heliXcyto: Real-time binding kinetics on live cells!
Experience a groundbreaking approach and delve into measuring binding kinetics directly on living cells! The new Real-Time Interaction Cytometry (RT-IC), provides real-time insights into binding to cell surface receptors, for example GPCRs. RT-IC not only explores the dynamic binding kinetics but also sheds light onto the influence of membrane fluidity and third-molecule interactions on binding kinetics. This innovative method opens new dimensions in binding characterizations, offering unmatched insights and a deeper understanding of molecular interactions occurring on the cell surface.
Affinity
LIGAND BINDING
KD
Equilibrium dissociation constant. Can be obtained by kinetic or classical equilibrium binding analysis. Provides information about the strength but not the dynamics of an interaction.
Avidity
Functional affinity, representing the overall strength of an interaction. Influenced by the binding affinity, binding valency, and the structural arrangement of target and ligand.
EC50/IC50
Dose-response data. Ligand concentration that gives half-maximal response or half-maximal activity.
Kinetics
BINDING KINETICS
ka (kon)
Association rate constant. Provides information on how fast complexes form; can be used for KD determination.
kd (koff)
Dissociation rate constant. Provides information on how fast complexes dissociate; can be used for KD determination.
KD
Equilibrium dissociation constant. Can be obtained by kinetic or classical equilibrium binding analysis. Provides information about the strength but not the dynamics of an interaction.
Avidity
Functional affinity, representing the overall strength of an interaction. Influenced by the binding affinity, binding valency, and the structural arrangement of target and ligand.
Other
BI-PHASIC INTERACTIONS
Relative amplitudes for bi-phasic interactions
Bivalent binding interactions frequently result in biphasic kinetics and delivering two KD values. Relative amplitudes reveal which KD is more relevant.
HALF-LIFE DETERMINATION
Half-life (t1/2) on the cell surface
The time required until the antibody amount bound to the cell surface is reduced to 50%.
Besides KD values, the cell surface antibody retention time should be considered for comparing different antibody candidates.
ANALYSIS OF COMPETITION
Competition experiments
Analyze the concentration-dependent effect of an inhibitor on the interaction of the analyte with the cell surface.
The heliXcyto Biosensor is the go-to tool for measuring binding kinetics on live cells
Meet the heliXCyto system, a revolutionary tool designed for researchers seeking advanced molecular interaction analysis. This cutting-edge technology specializes in real-time studies directly within the native environment of living cells. With a focus on dynamic binding kinetics, particularly with G protein-coupled receptors (GPCRs), the HelixCytO stands out by not only capturing real-time interactions but also examining the influence of membrane fluidity and third-molecule interactions on binding kinetics. Offering unparalleled insights, the instrument allows us to provide specialized services for researchers to comprehend molecular interactions on the cell surface comprehensively. Ideal for drug discovery and cell signaling research, the heliXcyto opens new dimensions in binding characterizations, providing a potent tool for accelerating scientific studies.
The technology behind the heliXcyto
Fluorescently labeled analyte
• Red or green fluorophore
• Direct labeling via NHS conjugation or other (part of 2bind’s core expertise and service)
• Detection with secondary antibodies is possible
Cells trapped on the chip surface
- Living or fixed cells
- One or five cell traps per chip
- Cell traps are available in three different sizes
Measurement in heliXcyto biosensor
- Fluidic system with automated sample loading
- Two-color LEDs and dual-color photon detector
- Association is visible as increase in fluorescence, dissociation as decrease
Why Real-Time Interaction Cytometry (RT-IC)?
Native Folding
Integral membrane proteins, like GPCRs, exhibit folding intricacies influenced by the cell surface membrane. Real-Time Interaction Cytometry (RT-IC) proves indispensable for precisely measuring the binding kinetics of such proteins. RT-IC’s unique advantage lies in preserving proteins within their native environment, ensuring an accurate assessment of interactions. This approach, vital for membrane-bound proteins, captures real-time binding kinetics while considering the nuanced influence of the cell surface membrane. Researchers benefit from a comprehensive understanding of protein behavior in its natural setting.
Native density/mobility
The cell membrane, characterized by its fluidic and crowded nature, presents challenges unaddressed by surface-based technologies due to lateral movement and uneven receptor density distributions. To accurately capture these dynamic aspects, an innovative approach like Real-Time Interaction Cytometry (RT-IC) becomes crucial. By allowing a real-time analysis of molecular interactions within the native environment of living cells, RT-IC surpasses surface-based limitations, offering researchers unparalleled insights into the intricate dynamics of receptors influenced by the fluidic and crowded cellular milieu.
Native co-interactions
Consider the complexity where the target of interest interacts with additional molecules on the cell surface. Notably, heterodimerization, a frequent occurrence among cell surface receptors, introduces intricacies influencing binding kinetics. Traditional approaches may overlook such nuances, but Real-Time Interaction Cytometry (RT-IC) excels in this scenario. By facilitating real-time analysis within the native environment of living cells, RT-IC uniquely captures the impact of heterodimerization and other interactions on binding kinetics, offering researchers a comprehensive understanding of molecular dynamics at the cell surface.
