Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-22
2002-09-10
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007100, C436S501000, C436S514000, C436S518000
Reexamination Certificate
active
06448001
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an assay method for assaying an analyte as a biological assay subject or for detecting the presence or absence thereof, which is useful for simple clinical diagnosis, and a kit and an assay device to be used for the method; more specifically, the present invention relates to an assay method for assaying a great number of combinations of one or more species of analytes contained in a fluid sample or the presence or absence thereof, and a kit and an assay device therefor.
BACKGROUND ART
For determining the disease affecting a patient in a laboratory test, several types of laboratory test results should collectively be examined. In general, patients should undergo several types of such tests for appropriate diagnosis and therapeutic treatment. However, one laboratory reagent can assay or detect one item in most cases in the prior art, so the sample volume drawn from a patient is increased in proportion to the number of tests, which works a physical burden on the patient.
Alternatively, it is required to carry out conventional immunological tests by using automatic assay devices, so the sample drawn from a patient is delivered to an institute equipped with such automatic assay devices, where the tests are conducted, and then, the test results are reported to the doctor. In such manner, the doctor can make diagnosis based on the results and the clinical conditions of the patient. Therefore, such device works as one cause of delay treatment because the doctor cannot make a decision instantly.
So as to overcome such problem, a method comprising a combination of immune reaction and chromatography (abbreviated as “immunochromatography” herein below) has been developed in recent years. The standard principle of conventional immunochromatography will now be described below.
In the assay device to be used for the conventional immunochromatography, the following zones are arranged; a loading zone for loading a fluid sample containing an analyte, at one end of a developing element in the form of porous sheet such as nitrocellulose film, a water absorption zone for receiving the fluid transferred through capillary action in the developing element, at the other end, a sealing zone containing a marker-labeled immune substance, located on a side close to the loading zone between the water absorption zone and the loading zone, and a detection zone where an immune substance to bind a complex composed of the analyte and the labeled substance is immobilized, the zone being arranged on a side apart from the loading zone.
By the assay method by using such assay device, a fluid sample containing an analyte to be assayed is firstly loaded on the loading zone, and the fluid sample is then transferred through capillary action to the sealing zone containing a marker-labeled immune substance. In the sealing zone, the marker-labeled immune substance and the analyte are bound together through immunological affinity, to form a marker-labeled immunocomplex. The marker-labeled immunocomplex is developed and transferred, through capillary action and/or diffusion in the developing element, to the detection zone, where an immune substance immobilized in the detection zone captures the complex. The marker in the marker-labeled immunocomplex captured in the detection zone is assayed or detected, whereby the amount or presence of the analyte contained in the fluid sample can be assayed.
Compared with enzyme immunoassay, another assay for immunochemical active substances, the method is characteristic in that no rinsing procedure is required during an intermediate stage of assaying and the assay can be done under naked eyes, essentially never requiring any device to detect the marker, and in that the reagent contained in the assay device is kept at dry state so it can be stored at ambient temperature for a long term. According to the conventional immunochromatography, a doctor can instantly examine a sample collected by himself, and hence, the doctor can inclusively take into account clinical conditions of a patient and the immunological test results of the patient, to diagnose the patient in a short time. Accordingly, the delay in the treatment will be less, advantageously.
A number of patent application have been laid open concerning immunochromatography. For example, the immunochromatography described in Japanese Patent Publication No. Hei 7-13640 is essentially the same as the prior art immunochromatography described above, characterized in that a ligand bound to an insoluble vesicle marker is used and the insoluble vesicle marker is colored liposome, colored polymer bead, or metal or polymer dye particle. However, the publication does not include any description about the simultaneous assay or detection of one or more species of biological substances such as antigen or antibody.
Japanese Patent No. 2504923 describes an immunochromatography essentially the same as the prior art immunochromatography, suggesting an analysis by a sandwich method wherein a complex captured in a detection zone is a marker-labeled receptor-analyte-receptor as well as simultaneous detection of a first analyte and a second analyte, having biological affinities different from each other. However, the publication does not suggest that the marker-labeled immunocomplex is captured through the complementary binding between the bases of nucleic acids in a detection zone or the applicability of the method to two or more analytes or the assay sensitivity thereof.
Alternatively, immunoassay methods can yield higher sensitivity when a large amount of immunochemically active substances can be immobilized, and in that case, the methods can detect the same levels of immunochemically active substances in a shorter time. Hence, a more highly sensitive assay technique in the field of immunochromatography has been desired.
It is thus an object of the present invention to provide an assay method useful for clinical diagnosis, which can simultaneously assay one or more species of biological substances or detect the presence or absence thereof, at a higher sensitivity, by means of a single assay device in a simple fashion, and a kit and an assay device for the assay.
DISCLOSURE OF INVENTION
A first aspect of the assay method of the present invention is an assay method by means of a kit, wherein a reagent and an assay device are separately arranged. The assay method is an assay method for assaying the amounts of one or more species of analytes present in a fluid sample or detecting the presence or absence thereof, comprising:
(1) putting a fluid sample containing one or more species of analytes in contact to a reagent containing one or more species of marker-labeled ligands each produced by binding a marker to a first ligand, and one or more species of bond element-labeled ligands each produced by binding a bond element consisting of nucleic acids with a predetermined base sequence depending on the analyte species, to a second ligand, to generate one or more species of specific complexes each composed of a specific analyte species, a specific marker-labeled ligand species specifically binding to the specific analyte species, and a specific bond element-labeled ligand species specifically binding to the specific analyte species;
(2) developing one or more species of generated complexes through capillary action in a developing element in a sheet form;
(3) capturing a complex depending on the analyte species, through the complementary binding between the bond element and an anti-bond element, in the detection zone produced by immobilizing independently anti-bond elements consisting of nucleic acids each having a complementary sequence to the base sequence of one bond element species in the complexes, thereby forming an independent band; and
(4) assaying or detecting the marker formed in the band in the detection zone.
Another embodiment of the assay method of the present invention is a method using an assay device integrally containing a reagent. The assay method is an assay method for assaying the amounts of one
Oku Yuichi
Otsuka Yoko
Tanaka Yoshitatsu
Einsmann Juliet
Fredman Jeffrey
Lorusso & Loud
Nissui Pharmaceutical Co., Ltd.
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