BIAcore: Real-Time Biomolecule Interaction Analysis Applications in Life Science Research. 生物分子感測系統

Transcription

1 BIAcore: Real-Time Biomolecule Interaction Analysis. 生物分子感測系統 林怡欣 奇異亞洲醫療設備股份有限公司台灣分公司 Biosciences DNA/RNA Proteins Cells Clinical imaging Sequencing, genotyping, SNP analysis Toxicology DNA/RNA Gene expression Functional Biology Cell Biacore Drug screening Protein Sample preparation, Protein analysis Protein purification & scale-up Protein analysis & characterisation 1

2 *Publications Generating publishable results Publications in Biacore Reference Database Publish Reference Cell 43 Sciences 38 Nature 50 Immunity 56 EMBO J 132 PNAS 295 JBC

3 Information to enable better decisions Selection criteria Protein functionality - how fast, stable, strong Quantity Screening Characterization Concentration yes/no ranking selectivity kinetics affinity thermodynamics epitope mapping New Approach in Protein interactions OD End-point assay Highly parallel h Real-time Label free Traceable quality Rapid 3

4 Why use label-free protein interaction analysis? Label free No labels required Measures binding properties of unmodified substances Time and workload greatly reduced Real time Contact free See binding as it happens. Unique information on binding characteristics (on- and off-rates) for any size of biomolecule Facilitates study of weak and fast interactions Non-invasive Direct measurements of opaque or colored samples without loss of sensitivity or accuracy Cornerstones of Biacore SPR Technology SPR (Surface Plasmon Resonance) Detection System Gold-Dextran Surfaces Microfluidic System 4

5 Gold Dextran Surfaces Sensor Chips for a multitude of molecules Immobilization of interaction partners: Proteins Tagged proteins (His, biotin) LMW molecules Membrane associated molecules Nucleic acids Carbohydrates Viruses or intact cells 5

6 Capture kits for a wide range of analyses Mouse Antibody Capture Kit GST Capture Kit Anti-Mouse IgG Regeneration Immobilization Anti-GST Regeneration Immobilization Human Antibody Capture Kit Anti-Human IgG (Fc) Regeneration Immobilization Biotin CAPture Kit Analyte Biotinylated ligand Biotin CAPture conjugate Regeneration Sensor Chip CAP Microfluidics 6

7 See how it works Monitoring interactions in real time 7

8 The Sensorgram Continuous real-time monitoring is displayed as a sensorgram. 18 Resonance Signal (KRU) Association Kinetics Concentration Dissociation Regeneration Time (s) 8

9 Biacore Application 蛋白質組學研究 (Proteomics) 癌症研究 (Cancer Research) 新藥研發 (Drug Discovery) 信號傳遞 (Cell Signalling) 多分子複合物的結構和組裝 (Multi-molecular Complexes) 分子識別 (Molecular Recognition) 免疫調節 (Immune Regulation) 免疫測定法 (Immunoassay) 疫苗開發 (Vaccine Development) 瞬時結合 (Transient Binding) 配體垂釣 (Ligand Fishing) 結合特異性 (Binding Specificity) 結構與功能的關係 (Structure-function Relationship) 酶反應 (Enzyme Reaction) 180 Binding response, RU 0 Applications of Surface Plasmon Resonance Interaction screening or verification Pure inhibitor potato tomato Egg-plant Peber 0 Time, s 200 Binding response, RU potato egg-plant peber tomato CI Co-factor requirements and Stoichiometry ParM + Nucleotide ParR ATPγS ATP ADP Kinetics and thermodynamics Concentration analysis Response(RU) Pseudo first-order kinetics d[ AB ] = ka[ A][ B] kd[ AB ] dt dr = kacr max ( kac + kd) R dt Response dr/dt Time (seconds) 1 dr = kdr dt time Conc, nm 9

10 Applications of Surface Plasmon Resonance Quantitative Ranking Binding Conditions Kinetics Studies Drug screening Quantitative Ranking Concentration = 1000 nm Response 30 min 60 min Time 10

11 Isolation of chromosaponin I-specific antibody by affinity chromatography Applications of Surface Plasmon Resonance Quantitative Ranking Binding Conditions Kinetics Studies Drug screening 11

