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The specificity with which a radioligand interacts with a receptor is typically determined by competition studies. In these studies, membrane preparations or cells are incubated with a constant concentration of radioligand and increasing concentrations of unlabeled competitors. For any given receptor, detailed competition studies should be performed to demonstrate that the ligand selectivity expected for the receptor is exhibited for various relevant receptor agonists and antagonists. Generally, competition binding data are plotted as specific binding vs the log₁₀ of the concentration of competitor. Again, the potency of a competing compound to inhibit radioligand binding should correlate with the ability to promote (if it is an agonist) or inhibit if it is an antagonist) the appearance of a physiological response associated with the receptor.

determines the concentration of the competing compound that reduces radioligand binding by 50%. This concentration is referred to as the IC₅₀.The IC₅₀can be determined by nonlinear regression analysis using a suitable computer software package such as Prism (GraphPad, San Diego). After an IC₅₀value has been determined, the equilibrium dissociation constant for the competitor can be determined using the Cheng-Prussof equation:

Figure 41: Saturation binding of [125I]IL-8 to human neutrophil membranes. Assays contained 30 mg of membranes and incubations were performed for 60 minutes at room temperature. Data points represent the mean+/−SEM of triplicate determinations at each [125I]IL-8 concentration.

where K_DI=equilibrium dissociation constant for competitor, I and K

D=equilibrium dissociation constant for radioligand

A major assumption for use of the Cheng Prussof equation is that [R]« KD of the radioligand such that [*D] added=[*D] free. However, in certain cases the amount of added radioligand that binds to receptor may be greater than 5%. This could easily be the case in assays in which receptor concentration within the membrane is high and radioligand concentration added is of a similar magnitude. An alternative equation has been proposed by Linden (1982) for this situation in which greater than 5% added radioligand is bound:

Where R_TOT=calculated concentration of binding sites

It is important to carefully evaluate the shape of competition curves during assay development and validation. For competition in which a single population of receptors is involved, the competition curve will proceed from 10% to 90% inhibition of radioligand binding over a 81-fold concentration of the competing compound. Curves that are more shallow and proceed from 10% to 90% over a greater than 81-fold range of competitor, have a more shallow shape which may be indicative of either negative cooperativity or interaction with multiple populations of binding sites. Conversely, a competing compound that reduces radioligand binding from 10% to 90% in less than a 81-fold concentration range, would have a steeper shape indicating possible positive cooperativity. One parameter commonly calculated to quantitate competition curve shape is the pseudo-Hill coefficient. Computer software packages such as Prism (GraphPad, San Diego, CA) can be used to calculate this coefficient. A normal steepness competition curve will have a pseudo-Hill coefficient of 1, a shallow curve will have a pseudo-Hill coefficient of less than 1, and a steep curve will have a pseudo-Hill coefficient greater than 1. It is important to stress that incubations during competition experiments must be sufficient not only for radioligand binding to achieve equilibrium but also sufficient for the competing compound to reach equilibrium. If this condition is not met, deviations from the normal steepness competition curve may occur (Ehlert et al., 1981).

Several competition experiments were performed during development of the [125_I]IL-8 assay. Data from three experiments were pooled and the results are shown in Figure 42. Competition data were fit best to a one site competition equation using the nonlinear least-squares program Prism. The reduction in radioligand binding occurred over approximately two log units as expected for a simple one-site model of binding. An IC₅₀ value of 1.23+/−0.2 nM was determined which is in close agreement with saturation data. Several other cytokines were evaluated for their ability to compete for binding [125_I]IL-8 to neutrophil membranes. Saturating concentrations of IL-3, IL-5, IL-6, GM-CSF, FMLP, and C5a had no significant influence on [125_{I]IL-8 binding as expected (data not shown).}

Since the IL-8 receptor is a member of the seven transmembrane receptor superfamily, agonist binding should be reduced by addition of exogenous guanine nucleotides. Addition of the nonhydrolyzable GTP analog, GMP-PNP, was observed to reduce [125_I] IL-8 binding to human neutrophil membranes. However, only 60% of agonist binding was reduced suggesting that a subpopulation of the IL-8 receptor may not couple to G- protein or that membranes contain a larger population of receptors than G-protein (Figure

43). Similar effects of guanine nucleotides on [125I]IL-8 binding to human PMN membranes have been reported in the literature (Barnett et al, 1993).

Figure 42: Competition of IL-8 for [125I]IL-8 binding to human neutrophil membranes. 1 nM [125I]IL-8 and 30 µg of membranes were present and incubated for 60 min at room temperature in the presence of indicated concentrations of IL-8. Data points represent the mean+/−SEM of data from three separate experiments in which triplicate determinations were made at each IL-8 concentration.

Similar to most radioligand binding assays, the IL-8 binding to human neutrophil membranes was not adversely affected by high concentrations of DMSO. A final assay concentration of up to 10% DMSO had no significant influence on [125_{I]IL-8 binding} (data not shown).