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Affinity Measurement with Biomolecular Interaction Analysis Biacore What SPR Biosensors Measures Kinetics Affinity Specificity Concentration Thermodynamics How fast, strong & why… How specific & selective... Is the binding of a lead compound Is this drug binding to its receptor? How much... Biologically active compound is in a production batch? 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 SPR Detection Sensor Chips IFC Microfluidic 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 Result SPR detects refractive index changes close to the surface No need to separate bound from free E.g. accumulation of 1 pg/mm2 gives a change of 1 µRIU or 1 RU This facilitates real-time measurements as a basis for taking kinetic data All biomolecules have refractive properties, so no labeling required Work with un-altered analytes possible Sensor Chips Glass Sensor Chip specific matrix Gold 50 nm 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, with retention of lipid bilayer structure • Allows studies of transmembrane receptors in a membrane-like environment , for example. The Steps in the Biacore Assay Surface preparation Analysis Cycle Surface Preparation: Immobilization a n a l y te a n a ly te li g a n d lig a n d c a p t u r in g m o le c u l e Direct Covalent coupling of Regeneration down to ligand Capture capture molecule Direct Immobilization Various Coupling Chemistries Amine Coupling - Sensorgram • Activation = EDC/NHS injection surface esters • Ligand contact = reaction with amine groups on ligand • Blocking = deactivation of free esters with ethanolamine Blocking Activation High Affinity Capture Capture Surfaces and Molecules Type Product/Molecule Comment 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-Fc Protein A / G / L - Biotin-binding Avidin family StrepTactin Streptavidin / Neutravidin See IBA Oligos Sequence specific Home made Sensor Chip SA, NTA, L1 Available from Biacore Analysis Cycle Sample injection Regeneration Evaluation Generates the desired data Analysis Cycle • Done by Sample injection Regeneration • Buffer flow, pH shift, salt & chaotrophic ions, detergents • Similar concept as in affinity chromatography • Results Evaluation • 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 Multiple Binding Analyte Ligand 2nd Binder Response [RU] 31000 30000 29000 28000 27000 26000 50 100 150 200 Time [s] 250 300 350 400 Epitope Specificity of two mAbs against HIV1-p24 Immobilization of rabbit rabbit anti-mouse IgG1 A: baseline A-B: 1st mAb against HIV1-p24 B-C: blocking antibody C-D: HIV1-p24 D-E: 2nd 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 • 4 - 7 concentrations in duplicate • Calibrants and unknowns in same matrix • Moderate to high densities on sensor chip • Direct binding formats • Inhibition formats Calibration Curves Response x x xx x Sample Concentration Sample matrix for calibration curve = Sample matrix for unknown samples Affinity Analysis • How STRONG is the binding at equilibrium? • » Quantify KD • » 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] 20 15 10 5 0 Time [s] 0 60 120 Determining Affinity Constants • Plot Req against C • Steady state model • Concentration at 50% saturation is KD Kinetic Analysis How FAST is the binding ? » ka kon (recognition) » kd koff (stability) » KD = kd/ka » Ab selection; wash steps Same Affinity but different Kinetics • All four compounds have the same affinity KD = 10 nM = 10-8M • The same affinity can be the result from different kinetics ka kd [M-1s-1] [s-1] All target sites occupied 100 nM 30 min 60 min 106 10-2 105 10-3 104 10-4 103 10-5 KD 10 nM 1 µM 30 min 60 min Rate Constants Association rate constant ka Definition Unit Describes Typical range A+B ka AB Dissociation rate constant kd AB kd A+B [M-1s-1] [s-1] Rate 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. 1x10-3 – 1x107 1x10-1 – 5x10-6 Equilibrium Constants Equilibrium dissociation Equilibrium association constant KD constant KA Definition Unit Describes Typical range (A).(B) kd =k a (AB) [M] Dissociation tendency High KD = low affinity 1x10-5 – 1x10-12 k (AB) = ka (A).(B) d [M-1] Association tendency High KA = high affinity 1x105 – 1x1012 Equilibrium and Kinetic Constants are related A+B ka kd AB 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. dR = k . C . (R -R) – k . R a max d dt • Obtain values for the constants ka, kd, Rmax • Assess the fit overlay pots, residual plots acceptable statistics e.g. chi2 – curve fidelity Biological and experimental relevance of the calculated parameters Biacore and other Methods Conventional Biacore Assays Time Method Time Isotyping Day 1 ELISA One Day Affinity Day 1&2 RIA Weeks + labelling Kinetics Day 1&2 Na Na Epitope Map Overnight ELISA Weeks + labelling Biacore is much quicker than conventional methods Summary 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