We thank Cedric A. J. Hutter and Markus A. Seeger from the Institute of Medical Microbiology at the University of Zurich, and Gonzalo Cebrero from Biozentrum of the University of Basel for collaboration in the generation of synthetic nanobodies (sybodies). We thank Maria Barthmes and Andre Bazzone from NANION Technologies for technical assistance. This work was supported by the Swiss National Science Foundation (SNSF) (PP00P3_170607 and NANION Research Grant Initiative to C.P.).

<ol>
	<li>Braden, B. C., Goldman, E. R., Mariuzza, R. A., Poljak, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+an+antibody+molecule:+structure,+kinetics,+thermodynamics,+and+mutational+studies+of+the+antilysozyme+antibody+D1.3.">Anatomy an antibody molecule: structure, kinetics, thermodynamics, and mutational studies of the antilysozyme antibody D1.3.</a> Immunology Reviews. 163, 45-57 (1998).</li><li>Hamers-Casterman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+antibodies+devoid+of+light+chains.">Naturally occurring antibodies devoid of light chains.</a> Nature. 363, 446-448 (1993).</li><li>Perez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+inhibition+of+lipid-linked+oligosaccharide+flippase+PglK+by+a+conformational+nanobody.">Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.</a> Science Reports. 7, 46641 (2017).</li><li>Grahl, A., Abiko, L. A., Isogai, S., Sharpe, T., Grzesiek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+description+of+beta1-adrenergic+receptor+functional+dynamics+and+allosteric+coupling+from+backbone+NMR.">A high-resolution description of beta1-adrenergic receptor functional dynamics and allosteric coupling from backbone NMR.</a> Nature Communication. 11, 2216 (2020).</li><li>Schenck, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+anti-VGLUT+nanobodies+acting+as+inhibitors+of+transport.">Generation and characterization of anti-VGLUT nanobodies acting as inhibitors of transport.</a> Biochemistry. 56, 3962-3971 (2017).</li><li>Mireku, S. A., Sauer, M. M., Glockshuber, R., Locher, K. 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The authors declare no competing financial interests.

The technique presented here classifies nanobodies with inhibitory and non-inhibitory properties targeting electrogenic transporters. Assessing the substrate transport is possible due to the detection of the movement of charges through the transporter embedded in the membrane of proteoliposomes. Some of the critical steps during the setup of an experiment are reconstitution of active protein in liposomes, preparation of stable monolayers on SSM chips, and recovering of initial conditions after the application of the wash...

