WO2008053992A1 - Efficient method for detecting antigen, virus, cell and microorganism - Google Patents

Abstract

It is intended to provide a method which is simple and at low cost and is capable of easily detecting a virus or a microorganism even at a small level at which detection was impossible in the past. In particular, a method which is capable of detecting HCV at a small level at which a therapeutic effect of administration of interleukin can be sufficiently exhibited is provided. Further, a detection kit for achieving these methods is provided. Specifically, the detection method of the invention is characterized by comprising the steps of: binding a virus and/or a microorganism in a test sample to a solid-phase primary antibody; binding a secondary antibody to the virus and/or microorganism bound to the primary antibody; binding a binding substance to the secondary antibody labeled with an oligonucleic acid strand to the secondary antibody bound to the virus and/or microorganism; amplifying the oligonucleic acid strand; and detecting the amplified product.

Description

 Specification

 Highly efficient detection method for antigens, viruses, cells and microorganisms

 Technical field

 [0001] The present invention relates to a highly efficient method for detecting an antigen, a method for detecting violets, cells, and microorganisms in a test sample using the detection method, and a kit for detecting the virus and / or microorganism. . In particular, the present invention relates to a method for detecting hepatitis C virus in a test sample, and a kit for detecting the virus.

 Background art

 [0002] Viral hepatitis (hepatitis C) caused by hepatitis C virus (HCV) is chronic in 75% of those who develop the disease, and often develops liver cancer with a probability of about 25%. It is known that. At present, it is estimated that approximately 700,000 people exist in Japan as HCV carriers, but early hepatitis C has very few subjective symptoms and it is very difficult to predict the symptoms and progress. In many cases, the patient becomes serious and becomes aware of power and infection. Although the number of new infections is currently decreasing, HCV infection, or hepatitis C, can threaten the lives of infected people and have a major impact on Quality of Life (QOL).

 With the development of medicine, hepatitis C can often be healed by treatment with interferon α, / 3, an antiviral drug ribavirin. In reality, however, the correctness of treatment depends largely on the amount of virus possessed by patients with hepatitis C. Generally, when the amount of blood wine is above a certain value, the therapeutic effect may drop sharply. Are known. Currently, the amount of HCV is used for predicting the therapeutic effect of interleukin treatment and monitoring the therapeutic effect, and the virus quantification method occupies an important position not only in diagnosis but also in treatment.

 So far, HCV antibody measurement method, HCV-RNA qualitative / quantitative method, and Η CV core protein quantification method are known as HCV quantification methods.

[0003] The HCV antibody measurement method captures the amount of antibody against the HCV virus produced in the body using recombinant proteins in a region that is known to have high homology and high antigenicity to various HCV virus strains. It is a method that detects and quantifies, and is a useful method because it is relatively simple. Yes (Kuo, G. et al., An assay for circulation g antibodies to a major etiologic virus of human 顯 _A, 顯 -B hepatitis., Science, vol.244, p.302-304, 1989). However, in this method, since antibodies are not produced until several weeks after HCV infection, it is extremely difficult to determine the presence or absence of HCV virus immediately after infection. In addition, this method measures the amount of antibody against HCV, not HCV virus, and therefore cannot identify HCV species.

 [0004] The HCV-RNA qualitative / quantitative method is a method that detects and reconstitutes HCV genomic RNA by reverse transcription PCR, etc. (Okamoto, Tsuji, et al., Detection of hepatitis C virus RNA by a two -stage polymerase chain reaction with two pairs of primers deduced from the 5'_non cording region., Tpn. J. Exp. Med., vol.60, p.215-222, 1990), very sensitive, Even the amount of virus at the early stage of infection can be detected. However, this method is complicated in the handling and operation of the test sample, and the testing itself requires a long time, manpower, and a large amount of money, so the facilities that can be implemented are limited. Therefore, as a result that is difficult to use for primary screening, it is currently used as a confirmatory diagnostic method after infection has been confirmed by other screening methods that are simpler and less sensitive.

[0005] On the other hand, the HCV core protein quantification method is a method for detecting and quantifying HCV by various immunoassays using antibodies to the HCV core protein. For example, ELISA (Enzyme-Linked Immunosorbent Assay: enzyme immunity , / Law; ^ method Noya is known to be based on immunoassay methods such as IRMA (Immunoradiometric Assay), written by Eiji Tanaka et al. "Evaluation of usefulness", New drugs and clinical medicine, published by National Institute of Medical Information, vol.50 (8), p.109-119, 2001; written by Eiji Tanaka et al., "Evaluation of oso HCV antigen IRMA test, ... Pharmacy, published by Natural Sciences Co., Ltd., vol.46 (2), p.247-256, 2001) The HCV core protein quantification method has a correlation with other test methods that are inexpensive and sensitive, and can be operated. Has many advantages, such as being relatively easy, and has been put to practical use as various test reagents and clinical test kits. Therefore, even with this method, it is difficult to detect a trace level of HCV that can sufficiently exert the therapeutic effect of interleukin administration. When new HCV infection is confirmed, treatment with interleukin administration has already been performed. There are some cases where the amount of virus is impossible! [0006] From the above, there is a strong demand for the development of a method for detecting HCV that is simple, low-cost, and more sensitive.

 By the way, in the current HCV test, blood is usually used as a test sample. This is because it is possible to reliably detect winoles when blood is collected relatively quickly and in the presence of virus infection. In recent years, however, the importance of non-invasive testing methods aimed at improving patient quality of life has been recognized again.

 For this reason, it is desirable to develop a method for detecting HCV with higher sensitivity, which is desirable for methods that allow HCV testing even from test samples that can be collected noninvasively, such as saliva and urine. ing.

