Application of Biacore Technology

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1 Principles and typical results Application of Biacore Technology Common types of Biacore analyses Specificity analysis Is my molecule of interest specific for its target? Multiple binding analysis In which order do the different components in the complex bind to each other? How much analyte is there in the sample? Affinity analysis How strong is the binding between ligand and analyte? Kinetic analysis How fast are complex formation and decay? Thermodynamic analysis Why is the interaction this fast/strong? 2 / GE Biacore T100 training_applications / Specificity analysis Is my molecule of interest specific for its target? Flexibility in Biacore assay design allows assessment of crossreactivity and specificity Does my antibody recognize multiple derivatives? Which members of the protein family are detected? Screen for inhibitor specificity by using several ligands Determine the specificity of a drug for its target receptor 3 / GE Biacore T100 training_applications / 1 /

2 Multiple binding analysis To monitor stages in the formation of molecular complexes Determine the isotype of antibodies binding to an immobilized antigen Determine epitope specificity by testing sequential binding of multiple antibodies to an antigen 4 / GE Biacore T100 training_applications / How much analyte is there in my sample? Serum concentration of IgG Quality control of production batches Levels of streptomycin in bovine milk 5 / GE Biacore T100 training_applications / Calibration curves All concentration assays require a calibration curve 6 / GE Biacore T100 training_applications / 2 /

3 Different assay formats Direct binding assay Indirect binding assay 7 / GE Biacore T100 training_applications / - Typical results Indirect binding assay with anti-biotin as detecting molecule 8 / GE Biacore T100 training_applications / Affinity analysis by steady-state analysis How strong is the binding between analyte and ligand? Usually used when the observed binding rates are too fast to determine accurately Affinity of a monoclonal antibody for its antigenic epitope Affinity of the T cell receptor for MHC class II Affinity of a drug for its target receptor 9 / GE Biacore T100 training_applications / 3 /

4 Calculating affinity constants Select Curves Select Data Select Affinity Data Affinity analysis yields the equilibrium constants (K A and K D ) Fit Affinity 10 / GE Biacore T100 training_applications / Affinity analysis Typical results LMW binding to immobilized DNA ( µm) 11 / GE Biacore T100 training_applications / Kinetic analysis How fast do the ligand and analyte associate and dissociate? Same affinity, but different kinetics! 12 / GE Biacore T100 training_applications / 4 /

5 Kinetic analysis - Extracting constants Select Curves Select Data Fit Kinetics Kinetic analysis yields the rate constants (k a & k d ) and the equilibrium constants (K A & K D ) 13 / GE Biacore T100 training_applications / Kinetic analysis Typical results Binding beta-2-microglobulin to immobilized anti-beta-2- microglobulin (2 nm to 32 nm) 14 / GE Biacore T100 training_applications / Thermodynamic analysis Fit Kinetics and/or Affinity Select Kinetics Data Thermodynamic overview Create results Thermodynamic analysis yields van t Hoff and Eyring plots and thermodynamic constants 15 / GE Biacore T100 training_applications / 5 /

6 Thermodynamic analysis - Typical results Binding beta-2-microglobulin to immobilized anti-beta-2-microglobulin (5 temperatures) 16 / GE Biacore T100 training_applications / References to Biacore Applications More than 4000 peer-reviewed references in which Biacore systems have been used Refer to Use keywords or abstracts to search the peer-reviewed references Download Biacore Application Notes for useful information on different types of applications 17 / GE Biacore T100 training_applications / 6 /