Table 3.

Predictions of the impact of avidity on CFCA data

Parameter values used in simulations								Concentrations
ka1 (M−1s−1)	ka2 (RU−1s−1)	kd1 (s−1)	kd2 (s−1)	Rmax (RU)	Flow rates (μL/min)	kt at 1 μL/min (RU*M−1s−1)	Conc. (nM)	Expected (nM)	Found (nM)
7.59*103	5.06*10−4	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
7.59*103	5.06*10−3	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
7.59*103	5.06*10−5	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
8.65*104	5.77*10-3	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
8.65*104	5.77*10−2	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
8.65*104	5.77*10−4	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
1.48*106	9.87*10−2	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.1
1.48*106	9.87*10−1	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.0
1.48*106	9.87*10−3	1*10−2	1*10−2	2000	5 and 100	4.88*108	1, 2, and 4	4.0	4.1

Avidity was simulated with the bivalent analyte model and re-evaluation of data with the CFCA model (see the Supplementary data for rate equations) using BIAevaluation software v4.1.1. The second association rate constant reflects a binding event that occurs on the immobilized ligand. For each first association rate constant, three second association rate constants were tested to probe cooperative binding events. Low concentrations were used to ensure low ligand occupancy