NAA Affinity Measurement
NAA affinity measurement analysis is of great importance for scientists to study interactions between autoantigens and autoantibodies (NAA) in disease diagnosis. Creative Biolabs is a world’s leading service provider in the field of comprehensive NAA detection, NAA profiling, NAA affinity measurement, etc. With optimized strategies and advanced technology platform, our expert team offers the most professional solutions, the largest portfolio of premade or custom NAA products and services to help you get milestone development. Here, we focus on the introduction of NAA affinity measurement provided by our company.
Introduction of Antibody Affinity
Antibody affinity, which means the strength with which an antibody binds to its cognate antigen, is an equilibrium dissociation constant (KD), that is regulated by two kinetic constants, i.e., the rates of association (kon or ka) and dissociation (koff or kd). The rate of association is defined by the time required for the antibody to bind with its antigen. The dissociation rate, which is usually given as a half-life constant, defines the time the antibody-antigen complex remains intact. Several methods have been developed to measure these kinetic constants, which can be used to estimate kD. Surface Plasmon Resonance (SPR) is the most widely used method today.
NAA Affinity Measurement
NAA affinity measurement can be performed using the common detection method-Surface Plasmon Resonance (SPR). SPR is an extremely sensitive method for the detection of molecular interactions by tracking the changes of signal via sensor chips. SPR signal is established as refractive index changes, meaning that 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 can obtain a sensorgram, which could be visualized in real time. Generally, the binding response consists of 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). The measurement of antibody binding affinity based on equilibrium point is different from isothermal calorimetry (ITC) method, SPR could obtain full kinetic parameters to evaluate all the effects of association and dissociation as well as antibody affinity.
Fig.1 Schematic view of the surface plasmon resonance immunoassay technique. (Shankaran, 2007)
Workflow of NAA Affinity Measurement
In NAA affinity measurement, firstly, the autoantibodies of serum or other body fluids to be analyzed are captured by high-affinity anti-IgG antibody immobilized on the sensor chip surface. Then different concentrations antigens are sequentially added across the surface. We can rank the antibodies based on the sensorgram curve and kinetic parameters. In addition, according to the bivalent analyte model, we can identify autoantibodies binding to two distinct antigen epitopes. Firstly, a generic anti-antibody is covalently attached to the chip surface, and then the first antibody is captured by the generic antibody previously attached. After the blocking of unbound sites, the injected antigen will be combined by the first antibody with the formation of Ab-Ag complex. Followed by the implementation 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.
Features of Our Services
- Determination of direct NAA-antigen interactions;
- High throughput analysis;
- Full kinetics analysis, such as detailed binding kenitic data;
- Timely and cost-effective services.
Creative Biolabs is a professional service provider in antibody analysis, design and discovery. At present, we can provide a comprehensive set of NAA services to help clients detect NAA biomarkers, perform NAA affinity measurement, NAA profiling, etc. in a timely and cost-effective manner. Our proven and optimized platforms can help you quickly get satisfactory results without repeated trials, contributing greatly to the success of your projects. Please feel free to contact us for more information and a detailed quote.
Reference:
- Shankaran, D.R.; et al. Recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest. Sensors & Actuators B Chemical. 2007, 121(1): 158-177.