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

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

To assess the antibody-antigen affinity by mass photometry, apply a drop of microscope immersion oil to the instrument objective, and place the flow chamber onto the stage of the microscope. Add 10 microliters of filtered PBS to one end of the flow chamber channel. The buffer will enter the channel by capillary action.
In the Focus Control tab of the data collection software, use the coarse stage movement Up and Down buttons to make the initial adjustments, and click Sharpness to view the sharpness signal readout. Use the fine Up and Down adjustment buttons to maximize the Sharpness value, and click Set Focus and Lock Focus to activate the focus tracking function. An image of a clean slide should have a &#34;signal&#34; value equal to or below 0.05%.
Load 20 microliters of the first antibody-antigen sample as demonstrated, blotting the liquid from the other end with a small piece of blotting paper. Immediately upon loading, click Record to acquire the appropriate number of frames equivalent to 100 seconds of data. At the end of the data collection period, enter a file name for the data and click OK then Sample to save the file.
To analyze the mass photometry data, open the file of interest and click Analyze. Click Load to load the calibration function, and click File and Save Results As to save the analyzed data.
To obtain the relative concentrations of each species in the sample, open the &#34;eventsFitted.csv&#34; file, copy the data in column M into the appropriate plotting and analysis software, and use the Plot/Statistics/Histogram function to plot the molecular mass distribution. Double-click on the histogram to open the Plot Properties window, disable the automatic binning and select a bin size of 2.5 kiloDaltons.
To create the bin centers and counts data, click Apply and Go. To fit the histogram with Gaussian functions, select the Bin Centers and Counts columns and click the Analysis/Peaks and Baseline/Multiple Peak Fit menu functions. Double-click to indicate the approximate peak positions on the distribution plot, and click Open NLFit.
Check the Fixed checkboxes for the &#34;xc&#34; centers and set their values to the expected molecular masses of the free antibody and the single and double antigen-antibody complexes. Check the Share option for the width parameters and click Fit.
The fitted peak height values of the Gaussian components represent the relative concentration of each species in the sample. Then, use the equation to calculate the concentration fraction of each species from the peak height values.

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Biomolecular Interaction Detection Techniques

