Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-08-17
2002-12-31
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007940, C435S007800, C435S471000, C435S472000, C435S475000, C424S204100, C424S205100
Reexamination Certificate
active
06500611
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to a novel detection system for detecting small numbers of analytes, and a method for making said detection system.
BACKGROUND
The need for the detection of smaller amounts of target analytes requires the development of more sensitive detection systems. The most widely employed approach solves this problem simply by increasing the number of target analytes, for example, the PCR-like approach. A second general approach is to develop more sensitive methods for detecting limited numbers of target analytes. An example using this approach is the bead-based fiber-optical arrays. See Walt, D. R. (2000),
Science
287:451-452. A third general approach involves attaching a larger number of commonly used reporter groups to each target analyte. Since an analyte of interest may offer only a limited number of sites for attaching reporter groups, this third approach is limited by the number of independently available attachment sites, unless multiple reporter groups can be attached to each attachment site. Numerous methods applying this third approach have been reported. For example, multiple reporters may be attached to single attachment sites either by creating a branched structure on the attachment site and then attaching reporter molecules to the distal ends of the created branches, or by bundling the reporter groups together and then attaching the bundle to the individual attachment sites of an analyte of interest.
One example of a commercial technology using the third approach is the Quantigene system. This system uses branched DNA (bDNA) technology to detect target RNA molecules.
Another commercially available method using the third approach involves microspheres, which are small polystyrene particles enclosing multiple reporter groups. The microspheres are often covered with functional groups, thereby enabling the microspheres to be attached to particular targets. For instance, Molecular Probes (Eugene, Oreg.) produces polystyrene microspheres with diameters of as small as 20 nm. The 20 nm microspheres produced by Molecular Probes are reported to contain about 200 equivalents of fluorescein, and can be visualized as single particles in light microscopes, appearing as point sources of fluorescence. See Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition, page 111. However, it is generally recognized that polystyrene microspheres are not generally useful for detecting small numbers of target analytes because they tend to be too sticky, and often generate a high, nonspecific background signal.
Alternative methods and approaches for bundling reporter groups would provide new approaches for efforts to reduce nonspecific background binding to levels which would allow detection of fewer analytes than is possible with currently available reporter bundles. Thus, novel methods for bundling could be the central elements in detection systems which are capable of detecting smaller numbers of analytes of interest.
This disclosure provides a new approach for creating a detection system which is capable of linking multiple copies of reporter groups to virtually any target analyte of interest. In this disclosure, a viral particle, preferably a bacteriophage particle, more preferably a Bacteriophage T4 capsid, is used as a scaffold or container to bundle multiple copies of reporter groups.
Various molecules have been incorporated into a viral particle for different purposes, and individual virus particles have been stained with various agents and visualized in light microscopes. For example, see U.S. Pat. No. 5,403,484 and Matsumato, S., Morikawa, K. and Yanagida, M. (1981),
J. Mol. Biol
. 152:501-516. See also Doolittle, M. M., Cooney, J. J. and Caldwell, D. E. (1996),
J. Ind. Microbiol
. 16(6):331-341
. However, these previous uses of viral particles either rely on the infectiousness of the virus, or have a limited capability to detect a broad range of analytes of interest.
In contrast, a prominent feature of the presently described detection system is that it can detect almost any analyte of interest by incorporating multiple copies of reporter groups and a linking molecule into a non-infectious viral particle. A major practical advantage of a detection system thus formed is that it provides a simple, fast method for the detection of small numbers of analytes. It is especially true when the nature of the target analytes is such that they cannot be amplified and/or if they offer only a single or a few sites for attaching reporter groups.
SUMMARY OF THE INVENTION
Thus, it is an object of the invention to provide a detection system that is capable of detecting a small number of analytes of interest, and to provide a method for making such a detection system.
In achieving this object, there has been provided, in accordance with one aspect of the present invention, a detection system capable of binding to an analyte, wherein the detection system comprises a non-infectious viral particle, a reporter group linked to the viral particle, and a linking molecule which is capable of linking the viral particle to the analyte; and wherein the reporter group and the linking molecule are not components of a naturally occurring virus.
In one embodiment, the viral particle is a bacteriophage particle, preferably a bacteriophage of the T Even family, and more preferably a Bacteriophage T4.
In a preferred embodiment, the bacteriophage particle comprises a capsid and does not have a tail structure. More preferably, the capsid is a Bacteriophage T4 capsid.
In another embodiment, the detection system of the present invention comprises at least 50 reporter groups, preferably at least 100 reporter groups, and more preferably at least 200 reporter groups.
In yet another embodiment, the reporter group comprises a fluorescent molecule, which preferably comprises a fluorescent protein, such as a green fluorescent protein. The fluorescent protein may preferably be enclosed within a Bacteriophage T4 capsid.
In yet another aspect of the present invention, the reporter group comprises an outer capsid protein covalently linked to a fluorescent protein. Preferably, the outer capsid protein is a non-essential outer capsid protein.
In a preferred embodiment, the linking molecule comprises an outer capsid protein covalently linked to an extra amino acid sequence. The outer capsid protein preferably is a non-essential outer capsid protein, such as the hoc and soc proteins of Bacteriophage T4. The extra amino acid sequence preferably comprises a polypeptide selected from the group consisting of a single chain Fv molecule, an Annexin and a DNA methyltransferase.
In another embodiment, the detection system comprises at least two bacteriophage particles which are linked together, either covalently or non-covalently, which thereby permits an increased number of reporter groups to bind to a single analyte.
A detection system has also been provided, in accordance with one aspect of the present invention, wherein said detection system comprises a viral particle, a fluorescent protein which is enclosed within the viral particle, and a linking molecule which is capable of linking the viral particle to the analyte; and wherein the linking molecule is not a component of a naturally occurring virus.
In another aspect of the present invention, the detection system comprises a viral particle, a reporter group which is linked to the viral particle and comprises a first outer capsid protein covalently linked to a first extra amino acid sequence, and a linking molecule which is capable of linking the viral particle to the analyte and comprises a second outer capsid protein covalently linked to a second extra amino acid sequence; and wherein the first and second extra amino acid sequence are not identical.
There has been provided, in accordance with another aspect of the present invention, a method making a detection system, comprising the steps of:
(1) providing a viral particle;
(2) providing a reporter group comprising a first outer capsid protein;
(3) linking the
Brown Stacy S.
Foley & Lardner
Park Hankyel T.
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