Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
  • G01N21/553—Attenuated total reflection and using surface plasmons

Definitions

• the affinity (expressed as the affinity constant K ⁇ or the dissociation constant Kj)) can be calculated from the association and dissociation rate constants. Many times, however, it may be difficult to obtain definitive kinetic data, and it is therefore usually more reliable to measure the affinity by equilibrium binding analysis, which involves determining, for a series of analyte concentrations, the level of binding at equilibrium, or steady state, which is presumed to have been reached at or near the end of the association phase of the binding interaction. To ensure that the association phase of the binding curve is indeed likely to have reached steady state, one usually determines in advance the necessary time length of sample injection (i.e. sample contact time with the sensor chip surface) for the bound analyte to reach equilibrium under the conditions intended to be used for the affinity measurements. Since both the time taken to reach equilibrium and the time it takes for the anlyte to dissociate are governed primarily by the dissociation rate constant, approximate injection times may also be estimated from the dissociation constant.
• sample injection i.e. sample contact time with the sensor chip surface
• steps c) to e) comprise the following steps: selecting pluralities of other binding data from the plurality of experimental binding data sets; determining which ones of the pluralities of other binding data fall within a predetermined range, the pre-determined range representing the quality of the ones of the experimental binding data sets; excluding from the final plurality of experimental binding data sets poor-quality experimental binding data sets, whereby the poor-quality experimental binding data sets have pluralities of other binding data falling outside of the predetermined range; and determining the affinity from the steady state binding data from the final plurality of experimental binding data sets.
• representative methods include those that detect mass surface concentration, such as reflection-optical methods, including both external and internal reflection methods, which are angle, wavelength, polarization, or phase resolved, for example evanescent wave ellipsometry and evanescent wave spectroscopy (EWS, or Internal Reflection Spectroscopy), both of which may include evanescent field enhancement via surface plasmon resonance (SPR), Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR), scattered total internal reflection (STIR) (which may include scatter enhancing labels), optical wave guide sensors; external reflection imaging, evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR-angle resolved imaging, and the like.
• reflection-optical methods including both external and internal reflection methods, which are angle, wavelength, polarization, or phase resolved, for example evanescent wave ellipsometry and evanescent wave spectroscopy (EWS, or Internal Reflection Spectroscopy), both of which may
• photometric and imaging/microscopy methods “per se” or combined with reflection methods, based on for example surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS), evanescent wave fluorescence (TIRF) and phosphorescence may be mentioned, as well as waveguide interferometers, waveguide leaky mode spectroscopy, reflective interference spectroscopy (RIfS), transmission interferometry, holographic spectroscopy, and atomic force microscopy (AFR).
• SERS surface enhanced Raman spectroscopy
• SERRS surface enhanced resonance Raman spectroscopy
• TIRF evanescent wave fluorescence
• phosphorescence phosphorescence
• waveguide interferometers waveguide leaky mode spectroscopy
• RfS reflective interference spectroscopy
• transmission interferometry holographic spectroscopy
• AFR atomic force microscopy
• biosensors include the afore-mentioned BIACORE® system instruments, manufactured and marketed by Biacore AB, Uppsala, Sweden, which are based on surface plasmon resonance (SPR) and permit monitoring of surface binding interactions in real time between a bound ligand and an analyte of interest.
• ligand is a molecule that has a known or unknown affinity for a given analyte and includes any capturing or catching agent immobilized on the surface
• analyte includes any specific binding partner thereto.
• SPR The phenomenon of SPR is well known, suffice it to say that SPR arises when light is reflected under certain conditions at the interface between two media of different refractive indices, and the interface is coated by a metal film, typically silver or gold.
• the media are the sample and the glass of a sensor chip which is contacted with the sample by a microfluidic flow system.
• the metal film is a thin layer of gold on the chip surface.
• SPR causes a reduction in the intensity of the reflected light at a specific angle of reflection. This angle of minimum reflected light intensity varies with the refractive index close to the surface on the side opposite from the reflected light, in the BIACORE® system the sample side.
• FIG. 1 A schematic illustration of the BIACORE® system is shown in Fig. 1.
• Sensor chip 1 has a gold film 2 supporting capturing molecules (ligands) 3, e.g. antibodies, exposed to a sample flow with analytes 4, e.g. an antigen, through a flow channel 5.
