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In both drug development and fundamental research, the investigation of interactions between proteins and small molecules constitutes a crucial type of experiment. Small molecules are primarily characterized by their relatively low molecular weights, typically below 1000 Da. Moreover, they exhibit diverse structural properties, and some have poor solubility, necessitating the use of organic solvents (such as DMSO) for dissolution. These characteristics pose significant challenges for detecting their interactions. The Biacore system, based on the Surface Plasmon Resonance (SPR) principle, offers advantages such as high sensitivity and the ability to perform solvent corrections. It can detect even weak signals and accurately characterize interactions involving molecules with disparate molecular weights. Furthermore, it can mitigate the effects of organic solvents. As a result, an increasing number of researchers are opting for the Biacore system to study protein-small molecule interactions.
What is Biacore System?
Biacore is a classic bioanalytical sensing device developed based on the Surface Plasmon Resonance (SPR) principle. The device consists of three core components: a sensor chip, an SPR optical detection system, and a microfluidic cartridge. During experiments, a specific biomolecule is immobilized on the dextran-coated surface of the sensor chip, and molecules interacting with it are dissolved in a solution that flows over the chip's surface. The SPR detector tracks changes throughout the binding and dissociation processes between molecules in the solution and those on the chip's surface. These changes are recorded as sensorgrams, providing kinetic and affinity data."
How Does Biacore Work?
Biacore SPR Principle
SPR relies on the principle that changes in the refractive index near a sensor surface can alter the resonance conditions of light. When polarized light hits a gold-coated sensor chip at a certain angle, it induces electron oscillations (surface plasmons) in the gold layer.
These surface plasmons resonate with the incident light, creating a phenomenon known as surface plasmon resonance. However, this resonance condition is sensitive to changes in the refractive index near the sensor surface, which is influenced by the binding of molecules.
Figure 1. The principle of Biacore (SPR). Schematic representation of a sensorgram curve when the sample solution pass over the sensor surface
Sensor Chip Setup
The Biacore sensor chip surface is modified to immobilize one of the interaction partners, usually referred to as the "ligand," while the other molecule (the "analyte") is injected in a flow across the surface. The ligand-analyte pair can vary depending on the molecules under study.
Real-time Measurement
When the analyte flows over the surface, it interacts with the immobilized ligand. If binding occurs, the refractive index at the surface changes, altering the SPR signal. This change is recorded in real time as a "sensorgram," a plot showing the SPR response (typically in response units, or RU) over time.
Association and Dissociation Phases
- Association Phase: As the analyte binds to the ligand, the SPR signal increases, providing data on the association rate.
- Dissociation Phase: Once the analyte flow stops, it begins to dissociate, leading to a decrease in the SPR signal, which provides information on the dissociation rate.
Data Analysis
The data from these association and dissociation phases allow for the calculation of kinetic parameters: the association rate constant (ka) and dissociation rate constant (kd), and from these values, the affinity constant (KD) can be derived.
Surface Plasmon Resonance Service Based on Biacore System
Creative Proteomics has established a Biacore service platform, which features label-free samples, high sensitivity, rapid detection, real-time quantitative testing, and more. By detecting changes in SPR angles, we obtain information about the concentration, affinity, kinetic constants, specificity, and more of the analyte. This platform is extensively used to study the interactions among biomolecules such as proteins, small molecules, DNA/RNA, lipids/liposomes/biomembranes, polysaccharides, peptides, and whole cells.
We offer the following Biacore testing services, but not limited to:
Antibody screening and affinity maturation
Antibody-Fc receptor/complement affinity determination
Protein product concentration determination
Protein product activity determination
Protein product batch consistency determination
Small molecule compound screening, natural drug screening
Antibody epitope mapping
Antibody pairing analysis
Antibody characterization, antibody engineering
Drug, small molecule drug, natural drug characterization
Protein-protein interaction detection
Protein-peptide interaction detection
Protein-nucleic acid interaction detection
Protein-small molecule interaction detection
Nucleic acid aptamer screening
Ligand fishing
Fields of Study
Cellular signaling pathways, molecular structure-function relationships, nucleic acid-protein recognition and regulation, enzyme-substrate-inhibitor development, high-throughput antibody screening, receptor-ligand recognition, disease mechanisms, infectious disease pathogenesis, new drug discovery and development, and more.
Various Testing Methods
Creative Proteomics can flexibly choose ligand immobilization methods and analysis methods based on the sample conditions, aiming to achieve high-quality analytical results while minimizing sample consumption and reducing sample preparation complexity. Ligand proteins can be directly immobilized on the chip using amino coupling methods, or immobilized using capture methods based on protein tags. Analytes can be injected using single-cycle or multi-cycle modes. Depending on the different molecular interaction mechanisms, results can be analyzed using kinetic or steady-state affinity analysis modes.
