We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.
Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.
Antibodies have become ubiquitous tools of molecular biology and are used extensively in both medical and research applications. In medicine, they are critically important in diagnostics, but their therapeutic applications are also expanding and new antibody-based therapies are constantly being developed1,2,3,4. The scientific applications of antibodies include many indispensable laboratory techniques such as immunofluorescence5, immunoprecipitation6, flow cytometry7, ELISA, and western blotting. For each of these applications, obtaining accurate measurements of the antibody’s binding properties, including binding affinity and specificity, is of crucial importance.
Since the first commercial surface plasmon resonance (SPR) instrument was introduced in 1990, optical biosensors have become the “gold standard” of antibody characterization, but other techniques, including ELISA, are also routinely used to measure antibody affinities8,9. These methods usually require immobilization or labeling of the analyzed molecules, which can potentially affect the interaction of interest. They are also relatively slow, involving multiple assay steps before the results can be collected for data analysis. A recently developed single-molecule method, mass photometry (MP), detects molecules directly in solution when they land on the surface of the microscope coverslip10,11. The light scattering-based optical detection that MP employs does not require protein labeling or modification. Individual protein molecules are recorded by the interferometric scattering microscope as dark spots appearing in the image (Figure 1D), and several thousand molecules can be detected during the one-minute data acquisition12. The signal generated by each individual particle is quantified, and its contrast value (relative darkness) is calculated. The interferometric contrast values are proportional to the molecular masses of the proteins, which allows for the identification of bound and free species in the antigen-antibody mixture. At the same time, by counting molecular landing events, MP directly measures the species populations. This gives MP based methods a unique capability to independently quantify affinities of multiple binding sites.
Binding of the antigen (Ag) molecules to the two binding sites of the intact antibody (Ab) can be described as:
with the equilibrium association constants Ka1 and Ka2 defined as:
where ci and fi represent concentration and fraction of the component i, respectively. The total antigen concentration (cAg)tot can be expressed as:
Since the total concentrations of the antibody (cAb)tot and antigen (cAg)tot are known, this equation can be used to directly fit the experimental component fractions obtained from the MP measurements and calculate the equilibrium association constants Ka1 and Ka2 (see Supplementary Information).
The MP data can also be used to estimate cooperativity between the two antibody binding sites11. For two antibody paratopes with identical microscopic binding constants, the statistical factors describing the process of population of the Ab·Ag and Ab·Ag2 complexes dictate that the apparent macroscopic equilibrium constants Ka1 and Ka2 will not be numerically equal, and Ka1 = 4Ka2. Therefore, the experimental values of Ka1 < 4Ka2 indicate positive cooperativity between the two antibody binding sites. Similarly, Ka1 > 4Ka2 indicates negative cooperativity.
MP measurements of the antigen-antibody binding affinity are fast and require a small amount of material. The MP mass distributions used for equilibrium constant calculations provide additional information about the sample properties and enable the assessment of the sample purity, oligomerization, and aggregation in a single experiment. The same method can be used to measure high affinity protein-protein binding, and MP is particularly useful for studies of multi-valent protein interactions. Multi-protein complexes usually have large molecular masses, optimal for MP detection, and single-molecule data can be used to measure stoichiometry and calculate affinities of multiple binding sites simultaneously. This information is usually difficult to obtain using bulk-based methods.
Without modifications, the current protocol is suitable for measurements of relatively high-affinity, sub-micromolar interactions with antigens of a molecular mass of 20 kDa or larger. For optimal results, protein stocks should be of high purity, but there are no specific buffer requirements. By using MP, the antigen-antibody binding can be assessed in less than five minutes. The data collection and analysis required for accurate Kd calculations can be performed within 30 minutes.
1. Prepare the flow chambers
2. Prepare the antibody-antigen samples for the affinity measurements
3. Collect the Mass Photometry data
4. Analyze the MP data
5. Calculate equilibrium constant values
Figure 1: Mass photometry images. (A) Representative native view image of the imaging buffer collected on a clean coverslip and (B) on a coverslip with surface imperfections. (C) Differential ratiometric image of the imaging buffer and (D) the AHT·HT solution. Please click here to view a larger version of this figure.
Figure 2: MP flow chamber preparation and loading. (A) Coverslip holding position for the cleaning procedure. (B) Alignment of the 24 x 24 mm coverslip (middle layer) and the double-sided tape (top layer) on the surface of aluminum foil (bottom layer, not shown). Blue dashed lines show the location of cut lines. (C) Top and side view of the assembled flow chamber with two sample channels, and a picture of the assembled flow chamber. (D) Procedure for sample loading into a flow channel previously filled with buffer. Please click here to view a larger version of this figure.
