IVIg binds to CD22 on the surface of B cells. (A) Tonsillar B cells were incubated with IVIg (left panel), or with aggregate IgG (middle panel). The binding of IgG was revealed using FITC-conjugated goat Fab′2 anti–human IgG. The right panel shows binding of the secondary Ab alone with the TRITC-conjugated donkey anti–rabbit IgAb, used as negative control. (B) Tonsillar B cells were stained with rabbit anti-CD22 followed by TRITC conjugated anti–rabbit Ab. After several washes, the cells were incubated with IVIg and and followed by FITC-conjugated anti–human IgG F(ab′)2. (C) Sialylated IVIg (IVIg SA+) and asialylated (IVIg SA−) fractions were used to stain B cells followed by FITC-conjugated F(ab′)2 anti–human IgG antibody. B cells were also stained with anti-CD22. (D) IVIg was separated according to their SA content using SNA affinity chromatography. Purity of the fractions was assessed by lectin blotting. Staining for CD22 was followed by secondary HRP-conjugated Ab, and the blots developed using the ECL system. (E) Tonsillar B cells were incubated with IVIg before warming the cells up at 37°C (middle lane) or examined without warming (left lane). Control for warming up at 37°C without IVIg is also shown (right lane) is shown. After the incubation, cell lysates were prepared and immunoprecipates with protein G Sepharose beads and analyzed by WB using anti–human CD22 Ab followed by secondary HRP conjugated Ab. The blots were developed using the ECL system.
