How to detect and quantify the binding affinity of the antibody to the antigen?

The binding affinity of the antibody to the antigen can be quantified and detected by mass photometry (MP). MP does not require labels and immobilization, and is able to quantify populations of antibodies and antigen-antibody complexes on an extremely small scale (single-molecule level). It is a simple technique which requires only picomole amounts, and is a fast and accurate procedure. One must first prepare dilutions for the antibody and antigen in a buffer solution. The samples are then loaded onto a coverslip to form the antibody-antigen complex, and then incubated. After the mass photometer analyzes the data, the binding affinity is measured by titrating the antibody with the antigen. By fitting a model equation (e.g. Langmuir isotherm) for the binding data, the on- and off-rate constants of the antibody binding to the antigen can be determined and thus, the affinity. It is important to note protein concentrations are limited from 10nM-50nM when measuring their affinities using MP. 

In addition to MP, ELISA assays are very efficient techniques for the detection and quantification of the binding affinity of antibodies. All ELISAs share common steps in their procedures such as: coating, plate blocking, probing, and signal measurement. During coating, the antigen may be directly or indirectly immobilized to the surface of the microplate wells. In plate blocking, non-specific proteins or molecules are added to coat the unused binding sites on the wells. During probing, the microplate wells are able to interact with antibodies which affinity-bind to antigens through the process of incubation. In signal measurement, the signal is detected by the direct or secondary label bound to the specific antibody. The most commonly used ELISA assay is the sandwich ELISA, in which the target antigen is indirectly immobilized and detected. In this assay, the analyte of interest is bound between 2 primary antibodies - the capture antibody and the detection antibody. Each antibody detects a different epitope on the antigen. The sandwich ELISA is preferred over other types of ELISA due to its high specificity and sensitivity.