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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, a...
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 mole...
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.
Name | Company | Catalog Number | Comments |
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 24x24 mm | Globe Scientific | 1405-10 | |
Coverslips 24x50 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 |
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