Encyclopedia of Experiments: Biological Techniques
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Transcript
To detect antigen-antibody interactions using mass photometry, first, incubate the desired concentration of purified antigens and antibodies for the required period of time, facilitating antigen-antibody binding. Based on the stoichiometry of antigen binding, the paratope regions of these antibodies can have varying numbers of attached antigens.
Flow in the solution containing the free antibodies along with the antigen-antibody complexes through a pre-assembled flow chamber. Secure the chamber onto the microscope stage of a mass photometer. Then, direct the laser beam through the flow chamber for optimum imaging.
Depending upon the number of attached antigens to each antibody, the molecular mass of the complexes changes. These variations in mass cause the complexes to scatter light differently.
Complexes with higher molecular masses, where two antigens are bound to both paratopes of a single antibody, scatter more light compared to single antigen-containing complexes. In addition, free antibodies show the least amount of light scattering.
A detector collects the scattered light and measures its frequency and intensity. The photometer records fluctuations in the scattered light as an interference pattern, representing this as spots.
Faint spots correspond to free antibodies. In contrast, very dark spots represent complexes with two antigens, and the less dark spot contains one bound antigen.
The presence of dark spots conforms to the antigen-antibody interactions.
