Presentation on theme: "Affinity Measurement with Biomolecular Interaction Analysis Biacore."— Presentation transcript:
Affinity Measurement with Biomolecular Interaction Analysis Biacore
What SPR Biosensors Measures How specific & selective... Is this drug binding to its receptor? How much... Biologically active compound is in a production batch? SpecificitySpecificityConcentrationConcentration How fast, strong & why… Is the binding of a lead compound KineticsKineticsAffinityAffinity ThermodynamicsThermodynamics
Biacore History Founded 1984 as Pharmacia Biosensor AB Biacore System launched October 1990 Biacore Symposium 1991 Inline referencing started 1994 Became Biacore AB in 1996 Support of regulated environments from 2002 Entering the drug discovery market with S51 in 2002 Going into protein arrays with Biacore A100 and Flexchip in 2005
Probing Biological Affinities
The Corner-stones of the Technology
The SPR Detector
Total Internal Reflection & SPR Gold layer Evanescent field Total Internal Reflected light (TIR) TIR angle Incident Light High refractive index medium: Prism Low refractive index medium: Buffer
SPR detection Principle SPR detects refractive index changes close to the surface E.g. accumulation of 1 pg/mm 2 gives a change of 1 µRIU or 1 RU All biomolecules have refractive properties, so no labeling required Result No need to separate bound from free This facilitates real-time measurements as a basis for taking kinetic data Work with un-altered analytes possible
Sensor Chip CM5 Dextran matrix covered with carboxyl groupes (red circles) Captures ligands such as proteins, lipids, carbohydrates and nucleic acids (irreversible) Study of analytes ranging in size from small organic molecules, e.g. drug candidates, to large molecular assemblies or whole viruses.
Sensor Chip CM4 Similar to CM5 but with a lower degree of carboxymethylation resulting in low immobilization capacity and lower surface charge density. Allows to reduce non specific binding in case of complex mixture such as cell extract or culture medium. Advantageous for kinetic experiments where low immobilization levels are recommended.
Sensor Chip CM3 Similar to CM5 but with shorter dextran chains, giving a lower immobilization capacity of the surface. Allows the interaction to take place closer to the cell surface which can improve sensitivity when working with large molecules, molecular complexes, viruses or whole cells.
Sensor Chip SA CM dextran matrix pre-immobilized with streptavidin Captures biotinylated ligands such as carbohydrates, peptides, proteins and DNA (irreversible) Ideal for capture of large biotinylated DNA fragments and study of nucleic acid interactions
Sensor Chip NTA CM dextran matrix pre-immobilized with nitrilotriacetic acid (NTA) Capture of His-tagged ligands via metal chelation Controled steric orientation of ligand for optimal site exposure Regeneration by injection of EDTA to remove metal ions
Sensor Chip L1 CM dextran matrix modified with lipophilic anchor molecules For rapid and reproducible capture of lipid membrane vesicles such as liposomes, w i th retention of lipid bilayer structure Allows studies of transmembrane receptors in a membrane-like environment, for example.
Surface preparation Analysis Cycle The Steps in the Biacore Assay
Surface Preparation: Immobilization Direct Capture analyte ligand analyte ligand capturing molecule Covalent coupling of Regeneration down to ligandcapture molecule
Various Coupling Chemistries
Activation Ligand contact Blocking Amine Coupling - Sensorgram Activation = EDC/NHS injection surface esters Ligand contact = reaction with amine groups on ligand Blocking = deactivation of free esters with ethanolamine
High Affinity Capture
Capture Surfaces and Molecules TypeProduct/MoleculeComment Anti-Antibody RaM Fc anti-human Fc Available from Biacore Use affinity-pure products Anti-tag anti-GST anti-His Strep-MAB anti-Biotin Available from Biacore E.g. Penta-His See IBA Use affinity-pure products Anti-FcProtein A / G / L- Biotin-binding Avidin family StrepTactin Streptavidin / Neutravidin See IBA OligosSequence specificHome made Sensor ChipSA, NTA, L1Available from Biacore
Sample injection Regeneration Evaluation Analysis Cycle Generates the desired data
Sample injection Regeneration Evaluation Analysis Cycle Done by Buffer flow, pH shift, salt & chaotrophic ions, detergents Similar concept as in affinity chromatography Results Re-use of biospecific surface Low amount of ligand needed
The Result: the Sensorgram
Experiments without Kinetics Specificity Multi layer structure Concentration assays Affinity constants
Specificity Do two molecules interact with each other? Yes/No Answers. Different analytes are tested with the same ligand e.g. different lectins with immobilized thyroglobulin. Quantitative measurements, test a range of analyte concentration to determine the concentration dependency of the response.
