Figure 2

pIgA1 trap, a novel pIgA1 specific for O -glycan analysis. Serum pIgA1 was captured by mouse Fcα/μR transfectant and O-glycans of captured pIgA1 were stained with fluorescein-labeled HAA lectin. The fluorescence intensity of the transfectant was detected by flow cytometry. a. Dead cells were extracted from analysis. b. Mouse Fcα/μR transfectant can bind to pIgA1 from both human milk and myeloma serum but not bind mIgA1. c. IgM does not interfere in pIgA1 binding to mouse Fcα/μR. Mouse Fcα/μR transfectant was pretreated with or without human IgM. pIgA1 was added to these cells and pIgA1 binding on cell surface was detected by PE-conjugated anti-human IgA. IgM had no effect for pIgA1 binding to mouse Fcα/μR transfectant (left panel). Similarly, pretreatment with pIgA1 did not interfere IgM binding to mouse Fcα/μR (right panel). d. HAA lectin only binds to pIgA1 but not IgM. Mouse Fcα/μR transfectant was pretreated with pIgA1. Confocal images collected from one mouse Fcα/μR transfectant triply stained for anti-human IgA antibody (pIgA1, green), degalactosylated O-glycans (HAA lectin, red), DAPI (nucleus, blue), and their merged images. A similar procedure was performed for BW5147 (parent) cells as a negative control. e. pIgA1 trap analysis. Mouse Fcα/μR transfectant was pretreated with human serum and washed. The cells were stained with biotin-conjugated HAA and PE-conjugated streptavidin. The healthy adult was a 44 year-old male and the IgAN patient was a 19 year-old female. A negative control was prepared without serum and a positive control was prepared with pIgA1 from myeloma which has been known as highly degalactosylated by ELISA.