We have previously examined the interaction of human α-thrombin (HT) and mouse monoclonal anti-human thrombin antibody (AHT) using the MP based assay11. Since the molecular mass of the HT (37 kDa) is below the 40 kDa detection limit, the maximum sample concentration can exceed the 50 nM MP concentration limitation without negatively affecting the resolution of mass distributions. The experiment was planned as a titration series with the AHT antibody at a fixed 25 nM concentration, and the HT at concentrations of 7.5 nM, 15 nM, 30 nM, 60 nM and 120 nM. Figure 3 shows the molecular mass distributions of the antigen-antibody mixtures and the antibody-only sample. Here we analyze the data using the method described in the protocol. A scientific graphing software was used to fit the mass distributions with three Gaussian components representing the free AHT, AHT·HT and AHT·HT2. The known molecular mass values of the three components were fixed, and a single peak width parameter was fitted for the three species. Best-fit peak height parameters of the Gaussian components were normalized using Eq. 3 to obtain species concentration fractions (Table 1). Those values, together with the total antibody and antigen concentration for each sample were entered into the “Kd calculation.xlsx”. The global fit in the spreadsheet yields Kd1 = 40 nM (68.3% confidence interval: 28 nM, 68 nM) and Kd2 = 28 nM (68.3% confidence interval: 17 nM, 45 nM). The experimental concentration fractions and the fit results are plotted in Figure 4. The dissociation constant values obtained here by fitting the integrated concentration fractions are in agreement with those obtained previously by directly fitting MP distributions, and with dissociation constant values obtained by Isothermal Titration Calorimetry (ITC)11.
Figure 3: MP molecular mass distributions of the 25 nM AHT mixed with HT at 0, 7.5, 15, 30, 60 and 120 nM (A-F, respectively). Black dots show the experimental MP data plotted with 2.5 kDa bin size. Cyan, green, and blue lines represent the best-fit Gaussian distributions of the free antibody, single bound antibody, and double bound antibody species, respectively. Red lines show the sum of the three Gaussian components. Please click here to view a larger version of this figure.
Figure 4: Fractions of the free AHT (blue), AHT·HT (red), and AHT·HT2 (black) as a function of HT concentration. Points represent experimental values obtained from the Gaussian fitting of the MP distributions. Solid lines represent the best-fit using Eq. 1 and Eq. 2. Please click here to view a larger version of this figure.
Figure 5: Technical replicates of the AHT molecular mass distribution measurements. Plots shows the reproducibility of the MP measurements and the purity of the antibody preparation. Please click here to view a larger version of this figure.
Ab conc (M) | Ag conc (M) | f_Ab | f_AbAg | f_AbAg2 |
2.50E-08 | 7.50E-09 | 0.88 | 0.10 | 0.02 |
2.50E-08 | 1.50E-08 | 0.81 | 0.15 | 0.04 |
2.50E-08 | 3.00E-08 | 0.72 | 0.21 | 0.07 |
2.50E-08 | 6.00E-08 | 0.27 | 0.37 | 0.35 |
2.50E-08 | 1.20E-07 | 0.05 | 0.23 | 0.72 |
Table 1: Normalized peak heights of Gaussian components obtained by fitting the MP distributions (Figure 3).
Supplementary Information: Implementation of the equilibrium constants fitting procedure in Excel and Affinity calculation worksheet. Please click here to download this file.
The Mass Photometry based protocol outlined here provides a fast and accurate method of measuring antigen-antibody binding affinities. MP analysis uses a very small amount of material, and additional information—including stoichiometry, oligomerization, and purity—can be assessed from the same data (Figure 5). Without modifications, this method is applicable to the measurements of dissociation constants in the approximately 5 nM to 500 nM range, and for ligand molecules with molecular mass of approximately 20 kDa or larger. The same protocol can be used not only to analyze the antigen-antibody binding, but also to measure protein-protein interaction affinities when the molecular mass of at least one of the binding partners is larger than 50 kDa. Since the MP detection is non-specific, MP measurements can’t be performed on samples containing a high concentration of carrier proteins or large molecular mass impurities.
