Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-09
2001-07-03
Minnifield, Nita (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007230, C435S007240, C435S007950, C436S518000, C436S906000, C436S828000
Reexamination Certificate
active
06255048
ABSTRACT:
This application is the national phase of international application PCT/ JP97/01960 filed Jun. 9, 1997 which designated the U.S.
TECHNICAL FIELD
The present invention relates to high sensitivity fluoroassay.
BACKGROUND ART
Many methods are known for detecting biological trace components, which are highly sensitive and specific. Briefly, in those methods a targeted trace analyte is detected or quantitatively determined by labeling the analyte with a suitable labeling material, immobilizing the labeled analyte on a suitable medium based on a specific binding reaction, thoroughly washing it, and then detecting the labeling material by suitable means. Examples of the specific binding reactions generally used are the antigen-antibody reaction, the avidin-biotin binding reaction, and the receptor-ligand binding reaction.
Labeling materials and methods for label detection are known to be of various types depending on the chemical and physical properties of the labeling materials. Usually, such methods are as follows: (1) The sum of signals, e.g., radiation doses, amounts of fluorescence, or amounts of luminescence (chemiluminescence, bioluminescence), from the trace analyte labeled with a labeling material and immobilized on a suitable solid phase is measured, and presence of the trace analyte is evaluated, and quantitatively determined, based on the correlation between measured values and concentrations of the trace analyte. (2) The number of the labeled analyte molecules immobilized on a suitable solid phase is counted by observing phenomena such as radiation, fluorescence, or luminescence (chemiluminescence, bioluminescence) from the labeling material, and presence of the trace analyte is evaluated, and quantitatively determined, based on the count. Method (2) requires that the trace labeled analytes immobilized on the solid phase are counted molecule by molecule. Compared with method (1), method (2) may have advantages, such as lower background noise, shorter measuring time, and decreased measurement errors (increased measurement sensitivity).
Thus, it is desirable to develop a method which comprises counting, individually, trace labeled analytes fixed to a suitable solid phase, based on presence or absence of signals from the labeling material, in order to evaluate the presence of the analyte and quantitatively determine the analyte.
The usual, known method of labeling an analyte with a fluorescent molecule should give single molecule detection under idealized conditions. However, it is difficult to use this method for the above-mentioned ultrahigh sensitivity detection under practical measuring conditions, which involves use of an ordinary fluorescence microscope. The amount of fluorescence emitted from a single fluorophore is usually very small partly because of rapid bleaching of the fluorochrome. That is, such signals from the labeled analyte are very small and weak. Since background fluorescence is also comparable with signal fluorescence, measurement of individual luminescence phenomena is virtually unpractical.
DISCLOSURE OF THE INVENTION
High sensitivity fluoroimmunoassay related to the present invention circumvents the above-described difficulties, and makes it possible to detect a fluorescent-labeled analyte with higher sensitivity. The first objective of the invention is to provide a fluoroimmunoassay which comprises the step of labeling an analyte with a labeling fluorescent material having a nucleic acid portion stained with multiple fluorochromes by an ordinary detecting means, and a specifically binding reactive group specifically binding to the analyte, and the step of detecting the fluorescence of the labeling fluorescent material.
The second objective of the invention is to provide the above-mentioned fluoroimmunoassay which further includes the step of immobilizing the labeled analyte on a solid phase.
Still another objective of the invention is to provide the above-mentioned fluoroimmunoassay wherein the step of detecting the fluorescence further uses optically magnifying means.
A further objective of the invention is to provide the above-mentioned fluoroimmunoassay wherein the optically magnifying means is a fluorescence microscope.
A still further objective of the invention is to provide the above-mentioned fluoroimmunoassay wherein the fluorescence of the labeling fluorescent material is counted as fluorescent spots in the microscope images obtained.
Further, an objective of the invention is to provide fluoroimmunoassay which comprises the step of labeling an analyte with a labeling fluorescent material, or probe, having a nucleic acid portion stained with fluorochromes, and a specifically binding reactive group specifically binding to the analyte, and the step of detecting the fluorescence of the labeling fluorescent material.
Another objective of the invention is to provide the fluoroimmunoassay which further includes the step of immobilizing the labeled analyte on a solid phase.
Still another objective of the invention is to provide the fluoroimmunoassay wherein the step of the fluorescence detection further uses optically enlarging means.
A further objective of the invention is to provide the fluoroimmunoassay wherein the optically enlarging means is a fluorescence microscope.
A still further objective of the invention is to provide the fluoroimmunoassay wherein the fluorescence of the labeling fluorescent material is counted as fluorescent spots in the microscope images obtained.
An additional objective of the invention is to provide the fluoroimmunoassay wherein the nucleic acid portion is a double-stranded nucleic acid having 100 to 50,000 bases, and the staining with the fluorochromes is performed with intercalating fluorochromes whose number is estimated to be 10 to 25% of the number of the bases.
A further additional objective of the invention is to provide the fluoroimmunoassay wherein the nucleic acid portion is a double-stranded nucleic acid having 1,000 to 5,000 bases, and the staining with the fluorochromes is performed with 100 to 1,200 intercalating fluorochromes.
A further additional objective of the invention is to provide the fluoroimmunoassay wherein the nucleic acid portion is a double-stranded nucleic acid having 100 to 50,000 bases, and the staining with the fluorochromes is performed by the fluorochromes binding to the minor groove of the double-helix, the number of the fluorochrome being 10 to 25% of the number of the bases.
A further additional objective of the invention is to provide the fluoroimmunoassay described above, wherein the nucleic acid portion is a single-stranded nucleic acid having 100 to 50,000 bases, and the staining with the fluorochromes, whose number is 10 to 70% of the number of the bases, is by covalently binding to the nucleic acid.
A further additional objective of the invention is to provide the fluoroimmunoassay wherein the nucleic acid portion is a double-stranded nucleic acid having 100 to 50,000 bases, and the staining with the fluorochromes, whose number is 10 to 70% of the number of the bases, is by covalently binding to the nucleic acid.
A further additional objective of the invention is to provide the fluoroimmunoassay wherein the specifically binding reactive group is biotin bound to a terminal of the nucleic acid.
A further additional objective of the invention is to provide the fluoroimmunoassay wherein the magnification of the fluorescence microscope is 20× to 100× magnification at the objective.
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“Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera”, M. Masu
Hosoi Shigeru
Kadouchi Sachiko
Kojima Makiko
Baskar Padma
Laboratory of Molecular Biophotonics
Minnifield Nita
Pillsbury & Winthrop LLP
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