TABLE 4.

Affinity and dissociation constants of VHH homodimers and heterodimers

VHH	ka	kd	KD	KD (calculated)a
	1/ms	1/s	m
JJX21	2.0 × 105	7.4 × 10−6	3.84 × 10−11	<7.5 × 10−11
JNA6	4.58 × 105	9.55 × 10−7	2.10 × 10−12	<6.22 × 10−12
JNA2	1.34 × 106	1.69 × 10−5	1.26 × 10−11	NAb
JNA3	3.96 × 105	1.26 × 10−5	3.20 × 10−11	NA
JNA10	4.40 × 105	1.20 × 10−6	2.70 × 10−12	<6.48 × 10−12
JNA11	5.77 × 105	9.66 × 10−7	1.67 × 10−12	<4.94 × 10−12
JNA8	3.43 × 105	1.47 × 10−6	4.27 × 10−12	<2.77 × 10−12
JNA14	7.67 × 105	7.96 × 10−7	1.04 × 10−12	<3.10 × 10−12

^a K_D calculated was calculated using the k_d limit and the k_a measured for those VHHs that did not dissociate from ricin within the k_d limit. In several instances little to no VHH dissociation was detected over a 6-h time course, suggesting that the dissociation rate constants were slower than 2 × 10⁻⁶ s⁻¹ (37). It was therefore not possible to generate a true K_D value. Instead, the K_D was calculated based on the so-called 5% rule that compensates for the limitations of SPR (38). Actual SPR sensorgrams are provided in supplemental Fig. S2.

^bNA, not applicable.