Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is particulate and the particles are of...
Reexamination Certificate
2001-08-16
2004-05-04
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is particulate and the particles are of...
C436S151000, C436S100000, C436S800000, C436S806000, C435S174000, C435S176000, C435S242000, C435S242000, C435S288300
Reexamination Certificate
active
06730521
ABSTRACT:
The present invention relates to a method and apparatus for performing chemical and biochemical assays.
DESCRIPTION OF RELATED ART
The ability to characterize processes at a cellular or sub-cellular level is important in both drug discovery and clinical diagnostics. One class of interactions frequently studied is the binding of one biological molecule to another molecule, cell or part of a cell. This may be for example the binding of antibodies to antigens, hormones to receptors, ligands to cell surface receptors, enzymes to substrates, nucleic acids to other nucleic acids, nucleic acids to proteins, and viruses to cell surfaces.
Another class of interactions important in the biology of the cell are diffusion or transport of molecules or cells across membranes. This may for example occur by osmosis; via special transport proteins or through phagocytosis.
Many diseases are characterised by binding or transport processes. In drug discovery the aim is to identify a means of enhancing or blocking the process. In clinical diagnostics the aim is to detect abnormal function of these processes; the presence of abnormal nucleic acid material; or to identify foreign bodies (such as viruses or bacteria) to diagnose a disease so that appropriate treatment may be given.
The present invention seeks to provide a rapid and simple assay to detect and quantify binding and transport processes important in drug discovery and clinical diagnostics.
For the purpose of the following discussion “receptor” shall mean any biological molecule, cell or structure that binds another molecule, cell or structure. Similarly “ligand” shall mean any organic or inorganic molecule that binds to the “receptor”. The discussion and examples will focus on the assay of a labelled ligand binding to a receptor. The prior art described and the invention can be extended to include the interaction of a non-labelled agonist or antagonist in a competition assay as commonly used in drug discovery.
The basic principle of a reversible binding reaction is described by the equation:
Dissociation Constant (
K
d
)=[
L]×[R]/[L.R ]
Where [L]=concentration of unbound ligand at equilibrium, [R]=concentration of unbound receptor at equilibrium, and [L.R]=concentration of bound ligand/receptor complex at equilibrium
The concentration is commonly measured in molar, and K
d
for ligand; protein interactions is typically in the range 10
−4
to 10
−15
M
−1
.
The classical assay used in drug discovery and diagnostics is the separation assay. In this assay one component (for example the ligand) is dissolved or suspended in solution. The other component (for example the receptor) may be immobilised to a surface such as the walls of a well in a microtitre plate, or may be present on the surface of a cell. One or both components may have a label such as a fluorescent or radioactive marker attached to it to assist measurement with an instrument. The assay is performed by adding the soluble component to a well containing the immobilised component and allowing the binding of the components to come to equilibrium. It is not possible with conventional detectors such as colourimetric, fluorescent or radioactivity plate readers to directly determine the amount of bound labelled ligand in the presence of free labelled ligand. This problem is overcome by separating the free ligand from the bound ligand by decanting off the solution containing the free ligand. One or more washes with fresh solvent may be performed to remove any excess free ligand. A measurement of the remaining label is assumed to represent the concentration of bound complex in the original solution. This process may also be performed where the receptor is on a cell. If the cells are not attached to the well the washing process is performed in special filter plates that retain the cells, but allow the wash solvent to pass through.
This method works well where the rate of dissociation of the bound complex is slow, and indeed it is used with success in many assays. However, there are significant disadvantages to this assay method when applied more widely.
If the rate of dissociation is fast some of the bound label will be released back into the wash solution resulting in an error during reading. The efficiency of washing itself may vary from one sample to the next, reducing the repeatability of the assay. It is desirable to reduce or eliminate washing steps in automated systems to increase throughput, reduce complexity and eliminate the risk of cross-contamination between samples.
A number of non-separation assay techniques have been developed in recent years to overcome these problems including Scintillation Proximity Assay (SPA), Fluorescence Polarisation (FP), Fluorescence Correlation Spectroscopy (FCS), and Time Resolved Fluorescence (TRF). However, each of these techniques has disadvantages that limit their applicability.
SPA relies on the transfer of energy from a radiolabelled ligand to a scintillant bead onto which the receptor is attached. The assay has to be conducted at relatively high concentrations to produce enough signal. Legislation on the disposal of radioactive material and the risk of exposure to operators has led to companies seeking alternatives. SPA is not suited to some assays using whole cells and cannot be used to assay receptors or proteins inside cells. This means that functional receptor must be isolated from the cell to perform the assay, and this is costly, difficult and in some cases cannot be achieved.
FP is a technique for estimating the mass of a fluorescent object from its speed of rotation or translocation through diffusion. The sample is illuminated by a burst of polarised light and emitted fluorescence is measured in the same or other polarisation plane. If the label is bound to a large object, rotation or translocation will be slower and emission will be in the same polarisation plane as the excitation for some time after the illumination. If the free label is much smaller than the bound complex the molecule may more rapidly move out of the plane of the incident polarised light and emit in another plane. Provided that the fluorophore has a sufficiently long decay time, the light reaching the detector will take longer to decay after excitation if a substantial number of fluorophore-labelled ligands in the solution are bound to larger molecules.
The method is a correlation rather than a direct measurement of bound to free label. Some of the free label will emit in the same plane as the excitation. It also requires that the labelled ligand be very much smaller than the receptor and that decay time for the fluorophore be longer than the speed of rotation of target molecules. This technique has many drawbacks: it is difficult to differentiate non-specific binding and contaminating background fluorescence from specifically bound labelled ligand; it cannot be used to study intracellular interactions; the sensitivity of the method is reduced by relying on the decay of the signal rather than peak fluorescence and it is limited to the use of certain fluorophores.
FCS is similar to FP with the exception that FCS performs correlations on single molecules. The technique predicts the size of a fluorescent particle or molecule from its speed of translocation through a fixed laser beam by brownian motion. To perform the technique it is desirable that only a single molecule of fluorophore be present in the laser beam at one time. There is a practical limit to how narrow the laser beam can be (typically of the order of a few microns in diameter). It is also impractical to have extremely short path lengths through the fluid. For this reason FCS is usually performed with very low concentrations of label. This technique is highly susceptible to contaminating background fluorescence typical in practical assays. It is also comparatively slow, taking up to half an hour of continuous measurement to detect binding to larger molecules.
The technique of FCS has been kno
Cantor & Colburn LLP
Cheu Jacob
Le Long V.
The Technology Partnership PLC
LandOfFree
Chemical and biochemical assay method and apparatus does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Chemical and biochemical assay method and apparatus, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Chemical and biochemical assay method and apparatus will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3203999