Pierce jacalin agarose

Hybridoma methods were performed with cultured PBMCs as described previously, with minor modifications [32 (link)]. PBMCs were isolated using ficoll-Paque (17-1440-02; GE Healthcare, Chicago, IL, USA) density gradient centrifugation and cryopreserved. CD27+ cells were MACS-isolated using CD27 microbeads (130-051-601; Miltenyi Biotec, Cambridge, MA, USA). CD27+ cells (2 × 10⁵ cells/well) were plated in a 24-well plate and cultured for eight days in advanced RPMI supplemented with 10% FBS, 100 IU/mL penicillin, 50 μg/mL streptomycin, 5 ng/mL human IL-2, 50 ng/mL human IL-10 (Peprotech, Rocky Hill, NJ, USA), 10% CHO cell conditioned medium containing Ultra CD40L (Multimeric Biotherapeutics, La Jolla, CA, USA), 1 μg/mL CpG (ODN2006, Invivogen, San Diego, CA, USA), 100 ng/mL rhBAFF, 1 μg/mL cyclosporin, 0.5 ng/mL rhTGF-β and 100 ng/mL rhAPRIL (R&D Systems, Minneapolis, MN, USA). On day 8, cells were harvested and fused with the LCX OCMS fusion partner cell line by electrofusion [33 (link)]. Positive wells were subjected to three rounds of single cell cloning to establish stable hybridomas secreting anti-PV IgA mAbs. For scale up, hybridoma clones were adapted to 5% Ultra Low IgG FBS (Thermo Fisher Scientific, Waltham, MA, USA) and purified with columns packed with Pierce Jacalin Agarose (20395; Thermo Fisher) following the manufacturer’s recommendations.