• Monochromatic p-polarised light 6 from a light source 7 (LED) is coupled by a prism 8 to the glass/metal interface 9 where the light is totally reflected.
• the intensity of the reflected light beam 10 is detected by an optical detection unit 11 (photodetector array).
• An optical detection unit 11 photodetector array
• the concentration, and therefore the refractive index at the surface changes and an SPR response is detected. Plotting the response against time during the course of an interaction will provide a quantitative measure of the progress of the interaction.
• Such a plot, or kinetic or binding curve (binding isotherm) is usually called a sensorgram, also sometimes referred to in the art as "affinity trace” or "affmogram”.
• affinity trace or "affmogram”.
• the SPR response values are expressed in resonance units (RU).
• One RU represents a change of 0.0001° in the angle of minimum reflected light intensity, which for most proteins and other biomolecules correspond to a change in concentration of about 1 pg/mm ⁇ on the sensor surface.
• association As sample containing an analyte contacts the sensor surface, the capturing molecule (ligand) bound to the sensor surface interacts with the analyte in a step referred to as "association.” This step is indicated on the sensorgram by an increase in RU as the sample is initially brought into contact with the sensor surface. Conversely, “dissociation” normally occurs when the sample flow is replaced by, for example, a buffer flow. This step is indicated on the sensorgram by a drop in RU over time as analyte dissociates from the surface-bound ligand.
• association phase This part B of the binding curve is usually referred to as the "association phase".
• association phase Eventually, a steady state condition is reached at or near the end of the association phase where the resonance signal plateaus at C (this state may, however, not always be achieved).
• steady state is used synonymously with the term “equilibrium” (in other contexts the term “equilibrium” may be reserved to describe the ideal interaction model, since in practice binding could be constant over time even if a system is not in equilibrium).
• the sample is replaced with a continuous flow of buffer and a decrease in signal reflects the dissociation, or release, of analyte from the surface.
• This part D of the binding curve is usually referred to as the "dissociation phase".
• the analysis is ended by a regeneration step where a solution capable of removing bound analyte from the surface, while (ideally) maintaining the activity of the ligand, is injected over the sensor surface. This is indicated in part E of the sensorgram. Injection of buffer restores the baseline A and the surface is now ready for a new analysis.
• Equation (1) K[A] ⁇ [B T ]- [AB ⁇ -kAAB] (2) at
• Equation (3) R is the response at time t in resonance units (RU), C is the initial, or bulk, concentration of free analyte (A) in solution, and R max is the response (in RU) obtained if analyte (A) had bound to all ligand (B) on the surface.
• R is the response in resonance units (RU).
• RU resonance units
• R R 0 - e ⁇ k> " (7)
• Ro is the response at the beginning of the dissociation phase (when the buffer wash of the surface starts).
• the above described analysis is usually repeated for a number of different analyte concentrations and, suitably, also at at least one other ligand density at the sensor surface.
• the chi2 value is in the same magnitude as the noise in RtA
• "residual plots” are also provided which give a graphical indication of how the experimental data deviate from the fitted curve showing the difference between the experimental and fitted data for each curve. The operator then decides if the fit is good enough. If not, the sensorgram or sensorgrams exhibiting the poorest fit are excluded and the fitting procedure is run again with the reduced set of sensorgrams. This procedure is repeated until the fit is satisfactory. Determining affinity constants from measured steady state binding levels with the BIAevaluation software involves the following steps:
• the first set of rules may further include:
• Batches without penalties or with a single penalty are accepted for analysis without further action. If a batch has any penalty 3, or if the sum of all penalties for a batch is 5 or greater, the batch is automatically excluded. Batches with a sum of penalties equaling 2, 3 or 4 are marked for user inspection.
• a second set of rules are applied to the data resulting from step 405 or 405c, which rules may include: (2a) If the standard deviation of the Kjj 's calculated in the cross-validation procedure in steps 405 or 405c is too large, a penalty is attached to the batch.

Landscapes

• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)

Priority Applications (2)

Applications Claiming Priority (4)

Publications (2)

Family

ID=37532718

Family Applications (1)

Country Status (3)

Families Citing this family (13)

Family Cites Families (4)

• 2006
  • 2006-06-12 EP EP06747872.7A patent/EP1893978A4/fr not_active Withdrawn
  • 2006-06-12 JP JP2008516784A patent/JP5052509B2/ja not_active Expired - Fee Related
  • 2006-06-12 WO PCT/SE2006/000679 patent/WO2006135309A2/fr not_active Ceased

Also Published As

Similar Documents

Legal Events