BINDING ASSAY

Abstract

What has been developed is an improved method of determining the binding constants between two molecules that requires significantly fewer materials and potentially less time (in the case of a whole cell analysis less time to grow cell cultures as fewer cells are required in the same analysis) to undertake. The method involves utilizing an NSB measurement in preferably an n-curve analysis in order to determine the K.sub.d and/or R.sub.t without having to complete actual measurements to determine the lower knee of the curve(s) in the n-curve analysis. Preparing the experiment utilizing the additional samples allows an experiment represented in a binding curve having an upper and a lower knee to no longer be required to run through the lower knee to a point of completion.

Claims

1. A method for determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B, wherein said first binding partner and said second binding partner form complex AB, wherein said method comprises the following steps: the step of providing at least a first solution comprising molecule B and at least a second solution comprising molecule B; the step of providing a first NSB solution, wherein said first NSB solution is free of molecule B; the step of adding a known amount of molecule A to said first solution and adding a known amount of molecule A to said second solution and incubating said first solutions and said second solution after adding said molecule A; the step of measuring the signal of free molecule B in said first solution and said second solution after allowing said first solutions and said second solutions to incubate; the step of measuring the signal of free molecule B in said first NSB solution; and the step of calculating the binding constants for the molecular interaction between said molecule A and said molecule B wherein said binding constants are calculated by comparing a function comprised of the binding constants between molecule A and molecule B, the concentration of molecule B in said first solution and said second solution, the concentration of molecule A added to each of said first solution and said second solutions, the signal of the first solution and the second solution at 100% free molecule B (Sig100), and the signal of the non specific binding (NSB) from said first solution and said second solution by varying the actual and/or theoretical values of the concentration of free or bound molecule B, Sig.sub.100, NSB, and the binding constants to the obtained results to obtain the binding constants of best fit in said calculation, wherein said step further comprises utilizing the signal measured for free or bound molecule B in said first NSB solution to define the signal from solutions 1 and 2 in the presence of saturating quantity of molecule A.

2. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said step of providing a first solution comprising molecule B and a second solution comprising molecule B comprises providing a plurality of first solutions comprising molecule B and a plurality of second solutions comprising molecule B, wherein said plurality of first solutions of molecule B comprise the same known concentration of molecule B, wherein said plurality of second solutions comprise the same known concentrations of molecule B, wherein said concentration of molecule B is non-identical in said first solution and said second solution.

3. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 2 wherein the step of adding a known amount of molecule A to said first solution and a known amount of molecule A to said second solution comprises adding varying known amounts of molecule A to each of said first solutions in said plurality of first solutions and varying known amounts of molecule A to each of said second solutions in said plurality of second solutions and incubating said plurality of first solutions and said plurality of second solutions after adding said molecule A.

4. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said molecule A or said molecule B comprises a molecule expressed on a surface of a cell.

5. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said step comprising incubating said first solution and said second solution comprises incubating said first solution and said second solution until the molecular interaction between molecule A and molecule B in each of said solutions reaches equilibrium.

6. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said step of calculating the binding constants for the molecular interaction between said molecule A and said molecule B by including the signal measured for free molecule B comprises utilizing said signal of free molecule B from said first NSB solution as an actual NSB measurement in analyzing the signals from said first and second solutions.

7. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said method comprises the step of adding a second NSB solution, wherein said second NSB solution is free of molecule B, wherein said first NSB solution and said second NSB solution are identical to said first solution and said second solution, respectively, but for said first NSB solution and said second NSB solution are free of molecule B; and wherein said step of calculating said binding constants comprises utilizing the signal of free molecule B from said first NSB solution and said second NSB solution as the actual NSB value for the first solution and the second solution, respectively.

8. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said step of calculating binding constants comprises utilizing said signal measured from said first NSB solution as a data point to compare to the theoretical NSB value to determine an NSB value of best fit in calculating said binding constants.

9. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 8, wherein said method comprises the step of providing a second NSB solution free of molecule B, wherein said second NSB solution comprises an identical solution to said second solution but for said second NSB solution is free of molecule B, wherein said first NSB solution comprises an identical solution to said first solution but for said first NSB solution is free of molecule B, wherein said step of calculating binding constants comprises utilizing said signal measured from said first NSB solution as a data point to compare to the theoretical NSB value to determine an NSB value of best fit for said first solution, and wherein said step of calculating binding constants comprises utilizing said signal measured from said second NSB solution as a data point to compare to the theoretical NSB value to determine an NSB value of best fit for said second solution.

10. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 1, wherein said step of calculating the binding constants for the molecular interaction between said molecule A and said molecule B further comprises utilizing the signal measured for free molecule B in said first NSB solution to define a lower plateau of measurement lacking free molecule B, wherein the NSB is calculated by assigning a significantly greater concentration of molecule A than was added to said solutions in said step of adding a known amount of molecule A to said first and second solutions to simulate a solution in which 100% of molecule B is bound to molecule A.

11. The method of determining binding constants for a molecular interaction between a first binding partner A and a second binding partner B of claim 10, wherein said step of providing a first NSB solution comprises providing at least a second NSB solution, wherein said step of calculating the binding constants further comprises utilizing the signal measured for free molecule B in said NSB solutions to define a lower plateau of measurement lacking free molecule B, wherein the NSB is calculated by assigning a significantly greater concentration of molecule A than was added to said solutions in said step of adding a known amount of molecule A to said first and second solutions to simulate a solution in which 100% of molecule B is bound to molecule A

Description

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1.1 is a table of a sample of binding signal obtained from free receptor after a series of solutions of ligand receptor of uniform concentration have been combined and allowed to equilibrate with a series of solutions with varying receptor concentration.

[0033] FIG. 1.2 is a graph of the information provided in table 1.1 FIG. 2.1 is a chart of theoretical signals and associated error calculated from the actual signal recorded in FIG. 1.1.

[0034] FIG. 2.2 is a graph of the theoretical and actual signals from FIG. 2.1.

[0035] FIG. 3.1 is a table illustrating further iterative analysis of the data presented in FIG. 2.1.

[0036] FIG. 3.2 is a table illustrating further iterative analysis of the data presented in FIG. 2.1 and resulting in a line graph presenting the lowest RMS error of the combined theoretical data.

[0037] FIG. 3.3 illustrates the results of the theoretical data with the lowest RMS error.

[0038] FIG. 4.1 illustrates the first step in plotting an error curve for the Kd data obtained in the iterative analysis described above.

[0039] FIG. 4.2 illustrates the data of the first K.sub.d value plotted in FIG. 4.1.

[0040] FIG. 5.1 illustrates the actual data versus theoretical signal curve derived from the data in FIG. 3.1 but in which the K.sub.d value has been varied to provide a theoretical error percent.

[0041] FIG. 5.2 illustrates the plotting of the second K.sub.d value on the K.sub.d error graph of FIG. 4.1.

[0042] FIG. 5.3 illustrates a second K.sub.d value plotting the error graph as shown in FIG. 5.2.

[0043] FIG. 6 illustrates a K.sub.d value error graph for determining a range in which the K.sub.d has been determined to exist and the error associated with the values in that range.

[0044] FIG. 7 illustrates an example determination of a K.sub.d for a single curve analysis and the determination of the K.sub.d and R.sub.t from the data depicted in the line graph. The K.sub.d has been resolved with reasonable certainty while the R.sub.t has not been resolved with reasonable certainty as evidenced by the lack of curvature in the left side of the graph.

[0045] FIG. 8 illustrates an example determination of a K.sub.d for a single curve analysis and the determination of the K.sub.d and R.sub.t from the data depicted in the line graph. The R.sub.t has been resolved with reasonable certainty while the K.sub.d has not been resolved with reasonable certainty as evidenced by the lack of curvature in the left side of the graph.

