The overall goal of this titration ELISA is to determine the affinity constant of two binding partners in a reproducible and efficient manner using a novel algorithm. This method can help answer key questions not only in the field of protein ligand interaction and receptor research but also in applied life sciences such as pharmaceutical research and pharmacology. The main advantage of this technique is that the affinity constant of a receptor ligand interaction can be determined easily by titrating a receptor with a ligand on an ELISA plate and by using a novel evaluation algorithm.
Begin this procedure by diluting the stock solution of Integrin Alpha 2 A Domain to a final concentration of five micrograms per milliliter in TBS magnesium chloride buffer. Both a wild type and a mutant Integrin Alpha 2 A Domain will be used in this demonstration. Fill each well of a row on a half area microtiter plate with 50 microliters of the Integrin Alpha 2 A Domain coating solution.
Perform every titration row at least in duplicates. In this example, every titration row is performed in quadruplets. Seal the plate with foil and leave the plate at four degrees Celsius overnight.
On the following day, remove the coating solution and fill each well with 15 microliters of TBS magnesium chloride buffer. Remove the wash solution and fill the wells with new wash solution. The washes will remove soluble receptor molecules which have not been immobilized to the plastic surface by physical absorption.
The next step is to block non-specific binding sites by adding 50 microliters of one percent BSA solution in TBS magnesium chloride buffer to each well. Reseal the plate and incubate at room temperature for one hour. While the wells of the microtiter plate are being blocked with BSA, prepare a serial dilution of the ligand Rhodocetin.
A start concentration of 243 enamel-eroded Rhodocetin and a dilution factor of 2.3 are used in this case. For the eight titration curves in this experiment, prepare 920 microliters of Rhodocetin solution at the highest concentration in one per cent BSA in TBS magnesium chloride in test tube number one. Pipette 520 microliters of one per cent BSA solution in TBS magnesium chloride into each of the other ten test tubes labeled two through eleven.
Transfer 400 microliters of the Rhodocetin solution from test tube to test tube two. Mix both solutions by chut-ter-a-tion and then transfer 400 microliters from this mixture to test tube three. Continue this serial dilution until test tube eleven.
For the success of this procedure, it is important to always fill every well quickly with solution to make sure the wells do not dry out. At the completion of the one hour incubation, remove the blocking solution from the microtiter plate wells. Immediately add 50 microliters of the Rhodocetin solution from test tube one into the wells of column one, the solution from test tube two to the wells of column two, and continue through column eleven.
Add 50 microliters of one per cent BSA and TBS magnesium chloride as a ligand-free control to the wells of column twelve. Reseal the plate and incubate at room temperature for one and a half hours. To remove non-bound ligand molecules, remove the binding solution and fill each well with 50 microliters of HBS magnesium chloride buffer.
Then remove the HBS buffer and add new HBS buffer. To chemically fix the receptor and bound ligand, fill each well of the microtiter plate with 50 microliters of a 2.5 per cent glutaraldehyde solution in HBS magnesium chloride buffer. Incubate the microtiter plate at room temperature for ten minutes.
Remove and in inactivate excess glutaraldehyde by removing the fixation solution and filling each well with 50 microliters of TBS magnesium chloride buffer. Remove the wash buffer and add new wash buffer to each well. To begin this assay, remove the wash buffer from all wells of the microtiter plate and add to each well 50 microliters of the primary antibody solution, diluted one to two thousand in one per cent BSA TBS magnesium chloride.
Reseal the plate and incubate it at room temperature. Remove the primary antibody solution from all wells and add to each well 50 microliters of TBS magnesium chloride buffer. Wash three times with TBS magnesium chloride buffer.
Next, add to each well 50 microliters of the secondary antibody solution, diluted one to two thousand in one per cent BSA in TBS magnesium chloride. Reseal the plate, and leave it standing at room temperature. Remove the secondary antibody solution from the wells, and add to each well 50 microliters of TBS magnesium chloride buffer.
Wash three times. After the third wash, tap the microtiter plate onto a tissue cloth to remove all traces of liquid. Using a multi-channel pipette, promptly add 50 microliters of the alkaline phosphatase detecting solution to each well of the microtiter plate to start the enzymatic conversion as simultaneously as possible.