Detectable Samples by Biacore
The range of samples detectable by Biacore is quite extensive, including proteins, peptides, DNA/RNA, lipids/liposomes/biomembranes, polysaccharides, peptides, small molecules, nanoparticles, polymer materials, bacteria, viruses, as well as samples like tissue lysates, serum, ascites, and more.
Applications of Biacore
Biological Macromolecule Screening
Capable of screening around 1000-30000 samples, providing information on specificity, affinity, kinetics, and ranking order.
Biological Macromolecule Characterization
Able to characterize 10-100 samples, providing information on affinity, kinetics, active concentration, epitope mapping, stability, efficacy, pharmacokinetics, and more.
Small Molecule Drug Screening
Capable of screening around 1000-30000 samples, providing information on specificity, affinity, kinetics, and dose dependency.
Small Molecule Drug Characterization
Able to characterize 10-150 samples, serving affinity, selectivity, MOA (Mechanism of Action), structural optimization, efficacy, pharmacokinetics, and more.
Advantages of Surface Plasmon Resonance Assays
- High sensitivity: The detection limit can be as low as picomolar to nanomolar range for the analyte.
- High specificity: The specificity is determined by the properties of the biomolecules attached to the sensor surface.
- Label-free analysis: It does not require the analytes to be labelled with a colorimetric or fluorescent label.
- Real-time monitoring: The interaction between the ligand and analyte resulting in the change of the sensor surface can be monitored in real time.
- Customized service: We can customize the service based on the needs of your research plan and provide professional solutions to you. You can determine or recommend the required items for analysis.
Sample Requirements for SPR Services
| Interaction Type | Sample Requirements |
|---|---|
| Protein-Small Molecule | Protein concentration > 200 μg/ml, volume > 100 μl (estimated at the lowest concentration); Provide small molecule molecular weight and structure, preferably as powder; Provide small molecule dissolution information, ability to dissolve in water or DMSO, DMSO dissolution concentration ≥ 10 mM, water dissolution concentration ≥ 1 mM. |
| Protein-Protein | For antigen-antibody interaction, provide specific details; Ligand protein concentration > 200 μg/ml, volume > 100 μl (estimated at the lowest concentration); Analyte protein concentration > 200 μg/ml, volume > 500 μl (estimated at the lowest concentration). |
| Protein-Nucleic Acid | Nucleic acid must be labeled with Biotin, concentration ≥ 100 nM, volume > 50 μl (estimated at the lowest concentration); Protein concentration > 200 μg/ml, volume > 500 μl (total > 20 μg). |
| Protein-Other | Protein-cell surface interaction: cell size must not exceed 30 μm; For other personalized tests, provide information about analyte properties, concentration, molecular weight, etc., and customize the protocol based on sample characteristics. |
Biacore Empowers HER2 Bispecific Antibody Development
An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity
Journal: Nature Communications
Published: 2023
This paper introduces a novel HER2 IgG1-specific bispecific antibody, zanidatamab, which exhibits superior anti-tumor activity and potent cytotoxic effects against various transplant tumor models and in vitro high-expressing HER2 tumor cells through multiple mechanisms of action.
Figure 1: Zanidatamab Structure
The structure of zanidatamab is relatively straightforward, consisting of single-chain variable fragments (scFv) and Fab fragments linked to two heavy chains. The scFv binds to HER2 ECD4, while the Fab binds to HER2 ECD2. Utilizing Biacore technology, the original scFv and Fab were measured to have binding KD values of 1 nM and 15 nM, respectively, to HER2 ECD. By modifying and screening the low-affinity Fab, a thermally stable Fab with an 8.8-fold increase in affinity was identified. This enhanced Fab was incorporated into the final zanidatamab antibody, exhibiting an affinity of 0.74 nM (Table 1).
Table 1: Biacore Determination of Affinities for scFv, Fab, and OAA-Fab
The research team designed ingenious experiments to decipher the binding patterns of HER2 with Pertuzumab, Pertuzumab precursor, and Trastuzumab using Biacore. The experiment utilized a CM5 chip immobilized with sheep anti-human antibodies to capture the three types of antibodies. Simultaneously, the team employed varying capture levels (15~400 RU) to determine if the binding mode between the antibodies and HER2 changes at different concentrations. The results revealed that, unlike Trastuzumab, both the dissociation rate Kd of Pertuzumab and its precursor increase with higher antibody concentrations.
Figure 2: Biacore Analysis of the Binding of Three Antibodies to HER ECD at Different Antibody Concentrations
Through Biacore experiments, the research team revealed the binding patterns of Pertuzumab at different concentrations. At low antibody concentrations, Pertuzumab binds to HER2 ECD in a 1:1 cis conformation. However, at high antibody concentrations, HER2 ECD can be cross-linked by two Pertuzumab molecules through ECD2 and ECD4 (Figure 3).
Figure 3: Biacore Validation of the Binding Mode between Pertuzumab and HER2
In this study, the research team not only enhanced the binding affinity of Pertuzumab using Biacore technology but also conducted in-depth investigations. They employed a capture-based approach to unravel the binding model of Pertuzumab with HER2 at the molecular level. This allowed them to determine both cis and trans binding models at different antibody concentrations.
