Table 2.

Binding evaluation of the scFv mutants to 3 by SPR spectroscopy.^[a]

	CDR	Mutant	KD [m] equilibrium[b]	KD [m] koff/kon[a]	kon [m−1 s−1]	koff [s−1]
1	L1	Ser31Ala	NB[c]	NB	–	–
2	H1	Trp33Ala	NB	NB	–	–
3	L1	Asn34Ala	NB	NB	–	–
4	H3	Phe95Ala	NB	NB	–	–
5	H3	Asn97Asp	NB	NB	–	–
6	H3	Tyr98Ala	NB	NB	–	–
7	H1	His35Ala	<103	–	–	–
8	H1	Asn58Ala	<103	–	–	–
9	H2	Ser50Ala	<103	–	–	–
10	H3	Tyr98Phe	1.8 ± 0.21×10−6	1.85×10−6	2.66×103	4.91×10−3
11	L1	Asn34Asp	4.02 ± 4.2 ×10−6	1.85×10−6	40	1.21×10−4
12	L2	Tyr50Ala	7.7 ± 0.85 ×10−6	7.40 ×10−6	56.7	4.35×10−4
–	–	wild-type scFv	5.6 ± 0.05×10−7	5.0×10−7	3.86×103	3.56×10−3

^[a]K_D was calculated from k_off/k_on values obtained from BIAcore.

^[b]K_D was calculated from equilibrium binding analysis, Equation (1), Figure S3 in the Supporting Information.

^[c]Non-binding.