Protein-protein interactions

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1. Prepare the flow chambers
<ol>
	<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 (Figure 1A).</li>
		<li>Similarly, rinse the 24 mm x 24 mm coverslips with distilled water, ethanol, and distilled water. Dry the coverslips with a stream of clean nitrogen.</li>
		<li>Identify the working side of the coverslip, place a drop of distilled water on the surface of the clean coverslip, and follow steps 3.1&#8211;3.2 of the protocol. Usually, only one side of the 24 mm x 50 mm coverslip has the optical quality suitable for MP measurements.&#160;&#160; 
		NOTE: After focusing, no significant surface imperfections should be detectable, and the &#34;signal&#34; value shown in the data collection software should be less than 0.05% (Figure 2A-C). The working sides of all coverslips in the box are oriented in the same direction. The same procedure should be used to test the efficiency of the coverslip cleaning.</li>
	</ol>
	</li>
	<li value="2">Assemble the flow chamber
	<ol>
		<li>Position the 24 mm x 24 mm coverslip on a piece of aluminum foil. Place strips of double-sided tape on top of the 24 mm x 24 mm coverslip as shown in&#160;Figure 1B&#160;and cut the tape along the edge of the glass. Separate the coverslip from the aluminum foil and attach it to the working side of the 24 mm x 50 mm coverslip (Figure 1C).&#160;&#160;&#160; 
		NOTE: Channel size can vary, but a width of 3 mm&#8211;5 mm is recommended. Wider channels require larger sample volumes and very narrow channels may be difficult to load. Usually, two parallel channels can easily be created on the 24 mm x 24 mm coverslip. Protocol can be paused here.</li>
	</ol>
	</li>
</ol>
2. Prepare the antibody-antigen samples for the affinity measurements
<ol>
	<li>Filter at least 2 mL of the PBS buffer using 0.22 &#181;m syringe filters to remove dust particles or aggregates. Centrifuge the protein stock for 10 minutes at the maximum speed of the tabletop centrifuge (approximately 16,000 x&#160;g). 
	NOTE: PBS is the recommended buffer for this protocol, but MP has no particular buffer requirements, and other biological buffers are also acceptable. However, high glycerol concentrations (&#62;10%) and very low ionic strengths (salt concentration &#60;10 mM) may affect the image and data quality and are not recommended.</li>
	<li>Determine the actual concentrations of the antibody and antigen stocks by measuring their 280 nm UV absorbance.</li>
	<li>Calculate the measured concentrations of the antigen-antibody mixture. If the estimated value of the antibody binding affinity is not known, plan to prepare a sample with 30 nM antigen and 20 nM antibody concentration. When the approximate affinity is known, the antibody-to-antigen ratio and their concentrations should be optimized according to the expected&#160;Kd&#160;values. Use the total antigen concentration in the mixture equal to the sum of the expected&#160;Kd&#160;and the total antibody concentration in the equation below. Assuming&#160;Kd1&#160;=&#160;Kd2&#160;for the two paratopes of the antibody, this will result in comparable concentrations of the free antibody and the antibody-antigen complexes in the sample. 
	<img alt="Equation 1" src="/files/ftp_upload/21393/21393_Equation1.jpg" style="margin: auto;" /> 
	Adjust the antibody concentration to keep the total protein concentration in the sample within the 10&#160;nM and 50 nM range. Best results are obtained using mixtures with antibody concentrations between 5 nM and 25 nM. 
	NOTE: MP detects proteins with a molecular mass larger than 40 kDa. Consequently, sample concentrations of antigens with a molecular mass smaller than 40&#160;kDa can exceed the typical 50 nM limit. However, at concentrations higher than approximately 100 nM, even low molecular mass antigens might affect the image quality and accuracy of the&#160;Kd&#160;determination.</li>
	<li>Prepare 50 &#181;L of the antibody-antigen mixture at its final measurement concentration calculated in step 2.3.&#160;&#160;&#160;&#160; 
	NOTE: Only one sample of the antigen-antibody mixture is required for the&#160;Kd&#160;determination. However, preparing several samples with different antigen-to-antibody ratios can help optimize sample concentration. If data from several samples are collected, they can be analyzed via a global fit.</li>
	<li>Incubate the antigen-antibody mixture(s) for approximately 10 min at room temperature to allow the binding reaction to reach chemical equilibrium. Avoid unnecessarily long incubation times. 
	NOTE: Incubation time may vary depending on the binding kinetics. To confirm that the chemical equilibrium has been reached, sample measurements can be repeated at different incubation times. Time-invariant&#160;Kd&#160;values indicate sufficiently long incubation. Prolonged incubation may lead to significant protein adsorption to the surface of the plastic labware and, consequently, to significant errors in the protein concentration determination. For this reason, low-adhesion labware is strongly recommended for MP sample preparation.</li>
</ol>
3. Collect the Mass Photometry data
<ol>
	<li>Apply a drop of microscope immersion oil on the MP instrument objective and place the assembled flow chamber on the microscope stage. Make sure the oil spans the gap between the coverslip and the objective.</li>
	<li>Load the flow chamber and focus the mass photometer.
	<ol>
		<li>Deposit 10 &#181;L of a clean, filtered buffer solution at one end of the flow chamber channel prepared in step 1. Liquid will enter the channel by capillary action.</li>
		<li>Adjust the stage&#39;s Z-position to focus the microscope on the working surface of the 24 x 50 mm coverslip.
		<ol>
			<li>In the&#160;Focus Control&#160;tab of the data collection software, use the coarse stage movement&#160;Up&#160;and&#160;Down&#160;buttons to make the initial adjustments.</li>
			<li>Click the&#160;Sharpness&#160;button to show the sharpness signal readout and use the fine&#160;Up&#160;and&#160;Down&#160;adjustment buttons to maximize the&#160;Sharpness&#160;value.</li>
			<li>Click the&#160;Set Focus&#160;and&#160;Lock Focus&#160;buttons to activate the focus tracking function. A properly focused image (Figure 2A, C) should have a &#34;signal&#34; value below 0.05%. 
			NOTE: If the &#34;signal&#34; value at the maximum sharpness position is above 0.05%, this may indicate impurities on the glass surface or in the buffer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Using the same channel, load 20 &#181;L of the antibody-antigen sample by depositing it on one side of the channel and blotting the liquid from the other end with a small piece of blotting paper (Figure 1D). 
	NOTE: The volume of a 3&#8211;5 mm wide channel is approximately 10 &#181;L. The additional sample volume is recommended to completely replace the buffer present in the channel and to avoid sample dilution.</li>
	<li>After loading the sample, immediately click the&#160;Record&#160;button to start data collection, acquiring a 100 s video (Figure 2D).</li>
	<li>At the end of the data collection enter the file name and click&#160;OK&#160;to save the data file.</li>
	<li>Discard the coverslips and wipe the oil from the objective lens with cotton optical swabs wet with isopropanol. 
	NOTE: The protocol can be paused here.</li>
</ol>
4. Analyze the MP data
<ol>
	<li>Process the collected video file using the MP data processing software to identify the landing events.
	<ol>
		<li>Use the&#160;File/Open&#160;menu option to load the file for the analysis and click&#160;Analyze.</li>
		<li>Click the&#160;Load&#160;button to load the calibration function and save the analyzed data using the&#160;File/Save Results As&#160;menu option.</li>
	</ol>
	</li>
	<li>Fit the molecular mass distribution with Gaussian functions to obtain relative concentrations of each species in the sample. This analysis can be performed using a common scientific graphing software (see&#160;Table of Materials).
	<ol>
		<li>Import the &#34;eventsFitted.csv&#34; file into the software and plot the molecular mass distribution (column M in the .csv file) using the&#160;Plot/Statistics/Histogram&#160;function.</li>
		<li>Double-click on the histogram to open the&#160;Plot Properties&#160;window. Disable automatic binning and select a bin size of 2.5 kDa. Click the&#160;Apply&#160;and the&#160;Go&#160;buttons to create the&#160;Bin Centers&#160;and&#160;Counts&#160;data.</li>
		<li>Select the&#160;Bin Centers&#160;and&#160;Counts&#160;columns and use the&#160;Analysis/Peaks and Baseline/Multiple Peak Fit&#160;menu function to fit the histogram with Gaussian functions. Double-click to indicate the approximate peak positions on the distribution plot and then click the&#160;Open NLFit&#160;button.</li>
		<li>Check the&#160;Fixed&#160;checkboxes for the &#34;xc&#34; peak centers and set their values to the expected molecular masses of the free antibody and the single and double antigen-antibody complexes. Check the&#160;Share&#160;option for the width parameters. Click the&#160;Fit&#160;button. The fitted peak height values of the Gaussian components represent the relative concentration of each species in the sample.&#160; 
		NOTE: Bin size may be adjusted to optimize the resolution of the mass distribution plot. The MP precision limit is approximately 1&#160;kDa, and smaller bin sizes might amplify the noise of the distribution, while not revealing any additional information. Very large bin sizes will obscure the fine details of mass distributions.</li>
	</ol>
	</li>
	<li>Calculate the concentration fraction of each species using the following equation: 
	<img alt="Equation 2" src="/files/ftp_upload/21393/21393_Equation2v2.jpg" style="margin: auto;" /> 
	where the&#160;hi&#160;and&#160;fi&#160;values represent peak heights and concentration fractions of the free antibody and the single- and double-bound antibody in the sample, respectively.</li>
</ol>

