eoe sub articles

EoE Sub Articles

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.

mass-photometry-to-study-antigen-antibody-interactions-on-single

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

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.

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

JoVE Journal

Biochemistry

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.

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

Cancer Research

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.

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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