Biacore™ System Introduction

Introduction Applications Published Data FAQ Resources

Scientists from Creative Biolabs are willing to provide custom antibody affinity measurement using Biacore system, which is a real-time biomolecular interaction analysis system based on surface plasmon resonance (SPR) technology. Our Biacore system can achieve custom services:

Scouting analysis-rank the monoclonal antibodies based on K_a/K_d and K_D.
Epitope mapping-identify monoclonal antibodies binding to different epitopes of a certain antigen.
Full kinetics analysis-obtain detailed binding kinetic data.

Introduction

Antibody screening aims to identify cell clones that produce specific antibodies for a certain purpose. In Biacore system, crude and complexed samples such as cell culture medium or ascitic fluids can be measured without the purification step which makes the whole process much faster so that clones can be selected before they become unstable. This is mainly attributed to the specific interaction between antibodies and ligands attached on the surface.

Compared with the strategy of immobilizing antigens as ligands that only captures specific antibodies, immobilizing antibodies as ligands can discriminate different epitopes and avoid the complex regeneration step.

Figure 1. Standard procedure of Biacore system for affinity measurement and kinetic determination.

Kinetic constants could be obtained over a range of analyte concentrations in real time. These data could be obtained by combining the sensorgram curve in relation to a mathematical model. Though it is unnecessary to know the exact concentration of ligand immobilized on the surface, the immobilization level affects the kinetic determination. Rmax is defined as the maximum response of analyte binding capacity. To evaluate the efficiency of immobilization, a concentrated analyte is injected to saturate the surface for measuring the activity level.

Biacore System

Generally, the amount of ligand is maintained at a relative low level to reduce mass transfer process and avoid the crowding of analytes. During the kinetic measurement, a capturing ligand provides on-chip purification that improves signal-to-noise ratio in order to eliminate the non-specific binding of sample components to the surface. While the dissociation rate (Kd) does not depend on the analyte concentration, it is crucial for association rate (Ka) and affinity constant (KD) measurement to be determined as accurately as possible.

Figure 2. Analyte concentrations for kinetic analysis should cover a wide range (center panel). If the concentrations are too low (left panel) or too high (right panel) the sensorgrams may be tightly grouped and evaluation will be less robust.

Different antibodies can bind to distinct and separated regions of the same antigen simultaneously, such antibody specificity study is named epitope mapping. Biacore system provides pair-wise binding to test the ability of pairs of antibodies binding with the same antigen simultaneously. For those antibodies who have overlapped epitopes or the conformation change caused by the first antibody affecting the following binding, peptide inhibition study can solve the problem. A peptide could represent one of the physical epitope for the blocking interactions test.

Applications of Biacore System

As it is reported, Biacore system is not limited to the characterization of antigen-antibody interaction. This system can also be used for the direct determination of real-time analysis and label-free binding strength of any molecular (molecular weight above 1,200 D). The utility of Biacore assay includes (but not limited to):

Determination of direct molecular interaction.
Receptor-ligand interaction.
Enzyme-substrate interaction.
Protein-DNA interaction.
Protein-protein interaction.
Batch record and drug stability assay.

Other optional Antibody Affinity Measurement Services:

Published Data

Interaction of Gal-7 and HMGCS1 Detection by the Biacore System

Fig. 3 In vitro interaction of Gal-7 with HMGCS1 as detected by various procedures. (Norihiro Fujimoto, 2021)

The interaction between HMGCS1 and the pro-apoptotic β-galactoside-binding protein Gal-7 was identified by the yeast two-hybrid system. The interactions were confirmed by in vitro β-galactosidase, Biacore, and immunoprecipitation, and a unique interaction site for HMGCS1 was found. Exogenous Gal-7 upregulated the expression of HMGCS1 in cultured keratinocytes, and it was found that the overexpression of HMGCS1 could induce the expression of Gal-7, indicating that the expression of Gal-7 and HMGCS1 was stimulated by positive feedback regulation.

References

Katsamba P S, Navratilova I, Calderon-Cacia M, et al. Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users[J]. Analytical biochemistry, 2006, 352(2): 208-221.
Biacore Assay Handbook, GE Healthcare
Fujimoto, Norihiro, et al. "Interaction of Gal-7 with HMGCS1 In Vitro May Facilitate Cholesterol Deposition in Cultured Keratinocytes." Journal of Investigative Dermatology 142.3 (2022): 539-548.

FAQ

What types of molecules can be analyzed using the Biacore system?
The Biacore system is versatile and can analyze a wide range of molecules including proteins, nucleic acids, lipids, carbohydrates, and small molecules. This system is particularly renowned for its application in studying protein-protein interactions, antibody-antigen bindings, and drug-target interactions. It's also useful for kinetic, affinity, concentration, and thermodynamic analyses.
Can Biacore system be used for vaccine development?
Biacore system is extensively used in vaccine development to analyze the interaction between antigens and antibodies. It helps researchers determine the binding kinetics and affinity of antibodies towards various antigens, which is crucial for developing effective vaccines. This system provides real-time, label-free data that can accelerate the evaluation and optimization of vaccine candidates.
How do I interpret the sensorgram obtained from a Biacore assay?
Interpreting a sensorgram involves analyzing the real-time binding and dissociation curves of the molecules involved. The upward curve represents the association phase where molecules bind to the sensor surface, and the downward curve represents the dissociation phase. Key parameters to focus on include the association rate (ka), dissociation rate (kd), and the equilibrium dissociation constant (KD), which can be calculated from the ratio of kd/ka. These parameters help understand the interaction dynamics and strength between the analyte and ligand.
How to improve the reproducibility of Biacore assays?
Improving reproducibility in Biacore assays involves several practices: maintaining consistent experimental conditions such as temperature and flow rate, using freshly prepared and well-characterized reagents, calibrating the system regularly, and ensuring rigorous data analysis protocols. Also, replicate measurements and control experiments are crucial for assessing the reliability of the data.
Can Biacore systems detect conformational changes in proteins during binding events?
Biacore systems can be used to detect conformational changes in proteins during binding events. This is possible by observing alterations in the response units on the sensorgram, which may indicate changes in the mass distribution or structural properties of the protein upon ligand binding. Advanced analysis techniques and experimental design are required to interpret these changes accurately, which can provide valuable insights into the functional mechanisms of the protein.