Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Abstract

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.

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.

Figure 2.. MP flow chamber preparation and loading.

(A) Coverslip holding position for the cleaning procedure. (B) Alignment of the 24 × 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.

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.

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.

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.