Incubate the plate at room temperature until the solution in the wells with the highest ligand concentration turns yellow. Depending on signal intensity, the incubation time may vary between five minutes and one hour. Stop the conversion of the phosphatase substrate by using a multi-channel pipette to add 50 microliters of a 1.5 molar sodium hydroxide solution to each well in the same order of the alkaline phosphatase detecting solution was added.
Leave the plate for several minutes to ensure streak-free mixing of both solutions. Measure the optical density at 405 nanometers of each well using an ELISA reader. Start the data analysis by opening the table of raw data.
Copy the columns from the Excel file, open Graph Pad Prism Five, open a new project file in the Main Menu and under new data and graph, choose the X Y format. Choose the option Enter and Plot a Single Point for each value for the Y axis and paste them into the data sheet of Graph Pad Prism as X and y values respectively. Transpose the optical density at 405 nanometer values into a column format.
In the data sheet, the first column represents the concentrations as added ligand as X values, while the columns to the right contain the signal values of the eight titration curves as Y values. As a semi-logarithmic plot will be drawn, the last row, which counts the signals of the ligand-free controls, concentration zero, is deleted. Graph Pad Prism draws the plot which is seen in the graph section.
For the X axis, a semi-logarithmic scaling is selected. The X axis shows the signal values measured as OD values at 405 nanometers. By double-clicking any of the symbols, they can be selected and marked by simple shape and color.
The titration values for the wild-type form are highlighted with circles in different red shadings, whereas the titration signals for the mutant form are marked with blue squares. Open the analysis sub-program and under X Y Analysis, choose the option Non-linear Regression and press the New button to create a new equation. Type the titration curve equation into the newly opened template sheet and define the appropriate constraints.
Analyze the values of the data sheet by choosing the newly created user-defined equation. Open the table with the calculated approximation values, which are shown under the results section on the left side of the software screen. In this representative microtiter plate for a Titration ELISA, the yellow color of the converted alkaline phosphatase substrate indicates that the amount of bound-ward ascetant ligand decreases with decreasing concentrations of added Rhodocetin.
The lack of color in the Rhodocetin-free wells indicates a low background signal. A novel algorithm processes the raw data from photometric quantification at 405 nanometers to calculate the signal as a function of the total concentration of added Rhodocetin. The four titration curves for each receptor form while type and mutant are highly reproducible and almost superpose each other.
In contrast, the two groups of titration curves are clearly separated from each other. The titration curves of the mutant alpha 2 A domain are shifted to higher Rhodocetin concentrations, indicating that Rhodocetin binds with lower affinity to the mutated receptor. The dissociation constants of the eight titration curves were plotted and grouped for the wild type and mutant form of the alpha 2 A domain.
The affinity constant for the binding of Rhodocetin to the wild type alpha 2 A domain is 5.80 plus or minus 0.15 nanomolar, whereas Rhodocetin binds to the mutated receptor with a significantly lower affinity of 9.68 plus or minus 0.18 nanomolar. Once optimized for routine, this technique can be done within six to seven hours. While attempting this procedure, it is important to remember that the glutaraldehyde might affect ligand binding to the receptor and might harm the epitopes of the ligand, thereby compromising antibody detection.
This must be tested beforehand. If the equipment for surface plasma resonance is available, the SPR method could be used as an alternative, however, SPR evaluates kinetics data to determine the affinity constant. This Titration ELISA in combination with the novel evaluation algorithm is versatile and can, for instance, be used to compare the affinities of different ligands to analyze structure activity relations and to unravel the molecule mechanism of protein-ligand interactions.
After watching this video, you should have a good understand of how to perform the Titration ELISA and how to implement this novel evaluation algorithm so that the affinity constant for any protein-ligand interaction can be determined easily and routinely, even for multiple ligands simultaneously. Don't forget that working with glutaraldehyde can be hazardous. Therefore, safety measures such as wearing gloves and goggles should always be taken while performing the fixation step of this protocol.
