( I-labeled TGF-1 (Specific activity 30...)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

I-labeled TGF-1 (Specific activity 3,000-4,500 Ci/mmole, NEX267) was diluted in binding buffer just prior to its addition into the wells. The final concentrations of ¹²⁵I-labeled TGF-1 ranged from 50 pM to 2.5 nM. The plates were incubated at room temperature for two hours, then sealed and counted on a Packard Top Count Microplate Scintillation Counter. A one minute counting period was used. All determinations were carried out at least in duplicate. Nonspecific binding was determined with an excess of unlabeled TGF-1 (100-fold). The counting efficiency was determined through the use of 125I-labeled IgG directly coated into Protein A FlashPlates. The cpm obtained at equilibrium, divided by the dpm added to the well, gave a measure of the counting efficiency, which was evaluated at 5%. For the competition binding assay, the final concentration of radiolabeled ligand was 500 pM (specific activity 300-450 Ci/mmole), with final concentrations ranging from 0.05 to 200 nM for TGF-1, TGF-2 and TGF-3.

Binding of ¹²⁵I-labeled TGF-1 to cell: Ligand binding of cell monolayers with ¹²⁵I-labeled TGF-1 (20 pM to 1 nM) were carried out as previously described³, then scatchard plotted with values obtained after gamma scintillation counting. Scatchard experiments were performed on Cos-1 cells (American Type Culture Collection CRL1650) transiently transfected either with the TRII (pCMV5 TRII), or with both TRI and TRII containing plasmids (pCMV5 TRI + pCMV5 TRII), and compared to Cos-1 cells transfected with the empty pCMV5 (Invitrogen) in order to measure the nonspecific binding.

Results

Comparative studies on binding of ¹²⁵I-labeled TGF-1 to human TRIIs-Fc and wild type TRII: Ligand binding activity of the recombinant hTRIIs-Fc receptor was tested in a Protein A FlashPlate binding assay. Protein A precoated plastic surface
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