Cell Line Affinity Measurement Service

Background Services Published Data FAQ Resources

Scientists from Creative Biolabs are willing to provide custom cell line affinity measurement using Surface Plasmon Resonance microscopy (SPR microscopy). It is a state of the art label-free technique, which combines the advantages of SPR with electron microscopy to study the ligand-receptor interaction of living cells in real-time monitor.

Background

Technically, it is challenging to measure the binding kinetics of membrane proteins with living cells. Traditional methods such as fluorescence-based technology is a label based end-point assay that measures the changes of fluorescence signals before and after binding without the obtaining of binding kinetics. However, the label-free techniques, such as SPR, can measure protein-binding kinetics by immobilizing purified proteins onto a SPR chip. But for membrane proteins, an amphiphilic lipid bilayer has to be introduced to keep membrane proteins embedded into the liposome to preserve their activity. Meanwhile, atomic force microscopy can provide high spatial resolution for the study of membrane proteins, however, it is not suitable for the study of binding kinetics.

Nowadays, surface plasmon resonance microscopy (SPRM), a label-free imaging method, allows both optical and fluorescence imaging of the same sample without extracting membrane proteins from living cells. Briefly, cells are first cultured on a gold film, which is placed in an inverted microscope for simultaneous bright-field, fluorescence and SPRM imaging. Then, a p-polarized laser beam is casted onto a gold-coated glass coverslip through an oil-immersion objective to create SPR on the gold medium surface, which is imaged by a CCD camera. Additionally, a mercury lamp and appropriate optical filters are used to perform epifluorescence measurements. Furthermore, conventional bright-field images of the same sample can also be taken by illumination from the top of the sample.

Figure 1. Surface plasmon resonance microscopy

Like traditional SPR approaches, ligand-binding can result in the changes of mass concentration close to the surface. Therefore, the associate constant (K_a), disassociated constant (K_d) and binding affinity (K_D) can be determined from the signal response unit (RU) changes.

SPR microscopy is capable of working out the measurement of binding kinetics between ligands and receptors in single living cells. The sensorgrams can be fitted pixel by pixel with a mathematic model to map the local association and disassociation constant. Thus, the real-time distribution images of ligands’ interaction with cells can be obtained in a non-invasive way to get the insight of dynamic characteristics of the molecules of interest.

Figure 2. SPR microscopy images and immunofluorescence images of the same cell

Cell Line Affinity Measurement Using SPR Microscopy

Creative Biolabs can provide custom cell line affinity measurement using SPR microscopy and also membrane protein studies using an artificial bilayer to keep membrane proteins in their native structures. Compared with ex situ, SPR microscopy enables the direct in situ detection of ligand-receptor interaction more accurate. Therefore, SPR microscopy shows great potential in the discovery of drugs, including monoclonal antibodies and small-molecule drugs that target membrane proteins.

Other optional Antibody Affinity Measurement Services:

Published Data

Kinetic Measurements of Biomolecular Interactions with SPR Microscopy

Fig. 3 Bright-field (left) and SPRM (right) images of fixated SKBR3 cells. (Tianbao Dong, 2023)

The affinity and kinetic values based on the kinetic study of the binding of cell ligands or candidate drugs to membrane proteins are different from those measured using purified proteins. Here, under the same experimental conditions, the researchers measured the interaction of fluorophore-labeled lectins and antibodies with polysaccharides in HFF cells and human epithelial growth receptor 2 with SKBR3 cells. Combined with SPR microscopy, they studied the effects of labeling and cell/sub-cell heterogeneity on binding kinetics. The results showed that for the cell components whose structure and function were not closely dependent on cell activity, the ligand binding kinetics in fixed cells was only slightly different from that in living cells. The change in kinetics is due to the low mobility of the receptor, which is limited to the local environment produced by partially interconnected protein molecules. Cell/sub-cell heterogeneity and labeling of ligands can change the binding reaction more significantly. Therefore, fixed cells not only simplify the experimental procedure of drug screening and make detection more reliable, but also provide reliable kinetic information about the binding of drugs to cellular components whose structure will not be changed by chemical fixation.

References

Wang W, Yang Y, Wang S, et al. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells[J]. Nature chemistry, 2012, 4(10): 846-853.
Früh V, IJzerman A P, Siegal G. How to catch a membrane protein in action: a review of functional membrane protein immobilization strategies and their applications[J]. Chemical reviews, 2010, 111(2): 640-656.
Dong, Tianbao, et al. "Live cells versus fixated cells: kinetic measurements of biomolecular interactions with the LigandTracer method and surface plasmon resonance microscopy." Molecular Pharmaceutics 20.4 (2023): 2094-2104.

FAQ

What is the principle behind using SPR microscopy for measuring cell line affinity?
SPR microscopy measures the binding interactions between biomolecules by detecting changes in the refractive index near the sensor surface when biomolecules bind. This technique is particularly useful for assessing the affinity between a cell line expressing a particular receptor and its ligand. The real-time, label-free nature of SPR allows for the direct observation of the binding kinetics, providing insights into the strength and specificity of the interaction.
How can SPR microscopy help in drug development processes involving cell lines?
SPR microscopy is instrumental in drug development for screening and characterizing the interactions of drugs with target proteins expressed by cell lines. It enables the quantification of binding affinities and kinetics, which are crucial for evaluating the potential efficacy and specificity of drug candidates. By facilitating the rapid assessment of multiple interactions under various conditions, SPR microscopy accelerates the selection and optimization of the most promising therapeutic agents.
What are the advantages of using SPR microscopy for affinity measurements in cell lines over other methods?
Firstly, it allows for real-time monitoring of interactions, providing immediate insights into both the kinetics and the strength of the binding. Secondly, it does not require any labeling of the biomolecules, which can potentially alter their properties or functionality. Additionally, SPR can measure low-affinity interactions and requires only small amounts of sample, making it highly efficient and less resource-intensive.
Can SPR microscopy be used to measure the affinity of small molecule drugs to receptors on live cells?
SPR microscopy can be adapted to measure the binding affinity of small molecule drugs to receptors on the surface of live cells. This application is particularly useful in pharmacology for directly observing the drug-receptor interactions in a dynamic, live-cell environment. It helps in understanding how drugs interact with cellular receptors in real time, which is critical for assessing drug efficacy and safety profiles in early drug development stages.
How does temperature affect measurements in SPR microscopy when assessing cell line affinities?
Temperature can significantly impact the kinetics of molecular interactions. Higher temperatures can increase the rate of molecular movement, potentially leading to faster binding and dissociation rates. Conversely, lower temperatures might stabilize interactions but slow down the kinetics. Accurate temperature control during SPR experiments is essential to obtain reliable and reproducible data, as well as to mimic physiological conditions that are relevant to the biological processes being studied.
What is the sensitivity of SPR microscopy in detecting low-affinity interactions in cell lines?
SPR microscopy is highly sensitive and capable of detecting low-affinity interactions, which are often challenging to measure with other methods. It can detect interactions with dissociation constants (Kd) in the range of mM to pM, making it suitable for a wide array of biological processes. The technology's ability to provide quantitative data on binding affinity and kinetics even at low ligand concentrations is particularly valuable in drug discovery and basic research.
Can SPR microscopy measure competitive binding interactions in a cell line?
SPR microscopy is an excellent tool for studying competitive binding interactions within cell lines. By introducing a competitor molecule into the assay, researchers can observe how it affects the binding of a primary ligand to a receptor on the cell surface. This application is crucial for identifying potential inhibitors in drug development, as it allows for the evaluation of how different molecules interfere with or enhance each other's binding in real time.