SPR is commonly used for the characterization of the antigen-antibody binding, and direct comparison of both methods may help with the assay selection. In comparison with SPR, the MP binding assay is faster and does not require protein immobilization. For a typical binding experiment, the MP requires less material than SPR. MP data reveal the stoichiometry of the complexes which is not readily available from the SPR10,11. Kinetic parameters of binding can be obtained from the MP data13, but SPR measures the binding kinetics directly, and is able to measure a broader range of association and dissociation rates. The association constants of two separate binding sites can only be obtained from the SPR data when the association and dissociation rates of both sites are sufficiently different. On the other hand, the MP can precisely characterize molecular complexes with multiple binding sites10,11. SPR is able to measure binding affinity for small molecular mass ligands, and MP works best for ligands with molecular mass of approximately 20 kDa or larger.
Collecting good quality MP data is a critical step in obtaining accurate results from this protocol. Impurities in the buffer or on the surface of the coverslips will interfere with MP data collection. Improper focusing distorts the MP signal leading to errors in molecular mass estimates and equilibrium constants calculations. Both these factors should be examined when troubleshooting the protocol. An important consideration when working with low concentration protein solutions is that surface adsorption can lead to the loss of material and changes in the sample concentration. The resulting errors can be minimized by using low adsorption labware and by applying surface passivation. To obtain accurate results, it is important to allow all protein dilutions and mixtures to reach chemical equilibrium, but unnecessarily long incubation times should be avoided. For each protein system, it is recommended that the rates of the non-specific protein binding to the coverslip glass be assessed from the MP data. If significantly different rates are observed for different species, MP data can easily be corrected to avoid potential errors in the Kd calculations10,11.
Several factors should be considered when planning sample preparation. Accurate results can be obtained by measuring a single sample, but the antigen and antibody concentrations have to be adjusted to obtain a comparable concentration of the free and bound species. Analysis of a single mass distribution dominated by one of the reaction species may provide acceptable Kd values estimates but usually yields relatively large fitting errors11. An alternative strategy involves a global analysis of titration data. The spreadsheet software based fitting tool provided with this protocol can be used for fitting both individual data and for global analysis. If a single experiment or data set is analyzed, the confidence intervals of best fit parameters can be estimated using the error projection method. A detailed description of the confidence intervals calculation procedure in the spreadsheet software is provided elsewhere14. When designing the assay, it is recommended to plan for replicate experiments. When data from replicate experiments are analyzed, standard deviation can be used to report errors of the Kd values.
The protocol described here can be modified to extend its applicability beyond the typical molecular mass and affinity range limitations. The range of measurable affinities is limited by the protein concentration range accessible by MP, typically from 10 nM to 50 nM. This range can be extended and protein samples at concentrations below 10 nM can be measured by using perfused flow cells and longer data acquisition times. Protein samples at higher concentrations can potentially be measured by using passivated coverslips for the MP measurements. For antigens with a small molecular mass, the free antibody and the antigen-antibody complex peaks in the MP mass distributions will be unresolved. This will preclude the use of the Gaussian peak fitting analysis described in the protocol. In that case, the binding affinity can still be measured by titrating the antibody with the antigen and averaging the MP distributions to obtain the average molecular mass of all species for each sample. The binding equation can then be fit to this data to obtain the antigen-antibody binding affinity11.
When accurate affinity information is not required, the protocol can be simplified and used for fast antibody interaction screening. In that case, the commercially available gasket wells can be used instead of sample channels to further simplify the experimental procedure.
The authors have nothing to disclose.
We thank Keir Neuman for his critical reading of the manuscript. This work was supported by the intramural program of the NHLBI, NIH.
AcquireMP | Refeyn | MP data collection software | |
Anti-human thrombin | Haematologic Technologies | AHT-5020 | RRID: AB_2864302 |
Cotton-tipped applicators | Thorlabs | CTA10 | cotton optical swabs for lens cleaning |
Coverslips 24×24 mm | Globe Scientific | 1405-10 | |
Coverslips 24×50 mm | Fisher Scientific | 12-544-EP | |
DiscoverMP | Refeyn | MP data processing software | |
Forceps | Electron Microscopy Sciences | 78080-CF | soft-tipped forceps for coverslips handling |
Human α-thrombin | Haematologic Technologies | HCT-0020 | |
Immersion oil | Thorlabs | MOIL-30 | |
Isopropanol | Alfa Aesar | 36644 | |
Microsoft Excel | Microsoft | spreadsheet | |
OneMP | Refeyn | Mass Photometry instrument | |
Origin | OriginLab | scientific graphing software | |
PBS | Corning | 46-013-CM | 10x stock |
Syringe filter | Millipore | SLGSR33SS | buffer and sample filtering |