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

1. Prepare the flow chambers

1. Clean the glass coverslips
   1. Using wash bottles with distilled water, ethanol, and isopropanol, rinse the 24 mm x 50 mm coverslips in the following order: water, ethanol, water, isopropanol, water. Dry the coverslips with a stream of clean nitrogen. It is important to rinse the coverslips from top to bottom, holding the bottom corner with soft-tipped forceps. Dry the coverslip in the same direction to avoid transferring contamination from the forceps (Figure 1A).
   2. Similarly, rinse the 24 mm x 24 mm coverslips with distilled water, ethanol, and distilled water. Dry the coverslips with a stream of clean nitrogen.
   3. Identify the working side of the coverslip, place a drop of distilled water on the surface of the clean coverslip, and follow steps 3.1–3.2 of the protocol. Usually, only one side of the 24 mm x 50 mm coverslip has the optical quality suitable for MP measurements.  
      NOTE: After focusing, no significant surface imperfections should be detectable, and the "signal" value shown in the data collection software should be less than 0.05% (Figure 2A-C). The working sides of all coverslips in the box are oriented in the same direction. The same procedure should be used to test the efficiency of the coverslip cleaning.
2. Assemble the flow chamber
   1. Position the 24 mm x 24 mm coverslip on a piece of aluminum foil. Place strips of double-sided tape on top of the 24 mm x 24 mm coverslip as shown in Figure 1B and cut the tape along the edge of the glass. Separate the coverslip from the aluminum foil and attach it to the working side of the 24 mm x 50 mm coverslip (Figure 1C).   
      NOTE: Channel size can vary, but a width of 3 mm–5 mm is recommended. Wider channels require larger sample volumes and very narrow channels may be difficult to load. Usually, two parallel channels can easily be created on the 24 mm x 24 mm coverslip. Protocol can be paused here.

2. Prepare the antibody-antigen samples for the affinity measurements

1. Filter at least 2 mL of the PBS buffer using 0.22 µm syringe filters to remove dust particles or aggregates. Centrifuge the protein stock for 10 minutes at the maximum speed of the tabletop centrifuge (approximately 16,000 x g).
   NOTE: PBS is the recommended buffer for this protocol, but MP has no particular buffer requirements, and other biological buffers are also acceptable. However, high glycerol concentrations (>10%) and very low ionic strengths (salt concentration <10 mM) may affect the image and data quality and are not recommended.
2. Determine the actual concentrations of the antibody and antigen stocks by measuring their 280 nm UV absorbance.
3. Calculate the measured concentrations of the antigen-antibody mixture. If the estimated value of the antibody binding affinity is not known, plan to prepare a sample with 30 nM antigen and 20 nM antibody concentration. When the approximate affinity is known, the antibody-to-antigen ratio and their concentrations should be optimized according to the expected K_d values. Use the total antigen concentration in the mixture equal to the sum of the expected K_d and the total antibody concentration in the equation below. Assuming K_d1 = K_d2 for the two paratopes of the antibody, this will result in comparable concentrations of the free antibody and the antibody-antigen complexes in the sample.
   
   Adjust the antibody concentration to keep the total protein concentration in the sample within the 10 nM and 50 nM range. Best results are obtained using mixtures with antibody concentrations between 5 nM and 25 nM.
   NOTE: MP detects proteins with a molecular mass larger than 40 kDa. Consequently, sample concentrations of antigens with a molecular mass smaller than 40 kDa can exceed the typical 50 nM limit. However, at concentrations higher than approximately 100 nM, even low molecular mass antigens might affect the image quality and accuracy of the K_d determination.
4. Prepare 50 µL of the antibody-antigen mixture at its final measurement concentration calculated in step 2.3.    
   NOTE: Only one sample of the antigen-antibody mixture is required for the K_d determination. However, preparing several samples with different antigen-to-antibody ratios can help optimize sample concentration. If data from several samples are collected, they can be analyzed via a global fit.
5. Incubate the antigen-antibody mixture(s) for approximately 10 min at room temperature to allow the binding reaction to reach chemical equilibrium. Avoid unnecessarily long incubation times.
   NOTE: Incubation time may vary depending on the binding kinetics. To confirm that the chemical equilibrium has been reached, sample measurements can be repeated at different incubation times. Time-invariant K_d values indicate sufficiently long incubation. Prolonged incubation may lead to significant protein adsorption to the surface of the plastic labware and, consequently, to significant errors in the protein concentration determination. For this reason, low-adhesion labware is strongly recommended for MP sample preparation.