Specificity Analysis Overplay plot of sensorgrams showing interaction between different lectins and immobilized thyroglobulin.
Multiple Binding Enhancement Enhancing lower detection limit of assays Sandwich assays Enhancing selectivity of test Epitope mapping Charting the surface of antigens with antibodies Multimolecular complexes Identify the logical sequence of binding events
Epitope Specificity of two mAbs against HIV1-p24 Immobilization of rabbit rabbit anti-mouse IgG1 A: baseline A-B: 1 st mAb against HIV1-p24 B-C: blocking antibody C-D: HIV1-p24 D-E: 2 nd mAb against HIV1-p24
Concentration Assays Concentration based on biological activity All concentration assays require a calibration curve Concentrations of unknowns samples are calculated from this concentrations in duplicate Calibrants and unknowns in same matrix Moderate to high densities on sensor chip Direct binding formats Inhibition formats
Calibration Curves x x x x x Response Concentration Sample Sample matrix for calibration curve Sample matrix for unknown samples =
Affinity Analysis How STRONG is the binding at equilibrium? » Quantify K D » Rank Antibodies » Find best Ab pairs
Affinity and Equilibrium Furosemide binding to carbonic anhydrase Referenced data Report Point towards end of injection Do secondary plot Signal [RU] Time [s]
Determining Affinity Constants Plot Req against C Steady state model Concentration at 50% saturation is K D
How FAST is the binding ? » k a k on (recognition) » k d k off (stability) » K D = k d /k a » Ab selection; wash steps Kinetic Analysis
Same Affinity but different Kinetics All four compounds have the same affinity K D = 10 nM = M The same affinity can be the result from different kinetics All target sites occupied 30 min 60 min 100 nM 1 µM 30 min 60 min k d [s -1 ] k a [M -1 s -1 ] K D 10 nM
Rate Constants Association rate constant k a Dissociation rate constant k d Definition k a A + B AB k d AB A + B Unit[M -1 s -1 ][s -1 ] DescribesRate of complex formation, i.e. the number of AB formed per second in a 1 molar solution of A and B Stability of the complex i.e. the fraction of complexes that decays per second. Typical range1x10 -3 – 1x10 7 1x10 -1 – 5x10 -6
Equilibrium Constants Equilibrium dissociation constant K D Equilibrium association constant K A Definition Unit[M][M -1 ] DescribesDissociation tendency High K D = low affinity Association tendency High K A = high affinity Typical range1x10 -5 – 1x x10 5 – 1x10 12 kdkd (A).(B) (AB) = kaka kaka (A).(B) = kdkd
Equilibrium and Kinetic Constants are related
Equilibrium and Kinetics in Biacore
Information in a Sensorgram
Extracting Rate Constants from Sensograms Measure binding curves Decide on a model to describe the interaction Fit the curve to a mathematical rate equation describing the model e.g. Obtain values for the constants ka, kd, Rmax Assess the fit overlay pots, residual plots acceptable statistics e.g. chi 2 – curve fidelity Biological and experimental relevance of the calculated parameters dR dt = k a. C. (R max -R) – k d. R
Biacore and other Methods Biacore Assays Isotyping Affinity Kinetics Epitope Map Time Conventional MethodTime Day 1 Day 1&2 Overnight ELISA One Day RIAWeeks + labelling Na ELISA Weeks + labelling Biacore is much quicker than conventional methods
Surface plasmon resonance detects binding events as changes in mass at the chip surface Real-time kinetic measurements Qualitative rankings Measurement of concentrations Information about structure-activity relationships No labeling and low volumes samples needed Summary