Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-02-22
2001-07-10
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C436S516000, C436S536000, C436S829000
Reexamination Certificate
active
06258528
ABSTRACT:
The present invention relates to a method for the amplification of or creation of a signalling event for detection of a probe which reacts with a test substance, the method comprising causing the test substance to react with the probe and identifying the reaction of the test substance with the probe by the release of a signalling moiety from a vesicle. Preferably the signalling moiety is in an active but ineffective form and/or the signalling moiety is deposited on a solid phase in the vicinity of the probe. The invention also relates to a kit for the detection of a probe which reacts with a test substance, the kit comprising a vesicle which contains a signalling moiety.
Many systems for the quantitative and qualitative analysis of target substances are known. However, the need for simpler diagnostic assays with improved sensitivity is an ongoing aim and desire in the art.
U.S. Pat. No. 5,196,306 describes a method for detection of a probe through amplifying the signal through an enzyme activation system. In this system, the enzyme which comes into contact with the probe converts an inactive substrate (“conjugate”) into an activated conjugate which can then be deposited onto a solid phase or wherever a receptor for the activated conjugate is immobilised. This invention therefore provides for enzymatic generation of multiple signalling molecules for each probe although the choice of signalling molecules is limited to those generated by specific substrates which can yield a reactive product which binds to a solid phase. Potential disadvantages of U.S. Pat. No. 5,196,306 relate to the enzyme having a rate-limiting turnover of conjugate and the enzyme being subject to inhibition by the sample or by the accumulating active conjugate. In addition, specific inactive enzyme substrates are needed for conversion into activated conjugates for deposition onto a receptor thus reducing the flexibility of the invention for use in many other ligand-receptor systems. Thus, simpler amplification methods are required which do not require an enzymic conversion step to generate a reactive conjugate.
The present invention has the advantage over U.S. Pat. No. 5,196,306 that an enzyme and enzymatic substrate is not needed for direct generation of an activated signalling molecule. Indeed, in the present invention, the signalling molecule is presented in its activated form and not altered by an enzyme but rather is presented within a vesicle which is brought into the proximity of the prove by the binding molecule. This provides great flexibility in the range and types of signalling molecules which can be used in the present invention and also potentially provides more rapid reaction kinetics whereby generation of multiple signalling molecules are not directly dependant on the turnover rate of an enzyme.
Accordingly, a first aspect of the invention provides a method for the amplification and/or creation of a signalling event for the detection of a probe which reacts with a test substance, the method comprising causing the test substance to react with a probe, and identifying the reaction of the test substance and the probe by the release of a signalling moiety from a vesicle.
For the purpose of this invention, the term “probe” means a molecule or molecules with the ability to react with a test substance in such a way as to provide a means for detection or quantitation of said test substance.
Whilst the probe might be a nucleic acid probe which hybridises to a target nucleic acid or an antibody which binds to a target antigen, it will be understood by those skilled in the art that the probe could also be any molecule which constitutes one element of a binding pair whereby the probe binds to the other element of the binding pair as part of the test.
In order for the release of signalling moieties from the vesicle to indicate the presence and quantity of the target substance, either the vesicle or the element which causes release of the signalling moiety from the vesicle is preferably in close proximity to the target substance and/or the probe. This can be effected in many ways, all of which are covered by the present invention. In particular, the vesicle or the element which causes release of the signalling moiety may be brought into close proximity to the target substance and/or the probe by one or more binding molecules. “Binding molecule” means a second molecule or secondary molecules which bind(s) to the probe and/or target substance in such a way as to introduce, in close proximity to the probe and/or test substance, a source of detectable signalling moiety. Suitable binding molecules include antibodies, which may or may not be modified with further ligands (including further binding molecules), nucleic acid sequences, biotin, avidin or streptavidin, or any ligand (and may also be a binding molecule) which can be conjugated to either the test substance, the probe, or to a molecule which binds, directly or indirectly to either the test substance or the probe. The ligand may also bind to either the vesicle or the element which causes release of the signalling moiety.
The probe may be one part of a non-immune or immune pair (the test substance being the other part). Preferably the probe is a nucleic acid sequence such as DNA, or an antibody. Most preferably the probe is modified to include a ligand (also optionally a binding molecule), to which can be subsequently bound another molecule through which a signalling moiety can come into close contact with the probe and/or the test substance. The ligand may be natural or unnatural (synthetic molecule). Examples of ligands include fluorescein, a lipase, an antibody, biotin, streptavidin etc. In the context of the invention, the ligand on the probe may be either undetectable itself or, as with fluorescein, detectable in its own right. As a result of contact with the binding molecule and release of a signalling moiety from a vesicle, the probe should become respectively either detectable or should become more easily detectable as a result of introducing a stronger or more easily measurable signal. The signal may be amplified by virtue of introduction of multiple signalling molecules for each single probe.
In the method of the present invention, a probe is allowed to react with a test substance. The former, or the latter may be immobilised onto a solid phase. Where the test substance is immobilised onto a solid support, excess probe can be washed away from the reaction before further steps in the method are carried out.
The method can follow by the addition of a molecule (may be considered as a binding molecule) which comprises an element which binds and/or reacts in some way with the probe and/or with a ligand on the probe. The binding molecule may comprise either i) an element which either directly or indirectly can causes release of a signalling molecule from a vesicle, or ii) a vesicle which comprises a signalling moiety. The binding molecule which comprises either of components i) or ii) above need not bind directly to the probe or to the test substance. It may bind indirectly, i.e. via other binding molecules, as long as the ultimate aim of bringing the vesicle into close proximity to the probe and/ or test substance is obtained.
The next step in the method is to cause the release of a signalling moiety from the vesicle. This step is preferably sequential to the previous step. However, it may be that the probe already had bound to it a molecule which either comprised a vesicle which comprises a signalling moiety or an element which directly causes release of a signalling moiety from a vesicle. Preferably, the vesicle comprises more than one signalling moiety which may be the same or different types of molecules. Where the probe/test substance reaction, after addition of one or more binding molecules, comprises a vesicle which contains the signalling moiety, it is usually necessary to add to the system an element which causes release of the signalling moiety from the vesicle. The selection of such elements will depend on the composition of the vesicle. Th
Brusca John S.
Hale and Dorr LLP
Scancell Limited
Siu Stephen
LandOfFree
Signal amplification method does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Signal amplification method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Signal amplification method will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2538609