World Leader in SPR Technology Objectives of the
World Leader in SPR Technology
Objectives of the Biacore Experiment • • Kinetic Rate Analysis: How FAST? » » ka, kd KD = kd/ka, KA = ka/kd Affinity Analysis: HOW STRONG? KD, KA Relative Ranking Concentration Analysis: How MUCH? Active Concentration Solution Equilibrium Inhibition • Yes/No Data » Ligand Fishing
Biacore’s proprietary SPR technology • • Non-label Real-time Unique, high quality data on molecular interactions Simple assay design Robust and reproducible Walk-away automation Small amount of sample required
The Corner Stones of Biacore Technology SPR (Surface Plasmon Resonance) Detection System Gold-Dextran Surfaces Microfluidic System
Biomolecular Binding in Real Time Principle of Detection
The Sensorgram is Information Rich
Biacore SPR: binding event ==>real time sensorgra • SPR monitors Binding • Response Proportional to Mass Bound • Real Time
Correlation between SPR Response and Surface Concentration Signal proportional to mass Same specific response for different proteins
User-defined Biospecific Surface Specific layer Dextran layer Linker layer Gold Glass • • Biocompatible Low nonspecific binding Robust More than 100 runs on the same surface
Flexibility to Create Your Biospecific Surfaces • Direct: covalent coupling » » Amine Thiol Aldehyde Carboxyl • Capture » » » Streptavidin - Biotin NTA-Ni 2+-his Anti-his Ra. M Fc - MAb Anti-GST- GST
Flexibility in Assay Design • Multiple assay formats providing complementary data Direct measurement Indirect measurement Direct Binding Assay (DBA) Surface competition assay (SCA) Inhibition in solution assay (ISA) D 102
BIACORE® 3000 & 2000 Series • Flexible research system » Low sample consumption » Early screening – cell lines » Assay development » Screen validation
Areas where Kinetic Information is Needed Quantification of effects of structural changes on interactions Understanding of structure-function relations Design of affinity pairs Characterization of biopharmaceutical products Recombinant proteins Characterization of the immune response in vaccine development Development of assays based on affinity Selection of reagents Development of purification schemes Selection of affinity ligands and conditions for use Study the effect on function of conditions used
• Same affinity but different kinetics All 4 compounds have the same affinity KD = 10 n. M = 10 -8 M • The binding kinetic constants vary by 4 orders of magnitude Concentration = 100 n. M Completely blocked target - all target sites occupied Response 1035 Time Concentration = 1000 n. M kon koff M-1 s-1 106 10 -2 105 10 -3 104 10 -4 103 10 -5 Compounds with slow off-rates occupy the target for a longer time 1035 Time
HIV-p inhibitors: on-off rate map 10 p. M 100 p. M 1 n. M 10 n. M KD 100 n. M 107 1 M 106 kon (M-1 s-1) 10 M 105 100 M 104 1 m. M 103 102 0. 0001 0. 01 koff (s-1) 0. 1 1
HIV-p inhibitors: on-off rate map 10 p. M 100 p. M 1 n. M 10 B 376 n. M KD 27 n. M 107 U 75875 B 369 B 388 Rit. Amp B 322 Ind kon (M-1 s-1) 106 105 Saq Nelf B 425 B 408 B 4 40 B 409 B 268 B 365 B 429 B 412 A 03 B 439 B 435 7 A 038 B 355 B 295 A 018 1 M 10 M A 015 A 017 B 249 100 M B 347 104 1 m. M 103 102 0. 0001 0. 001 100 n. M A 016 B 277 0. 01 koff (s-1) 0. 1 1
SH 2 Domains Binds to Tyrosine Phosphorylated PDGF b-Receptor Sequences Biotinylated peptide GST-SH 2 Avidin Panayotou, G. et al. (1993) Molecular and Cellular Biology 13: 3567 -3576. 535
SH 2 Domains Binds to Tyrosine Phosphorylated PDGF b-Receptor Sequences Specificity of binding to phosphorylated and nonphosphorylated immobilized peptide Addition of competing peptide Panayotou, G. et al. (1993) Molecular and Cellular Biology 13: 3567 -3576. 536
SH 2 Domains Binds to Tyrosine Phosphorylated PDGF b-Receptor Sequences Kinetic constants of the interactions between SH 2 -domain containing proteins and phosphopeptides Panayotou, G. et al. (1993) Molecular and Cellular Biology 13: 3567 -3576. 537
Antibody Characterization BIACORE® No purification No labelling Earlier characterization Kinetic information BIACORE® Conventional 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 Assay Day 2 Various EIA Days - Weeks Extended map Day 3 ELISA TOTAL 2 - 3 days One day + labelling Weeks - Months Johne, B. et al. (1993) Journal of Immunological Methods 160: 191 -198.