<table><tbody><tr><td>AcquireMP</td><td>Refeyn</td><td></td><td>MP data collection software</td></tr><tr><td>Anti-human thrombin</td><td>Haematologic Technologies</td><td>AHT-5020</td><td>RRID: AB_2864302</td></tr><tr><td>Cotton-tipped applicators</td><td>Thorlabs</td><td>CTA10</td><td>Cotton optical swabs for lens cleaning</td></tr><tr><td>Coverslips 24x24 mm</td><td>Globe Scientific</td><td>1405-10</td><td></td></tr><tr><td>Coverslips 24x50 mm</td><td>Fisher Scientific</td><td>12-544-EP</td><td></td></tr><tr><td>DiscoverMP</td><td>Refeyn</td><td></td><td>MP data processing software</td></tr><tr><td>Forceps</td><td>Electron Microscopy Sciences</td><td>78080-CF</td><td>Soft-tipped forceps for coverslips handling</td></tr><tr><td>Human &amp;alpha;-thrombin</td><td>Haematologic Technologies</td><td>HCT-0020</td><td></td></tr><tr><td>Immersion oil</td><td>Thorlabs</td><td>MOIL-30</td><td></td></tr><tr><td>Isopropanol</td><td>Alfa Aesar</td><td>36644</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td>Spreadsheet</td></tr><tr><td>OneMP</td><td>Refeyn</td><td></td><td>Mass Photometry instrument</td></tr><tr><td>Origin</td><td>OriginLab</td><td></td><td>Scientific graphing software</td></tr><tr><td>PBS</td><td>Corning</td><td>46-013-CM</td><td>10x stock</td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLGSR33SS</td><td>Buffer and sample filtering</td></tr></tbody></table>

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 ...

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.

83 ビュー. 抗原-抗体相互作用を単一分子レベルで研究するための質量測光

ビデオ: 抗原-抗体相互作用を単一分子レベルで研究するための質量測光 - 実験

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

四バイオセンサープラットフォームを用いた高親和性抗体の抗原結合速度の決意

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

JoVE Journal

生化学

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

消化管間質腫瘍の遺伝子操作マウスモデルにおける分子および免疫学的技術

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

がん研究

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

カッパ（Vκ）に対する抗体の結合特異性軽鎖を含むヒト（IgM抗体）抗体：ポリシアル酸（PSA）ケーススタディとしてNCAMに添付

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

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

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