AF6-88.5.5.3 potently binds to H-2K^b, and 34-1-2S cross-reacts to H-2K^b. Binding affinities of IgG2a anti-MHC class I antibodies AF6-88.5.5.3 and 34-1-2S toward H-2K^b were determined by using SPR. C1.18.4 was used as IgG2a isotype control. (A-C) Binding affinity (K_D) with association–dissociation curves (RU) is shown in which antibodies were spotted in twofold dilutions in duplicate, ranging from 60 nM to 7.5 nM. (D) Association–dissociation curves (RU) of a setup in which AF6-88.5.5.3 was spotted on the sensor, and H-2K^b was allowed to bind first, followed by infusion of either 34-1-2S or C1.18.4. A representative of n = 6 experiments is shown in each figure panel.