Multisite Binding Analysis of Troponin using BIACORE®
Qualitative Ranking Three HIV-1 p 24 MAbs Relative response (RU) 400 300 200 100 0 300 700 50 n. M p 24 injection 1100 1500 Time (s) Dissociation of bound p 24 in buffer flow
Detection of BMP Receptor Expressed in E. Coli SDS-PAGE Fr. App 1 2 3 4 5 6 Western blot Isolated receptor shows no binding to BMP
BIA Detects the Active Receptor SDS-PAGE Fr. App 1 2 3 4 5 6 Western blot
Kinetic Effects of Alterations in the Z-domain of Protein A Affinity measurements Kaff*1 e-7 1/M 3, 5 3 2, 5 2 Resonance signal (RU) 1, 5 1 1850 0, 5 1830 0 1810 Native 1790 L 17 D 1770 1750 100 200 300 400 Time (s) Z ) Z( 7 D L 1 N Z( A) 8 2 BIACORE ) Z( F A 30 A) ) 1 I 3 Z( Z( K A 35 Binding assay
Membrane Protein Preparations Crude preparations: Cells Virus particles Cell membrane preparations Proteoliposomes: Enriched or purified membrane proteins reconstituted in liposomes. Solubilized membrane proteins: Enriched or purified membrane proteins that are stable in detergent micelles.
Theoretical levels of Rmax Assuming a 500 Da analyte: Cell membrane vesicles: Pioneer Chip L 1: 0. 04 RU Sensor Chip CM 5: 0. 08 RU Proteoliposomes: Pioneer Chip L 1: 10 RU Sensor Chip CM 5: 23 RU On-Surface Reconstitution: Pioneer Chip L 1: 114 RU
Immobilize a GPCR-specific m. Ab on a L 1 chip.
Capture a detergent-solublized GPCR on the immobilized m. Ab surface.
Reconstitute a lipid bilayer around the receptor Inject lipid/ detergent mixed micelles across the surface.
Wash the surface with buffer Dissociates the detergent from the micelles.
Establish the integrity of the reconstituted GPCR Use conformationally sensitive anti-bodies.
Study the kinetics of ligand/receptor interactions
Binding of the chemokine SDF 1 to the reconstituted CXCR 4 receptor KD = 288 ± 11 n. M
On-surface reconstitution approach üA very quick and easy method for functional reconstitution of immobilized membrane proteins with lipids. üConventional immobilization techniques are applicable on membrane proteins with OSR. üSurfaces with high density of receptor can be prepared. üThe lipid matrix can be renewed after every cycle. ü“Lipid bilayers” can be very rapidly and easily built and rebuilt on Pioneer Chip L 1.
BIACORE® in Proteomics • • • Fast, simple and compatible with any biological sample Monitors binding of native proteins from crude or purified samples Detects even low affinity binding events Recovers samples for MS analysis and identification Confirms results from other techniques Provides functional (interaction) data
Biacore Proteomics Study Ligand Fishing SPR/MS Recovery Enzymatic digestion HPLC separation MS Receptor Express 6 -24 months Kinetics, Affinity Structure-Activity studies BIACORE “Classical Applications”
SPR/MS Approaches Two Options : » Direct On-Chip » Elution/Microrecovery BIA + MS Direct detection of bound molecules on the surface (Krone et al 1997) Recover bound molecules and analyze with MS (Fitz et al 1997)
Summary • • • SPR detects binding events as changes in mass at the chip surface Real-time kinetic measurements Qualitative rankings Measurement of active concentration Information about structure-activity relationships Low volumes of precious samples needed
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