Millipore ultrafiltration device

Ovine anti-IL-1β mAb was generated using mouse hybridoma cells as previously described (Rothel et al., 1997 (link)) and purified using techniques that we reported (Chen et al., 2013 (link)) with some additional modifications. The immunoglobulin G (IgG) from the anti-IL-1β mAb was purified from cell culture supernatants by affinity chromatography on Protein G Sepharose (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Bound antibodies were eluted with 0.1 M glycine-HCl (pH 2.3) and neutralized to pH 7.4 by adding 1 M Tris buffer. The eluted antibodies were passed through an anion-exchange column (CIMmultus QA, BIASeparations, Wilmington, DE, USA) to remove potential endotoxin contamination. Bound antibodies were eluted from the column by a buffer containing 200 mM NaCl, whereas the endotoxin was retained on the column. Finally, the antibody solution was concentrated using a Millipore ultrafiltration device with 30-kDa exclusion membrane and passed to 0.2-micron syringe sterile filter (Millipore Corp., Chicago, IL, USA) (Chen et al., 2015 (link)). The pGEX-2T vectors and mouse hybridoma cells were kindly provided by the Commonwealth Scientific and Industrial Research Organization (CSIRO, Livestock Industries, Victoria, Australia).