3. Collect the Mass Photometry data

1. Apply a drop of microscope immersion oil on the MP instrument objective and place the assembled flow chamber on the microscope stage. Make sure the oil spans the gap between the coverslip and the objective.
2. Load the flow chamber and focus the mass photometer.
   1. Deposit 10 µL of a clean, filtered buffer solution at one end of the flow chamber channel prepared in step 1. Liquid will enter the channel by capillary action.
   2. Adjust the stage's Z-position to focus the microscope on the working surface of the 24 x 50 mm coverslip.
      1. In the Focus Control tab of the data collection software, use the coarse stage movement Up and Down buttons to make the initial adjustments.
      2. Click the Sharpness button to show the sharpness signal readout and use the fine Up and Down adjustment buttons to maximize the Sharpness value.
      3. Click the Set Focus and Lock Focus buttons to activate the focus tracking function. A properly focused image (Figure 2A, C) should have a "signal" value below 0.05%.
         NOTE: If the "signal" value at the maximum sharpness position is above 0.05%, this may indicate impurities on the glass surface or in the buffer.
3. Using the same channel, load 20 µL of the antibody-antigen sample by depositing it on one side of the channel and blotting the liquid from the other end with a small piece of blotting paper (Figure 1D).
   NOTE: The volume of a 3–5 mm wide channel is approximately 10 µL. The additional sample volume is recommended to completely replace the buffer present in the channel and to avoid sample dilution.
4. After loading the sample, immediately click the Record button to start data collection, acquiring a 100 s video (Figure 2D).
5. At the end of the data collection enter the file name and click OK to save the data file.
6. Discard the coverslips and wipe the oil from the objective lens with cotton optical swabs wet with isopropanol.
   NOTE: The protocol can be paused here.

4. Analyze the MP data

1. Process the collected video file using the MP data processing software to identify the landing events.
   1. Use the File/Open menu option to load the file for the analysis and click Analyze.
   2. Click the Load button to load the calibration function and save the analyzed data using the File/Save Results As menu option.
2. Fit the molecular mass distribution with Gaussian functions to obtain relative concentrations of each species in the sample. This analysis can be performed using a common scientific graphing software (see Table of Materials).
   1. Import the "eventsFitted.csv" file into the software and plot the molecular mass distribution (column M in the .csv file) using the Plot/Statistics/Histogram function.
   2. Double-click on the histogram to open the Plot Properties window. Disable automatic binning and select a bin size of 2.5 kDa. Click the Apply and the Go buttons to create the Bin Centers and Counts data.
   3. Select the Bin Centers and Counts columns and use the Analysis/Peaks and Baseline/Multiple Peak Fit menu function to fit the histogram with Gaussian functions. Double-click to indicate the approximate peak positions on the distribution plot and then click the Open NLFit button.
   4. 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 Compartir option for the width parameters. Click the Fit button. The fitted peak height values of the Gaussian components represent the relative concentration of each species in the sample. 
      NOTE: Bin size may be adjusted to optimize the resolution of the mass distribution plot. The MP precision limit is approximately 1 kDa, and smaller bin sizes might amplify the noise of the distribution, while not revealing any additional information. Very large bin sizes will obscure the fine details of mass distributions.
3. Calculate the concentration fraction of each species using the following equation:
   
   where the h_i and f_i values represent peak heights and concentration fractions of the free antibody and the single- and double-bound antibody in the sample, respectively.