- Service Details
- Case Study
Biacore System Based on the SPR (Surface Plasmon Resonance)
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.
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."
Surface Plasmon Resonance (SPR) Principle
The apparatus generating the SPR effect comprises four mediums: a thin metal film, a prism (which facilitates total internal reflection), the test sample, and air. When the incident light undergoes total internal reflection on the surface of the metal film, evanescent waves are produced. Under certain conditions, these evanescent waves excite the free electrons on the metal surface, creating a plasmonic state. Resonance occurs when the frequency and wave number of the surface plasmon match those of the evanescent wave, resulting in a resonance peak. If the refractive index of the sample on the metal surface changes, the position of the resonance peak shifts, establishing a correspondence between the refractive index and the resonance peak position.
Through this correspondence, the corresponding changes are reflected in the sensorgram. Information regarding binding reaction kinetics is extracted from the sensorgram, and the magnitude of dynamic parameters of interactions between molecules is used to assess the interactions between biomolecules.
Figure 1. The principle of Biacore (SPR). Schematic representation of a sensorgram curve when the sample solution pass over the sensor surface
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.
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.
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.
Our Biacore Service
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
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.
Advantages of Biacore Service
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.
|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.|
Creative Proteomics's research team can provide customers with professional and high-quality Biacore service. We will provide you with an accurate and sensitive Biacore platform, analysis reports, and other data collection and processing process related experimental reports. We are committed to providing you with expertise in a variety of analyses to help you solve problems related to technology and analysis.
Biacore Empowers HER2 Bispecific Antibody Development
An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity
Journal: Nature Communications
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.