생물물리 화학 방법론 -spectrochromatography

Chap 3. Spectroscopy
Part 5 – Surface plasmon resonance (SPR) and biolayer interferometry (BLI)

Biological applications of SPR – Biacore biosensors
SPR functions as biosensors for specific biological interactions including adsorption and desorption kinetics such as antigen-antibody binding.
=> Biacore SPR biosensors

Kinetics Affinity Thermodynamics
How fast, strong & why...
Is the binding of a lead compound
How specific & selective...
Is this drug binding to its receptor?
Concentration
How much...
Biologically active compound is in a production batch?

The corner-stones of the technology

• Dextran matrix covered with carboxyl groupes (red circles)
액티베이션
• 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.

The Steps in the Biacore Assay
Surface preparation
=intracrion -protein

Analysis Cycle
모니터

Surface Preparation: Immobilization
Surface에고정
• Ligand: the interactant immobilized on the sensor surface
 The ligand can be immobilized directly to the sensor surface,
다른프로틴을 흘려줌 부착or
indirectly via an immobilized capturing molecule • Analyte: the interactant in free solution 리간드 - 부착

Direct immobilization - various coupling chemistries

Immobilization by capture Capture Surfaces and Molecules
analyte ligand
capturing molecule
• Capturingmolecules
 Protein A or G (for antibody capturing)  Glutathione (for GST capturing)
 Anti-His antibody (for his tag capturing)  NTA (for his-tag capturing)
 Streptavidin (for biotin capturing)
 Antibodies specific to diverse proteins

Analysis Cycle

Result of analysis cycle : the sensorgram

Application of biosensors - specificity
• Do two molecules interact with each other? - Yes/No Answers.
• Different analytes are tested with the same ligand e.g. different antigens with immobilized antibody.

Application of biosensors – epitope mapping-antibody 결합하는 부위

Application of biosensors
– Affinity analysis by kinetic binding
=> real-time kinetics-실시간으로 antibody가 어떻게 변하는지
• How FAST is the binding ?
» ka, kon (association rate constant, on-rate constant) 얼마나 빨리 결합» kd, koff (dissociation rate constant, off-rate constant) » KD = kd/ka (dissociation equilibrium constant)분리되는 속도
Bi-molecular binding

Equilibrium and kinetic constants are related

Rate Constants

Equilibrium Constants

Same affinity but different kinetics
• All four compounds have the same affinity KD = 10 nM = 10-8 M • The same affinity can be the result from different kinetics

Biacore kinetic data

Extracting rate constants from sensorgrams
• Measure binding curves
• Decide on a model to describe the interaction
• Fit the curve to a mathematical rate equation describing the model
 e.g.
dt
dR
=ka.C.(Rmax-R)–kd .R
• Obtain values for the constants ka, kd, Rmax
• Assess the fit
 Overlay plots, residual plots
Acceptable statistics e.g. chi2 – curve fidelity
 Biological and experimental relevance of the calculated parameters

Application of biosensors
– affinity analysis by equilibrium binding
• Determine the equilibrium response when response goes flat (at equilibrium).
• Plot concentration-response.
• Fit the curve to a steady state model to derive equilibrium constant.

Advantages and limitations of biosensor
• Advantages 장번
 Once optimized – quick data acquisition
 Sensitive
 Label-free in many cases
 Ability to perform real-time measurement
 Can re-use chip surface as long as the surface is not damaged
 Can link up to mass spec to determine the identify of interactants  Low amounts of analyte/ligand required
• Limitations
 Mass transport limitations
 Can take time to optimize regeneration conditions
 Lack of sensitivity when monitoring low molecular weight analytes

3.11. Bio-layer interferometry (BLI)
• Bio-layer Interferometry (BLI) monitors the binding of proteins or other biomolecules to their partners directly in real time as for SPR.
• Label-free, fluidics-free, real-time detection based on an optically-coated biosensor
• Analyzes the interference pattern of white light reflected from two surfaces: 1 a layer of immobilized protein on the biosensor tip (glass fiber)
2 an internal reference layer
• A layer of molecules attached to the tip
• Any change in the number of molecules bound causes a measured shift in the pattern (both density and thickness contribute a shift)

Octet application
• Kd determination: The experimental binding curves for the interaction between
antibody Fab and antigen for fitting kon and koff 1 Base line
2 Load antibody Fab onto the biosensor 3 Base line for washing
4 Sample application - association
5 Buffer - dissociation
Fab1 + Ag, Kd = 0.40 nM
Fab2+Ag,Kd =590nM