Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-31
2004-11-30
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007710, C435S007930, C435S040500, C435S175000, C435S177000, C435S180000, C435S964000, C435S973000, C435S975000, C436S518000, C436S519000, C436S520000, C436S526000, C436S528000, C436S548000, C530S388260, C530S391100, C530S815000
Reexamination Certificate
active
06825000
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an immunoassay reagent utilizing an insoluble carrier and an immunoassay method, and more particularly to an immunoassay reagent and an immunoassay method capable of high-sensitive detection of a target substance in a subject.
BACKGROUND ART
In the field of clinical testing, diagnoses of diseases are carried out using biosamples (such as blood and urine). For these diagnoses, a variety of assays have been developed and utilized. Typical assays include biochemical assays such as utilizing an enzyme reaction, and immunoassays such as utilizing an antigen-antibody reaction. The recent demand to perform precise assaying of components present in biosamples has led to the wide-spread use of immunoassay methods utilizing highly-specific antigen-antibody reactions.
Examples of immunoassay methods include immuno-turbidimetry (TIA method), latex immunoassay (LIA method), enzyme immunoassay (EIA method) and radio-immunoassay (RIA method). Selection is made depending upon the particular purpose contemplated. That is, the TIA or LIA method may be employed when a biosample contains a target substance in a relatively large quantity. The TIA and LIA methods are generally employed to assay a substance, such as C-reactive protein (CRP), anti-streptolysin O antibody (ASO) or fibrin degradation product (FDP), particularly when it is contained in a biosample in concentrations of not below several ng/ml. On the other hand, the EIA or RIA method may be used when a biosample contains a target substance in a relatively small quantity. These TIA and LIA methods are generally employed in assaying substances, such as cancer markers represented by &agr;-fetoprotein (AFP) and hormones represented by insulin, particularly when they are contained in a biosample in concentrations of not above several ng/ml.
The recent trend of placing greater importance on assay of microscale substances in a biosample further increases frequencies in use of EIA and RIA methods. In contrast to the TIA and LIA methods which can enjoy shortend testing time and simplified operation and find applications to various types of autoanalyzers (hereinafter referred to as clinical chemistry autoanalyzers), the EIA and RIA methods suffer from the following dificiencies: they need prolonged reaction periods; they require complex operations; and they employ diverse enzymes and radioisotopes. Because of these dificiencies, the EIA and RIA methods are frequently limited to use with specific autoanlyzers (hereinafter referred to as specialized autoanalyzers). The RIA method further requires special facilities because of its utilization of radioisotopes.
A need has arisen for a technique which allows assaying of ultramicro-scale substances in a biosample to thereby enable early detection of cancers and early diagnosis of infection with AIDS virus. There are two groups of techniques which have been found to enable assaying of ultramicro-scale substances. One group of techniques is directed to increase precisions of conventional assay methods, including modifications and improvements of the LIA and EIA methods. Another group of techniques is directed to improve performances of conventional devices used for the LIA and EIA methods. Some of such techniques have been put into practice.
Examples of techniques contemplated to increase the precision of assay methods themselves include a technique which colors insoluble carriers for use in the LIA method (Japanese Patent Laying-Open No. Hei 1-214760) and a technique which utilizes fluorescent materials, instead of enzymes, for labeling antigens or antibodies for use in the EIA method (Japanese Patent Laying-Open No. Hei 5-34346). Also, examples of techniques contemplated to improve performances of deviced include a technique proposed in Japanese Patent Laying-Open No. Hei 3-167475.
However, neither of these techniques are applicable to clinical chemistry autoanalyzers and the problem of requiring specialized autoanalyzers remains unsolved. The need of such specialized autoanalyzers arises because the reaction time, procedure and type of enzyme or radioisotope for use in micro-scale assay methods, as represented by the EIA and RIA methods, are varied depending upon the particular method used, as stated earlier. Other major reason is based on the fact that the micro-scale assays as currently developed or heretofore marketed always require an operation called B/F separation (B is a bound component via an immune reaction and F is a free component). This makes them unapplicable to clinical chemistry autoanalyzers incapable of B/F separation and necessitates specialized autoanalyzers capable of B/F separation.
Assay methods which do not require B/F separation have been recently proposed and developed, as seen in Japanese Patent Laying-Open Nos. Hei 5-249112 and Hei 7-179495. Due to the insufficient sensitivity and extended determination period, they in some cases require specialized autoanalyzers and in other cases find a limited application to clinical chemistry autoanalyzers, which have been problems.
In order to perform ultramicro-scale analysis in the scene of actual clinical examinations, it is required to purchase an expensive special autoanalyzer and secure a place for its installation. There accordingly remains a strong demand for a method which can assay ultramicro-scale substances with the use of clinical chemistry autoanalyzers.
As stated above, the methods currently developed or marketed for assaying ultramicro-scale substances require the B/F separation, as against such a strong demand from users. This presents a major problem, i.e., their practices are limited on special autoanalyzers.
In the scene of clinical examinations, it is frequent to test several items using a single biosample. In such an instance, the single biosample is repeatedly subjected to different assay methods. This not only prolongs the time necessary to complete the testing, but also increases a chance for a tester to contact the biosample and thereby increases a risk of infection, which have been problems.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an immunoassay reagent and an immunoassay method which can solve the above-described problems, which can assay an ultramicro-scale substance in a sample, such as an antigen or antibody, with a high level of sensitivity, and which can either eliminate the need to carry out the B/F separation or simplify the B/F separation in assaying ultramicro-scale substances.
A first invention of the present application is an immunoassay reagent for quantitatively determining a target material, i.e., an antigen or antibody in a sample, which is characterized as containing (a) an insoluble carrier for carrying an enzyme and an antibody or antigen corresponding to the aforementioned antigen or antibody, the aforementioned insoluble carrier comprising at least one selected from the group consisting of an organic polymer powder particle, microorganism, blood cell and cell membrane fragment, (b) an enzyme inhibitor for inhibiting the activity of the aforementioned enzyme and (c) a substrate with which the aforementioned enzyme reacts.
In a particular aspect of the first invention, there are provided a first reagent containing the aforementioned insoluble carrier and a second reagent containing the aforementioned enzyme inhibitor and substrate.
A second invention of the present application is an immunoassay reagent for quantitively determining a target material, i.e., an antigen or antibody in a sample, which is characterized as containing (a) an insoluble carrier for carrying an enzyme inhibitor and an antibody or antigen corresponding to the aforementioned antigen or antibody, the aforementioned insoluble carrier comprising at least one selected from the group consisting of an organic polymer powder particle, microorganism, blood cell and cell membrane fragment, (b) an enzyme whose activity is inhibited by the aforementioned enzyme inhibitor and (c) a substrate with which the aforementioned enzyme reacts.
In a particu
Akamine Takayuki
Yokoi Masayuki
Yoshikawa Katsumi
Chin Christopher L.
Grun James L.
Sekisui Chemical Co. Ltd.
Townsend & Banta
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