( or TRIIs-Fc: It has previously been sh...)Use of FlashPlate Technology for In Vitro Measurement of 125I-Labeled TGF-1 Binding on Chimeric Extracellular Domain of Type II Transforming Growth Factor Receptor

or TRIIs-Fc: It has previously been shown that TGF-1 and TGF-3 bind with much higher affinities than TGF-2 to TRII. To identify the selectivity of hTRIIs-Fc, TGF- isoforms were compared in competition experiments with ¹²⁵I-labeled TGF-1 binding in the Protein A FlashPlate assay, where ¹²⁵I-labeled TGF-1 (final concentration 500 pM) was mixed with a serial dilution of recombinant TGF- isoforms. Figure 5 shows the displacement curve of three different TGF- isoforms: TGF-1, TGF-2, and TGF-3. The relative inhibitory concentration (IC₅₀) was determined as the TGF- concentration which inhibited 50% of ¹²⁵I-labeled TGF-1 binding. The IC₅₀ values obtained in this test were 3.3 + 0.06 nM and 3.9 + 0.09 nM for TGF-1 and TGF-3, respectively, whereas TGF-2 was unable to compete, even at a concentration of 200 nM, with the binding of 500 pM of ¹²⁵I-labeled TGF-1 to recombinant TRIIs-Fc. These results indicated that the selectivity for binding TGF- isoforms was maintained by this chimeric recombinant receptor.

Discussion

In this study, we have analyzed the binding properties of recombinant hTRIIs-Fc for TGF-1 using FlashPlate technology. For our experiments, the hTRIIs-Fc component was coated onto the wells of Protein A FlashPlates and incubated with ¹²⁵I-labeled TGF-1. The key feature of this Protein A FlashPlate binding assay, as described here, is its simplicity comprising only the addition of the ¹²⁵I-labeled TGF-1, incubation, and measurement. Unlike separation assays, this assay does not disturb the equilibrium, while the CPM obtained are directly correlated to the bound ¹²⁵I-labeled TGF-1. In our assay, we have determined a counting efficiency of about 5%; this result may be explained by the increased distance between ¹²⁵I-labeled TGF-1 and solid scintillant, due to the coating of protein A and hTRIIs-Fc on t
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