Monoclonal Antibody Sequence Determination

RNA was isolated from hybridoma cell lines according to manufacturer's protocol using TRIzol (Invitrogen). Contaminating DNA was then removed from RNA samples using Ambion DNA-free kit (Ambion). cDNA was generated using SuperScript IV Reverse Transcriptase (Invitrogen) and Oligo(dT) primers (Invitrogen) according to manufacturer's protocol. Igh genes were amplified by PCR with the Platinum Taq DNA Polymerase High Fidelity kit (Invitrogen) using a degenerate forward VH primer and a mixture of reverse primers for JH segment primers as previously described (36 (link)). PCR products were purified by gel-electrophoresis and extracted using the QIAquick gel extraction kit (Qiagen). Igh PCR products were expanded using TOPO cloning (Invitrogen) and several clones from each hybridoma amplification were submitted for sequencing (Eton Bioscience Inc.) to ensure hybridoma cell lines were monoclonal. Igh gene segments were identified using the NCBI IgBlast tool.