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FCS Application: Antibody - Antigen Binding

Fluorescence Correlation Spectroscopy (FCS) is an ideal technique for measuring and assaying molecular interactions such as antibody-antigen binding. FCS distinguishes components in your sample based on molecular weight, so it can easily differentiate bound antibody-antigen complexes from unbound antibody or antigen in solution.

No Washing or Immobilization

Unlike ELISAs, which require immobilization and several wash steps, FCS allows you to measure antibody-antigen binding in solution, reducing assay length and allowing you to measure freely diffusing samples in a more physiologically-relevant state.

No Standard Curves

FCS measures the number of fluorescent particles in its detection volume, providing a direct measure of concentration without the need for standard curves. In addition to total concentration, FCS also gives you the relative concentration of bound and unbound antibody or antigen, so you will know exactly what concentration of your antibody or antigen is bound and what concentration is unbound.

Measure Binding Kinetics

By measuring the change in the concentration of the bound antibody-antigen complex over time, you can use FCS to obtain information on binding kinetics (k_on, k_off) and binding affinities (K_d, K_a).

Analyze Competition/Inhibition

By measuring the decrease in concentration of antibody-antigen complex upon inhibitor/competitor addition, you can determine inhibitor binding affinities (K_i, IC₅₀).

Obtain Stoichiometry Information

By measuring the change in particle number and/or molecular size, FCS can tell you the antigen-to-antibody ratio of the bound complexes.

Create Multiplex Assays

If you use two different fluorescent labels, FCS can measure two separate interactions in a single sample.