Microarray-based Immunogenicity Profiling

Each of the purified rSEPs diluted in PBS to a concentration of 0.3 mg/ml was printed on epoxy slides (CapitalBio, Beijing, China) in 5 replicate spots as described previously [16 (link)]. Human IgG with serial dilutions (2.5, 5, 10 and 20 μg/ml) was used to fit the internal calibration curves or as positive controls. BSA in PBS or lysate of E. coli cells transformed with pET-32a plasmids at a concentration of 0.3 mg/ml was used as negative controls [16 (link)]. For quality control, the microarray slides were incubated with mouse anti-His tag IgG-Cy5 (SBA, Birmingham, AL) and the fluorescence intensity (FI) of each protein on the slides was scanned by GenePix Personal 4100A (Molecular Devices, Sunnyvale, CA) and analyzed by GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA) [16 (link)]. Proteins with a signal-to-background ratio over 3.0 were used for further analysis [16 (link)].