Extended Data Fig. 8: Recombinant antibody cloning, isotype switching and mutation of antibody effector recruitment sites of the anti-IFX 8E12 hybridoma.

a, The rearranged variable light and heavy regions of the anti-IFX 8E12 monoclonal antibody were amplified and assembled by fusion PCR using a ‘joining’ fragment before being subcloned into a mammalian protein expression plasmid containing the mouse IgG2a heavy chain. Twelve of fifteen colonies expressed functional anti-IFX antibodies and three selected clones contained identical V_H and V_L sequences. The 8E12–IgG2a antibody was produced by transfection of HEK293 cells. For uncropped gel images see Supplementary Fig. 1. b, The binding affinity of the 8E12 monoclonal antibody for IFX is unaffected after isotype switching. The biophysical binding parameters of the 8E12 monoclonal antibody for IFX were determined by SPR as both the hybridoma-expressed IgG1 (left) and recombinant IgG2a (right). Serial dilutions of the purified complete ectodomain of IFX were injected for two minutes over the biotinylated antibodies immobilized on a streptavidin-coated sensor chip and left to dissociate. Equilibrium binding constants were calculated by fitting the binding data to a Langmuir binding isotherm and found to be essentially equivalent. c, Mutation of the C1q and FcR recruitment sites on the 8E12–IgG2a heavy chain. The specified mutations that are known to abrogate binding to either C1q or FcR were made on the recombinant 8E12–IgG2a plasmid using site-directed mutagenesis. Mutations were made individually (ΔC1q and ΔFcR) and together (ΔC1qΔFcR). Each of the three mutant antibodies were expressed, purified and IFX-binding activity normalized to the parent 8E12–IgG2a and 8E12–IgG1 by ELISA.

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