After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines.
Synthetic IRNA was found capable of replacing the radiolabeled 5'UTR portion of GLV IRES from binding to recombinant GlLa protein in a dose dependent manner, whereas a non-specific RNA of similar size did not exert any detectable effect (Fig. 3B).
The FLAG-ECRG1 protein exhibited binding to recombinant His-ECRG4 protein.
To confirm the specificity of FG-3019 towards CTGF, as opposed to the structurally related CCN proteins CYR61 or NOV, binding of FG-3019 to recombinant proteins was compared by immunoblot analysis; FG-3019 was indeed avidly associated with CTGF, but not with CYR61 or NOV. This result was also confirmed by ELISA (data not shown).
The results showed that the recombinant phage D1 clone (with EHWS HDMFSPGD sequence) and phage H1 clone (with EKLK HSM sequence) all inhibited the mAb U001 binding to recombinant urease B protein, whereas wild-type M13 phage had no effect.
Presumptive evidence that CEP-PE induces this effect through binding with TLR2 was provided by the observation that CEP-PE competes with CEP-protein for binding to recombinant mouse TLR2-Fc protein.
FcRn−, Protein A and CD16a-binding to recombinant IgG1-Fc proteins were analyzed by surface plasmon resonance (SPR) spectroscopy using a Biacore 3000 instrument (GE Healthcare, Waukesha, WI).
Plasma IgG levels binding to a recombinant protein representing the 19-kilodalton C-terminal part of merozoite surface protein-1 (MSP-119 Wellcome allele fused to glutathione S transferase) were measured by indirect ELISA, using an assay protocol previously described [20], [26].
The specificity of binding of recombinant proteins to these sequences was observed, however, when increasing amounts of unlabeled probes were added and showed to compete with themselves in the presence of large excess of competitor (Fig. 5D, left and right panels).
This effect may also increase competitive binding of recombinant proteins to histidine-rich contaminant E. coli proteins.
