-matched, healthy volunteers, sera diluted 1/20 (dark shaded columns) and 1/40 (light shaded columns). For all samples, including the spiked serum samples, the mouse IgG2aκ isotype control is shown (open columns). Columns, mean of duplicate measurements; bars, ±SD.