Antibody Affinity Testing(SPR)-Special Topic on Testing Services-Tekbiotech-Yeast Display Service,Phage display technology

Antibody Affinity Testing(SPR)

The detection of bioactive compounds is one of the important topics in the field of molecular biology and biochemistry. One of the most promising detection methods is the label-free detection of bioactive compounds using surface plasmon resonance technology. This method allows real-time monitoring of binding without the need for labeling. Therefore, the system can be used to determine the affinity and rate constants of interactions between various types of molecules. Monoclonal antibodies (MAbs) are powerful tools for examining the structure and function of biomolecules. Knowledge of the distribution and structural characteristics of epitopes defined by MoAbs can be used to identify regions involved in biological activities.

I. Basic Principles of Antibody Affinity Detection

Surface plasmon resonance is a physical process that occurs when plane polarized light hits a metal film under conditions of total internal reflection. Typically, the surface that produces SPR consists of a 50nm thick metal layer deposited on glass. Since the SPR effect can only occur on thin metal layers, where electrons behave as a free electron gas, meaning that their movement is independent of the charge left behind when moving, the choice of metals is limited to copper, silver, aluminum, and gold.

Substrates for SPR measurements and single-molecule immobilization of bioactive molecules by self-assembly. SPR is detected by measuring the intensity of reflected light. At the total internal reflection angle, a sharp drop in the intensity of the reflected light is measured. The position of the SPR angle depends on the refractive index of the substance close to the sensing surface. The refractive index near the sensor surface changes due to the binding of macromolecules to the surface. Therefore, the total internal reflection angle will change depending on the number of bound macromolecules. There is a linear relationship between the amount of bound substances and the shift in the total internal reflection angle.

Analytes in aqueous buffer solution are induced to flow over the sensor surface by injecting them into the flow cell. The interaction of the analyte with the ligands attached to the metal surface induces changes in the metal surface and surface plasmon fields. The shift in the resonant wavelength of the incident light is measured. The light passes through the prism and slides, reflects from the gold, and returns to the detector through the prism. The high sensitivity of the optical response is due to a very efficient collective excitation of conduction electrons near the gold surface. Since SPR detection is independent of the chemical nature of the sample being analyzed, in principle, all types of molecules (such as proteins, lipids, nucleic acids) and small molecules (such as drugs, substrates and cofactors) can be used to monitor biomolecular interactions.

In SPR biosensing experiments, the entire gold surface containing the immobilized ligand is thoroughly washed with buffer. After the appearance of a stable baseline, a solution containing the analyte is introduced (binding phase). The interaction of the analyte with the ligand attached to the surface results in an increase in the signal. At the end of the analyte injection, the sample is replaced by a continuous flow of buffer and the decrease in the signal now reflects the dissociation of the analyte from the surface-bound complex. The surface is then regenerated with a specific regeneration reagent and the signal returns to a stable baseline in the presence of buffer.

Due to the very specific interaction between the immobilized bioactive material and the analyte, no other biomaterial is able to form complexes with the immobilized bioactive molecules and significantly affect the analytical signal of the SPR biosensor. The shift in the reflection angle of the reflected light is interpreted as the analytical signal. This signal is proportional to the concentration of the analyte forming the affinity complex. Therefore, by monitoring the shift in the minimum reflection angle, the extent of the formation of the affinity complex between the analyte present in the solution and the immobilized ligand can be easily observed and quantified.

One of the advantages of SPR is that the method allows one to study the interaction of biomolecules in real time. There is also no need for any labeling (e.g. radioactivity or fluorescence), which, in addition to saving time, helps preserve the native nature of the biomolecules. SPR biosensors have been used to study a wide range of biomolecular interactions, providing qualitative (identification, site specificity, epitope localization) and quantitative (kinetics, affinity and concentration analysis) information.

2. Antibody Affinity Detection Service Case

Sensor graph of the interaction between a certain protein and a small molecule - Tekbiotech.png

Figure 1 Sensor graph of the interaction between a certain protein and a small molecule

Tekbiotech provides customers with accurate and rapid affinity determination services based on platforms such as Octet and Biacore, and provides unique molecular interaction analysis services according to customer needs, including but not limited to commonly used detection technologies in the industry: surface plasmon resonance (SPR) and bio-layer interferometry (BLI), to ensure that the test results are accurate, objective and reliable. We focus on providing customers with high-quality one-stop technical services to meet the experimental needs of different customers.

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