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Akinetic exclusion assay (KinExA) is a type ofbioassay in which a solution containingreceptor,ligand, and receptor-ligand complex is briefly exposed to additional ligand immobilized on a solid phase.^[1][2]

During the assay, a fraction of the free receptor is captured by the solid phase ligand and subsequently labeled with afluorescent secondary molecule (Figure 1).^[1][2] The short contact time with the solid phase does not allow significant dissociation of the pre-formed complexes in the solution.^[3] Solution dissociation is thus "kinetically excluded" from contributing to the captured receptor and the resulting signal provides a measure of the free receptor in the solution.

Measuring the free receptor as a function of total ligand in a series of equilibrated solutions enables calculation of the equilibrium dissociation constant (K_d).^{[1][2][3][4][5][6][7][8]} Measuring the free receptor with several points beforeequilibrium enables measurement of the association rate constant (k_on). The off rate (k_off) can also be directly measured, however it is usually calculated from the measured K_d and measured k_on, (k_off = K_d * k_on).

Kinetic exclusion assays have been used to measure K_d's in thenanomolar tofemtomolar range.^{[4][5][6][7][9][10]}

Because the fluorescent secondary molecule is applied after capture of the free receptor from solution (Figure 2) the binding constants measured using a kinetic exclusion assay are for unmodified molecules in solution and thus more accurately reflectsendogenous binding interactions than methods requiring modification (typically labeling or immobilization) before measurement.^[1][2] Kinetic exclusion assays have been performed using unpurified molecules,^[4][5] in serum,^[7] and have measured binding tocell membrane proteins on intact whole cell^[8][11] which brings the measured binding interactions closer to their endogenous state.

Molecules suited for measurement by KinExA areantibodies,^{[4][7][12][13][14]}recombinant proteins,^[15][16][17] small molecules,^{[6][18][19][20]}aptamers,^[21][22] lipids,^[23][24]nanobodies,^[25] and toxins.^[12][26][27]

Kinetic exclusion assay have also been applied for concentrationimmunoassay, where it has proven capable of providing the maximum theoretical, K_d limited, sensitivity.^[28][29] An example of this technique has been employed for sensitive detection ofenvironmental contaminants in near real-time.^[30]

A series of samples are prepared with all the same receptor (R) concentration but in which the ligand (L) concentration istitrated. After equilibrium is reached each sample is measured by flowing it through the column (Figure 2).

For 1:1 reversible bindingEquilibrium Kd is defined as

(1)K_d≡k_off/k_on =R*L/RL

the binding is reversible soconservation of mass can be written as

(2)R_T = R+RL

(3)L_T = L +RL

Where:

K_d = equilibrium dissociation constant

k_on = forward rate constant

k_off = reverse rate constant

R = free receptor site concentration at equilibrium

L = free ligand site concentration at equilibrium

RL = concentration of complex at equilibrium

R_T= total concentration of receptors

L_T = total concentration of ligand

A simple equation^[1][2] relating the free fraction of R (=R/R_T) to the K_d and L_T is then fit to the measured data to find the K_d of the interaction.

To measure therate constants, known concentrations of receptor and ligand are mixed in solution and the quantity of free receptor is repeatedly measured over time as the solution phase reaction occurs. The time course of the free receptor depletion is then fit with a standardbimolecular rate equation.

(4)dLR/dt =k_on∙R∙L - K_d∙k_on∙RL

whereK_d * k_on has been substituted fork_off .

• Biochemistry methods
• Laboratory techniques

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