Presentation on theme: "Affinity Measurement with Biomolecular Interaction Analysis Biacore"— Presentation transcript:
1Affinity Measurement with Biomolecular Interaction Analysis Biacore
2What SPR Biosensors Measures KineticsAffinityThermodynamicsSpecificityConcentrationHow fast, strong & why…Is the binding of a lead compoundHow specific & selective...Is this drug binding to its receptor?How much...Biologically active compound is in a production batch?
3Biacore History Founded 1984 as Pharmacia Biosensor AB Biacore System launched October 1990Biacore Symposium 1991Inline referencing started 1994Became Biacore AB in 1996Support of regulated environments from 2002Entering the drug discovery market with S51 in 2002Going into protein arrays with Biacore A100 and Flexchip in 2005
8SPR detection Principle Result SPR detects refractive index changes close to the surfaceE.g. accumulation of 1 pg/mm2 gives a change of 1 µRIU or 1 RUAll biomolecules have refractive properties, so no labeling requiredResultNo need to separate bound from freeThis facilitates real-time measurements as a basis for taking kinetic dataWork with un-altered analytes possible
9Sensor ChipsSensor Chip specific matrixGlassGold 50 nm
10Sensor Chip CM5Dextran 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.
11Sensor Chip CM4Similar 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.
12Sensor Chip CM3Similar 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.
13Sensor 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
14Sensor Chip NTACM dextran matrix pre-immobilized with nitrilotriacetic acid (NTA)Capture of His-tagged ligands via metal chelationControled steric orientation of ligand for optimal site exposureRegeneration by injection of EDTA to remove metal ions
15Sensor Chip L1CM dextran matrix modified with lipophilic anchor moleculesFor rapid and reproducible capture of lipid membrane vesicles such as liposomes, with retention of lipid bilayer structureAllows studies of transmembrane receptors in a membrane-like environment , for example.
16The Steps in the Biacore Assay Surface preparationAnalysis Cycle
17Surface Preparation: Immobilization yteigdcpurmoanlyteigdDirectCaptureCovalent coupling ofRegeneration down toligandcapture molecule
22Capture Surfaces and Molecules TypeProduct/MoleculeCommentAnti-AntibodyRaM Fc anti-human FcAvailable from BiacoreUse affinity-pure productsAnti-taganti-GSTanti-HisStrep-MABanti-BiotinE.g. Penta-HisSee IBAAnti-FcProtein A / G / L-Biotin-bindingAvidin familyStrepTactinStreptavidin / NeutravidinOligosSequence specificHome madeSensor ChipSA, NTA, L1
23Analysis Cycle Generates the desired data Sample injection RegenerationEvaluation
24Analysis Cycle Done by Sample injection Results Regeneration Buffer flow, pH shift, salt & chaotrophic ions, detergentsSimilar concept as in affinity chromatographyResultsRe-use of biospecific surfaceLow amount of ligand neededSample injectionRegenerationEvaluation
26Experiments without Kinetics SpecificityMulti layer structureConcentration assaysAffinity constants
27Specificity 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.
28Specificity AnalysisOverplay plot of sensorgrams showing interaction between different lectins and immobilized thyroglobulin.
29Multiple Binding Enhancement Sandwich assays Epitope mapping Enhancing lower detection limit of assaysSandwich assaysEnhancing selectivity of testEpitope mappingCharting the surface of antigens with antibodiesMultimolecular complexesIdentify the logical sequence of binding events
31Epitope Specificity of two mAbs against HIV1-p24 Immobilization of rabbit rabbit anti-mouse IgG1A: baselineA-B: 1st mAb against HIV1-p24B-C: blocking antibodyC-D: HIV1-p24D-E: 2nd mAb against HIV1-p24
32Concentration Assays Concentration based on biological activity All concentration assays require a calibration curveConcentrations of unknowns samples are calculated from this4 - 7 concentrations in duplicateCalibrants and unknowns in same matrixModerate to high densities on sensor chipDirect binding formatsInhibition formats
34Affinity Analysis How STRONG is the binding at equilibrium? » Quantify KD» Rank Antibodies» Find best Ab pairs
35Affinity and Equilibrium Furosemide binding to carbonic anhydraseReferenced dataReport Point towards end of injectionDo secondary plot510152060120Signal [RU]Time [s]
36Determining Affinity Constants Plot Req against CSteady state modelConcentration at 50% saturation is KD
37Kinetic AnalysisHow FAST is the binding ? » ka kon (recognition) » kd koff (stability) » KD = kd/ka » Ab selection; wash steps
38Same Affinity but different Kinetics All four compounds have the same affinity KD = 10 nM = 10-8MThe same affinity can be the result from different kineticska[M-1s-1]kd[s-1]All target sites occupied30 min60 min100 nM1 µM30 min60 min10610-210510-310410-410310-5KD 10 nM
39Rate Constants Association rate constant ka Dissociation rate constant kdDefinitionkaA + B ABkdAB A + BUnit[M-1s-1][s-1]DescribesRate of complex formation, i.e. the number of AB formed per second in a 1 molar solution of A and BStability of the complex i.e. the fraction of complexes that decays per second.Typical range1x10-3 – 1x1071x10-1 – 5x10-6
40Equilibrium Constants Equilibrium dissociation constant KDEquilibrium association constant KADefinitionUnit[M][M-1]DescribesDissociation tendencyHigh KD = low affinityAssociation tendencyHigh KA = high affinityTypical range1x10-5 – 1x10-121x105 – 1x1012kd(A).(B)(AB)=ka(AB)ka=kd(A).(B)
44Extracting Rate Constants from Sensograms Measure binding curvesDecide on a model to describe the interactionFit the curve to a mathematical rate equation describing the modele.g.Obtain values for the constants ka, kd, RmaxAssess the fitoverlay pots, residual plotsacceptable statistics e.g. chi2 – curve fidelityBiological and experimental relevance of the calculated parametersdRdt= ka. C . (Rmax-R) – kd . R
45Biacore and other Methods ConventionalAssaysTimeMethodTimeIsotypingDay 1ELISAOne DayRIAWeeks + labellingAffinityDay 1&2KineticsDay 1&2NaNaELISAWeeks + labellingEpitope MapOvernightBiacore is much quicker than conventional methods
46SummarySurface plasmon resonance detects binding events as changes in mass at the chip surfaceReal-time kinetic measurementsQualitative rankingsMeasurement of concentrationsInformation about structure-activity relationshipsNo labeling and low volumes samples needed