Fig. 3.
Binding of soluble type A and type B TCRs to IAg7–peptide complexes parallels the peptide stimulation activities. (A) The affinities of soluble TCRs from two type A T cells (I.29 and PCR1-10) for IAg7 bearing various mutated insulin B:10–22 peptides were evaluated using SPR. Approximately 2,000 RUs of the biotinylated IAg7 peptides were immobilized in flow cells of a streptavidin containing BIAsensor chip. Biotinylated IAg7-HEL was immobilized in a separate flow cell to correct for the fluid phase SPR signal. The indicated concentrations of the soluble TCRs were injected for ∼75 s and the binding and dissociation of the TCRs followed by the SPR signal (RU). Where there was sufficient binding, kinetics (ka, kd, and Kd) were calculated using standard BIAevalution 4 software. B is similar to A, except the soluble TCRs came from two type B T cells (8-1.1 and 8F10). The 8F10 TCR showed second-order binding kinetics best represented by the equation shown. See Discussion.