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1. Prepare the flow chambers
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	<li>Clean the glass coverslips
	<ol>
		<li>Using wash bottles with distilled water, ethanol, and isopropanol, rinse the 24 mm&#160;x&#160;50&#160;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 (<str...

mass photometry to study antigen-antibody interactions on a single-molecule level

Source: Wu, D. et. al., <a href="https://app.jove.com/v/61784">Rapid Determination of Antibody-Antigen Affinity by Mass Photometry.</a> J. Vis. Exp.&#160;(2021)
In this video, we demonstrate the use of mass photometry to study antigen-antibody interactions in a flow chamber. The focused light scatters differently based on the mass of the molecules. A detector collects the scattered light and generates the interference pattern as spots of varied intensities, which correlate with the molecular masses.

<img alt="Figure 1" src="/files/ftp_upload/21393/21393_Figure1.jpg" /> 
Figure 1: MP flow chamber preparation and loading.&#160;(A) Coverslip holding position for the cleaning procedure. (B) Alignment of the 24 x 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 the cut lines. (C) Top and side view of ...

Mass Photometry to Study Antigen-Antibody Interactions on a Single-Molecule Level

Source: Wu, D. et. al., Rapid Determination of Antibody-Antigen Affinity by Mass Photometry. J. Vis. Exp.&#160;(2021)
In this video, we demonstrate the ...

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.

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Research

JoVE Encyclopedia of Experiments

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Biomolecular Interaction Detection Techniques

Protein-protein interactions

In this video, we demonstrate the use of mass photometry to study antigen-antibody interactions in a flow chamber. The focused light scatters differently based on the mass of the molecules. A detector collects the scattered light and generates the interference pattern as spots of varied intensities, which correlate with the molecular masses.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21393/21393_Figure1.jpg" /> 
Figure 1: MP flow chamber preparation and loading.&#160;(A) Coverslip holding position for the cleaning procedure. (B) Alignment of the 24 x 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 the cut lines. (C) Top and side view of ...

Mass Photometry to Study Antigen-Antibody Interactions on a Single-Molecule Level

In This Article

Overview

Protocol

Representative Results

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