Generation of Anti-Human MDA5 Antibodies

Full-length human MDA5 cDNA (GenBank Accession No. AF095844) with a 6X His tag, GST, and turbo 3C protease cleavage site of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro at the N-terminus was subcloned into the pPSC8 expression vector (Protein Sciences Corporation, Meriden, CT, USA); subsequently designated as pPSC8/human MDA5. Recombinant human MDA5 protein was isolated from SF9 cells co-transfected with baculovirus AcNPV and pPSC8/human MDA5. To generate antiserum, specific pathogen-free (Japanese White) rabbits were immunized with recombinant human MDA5 protein Purified antibody was generated from the antisera using a protein G column (Cytiva, Tokyo, Japan), as reported previously [22 (link), 23 (link)]. An anti-human MDA5 monoclonal antibody (mAb) clone H27 (mouse IgG1) and H46 (mouse IgG2b) was established by fusing mouse myeloma cell line X-63·Ag8/653 with spleen cells isolated from a BALB/c mouse immunized with the recombinant human MDA5 protein, as reported previously [22 (link), 23 (link)].