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Antibody Affinity Measurement

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Label-free interaction analysis is of great importance for scientists to study interactions between biomolecules. Scientists from Creative Biolabs are competent for performing antibody affinity and kinetics measurement with the most advanced Biacore, ProteOn, Octet systems, etc. These systems are mainly based on the optical phenomena technologies - surface plasmon resonance (SPR) and bio-layer interference (BLI), which enable a direct detection and measurement of molecular interactions in a real-time monitoring manner.

SPR is an extremely sensitive method for the detection of molecular interactions by tracking the change of signal via sensor chips. SPR signal is defined as refractive index changes, which refers the response unit (RU) is approximately equivalent to a surface concentration of protein at 1pg/mm2. Plotting the SPR signal over time during the interaction between two molecules results in a sensorgram, which could be visualized in real time (Figure 1). General binding response includes three phases: association, equilibrium and dissociation; fitting these sensorgram data with a mathematic model allows scientists to calculate the association (Ka) and dissociation (Kd) rate constants and ultimately determine the binding affinity (KD). Different from isothermal calorimetry (ITC) that measures antibody binding affinity based on equilibrium point, SPR could obtain full kinetic parameters to evaluate all the effects of association and dissociation as well as antibody affinity.

Antibody Affinity Measurement Figure 1. SPR imaging process

In antibody affinity measurement, the antibodies to be analyzed are first captured by high-affinity anti-IgG antibody immobilized on the sensor chip surface. Then a series of concentrations of antigens are sequentially injected across the surface. Based on the sensorgram curve, people can rank the antibodies according to the kinetic parameters and select monoclonal antibodies with the best characters of your preference. Compared to the discovery of tight-binding antibodies, the identification of specific region (epitope) is more promising in drug discovery since the binding affinity could be matured via standard protein engineering approaches. Thus, based on the bivalent analyte model, which refers to two binding responses, we are capable of providing scouting assay to identify antibodies binding to two distinct antigen epitopes. Firstly, a generic anti-antibody (such as rabbit anti-mouse immunoglobulins) is covalently attached to the chip surface, and then the first antibody is captured by the generic antibody previously attached. After the blocking of unsaturated sites, the injected antigen will be bonded by the first antibody with the formation of Ab-Ag complex. Followed by the administration of a second monoclonal antibody which is applied to evaluate the binding affinity, a unique sensorgram curve will be observed if the second antibody binds to a distinct separate epitope. (Figure 2)

Antibody Affinity Measurement Figure 2. Major steps in epitope mapping. ① First antibody capture. ② Block unsaturated site. ③ interact with antigen. ④ Introduce with the second antibody ⑤ Rinse and regeneration

Creative Biolabs can provide custom antibody affinity measurement, and also any molecular interactions (molecular mass not less than 100) can be determined in a real time manner without labeling. All the data analysis will be performed and documented. Please feel free to contact us for a detailed quote.

Reference:
Abdiche, Yasmina N., et al. "Exploring blocking assays using Octet, ProteOn, and Biacore biosensors." Analytical biochemistry 386.2 (2009): 172-180.
Hearty, Stephen, Paul Leonard, and Richard O’Kennedy. "Measuring antibody–antigen binding kinetics using surface plasmon resonance." Antibody Engineering: Methods and Protocols, Second Edition (2012): 411-442.




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