How does SPR accommodate organic solvents, and how can solvent effects be controlled?
Organic solvents, particularly DMSO, are often used to dissolve hydrophobic analytes in SPR experiments. The Biacore system is equipped to compensate for refractive index changes caused by solvents. During testing, we run solvent controls and create calibration curves to account for any effects on SPR signals. For best results, we recommend informing us of the solvent concentration in advance so we can perform appropriate corrections.
How much sample is typically required, and what is the minimum concentration for reliable data?
Sample requirements vary depending on the interaction type and molecule size. For protein-small molecule interactions, a minimum concentration of 200 µg/mL for proteins and a molecular weight of over 1000 Da for small molecules typically ensure reliable signal detection. However, if sample quantity is limited, we can explore alternate methods, such as high-sensitivity sensor chips, to work with lower concentrations without compromising data quality.
What is the expected sensitivity range for detecting low-affinity interactions?
Biacore SPR is highly sensitive, capable of detecting interactions with dissociation constants (KD) ranging from picomolar to millimolar levels. For low-affinity interactions (KD above micromolar), achieving sufficient analyte concentration is key to obtaining clear sensorgrams. Our team can assist in optimizing assay conditions, such as flow rate adjustments or alternative immobilization techniques, to capture low-affinity interactions effectively.
Can Biacore SPR be used for competitive binding assays, and what are the considerations for such experiments?
Yes, Biacore SPR can be effectively used for competitive binding assays to determine the binding affinities of competing analytes for the same ligand. In these assays, we often inject the competitor molecule after establishing a baseline with the primary analyte. It's crucial to maintain optimized analyte and competitor concentrations, as too high or low a concentration may mask true competitive interactions. We recommend pilot tests to fine-tune assay parameters for clear, reproducible results.
How are kinetic and affinity parameters determined, and which analysis model should I choose?
The Biacore system provides data on association (ka) and dissociation (kd) rates, allowing calculation of the affinity constant (KD). Depending on the molecular interaction, we use either a kinetic analysis model (for interactions where time-resolved binding and dissociation are important) or a steady-state affinity model (suitable when interactions rapidly reach equilibrium). For complex binding behaviors, such as those involving conformational changes, we may employ a bivalent or heterogeneous ligand model. Consulting with our experts can help you select the best model for your specific interaction.
What is sensorgram drift, and how can it affect my data?
Sensorgram drift is a gradual change in the baseline SPR response over time, often due to non-specific binding or instability in the flow cell. This drift can affect data accuracy, particularly in long experiments or with sensitive interactions. We address drift by performing solvent referencing and using buffer blanks to stabilize the baseline. Additionally, rigorous surface conditioning between samples minimizes drift and ensures more accurate measurements.
Can I reuse a Biacore sensor chip, and if so, how many times can it be regenerated?
Yes, sensor chips can be reused multiple times if they are properly regenerated after each run. The number of uses depends on the stability of the immobilized ligand and the effectiveness of the regeneration process. Commonly, chips can be regenerated 20-50 times, though this varies. Our team carefully selects regeneration solutions based on ligand and analyte properties to maximize chip longevity without compromising performance.
How can SPR data assist in understanding the mechanism of action (MOA) of a drug?
SPR provides real-time data on binding kinetics and affinities, which are critical for MOA studies. By analyzing how a drug interacts with its target at different concentrations, researchers can deduce whether binding is reversible or irreversible, cooperative or competitive. Detailed kinetic data can reveal interaction rates and binding sites, which can help predict in vivo behavior, optimize drug efficacy, and assess potential side effects.
What can be done if I observe non-specific binding during the SPR experiment?
Non-specific binding can be mitigated through several approaches, including optimizing buffer composition, adjusting pH, or using detergents and additives that reduce unwanted interactions. In cases where non-specific binding persists, we can suggest alternative immobilization methods or recommend blocking agents to minimize its impact. Custom assay optimization is available to address any persistent non-specific interactions.
How does temperature impact Biacore SPR experiments, and what considerations should be made?
Temperature significantly affects molecular interactions. The Biacore system offers temperature control to optimize assay conditions, as some interactions require specific temperatures for stability or optimal binding. Generally, room temperature (around 25°C) is standard, but certain protein-protein or enzyme-substrate interactions may perform better at physiological temperatures (37°C). Temperature effects are critical for kinetic analysis, as both association and dissociation rates are temperature-sensitive.
How long does a typical Biacore SPR experiment take, and is high-throughput analysis feasible?
A standard Biacore SPR experiment may range from a few minutes for simple binding studies to several hours for detailed kinetic or competition analyses. The Biacore system allows for high-throughput screening in multi-cycle analysis mode, making it possible to test thousands of samples per day. For high-throughput requirements, we optimize flow rates, regeneration times, and sample handling to maximize efficiency while maintaining data quality.
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