IgG Isotypes

Isotyping was performed using mouse anti-human antibodies that specifically recognize IgG1 (mouse anti-human IgG1 CH2 domain; Bio-Rad, Hercules, CA), IgG2 (purified mouse anti-human IgG2 clone G18-21 [RUO]; BD Biosciences, San Jose, CA), IgG3 (mouse anti-human IgG3; Bio-RAD), or IgG4 (purified mouse anti-human IgG4 clone G17-4 [RUO]; BD Biosciences). Either 100 or 200 μL (according to whether 96 or 24-well plate were used) of the IgG solution of interest (diluted 1/1,000 for IgG1 and IgG3 and 1/500 for IgG2 and IgG4) in DMEM-HEPES-NGS5%-BSA1% was used and incubated for 1 hour at room temperature in the dark. After aspiration, cells were washed with DMEM-HEPES (100/200 μL) and once again with PBS (100/200 μL). A secondary goat anti-mouse antibody (100/200 μL) coupled to a fluorochrome (Alexa Fluor 555; ThermoFisher Scientific, Waltham, MA), diluted 1/1,000 in DMEM-HEPES-NGS5%-BSA1%, was added and then incubated for 1 hour at room temperature in the dark. After aspiration, cells were washed with DMEM-HEPES (100/200 μL) and with PBS (100/200 μL). In addition, titration of IgG1 and IgG4 subclasses in serum and CSF was performed only in the double-positive samples and determined as the lowest dilution with positive signal by CBA. All the CBAs were read using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).

To validate the specificity of the secondary antibodies used for IgG isotyping, a Western blot was performed with 1 μg of every purified IgG subclass (IgG1, HCA192; IgG2, HCA193; IgG3, HCA194; IgG4, HCA195; Bio-Rad) load on each well. After migration, transfer, and saturation, membranes were separately incubated with each anti-human IgG subtype (1/1,000 dilution for anti-IgG1 and 3, 1/500 for anti-IgG2 and 4 antibodies). Revelation was performed with goat anti-mouse IgG (1/20,000; 115-036-003, Jackson ImmunoResearch, Suffolk, United Kingdom).