Table 1.

Summary of 24 h, 40 °C oxidations of h2E2 mAb in formulation buffer, using 5 mM AAPH or 5 mM AAPH plus 260 mM Met

Protein	Ligand added	Apo TmD (°C)	Apo TmB (°C)	Kd at TmB (μM)	RBA	Oxidation-induced increase in Kd
Control h2E2 mAb	None	70.33 ± 0.05	70.41 ± 0.03
Control h2E2 mAb	CE			0.0106 ± 0.00596	0.366 ± 0.065	—
Control h2E2 mAb	Cocaine			0.0228 ± 0.0132	(1.00)	—
Control h2E2 mAb	BE			0.1508 ± 0.07686	5.30 ± 1.30	—
AAPH-h2E2 mAb	None	67.97 ± 0.03	67.70 ± 0.03
AAPH-h2E2 mAb	CE			79.7 ± 20.6	0.325 ± 0.05	7519 X
AAPH-h2E2 mAb	Cocaine			242 ± 32	(1.00)	10,614 X
AAPH-h2E2 mAb	BE			946 ± 373	3.81 ± 1.13	6273 X
AAPH/Met-h2E2 mAb	None	68.60 ± 0.03	68.37 ± 0.05
AAPH/Met-h2E2 mAb	CE			72.1 ± 9.0	0.310 ± 0.011	6802 X
AAPH/Met-h2E2 mAb	Cocaine			233 ± 32	(1.00)	10,219 X
AAPH/Met-h2E2 mAb	BE			926 ± 144	4.00 ±0.64	6140 X

mAb samples were analyzed by differential scanning fluorimetry (DSF) in PBS buffer, using the fluorescent DASPMI reporting dye. Data are from three independent experiments, and the values reported are means ± standard deviations. “Apo” denotes no ligand (cocaine or cocaine metabolite) added to the mAb. RBA is the relative binding affinity, assigning the binding affinity for cocaine as 1.0 for each set of samples analyzed using three ligands. T_mD is the temperature at the peak maximum for the derivative of the fluorescence DSF curves, and T_mB is the temperature at the middle of the unfolding transition determined by Boltzmann fitting of the DSF fluorescence data. Affinities (K_d) at the T_mB for each sample are calculated from the DSF data via ΔG values derived from the DSF data, as previously reported (7). The oxidation-induced fold increases in K_d (decrease in affinity) are calculated by dividing the reported average K_d for the oxidized mAb by the average K_d for the control mAb for each of the three ligands, CE, cocaine, and BE.