[0046] FIG. 9 illustrates a dual curve analysis and the ability of the dual curve analysis to resolve both the R.sub.t and the K.sub.d.

[0047] FIG. 10 illustrates a dual curve whole cell analysis in which measurements have been obtained for data through both knees of the curve.

[0048] FIGS. 11.1-11.2 illustrate the data measurements obtained and displayed in the graphed curves of FIG. 10.

[0049] FIG. 12 illustrates a theoretical dual curve whole cell analysis in which the experiment has not run through the second knee in the curve. For illustration purposes the data are the data utilized in FIG. 10 but with data through the second knee of the curve ignored in the analysis. The error curves illustrate that neither the K.sub.d nor the R.sub.t of the receptor on the cell surface can be determined without obtaining a full curve.

[0050] FIGS. 13.1-13.2 illustrate the data plotted in the dual curve analysis of FIG. 12 illustrating the data from the second knee are ignored in the analysis.

[0051] FIG. 14 illustrates the dual curve whole cell analysis depicted in FIGS. 10 and 12 but in which a theoretical third solution has been measured to provide an NSB measurement utilized according to the inventive concept of this application. The use of the data point has allowed the K.sub.d and R.sub.t to be resolved.

[0052] FIGS. 15.1-15.2 illustrates the dual curve analysis data used in FIG. 14 but with NSB points 96, 97 utilized in the graph of FIG. 14 and associated analysis.

[0053] FIG. 16 further illustrates the dual curve analysis data of FIG. 14 but with an additional data point utilized that allows further resolution of the K.sub.d and R.sub.t.

[0054] FIGS. 17.1-17.2 illustrates the dual curve analysis data used in FIG. 16 but with additional actual data points 99, 101 considered in the analysis.

DETAILED DESCRIPTION OF THE FIGURES DEPICTING THE INVENTIVE CONCEPT

[0055] An example of the concept described in the above Background section is provided in FIGS. 1.1-9.

[0056] FIGS. 14-17.2 depict a whole cell dual n-curve analysis according to the above experimental description but in which the lower knee of each curve has not been determined through experimental measurement and subsequent iterative analysis as described above. Instead, an NSB measurement taken from a solution free of the receptor has been utilized in the iterative analysis described above. Utilizing the NSB measurement the analysis was able to resolve the curves 85, 86 as though actual measurements had been taken through the entire curve range and lower knee of the curve without actually having to utilize the full range of materials and time needed to conduct the experiment through these values. The analysis provided for a resolved K.sub.d 81 and R.sub.t 83 both with reasonable certainty and comparable to the K.sub.d and R.sub.t determined in FIG. 12. The benefit of this analysis is it allowed far fewer cells to be utilized in the experimental analysis. As illustrated in FIGS. 11.1 and 11.2, the experiment to determine full curve range required the experiment to proceed through a curve point having a cell concentration of 30.0M Cells/mL 65, 67. In contrast, experiments depicted in FIGS. 14-17.2 depict that it is thought that the experiment only need to run through a point to obtain the upper curve and including an NSB point in the analysis allows the analysis to resolve the full curve with a reasonable certainty.

[0057] The point to which the experiment ran in FIGS. 14 and 16 went through 234.38 k cells/mL 95, 97 or 468.75 k cells/mL 99, 101. This data shows that only approximately 0.8% or 1.6%, respectively, of the total number of cells was needed to resolve the experiment by including the measurement of the first NSB solution free of molecule B (which in this case was the ligand) and still obtaining a K.sub.d value 81 within reasonable certainty and R.sub.t within reasonable certainty 83. This data represent a potential huge savings in time and resources required to grow a cell culture (far fewer cells can be used in the new analysis), potential large monetary savings from a researcher having to purchase far fewer cells, and/or large savings in non-whole cell analysis in which either or both of the binding molecules are expensive, precious, or both. The data further represent the ability to measure a system in which the concentrations needed to reach the second knee cannot be achieved because of the low expression level of a molecule on a cell surface and/or weak interaction between the molecules.