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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:
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.
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.
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):
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
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