Mass Photometry to Study Antigen-Antibody Interactions on a Single-Molecule Level

To detect antigen-antibody interactions using mass photometry, first, incubate the desired concentration of purified antigens and antibodies for the required period of time, facilitating antigen-antibody binding. Based on the stoichiometry of antigen binding, the paratope regions of these antibodies can have varying numbers of attached antigens.

Flow in the solution containing the free antibodies along with the antigen-antibody complexes through a pre-assembled flow chamber. Secure the chamber onto the microscope stage of a mass photometer. Then, direct the laser beam through the flow chamber for optimum imaging.

Depending upon the number of attached antigens to each antibody, the molecular mass of the complexes changes. These variations in mass cause the complexes to scatter light differently.

Complexes with higher molecular masses, where two antigens are bound to both paratopes of a single antibody, scatter more light compared to single antigen-containing complexes. In addition, free antibodies show the least amount of light scattering.

A detector collects the scattered light and measures its frequency and intensity. The photometer records fluctuations in the scattered light as an interference pattern, representing this as spots.

Faint spots correspond to free antibodies. In contrast, very dark spots represent complexes with two antigens, and the less dark spot contains one bound antigen.

The presence of dark spots conforms to the antigen-antibody interactions.

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 file name 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 functions. Double-click to indicate the approximate peak positions on the distribution plot, and click Open NLFit.

Check the Fixed checkboxes for the "xc" 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.