Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-07-12
2004-06-01
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S164000, C436S172000, C436S501000, C436S512000, C436S524000, C436S528000, C436S532000, C436S533000, C436S534000, C436S823000, C436S829000, C435S004000, C435S006120, C435S007100, C435S007900, C435S007920, C435S174000, C435S176000, C435S177000, C435S182000, C435S287100, C435S287200, C435S287300, C435S287600, C435S287700, C435S808000, C435S810000, C435S975000, C428S402000, C428S402200, C422S068100
Reexamination Certificate
active
06743638
ABSTRACT:
The present invention relates to a method for the detection of analytes, particularly to analytes which are present in only small quantities, as well as to kits and reagents for use in the detection method.
Reactions are widely used in the assay, testing and measurement of chemical, biochemical and biological analytes (e.g. immunoassays). The reaction between an analyte and particular reagents is usually the basis of specificity of such assays. While chemical reagents are the basis for some assays, the higher affinity reaction between analytes and biological molecules (e.g. enzymes, antibodies, nucleic acids) introduces higher specificity and sensitivity into assay procedures. Those reactions providing the highest specificity and sensitivity assays often do not involve the covalent modification of the analyte (e.g. chemical or biochemical enzyme reaction), and instead rely on the binding of the analyte to the specific reagent (e.g. antibody). Many of these covalent and binding reactions are carried out in solution, so that the reaction product and the analyte are present in the same phase. Reactions are detected by various methods such as a change in the optical (e.g. a colour, fluorescent, luminescent, light scattering) or electrochemical (amperometric, potentiometric, conductimetric) properties of the assay or text mixture.
Recent improvements have included the immobilisation of assay reagents for example onto solid supports such as beads or other surfaces. This allows the signal generated to be localised in the same plane. It is therefore more concentrated at this position and so easier to detect. Furthermore, problems with background “noise” signals generated in the bulk of the analyte can be minimised. Examples of such surfaces include lipid films or membranes. Visible signals may be detected in these systems, for example as described in WO 98/00714 and WO 96/25665, or alternatively changes in conductance as a result of the use of ion gating may give rise to analyte detection, for example as described in WO 90/08783 or WO 89/01159.
Liposomes have been used in immunoassays, both as labels to carry a payload of signal molecules, and to release signal molecules in proportion to the level of analyte in the assay medium. In one such method, (W. J. Litchfield et al., Clin. Chem. (1984) 30(9), 1441-1445) liposomes have been lysed to release a signalling means such as a fluorescent dye or enzyme, into an assay reaction mixture in order to generate a signal. The signal however, is diluted throughout the reaction mixture.
The applicants have found an improved process for detecting analytes.
Furthermore, it is known that their are a plurality of methods of varying the output signal parameters of a marker by means of alteration of the energy state of the marker by means of application of a force or field to it. In many instances the force/field may be applied by the locality of a second, or more, entities. An example known in the art is the self quenching of the calcein fluorophore at high concentrations. A second example is the quenching of calcein, and similar fluorescent dyes, by the addition of ions. It is known in the art that cobalt ions quench many fluorophore species. However, the inventors have discovered new effects associated with the use of cobalt ions and other chemicals, which assist in signal generation both in the method of the invention and more generally.
The present invention provides a process for detecting an analyte which process comprises (a) contacting a sample suspected of containing said analyte with a containment means comprising a barrier which separates signal generating reagents from said sample, in the presence of an element which interacts specifically with said analyte, under conditions whereby interaction between the analyte and the said element results in activation of the signal generating reagents within the containment means on the side of the barrier opposite to the sample, and (b) detecting any signal generated and retained within the containment means from the sample side of the barrier.
In this process, although the analyte is free to interact directly or indirectly with the containment means, the signal is generated and retained by the barrier within the containment means. This provides concentration of the signal which is therefore easier to detect.
There is the possibility of amplification of a signal within the containment means. Very small numbers of analyte molecules or even a single analyte molecule, can generate a detectable signal within the containment means, making the system very sensitive for the detection of analyte which is present in only very small quantities.
In one embodiment of the invention, the said element is present on the barrier surface of said containment means prior to interaction with said analyte. In general, interaction with the analyte will result in removal of this element, and this in turn allows transport through the surface of signal activation reagents.
Alternatively, the element may be added to the sample. In this case the element may be associated with a reagent which permeabilises or otherwise allows transport of signal activating reagents through the surface of the containment means such that it blocks or competes with the activity of that reagent. Interaction of the analyte with the element releases this reagent which is then free to react with the containment means so as to allow activation of the signal generating reagents.
In yet a further embodiment, activation of the signal generating reagents may be dependent upon the formation of a complex between the analyte and the element. The thus formed complex may activate the signal generating means for example by interacting with the barrier of the containment means so as to allow signal activation to occur.
The containment means suitably comprises a solid or semi-solid structure whereas the sample may comprise a solid, semi-solid, liquid or a gaseous reagent. Suitably the containment means is in a different physical phase to the sample.
The containment means is suitably of particulate form, in which no one dimension of the 3-dimensional shape of each particle is greater than 4 times any other dimension. In other words, the ratio of the x:y or y:z or x:z or vice versa is not greater than 1:4. Examples of such particles are broadly spherical, such as polymer beads like nanospheres, nanoparticles of microparticles or membrane structures such as vesicles and liposomes. In this case the signal generating agents are contained inside the particle. They are suitably introduced during processes for the production of the particulate containment means.
Particulate containment means can be produced using conventional methods. For example, polymer particles can be produced using a range of processes including the use of phase separation in mixed phase emulsion systems, aggregation and agglutination reactions, extrusion as polymerising or setting beads, and from aerosols. Liposomes and vesicles can be produced by various encapsulation technologies which are known in the art, such as sonication, extrusion and detergent dialysis.
A suitable liposome composition comprises for example a phosphatidylcholine, cholesterol and dihexadecyl phosphate as illustrated hereinafter although other liposome compositions will be apparent to the skilled person. It may be preferable for stability purposes, for the liposomes to be biotinylated in the sense that they incorporate a biotin reagent such as biotinoyl dipalmitoyl phosphatidylethanolamine (biotin-DPPE).
Alternatively, the containment means may comprise a discontinuity, such as a pore structure in a solid or colloid surface, which may be closed to form a complete barrier to sample. Suitable solids include ceramic materials such as glass, metal oxides or silicon. Discontinuities can be introduced into such materials by phase separation or etching processes, or by adding a pattern lithographically or by anodisation. The signal generating agents are suitably introduced into the thus formed discontinuity. The opening to the discon
Aojula Harmesh S
Clarke David J
Lloyd Christopher J
Nicklin Stephen
Tsilosani Marina
Le Long V.
Nixon & Vanderhye P.C.
Padmanabhan Kartic
The Secretary of State for Defence
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