Determination of hIL-1RI, hIL-1RII, and hIL-1RAcP binding
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A recombinant version of wild type human IL-1β carrying a C-terminal hexahistidine tag and additional cysteine residue (hIL-1β-His-C) was site specifically biotinylated at its free cysteine residue using an EZ-link maleimide-PEG11-biotinylation kit .
- Biotinylated hIL-1β-His-C bound with the same affinity as wild-type human hIL-1β to immobilized recombinant human IL-1I and IL-II Fc chimeras , as shown byELISA using polyclonal goat antihuman IL-1β IgG for detection (data not shown).
- For determination of binding affinities, serial dilutions of nonlabeled wild-type human IL-1β or hIL-1b(145K) were premixed with a constant amount of 20 ng/ml (~1 nmol/l) of biotinylated hIL-1β-His-C and then transferred to ELISA plates ( eBioscience, Vienna, Austria) that had been coated with 1 µg/ml of human IL-1I or human IL-II Fc chimeras .
- Bound biotinylated hIL-1β-His-C was detected with horseradish peroxidase–conjugated streptavidin.
- Binding curves were fitted by four-parameter logistic equations using GraphPad Prism (GraphPad Software, La Jolla, CA), and inhibition data were reciprocally transformed…
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A recombinant version of wild type human IL-1β carrying a C-terminal hexahistidine tag and additional cysteine residue (hIL-1β-His-C) was site specifically biotinylated at its free cysteine residue using an EZ-link maleimide-PEG11-biotinylation kit .
- Biotinylated hIL-1β-His-C bound with the same affinity as wild-type human hIL-1β to immobilized recombinant human IL-1I and IL-II Fc chimeras , as shown byELISA using polyclonal goat antihuman IL-1β IgG for detection (data not shown).
- For determination of binding affinities, serial dilutions of nonlabeled wild-type human IL-1β or hIL-1b(145K) were premixed with a constant amount of 20 ng/ml (~1 nmol/l) of biotinylated hIL-1β-His-C and then transferred to ELISA plates ( eBioscience, Vienna, Austria) that had been coated with 1 µg/ml of human IL-1I or human IL-II Fc chimeras .
- Bound biotinylated hIL-1β-His-C was detected with horseradish peroxidase–conjugated streptavidin.
- Binding curves were fitted by four-parameter logistic equations using GraphPad Prism (GraphPad Software, La Jolla, CA), and inhibition data were reciprocally transformed to express % receptor binding of the respective nonbiotinylated protein.
- For determination of hIL-1AcP binding, ELISA plates were coated with 1 µg/ml of human IL-1I and incubated with 0.4 µg/ml wild-type human IL-1β or 100 µg/ml hIL-1b(145K) that had each been premixed with 1 µg/ml of hIL-1AcP-human Fc chimera .
- The concentrations of wild-type human IL-1β and hIL-1b(D145K) were chosen because based on the results of the IL-1RI binding assay, they were predicted to lead to saturation of all available hIL-1RI binding sites.
- Formation of the ternary complex consisting of hIL-1RI, hIL-1β wild type or hIL-1b(D145K), and hIL-1RAcP was detected by a polyclonal goat anti-hIL-1RAcP antibody and a polyclonal horseradish peroxidase-conjugated rabbit antigoat IgG antibody.
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