Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Summary

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.

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.

Introduction

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 developed^1,2,3,4. The scientific applications of antibodies include many indispensable laboratory techniques such as immunofluorescence⁵, immunoprecipitation⁶, flow cytometry⁷, EL....

Protocol

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 2A).
   2. Similarly, rinse the 24 mm x 24 mm coverslips with distilled water, ethanol, and distilled water. Dry....

Results

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.......

Discussion

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.......

Materials

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