Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-12-29
1997-06-24
Chin, Christopher L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
385 12, 385129, 385130, 356313, 356318, 356445, 422 8205, 422 8208, 422 8211, 435 79, 435 793, 435 794, 435808, 436164, 436165, 436518, 436524, 436525, 436527, 436805, G01N 33552, G01N 3353
Patent
active
056416407
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an improvement in assays of the type wherein the presence of the analyte is detected by determining a change in refractive index at a solid optical surface, which change is caused by the analyte involving or influencing the binding of a refractive index enhancing species to the optical surface, or the release therefrom, respectively.
One type of method for determining such refractive index changes at an optical surface is based upon surface plasmon resonance, hereinafter SPR. The phenomenon of SPR is well known. In brief, SPR is observed as a dip in intensity of light reflected at a specific angle from the interface between an optically transparent material, e.g. glass, and a thin metal film, usually silver or gold, and depends on among other factors the refractive index of the medium (e.g. a sample solution) close to the metal surface. A change of refractive index at the metal surface, such as by the adsorption or binding of material thereto, will cause a corresponding shift in the angle at which SPR occurs. To couple the light to the interface such that SPR arises, two alternative arrangements are used, either a metallized diffraction grating (Wood's effect), or a metallized glass prism or a prism in optical contact with a metallized glass substrate (Kretschmann effect). For further details on SPR, reference is made to our WO 90/05295. In an SPR-based immunoassay, a ligand may be bound to the metal surface, and the interaction thereof with an analyte of an aqueous sample in contact with the surface is monitored.
SPR assays have, however, certain fundamental limitations which restrict the technical performance thereof. One major limiting factor is the sensitivity, or signal strength. The SPR response depends on the volume and refractive index of the bound analyte, which volume is limited by mass transfer, reaction-kinetic and equilibrium parameters. For example, water and diluted aqueous buffers have a refractive index of about 1.33, whereas most proteins have a refractive index in the region of about 1.5 to 1.6. Since the SPR-measurement response is proportional to the change in refractive index caused when e.g. protein molecules are adsorbed to the surface and displace water therefrom, the refractive index difference between the protein and the buffer solution puts a theoretical limit to the strength of response that may be obtained. It is thus understood that SPR-based immunoassays for substances of low molecular weight or occurring in low concentrations, like, for instance, haptens, are problematic due to the very small changes in refractive index caused when the analyte binds to or dissociates from the antibody-coated sensing surface.
Another limiting factor is non-specific binding to the sensing surface, a problem common to all types of direct-measuring sensors, i.e. where no labelled reagent, such as an enzyme or a fluorophore, is used to provide the detected signal. Since SPR generates a signal for all material bound to the surface, the analyte can not be distinguished from non-specific material.
Still another limiting factor is the variation of the refractive index with temperature. The temperature difference between the base line reading and the reading of analyte uptake therefore puts a theoretical limit to the minimum amount of analyte that may be detected. Also other parameters than temperature, e.g. mechanical or chemical disturbances, that may vary between the time of the baseline reading and the reading of the analyte uptake will, of course, affect the measuring response.
Further limitations are, for example, non-ideal optics as well as electronic and other baseline fluctuations.
More or less unsuccessful attempts to avoid the sensitivity or signal strength problem are described in EP-A-276 142 and WO 90/11525 (the former specifically making use of the above mentioned Wood's effect, and the latter of the Kretschmann effect). Both publications disclose the conjugation of a reagent (for example the analyte or an analyte analogue in a competition assay) with a
REFERENCES:
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Biacore AB
Chin Christopher L.
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