Possible Analytes for heliXcyto RT-IC
Antibodies
Antibodies can be used as soluble analytes in RT-IC. Differences between binding kinetics measured with purified proteins and on cells can differ tremendously, reflecting changes in antigen conformation, avidity effects and/or the influence of third interaction partners on the cell surface.
Bi-specifics
Binding kinetics of bi-specifics targeting two cell surface receptors on the same cell can differ dependent on densities and ratios of the two target proteins, which are not necessarily evenly distributed on the cell surface. The effect can be measured in RT-IC.
Protein ligands
The measurement of any protein ligand to a cell surface receptor is possible in RT-IC. It might be of relevance for target validation or in a ligand-inhibition setup.
Small molecules
The signal is independent of the analyte’s size. Interactions of small molecules with cell surfaces can be measured as long as they are fluorescent and emit red or green light, or can be labeled with a fluorophore.
Possible Cell Types for heliXcyto RT-IC
Suspension and adherent cells
As in flow cytometry, suspension and adherent cells can be measured in RT-IC. Adherent cells have to be detached prior to the measurement.
Cells from 6 to 25 µm diameter
RT-IC chips are available with cages of different sizes to accommodate cells with diameters from 6 to 25 µm.
Tumor cell lines, recombinant cells, primary cells
RT-IC is possible with any cell expressing the target of interest. It can be tumor cell lines, transfected recombinant cells as well as primary cells, like T cells or NK cells.
Advantages of heliXcyto RT-IC
Cells as targets
RT-IC uses cells as targets and omits the need of high quality purified recombinant proteins, offering solutions in cases where the target proteins cannot be produced and purified in sufficient amounts and/or native folding conformation. Binding interactions are measured on the cell surface including all potential obstacles the analyte might face in the native surrounding and enable a comparison of candidates under conditions that might be more relevant for performance in vivo.
Kinetic data
RT-IC delivers kinetic data in contrast to titration experiments in flow cytometry. Association and dissociation rates are important parameters to be considered for planning concentrations, incubation times and dosing schedules for in vitro experiments or in vivo studies.
Automation
heliXcyto is equipped with an autosampler, reducing the hands-on time and minimizing manual operations.
Cell imaging
A built-in reflected light microscope and a CCD camera enable a strict observation and logging of cell appearance to improve the interpretability of collected measurement data.
Dual-color detection (coming soon)
Parallel detection of green and red fluorescence enables multiplexing experiments and reveals cooperative and competitive effects of two analytes labeled with different colors.
Frequently asked questions
FAQ – General
How often does NHS-labeling destroy the antibody binding?
The constant domains of antibodies contain many lysines that are accessible for amine coupling without impacting the binding to the target protein, which typically involves exclusively residues in the variable domains. However, exposed lysine residues in HCDR3 might represent a risk factor, and binding of such antibodies after labeling should be confirmed in GCI or FACS.
What about internalization?
Target-internalization is likely to influence the measured binding kinetics. To receive clean data, internalization can be chemically inhibited, or cells can be fixed by PFA prior to the measurement.
What is the detection limit in regard to target molecules on the cell surface?
The minimal number of target molecules on the cell surface depends on the binding kinetics, and in particular on the association rate of the analyte. Slow on-rates can be compensated by high concentrations of analyte. Analytes can also be labeled with more fluorophores, or detected with a secondary antibody, to increase the sensitivity. To reduced the analyte consumption it is possible to incubate cells and analyte offline for several hours or over night, and to monitor and analyze exclusively the dissociation.
Which affinities can be measured?
The detector can handle very fast on- and off-rates, and affinities from µM to pM range can be measured. Slow association rates in combination with low target expression levels might not lead to sufficient signal increase within the measurement time. In these rare cases, dissociation-only experiments should be considered, which allow the real time monitoring of analyte dissociation after an off-line incubation of analyte and cells.
Which dyes can be used?
There is one recommended red and one recommended green dye, which is included in the labeling kit sold by Dynamic Biosensors. However, RT-IC works with other fluorescent dyes as well.
What types of adapter strands have been designed up to now?
Ligand strands are compatible with standard adapters as well as with DNA nanostructures. Rigid DNA-origami nanolevers are recommended for sizing experiments with large molecules. Antibody-like Y-structures with flexible arms reduce the distance between attached ligands and enable bivalent binding of smaller bispecific molecules like Protacs. Ternary complex formation and dimerization can be followed by real-time FRET.
What dyes can be used for proximity sensing?
The two standard fluorescent probes for heliX® are the red and green dye Ra and Ga, respectively.
Fluorescence proximity sensing is based on the change in the local environment of the dye upon binding of an analyte to the ligand, which in turn results in a change of the fluorescence signal. This effect depends on the chemical nature of the dye and the interaction partner.
Therefore, a different dye may yield a higher signal response depending on the type of interaction. Dye scouting enables to screen for the most sensitive fluorophore for the respective application.
Three red and three green fluorophores with different chemical properties are available for dye scouting. The dyes differ in net charge and hydrophobicity as depicted below.
heliX® is equipped with LEDs and lasers to excite at 485 to 515 nm, or 600 to 630 nm, and to detect emissions at 520 to 580 nm, or 650 to 690 nm. Any dye respecting those characteristic can be used in heliX® applications.