Antibodies are composed of two identical heavy chains and two light chains that are responsible for the antigen binding. Camelids have heavy-chain only antibodies that exhibit similar affinity for their cognate antigen compared to conventional antibodies1,2. The single variable domain (VHH) of heavy-chain only antibodies retain the full antigen-binding potential and has been shown to be very stable1,2. These isolated VHH molecules or &#34;nanobodies&#34; have been implemented in studies related to membrane proteins biochemistry as tools for stabilizing conformations3,4, as inhibitors5,6, as stabilization agents7, and as gadgets for&#160;structure determination8,9,10. Nanobodies can be generated by the immunization of camelids for the pre-enrichment of B-cells that encode target-specific nanobodies and subsequent isolation of B cells, followed by cloning of the nanobody library and selection by phage display11,12,13. An alternate way to generate nanobodies is based on in vitro selection methods that rely on the construction of libraries and selection by phage display, ribosome display, or yeast display14,15,16,17,18,19,20. These in vitro methods require large library sizes but benefit from avoiding animal immunization and favor the selection of nanobodies targeting proteins with relatively low stability.
The small size of nanobodies, their high stability and solubility, strong antigen affinity, low immunogenicity, and relatively easy production, make them strong candidates for the development of therapeutics21,22,23. In particular, nanobodies inhibiting the activity of multiple membrane proteins are potential assets for clinical applications5,24,25,26. In the case of membrane transporters, to evaluate whether a nanobody has inhibitory activity, it is necessary to develop an assay that allows the detection of transported substrates and/or co-substrates. Such assays usually involve labeled molecules or the design of substrate-specific detection methods, which may lack a universal application. Furthermore, the identification of inhibitory nanobodies generally requires the screening of large numbers of binders. Thus, a method that can be used in a high-throughput mode and that does not rely on labeled substrates is essential for this selection.
SSM-based electrophysiology is an extremely sensitive, highly time-resolved technique that allows the detection of movement of charges across membranes (e.g., ion binding/transport)27,28. This technique has been applied to characterize electrogenic transporters, which are difficult to study using other electrophysiology techniques due to the relative low turnover of these proteins29,30,31,32,33,34,35. SSM electrophysiology does not require the use of labeled substrates, it is suitable for high-throughput screening, and either proteoliposomes or membrane vesicles containing the transporter of interest can be used. Here, we demonstrate that SSM-based electrophysiology can be used to classify transporter-targeted nanobodies with inhibitory and non-inhibitory properties. As a proof-of-principle, we describe the reconstitution of a bacterial choline transporter into liposomes, followed by detailed steps for immobilization of proteoliposomes on the SSM sensors. We next describe how to perform SSM-based electrophysiology measurements of choline transport and how to determine the half-maximal effective concentration (EC50). We then show how to use SSM-based electrophysiology to screen multiple nanobodies and to identify inhibitors of choline transport. Finally, we describe how to determine the half maximal inhibitory concentrations (IC50) of selected inhibitory nanobodies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-octadecanethiol solution</td>
			<td>Sigma Aldrich</td>
			<td>O1858-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-diphytanoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850356C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Beads SM-2 Adsorbent (Polystyrene adsorbent beads)</td>
			<td>BioRad</td>
			<td>#152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>PD 10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>GE17-0851-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter 200 nm membrane</td>
			<td>Whatman Nucleopore</td>
			<td>WHA800282</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Merck</td>
			<td>33539-1L-R</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Decane</td>
			<td>Sigma Aldrich</td>
			<td>8034051000</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecyl-&#223;-D-maltoside (DDM)</td>
			<td>Avanti Polar Lipids</td>
			<td>850520P-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>AppliChem</td>
			<td>131659.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>(SSM setup) SURFE2R N1</td>
			<td>Nanion</td>
			<td>-----</td>
			<td></td>
		</tr>
		<tr>
			<td>SURFE2R N1 Single Sensor Chips</td>
			<td>Nanion</td>
			<td># 161001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli Polar Lipid Extract</td>
			<td>Avanti Polar Lipids</td>
			<td>100600C</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg PC L-&#945;-phosphatidylcholine </td>
			<td>Avanti Polar Lipids</td>
			<td>840051C</td>
			<td></td>
		</tr>
	</tbody>
</table>

selection of transporter-targeted inhibitory nanobodies by solid-supported-membrane (ssm)-based electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

SSM-based electrophysiology has been extensively used for the characterization of electrogenic transporters. In the protocol presented here, we show how to use SSM-based electrophysiology to classify nanobodies targeting a secondary transporter (here a bacterial choline symporter) based on their inhibitory and non-inhibitory properties. One of the most useful features of this technique is that it allows for the high-throughput screening of multiple buffer conditions. This particular characteristic is beneficial for the a...

Watch this Scientific Journal Video about Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology at JoVE.com

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as ...

selection-transporter-targeted-inhibitory-nanobodies-solid-supported

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Biochemistry

2.5K Views.  Biozentrum, University of Basel. Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters. 

Video: Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Modification and Functionalization of the Guanidine Group by Tailor-made Precursors

The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. In this article, an easy method for the modification of the guanidine group in peptidic ligands is provided, with an example of RGD-binding integrin ligands. It was recently demonstrated that the distinct modification of the guanidine group in these ligands allows for the selective modulation of the subtype (e.g., between the subtypes &#945;v and &#945;5). Moreover, a formerly unknown strategy for the functionalization via the guanidine group was demonstrated, and the synthetic approach is reviewed in this document. The modifications described here involve terminally (N&#969;) alkylated and acetylated guanidine groups. For the synthesis, tailor-made precursor molecules are synthesized, which are then subjected to a reaction with an orthogonally deprotected amine to transfer the pre-modified guanidine group. For the synthesis of alkylated guanidines, precursors based on N,N&#8242;-Di-Boc-1H-pyrazole-1-carboxamidine are used to synthesize acylated compounds, the precursor of choice being a correspondingly acylated derivative of N-Boc-S-methylisothiourea, which can be obtained in one- and two-step reactions.

A protocol for the synthesis of alkyl-modified guanidines based on the use of the corresponding precursors is presented.

The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...