Antibodies are routinely used in laboratory techniques and their therapeutic and diagnostic applications are quickly expanding. Fast and accurate antigen-antibody binding characterization is critically important for these applications. Mass photometry quickly measures binding affinities using extremely small amounts of material.
No labeling or immobilization is required and information on protein oligomerization and purity can be obtained from the same experiment. This protocol can be used not only to study antibodies, but also to measure strong protein-protein bindings for proteins with a molecular mass larger than 50 kilodaltons. Begin by using soft-tip forceps and wash bottles to sequentially rinse 24 by 50 millimeter coverslips from top to bottom with distilled water, ethanol, distilled water, isopropanol, and distilled water.
After the last wash, dry the coverslips with a stream of clean nitrogen in the same direction to avoid transferring contamination from the forceps. Rinse 24 by 24 millimeter coverslips with distilled water and ethanol as demonstrated, followed by drying with a stream of clean nitrogen. After drying, place a 24 by 24 millimeter coverslip onto a piece of aluminum foil and place strips of double-sided tape onto the coverslip.
Then cut the 24 by 24 millimeter coverslip out of the foil and gently press the foil onto the working side of the 24 by 50 millimeter coverslip. To prepare antibody-antigen samples for the affinity measurements, filter at least two milliliters of PBS through a 0.22 micron syringe filter to remove dust particles and aggregates, and centrifuge the antibody and antigen stocks of interest. Measure the 280 nanometer UV absorbance of the antibody and antigen stocks to determine their actual concentrations, and prepare a titration series of antibody-antigen mixtures at the appropriate range of antigen concentrations in a final volume of 50 microliters per sample.
Then incubate the antigen-antibody mixtures for approximately 10 minutes at room temperature to allow the binding reaction to reach chemical equilibrium. To assess the antibody-antigen affinity by mass photometry, apply a drop of microscope immersion oil to the instrument objective and place the flow chamber onto the stage of the microscope. Add 10 microliters of filtered PBS to one end of the flow chamber channel.
The buffer will enter the channel by capillary action. In the focus control tab of the data collection software, use the coarse stage movement up and down buttons to make the initial adjustments and click sharpness to view the sharpness signal readout. Use the fine up and down adjustment buttons to maximize the sharpness value and click set focus and lock focus to activate the focus tracking function.
An image of a clean slide should have a signal value equal to or below 0.05%Load 20 microliters of the first antibody-antigen sample as demonstrated, blotting the liquid from the other end with a small piece of blotting paper. Immediately upon loading, click record to acquire the appropriate number of frames equivalent to 100 seconds of data. At the end of the data collection period, enter a filename for the data and click OK, then sample to save the file.
To analyze the mass photometry data, open the file of interest and click analyze. Click load to load the calibration function and click file and save results as to save the analyzed data. To obtain the relative concentrations of each species in the sample, open the eventsfitted.
csv file, copy the data in column M into the appropriate plotting and analysis software, and use the plot statistics histogram function to plot the molecular mass distribution. Double-click on the histogram to open the plot properties window, disable the automatic binning, and select a bin size of 2.5 kilodaltons. To create the bin centers and counts data, click apply and go.
To fit the histogram with Gaussian functions, select the bin centers and counts columns and click the analysis peaks and baseline multiple peak fit menu function. Double-click to indicate the approximate peak positions on the distribution plot and click open NL fit. Check the fixed checkboxes for the XC peak centers and set their values to the expected molecular masses of the free antibody and the single and double antigen-antibody complexes.
Check the share option for the width parameters and click fit. The fitted peak height values of the Gaussian components represent the relative concentration of each species in the sample. Then use the equation to calculate the concentration fraction of each species from the peak height values.
To calculate equilibrium constants using a spreadsheet program, open the dissociationconstantcalculation. xlsx worksheet. In this worksheet, the yellow highlighted cell values in rows 1 to 10 can be modified to perform the equilibrium constant calculations.
Enter the estimated dissociation constant values in nanomolar units into cells B1 and B2.If the estimated dissociation constant values are not known, leave the default values in cells B1 and B2 unchanged. Enter the total antibody and total antigen concentrations in nanomolar units into cells D2 and E2 and enter the calculated fraction values for each species into cells F2, G2, and H2.When performing global analysis of the titration, add the appropriate data fraction values into rows 2 through 10. In the solver parameters window, select cell B15 for the set objective box and cells B1 to B2 for the by changing variable cells box.
Select the min button for the to option and check the make unconstrained variables non-negative checkbox, then select GRG nonlinear as the solving method and click solve. The best fit dissociation constant values will be shown in cells B1 and B2.And the final sum of the squared errors will be shown in cell B15. In this representative experiment, a titration series with the anti-human thrombin antibody at a fixed 25 nanomolar concentration and 0, 7.5, 15, 30, 60, and 120 nanomolar human alpha-thrombin concentrations were performed to obtain the molecular mass distributions of the antigen-antibody mixtures and the antibody only sample.
Best fit peak height parameters of the Gaussian components were normalized to obtain species concentration fractions. The concentration fraction values were then fit globally to obtain the antigen-antibody binding affinities. The experimental concentration fractions and the global fit results for the free antibody, single antigen-antibody complex, and double antigen complex could then be plotted.
To obtain accurate KD values, protein concentrations have to be carefully selected to populate all of the reaction species. We recommend preparing a titration series using a range of antigen concentrations. After fitting the data, the error projection method can be used to obtain the fitting error, but it is better to repeat the experiment and calculate the standard deviations of the average binding affinity values.
