Protein a agarose chromatography

Antibodies were cloned as previously described^{44 (link)}. In brief, VH, Vκ and Vλ genes were amplified by reverse transcription PCR and nested PCR reactions from singly sorted germinal centre B cells using primer combinations specific for IgG, IgM, IgA, Igκ and Igλ from previously described primer sets^{45 (link)}, and then sequenced. To generate recombinant antibodies, restriction sites were incorporated via PCR with primers to the corresponding heavy and light chain V and J genes. The amplified VH, Vκ and Vλ genes were cloned into IgG1 and Igκ or Igλ expression vectors, respectively, as previously described^{45 (link)–47} . Heavy and light chain plasmids were co-transfected into Expi293F cells (Gibco) for expression, and antibody was purified using protein A agarose chromatography (Goldbio). Sequences were obtained from PCR reaction products and annotated using the ImMunoGeneTics (IMGT)/V-QUEST database (http://www.imgt.org/IMGT_vquest/)^{48 (link),49} . Mutation frequency was calculated by counting the number of nonsynonymous nucleotide mismatches from the germline sequence in the heavy chain variable segment leading up to the CDR3, while excluding the 5′ primer sequences that could be error-prone.