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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: J Autoimmun. 2018 Jan 4;89:171–185. doi: 10.1016/j.jaut.2017.12.017

Figure 2. FS IgG4 autoantibodies mainly recognize Dsg1 EC1 domain.

Figure 2

(A) The EC1 domain of Dsg1 (blue) was grafted on three molecular backbones: Dsg3 (red domains), Dsg4 (white domains) and Dsc1 (green domains). We also grafted the EC1 domain of Dsg4 on a Dsg1 backbone (blue domains) as a control hybrid. These constructs were expressed in the baculovirus system and the recombinant proteins were used to test against FS sera. (B) Affinity-purified FS IgG4 on the llama anti-human IgG4 column was tested against chimeric Dsg1-EC1/Dsg3 bb in the presence of calcium and EDTA. A stock of IgG4 (0.5 mg/ml) of each patient at a dilution of 1:300 was tested on microtiter wells coated with 50 ng/well of Dsg1-EC1/Dsg3 bb. Peroxidase-labeled mouse anti-human IgG4 (Southern Biotech) at a dilution of 1:2,000 was used as an indicator. The results are expressed in index values. Similar results were observed when testing the FS IgG4 on plates coated with Dsg1-EC1/Dsg4 bb (data not shown). (C) Affinity-purified FS IgG4 on the llama anti-human IgG4 column was tested against chimeric Dsg1-EC1/Dsg4 bb (blue), Dsg4-EC1/Dsg1 bb (red), wDsg4 (green) and wDsc1 (purple) in the presence of calcium. A stock of IgG4 (0.5 mg/ml) of each patient at a dilution of 1:300 was tested on microtiter wells coated with 50 ng/well of the chimeric protein. Peroxidase-labeled mouse anti-human IgG4 (Southern Biotech) at the dilution of 1:2,000 was used as an indicator. The results are expressed in index values (Scale of -20 to 100 is shown in the figure).