SPR screening of TNFSF binding to LDVICp016 and ectromelia virus CrmD. Purifed recombinant LDVICp016L-His or CrmD proteins (500 ng) were covalently coupled to CM4 Biacore chips. Potential ligands were injected at a standard concentration of 100 nM each in HBS buffer and passed over the LDVICp016-His or CrmD-containing sensor chip at 25°C at a flow rate of 30 μl/min for a period of three minutes and allowed to dissociate for an additional two minutes before regenerating the surface with a single 10 μl injection of glycine at pH 2.5. As a binding control, anti-penta His antibody (Life Technologies) was injected in the same conditions at the beginning and the end of each experiment. The signal from a reference cell of the same chip with no recombinant protein coupled and that from a blank injection was substracted in order to eliminate non-specific binding signals. Panels show sensorgrams of both chips with a selection of potential ligands of the TNFSF as indicated. Mouse TNFα (mTNFα), zebrafish (Danio rerio) TNF1 (ZfTNFα) pufferfish (Takifugu rubripes) TNFα (FuTNFα), sea bream (Sparus aurata) TNFα (Sb TNFα) and common carp (Cyprinus carpio) TNFα1 (Cc TNFα1), TNFα2 (Cc TNFα2) and TNFα3 (Cc TNFα3).