Generation of Anti-hIL-33 Monoclonal Antibodies

Nine Sprague Dawley rats (Charles River, Hollister, CA, USA) were immunized with hIL-33 (Genentech, PUR36805) at 100 µg/animal for the first dose and 50 µg/animal for the rest of the boosts, each divided among sites: intraperitoneal, s.c. (subcutaneous) at base of tail, s.c. at nape of neck, and s.c. in both hocks. A Toll-like receptor cocktail including monophosphoryl-lipid A (MPL) (Sigma-Aldrich, St. Louis, MO, USA), Poly (I:C), R848, and cytosine phosphodiester bond guanine (CpG) (InvivoGen, San Diego, CA, USA) was used as adjuvant for the first dose. Antigen-specific hybridomas were generated, sorted and screened as described [38 (link)]. Briefly, Immunoglobulin G (IgG)⁺ hIL-33⁺ hybridomas were single cell sorted using hIL-33 conjugated to PE (Novus Biological, Littleton, CO, USA) into 96-well plates and cultured for 7 days. Supernatants were screened by high throughput (HTP) ELISA using robotic platforms and hybridomas showing binding to hIL-33 by ELISA were scaled-up in 1 mL cultures. Supernatants were harvested and purified using a protein G affinity chromatography resin (GammaBind Plus, GE Healthcare, Pittsburgh, PA, USA). Anti-hIL-33 Abs were submitted for sequencing and cloned out for recombinant production.