Genes encoding Ig VH, Ig Vκ and Vλ from positive wells were recovered using RT-PCR (210210; QIAGEN). Nested PCR (203205; QIAGEN) was then performed to amplify genes encoding γ-chain, λ-chain and κ-chain with ‘cocktails’ of primers specific for human IgG. PCR products of genes encoding heavy and light chains were joined with the expression vector for human IgG1 or immunoglobulin κ-chain or λ-chain (gifts from H. Wardemann) by Gibson assembly. For the expression of antibodies, plasmids encoding heavy and light chains were co-transfected into the 293T cell line by the polyethylenimine method (408727; Sigma), and antibody-containing supernatants were harvested for further characterization.

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