12 Binding Conditions Epidermal Growth Factor Receptor p85 Bioorganic synthesis of lipid-modified proteins for the study of signal transduction 1. Raf 與 Ras-GTP 結合 2. Raf 不與 Ras-GDP 結合 12

13 Applications of Surface Plasmon Resonance Quantitative Ranking Binding Conditions Kinetics Studies Drug screening Kinetics Studies 13

14 Applications of Surface Plasmon Resonance Quantitative Ranking Binding Conditions Kinetics Studies Drug screening Antagonist Screening 14

15 Antagonist Screening Antagonist Screening: Multiplexing 15

16 Binding interaction of quercetin-3-β-galactoside and its synthetic derivatives with SARS-CoV 3CLpro: Structure activity relationship studies reveal salient pharmacophore features Bioorganic & Medicinal Chemistry Volume 14, Issue 24, 15 December 2006, Pages Proteomics Biacore s Contribution Functional or Interaction Proteomics Protein-Protein interactions Ligand fishing/orphan receptor studies from crude material SPR-MS interfacing 16

17 Protein-Protein Interactions Stoop et al Studying binding site structure Biacore SPR technology was used to investigate binding of plasminogen activation inhibitor 1(PAI-1), carrying point mutations within a defined binding site, to monoclonal anti-pai-1 Abs. PAI-1 immobilized on a sensor chip by binding to anti-pai-1 can still bind two further antibodies, indicating that the binding sites do not overlap. Thromb Haemost Mar;77(3): Angiogenesis Kinetic evaluation of VEGF antagonists One approach to inhibit angiogenesis is to use truncated rvegfr variants lacking the transmembrane domain sequesters VEGF and prevents angiogenic signalling 1) VEGF rvegfr2 V 2) V V V V rvegfr as competitor wt VEGFR2 affinity & kinetic data Establishes the credentials of a potential angiogenesis inhibitor and sets the basis for a screening assay for therapeutic variants rvegfr 0 x 40 x 200 x 1000 x Huang et al, Biochem. Biophys. Res. Commun. (1998) 17

18 Proteomics Biacore s Contribution Functional or Interaction Proteomics Protein-Protein interactions Ligand fishing/orphan receptor studies from crude material SPR-MS interfacing Surface plasmon resonance mass spectrometry 18

19 Immunogenicity Characterizing immune responses to viral gene therapy vectors Abad et al, (2002) Anal. Biochem., 310, Development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors. Background Gene therapy approaches to cancer treatment can involve viral delivery vectors efficient delivery, but viral proteins can be potent immunogens Abad et al used a non-replicating recombinant adenovirus (rad) vector to deliver wild-type p53 to cancer patients rad/p53 vectors promising, but viral capsid proteins are high-risk immunogens human adenoviral infections very common requires immune response monitoring before and during treatment 19

20 Methods anti-viral Ab from serum isotyped as IgG icosohedral viral capsid Results (2) These results showed a typical anamnestic response Ab titers as determined by Biacore time points at which Biacore assays were carried out 1st Ab production against rad/p53 memory B-cells primed by 1st treatment enable a stronger, sustained immune response to 2nd exposure 20

21 Conclusions Whole-virus immobilization is a valid approach for viral vector immunogenicity studies Biacore assay simple, rapid and consistent with existing ELISA method Simple and convenient isotyping within the single assay format described predominantly an IgG response, increases in IgA Abs in some later samples Shows that immunogenicity can be a problem for adenovirusbased gene delivery integrated Ab titer/isotyping Biacore assay is ideal for future adenovirus studies more efficient treatment & informed dosing BIAcore Advantages real-time and label-free analysis high quality and often unique data flexibility in surface chemistry protein-protein protein-dna RNA-carbohydrate protein-lipid interactions Yes/No data Specificity studies Ranking Early selection of binders Equilibrium analysis K D Determination of binding strength Kinetic rate analysis k a, k d Modelling binding reactions to determine the dynamic behaviour of a system 21

22 BIA Applications on the Web Immunology and Infectious Diseases Cancer Research CD4/gp120 Neuroscience Proteomics mab Drug Discovery natural chemokine LMW compound Detergent solubilized GPCRs Capture mab Assays to meet all research needs 22