2.6. FPLC Gel Filtration Analysis and Immunoblotting

Purified anti-PV IgA mAbs from hybridomas were dialyzed against PBS and fractionated by size exclusion chromatography (SEC) on a Superose 6 HR 10/300 column (17-0537-01; GE Healthcare) connected to an ÄKTA FPLC system (GE Healthcare). The column was calibrated with a wide range (143.7 to 443 kDa) of molecular weight markers (MWGF1000; Sigma Aldrich). Approximately 150 μg antibody in 100 μL was loaded onto the column at a flow rate of 0.5 mL/min in PBS and two 5 mL fractions containing dimeric IgA (fraction 1) and monomeric IgA (fraction 2) were collected. IgA mAbs from recombinant expression were analyzed by gel filtration of 20 µg mAb samples with a Superdex 200 Increase 10/300 GL column (28990944; GE Healthcare) and Gel Filtration Standards (1511901; Bio-Rad, Hercules, CA, USA).

For J-chain analysis of the hybridoma-expressed IgAs, FPLC fractionated human anti-PV IgA (10 μg) samples were resolved by reducing SDS-PAGE on a 4–12% (w/v) polyacrylamide gel with the Criterion system (Bio-Rad) and transferred to a nitrocellulose membrane (88024; Thermo Fisher). The membrane was blocked by incubation in 5% non-fat dry milk in TBS plus 0.05% Tween 20 (TBST) overnight at 4 °C. Subsequent incubations were all performed in TBST. After three washes with TBST, the blocked membrane was incubated with HRP conjugated goat anti-human J chain of dimeric IgA (MBS571594; MyBioSource, San Diego, CA) at 1:1000 dilution for 1 h, washed three times for 5 min, then incubated with HyGlo Quick Spray Chemiluminescent HRP detection solution (E2410; Thomas Scientific, Swedesboro, NJ, USA) for 1 min, and the substrate was detected with a light-sensitive camera (Chemidoc; Bio-Rad).