 Disclosure of the invention

 Problems to be solved by the invention

[0007] The problem to be solved by the present invention is to provide a method that is simple and low-cost, and that can easily detect trace levels of antigens, viruses, cells, and microorganisms that were not possible to detect in the past. There is to do. In particular, it is an object of the present invention to provide a method capable of detecting a trace level of HCV that can sufficiently exert the therapeutic effect of interleukin administration.

 Moreover, it is providing the kit for a detection which can implement such a detection method. Means for solving the problem

That is, the present invention is as follows.

 (1) a step of binding a first antibody that binds to an antigen to the antigen;

 Binding a second antibody that binds to the first antibody labeled with an oligonucleic acid chain to the first antibody;

 Amplifying the oligonucleic acid strand; and

 Process for detecting amplification products

 A method for detecting an antigen, comprising:

[0009] (2) a step of binding a virus and / or microorganism in a test sample to an immobilized primary antibody,

Binding a secondary antibody to the virus and / or microorganism bound to the primary antibody; A binding substance labeled with an oligonucleic acid chain is transferred to the virus and / or microorganism.

 / Toro mouth

 Binding to the prepared secondary antibody,

 Amplifying the oligonucleic acid strand; and

 Process for detecting amplification products

 A method for detecting viruses and / or microorganisms.

 The detection method of the present invention can further include, for example, a step of quantifying viruses and / or microorganisms in the test sample using the detection result of the amplification product as an index. In the detection method of the present invention, examples of the amplification method include a real-time PCR method, and examples of the binding substance include an antibody. Furthermore, examples of the virus include hepatitis C virus.

[0010] (3) A support on which a primary antibody against a virus and / or microorganism is immobilized,

 A secondary antibody against the virus and / or microorganism,

 A binding substance labeled with an oligonucleic acid strand, and

 Primers used for amplification of oligonucleic acid strands

 A kit for detecting viruses and / or microorganisms.

 In the kit of the present invention, examples of the virus include hepatitis C virus.

[0011] = = Cross reference with related literature = =

 This application claims the benefit of priority based on Japanese Patent Application No. 2006-298825 filed on November 2, 2006, and is incorporated herein by reference. Brief Description of Drawings

 FIG. 1 is a diagram showing a detection result (a) of HCV core protein by the detection method of the present invention, and an approximate curve (b) created based on the least square approximation method in a detectable range.

 FIG. 2 is a diagram showing the results of detection of HCV core protein by ELISA.

 FIG. 3 is a diagram showing a comparison between the detection result of the HCV core protein by the detection method of the present invention (FIG. 1 (a)) and the detection result of the HCV core protein by the ELISA method (FIG. 2).

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited by these explanations, but can be modified and implemented as appropriate without departing from the spirit of the present invention other than the following examples.

 [0014] 1. Method for detecting antigen

 The detection target in the detection method of the present invention is a force that is an antigen. The type thereof is not limited and may be any protein, sugar, glycoprotein, and the like. In addition, if the antigen is recognized by an antibody, its location and state are not limited, and it is an antigen present on the cell surface or in the cell, whether it is a purified antigen or a virus antigen. Even if the virus or cells remain alive, they may be fixed, destroyed, extracted or purified. For example, viruses include hepatitis C virus, hepatitis B virus, human immunodeficiency virus, cells include microorganisms such as Escherichia coli, tetanus, Staphylococcus aureus and chlamydia, cultured cells such as plants and animals, animals and Examples include cells derived from plant tissues. Among them, hepatitis C virus is preferable. In the case of a purified antigen, for example, it may be free in a liquid or immobilized on a support. In addition, when the antigen is solid-phased, for example, even if the antigen is directly bound to a support such as a coated plastic dish, the antibody of the antigen adheres to the support and the antigen is attached to the antibody. It may be indirectly bonded to the support, such as adhering. In the present embodiment, the support is not particularly limited.

 [0015] As described above, the method for detecting an antigen of the present invention includes the following steps (i) to (iv).

 (0 The step of binding the first antibody that binds to the antigen to the antigen (antigen-antibody binding step),

(ii) a step of binding a binding substance that binds to the first antibody labeled with an oligonucleic acid chain to the first antibody that binds to an antigen (binding substance binding step);

 (iii) Amplifying oligonucleic acid strand (amplification step),

 (iv) A step of detecting the amplification product (detection step).

 [0016] The detection method of the present invention may include other steps in addition to the above steps.

For example, (vi) the antigen is quantified using the detection result of the amplification product as an index. The process (quantitative process) is preferred.

 Below, the detail about each said process is demonstrated.

[0017] (1) Antigen-antibody binding step

 The first antibody used in this step is not particularly limited as long as it can bind to the target antigen, such as the type of antibody (eg, IgG, IgM, etc.) and the origin of animal species (eg, mouse, rat, rabbit, etc.). Nare ,.

 For example, a test sample containing viruses or cells is subjected to a disruption process, an extraction process, a purification process, etc. according to a conventional method, and the first antibody is administered to the obtained sample to bind to the antigen.

 [0018] (2) Binding substance binding step

 The binding substance used in this step means a substance that is labeled with an oligonucleic acid chain and that can specifically bind to the first antibody bound to the antigen. Here, the binding mode between the first antibody and the binding substance may be a mode in which they are directly bound to each other, or through another substance bound to the first antibody (that is, the binding substance is the relevant substance). There is no limitation as long as both are indirectly bonded (by binding to other substances). The other substances are not limited, but preferred examples include various proteins (for example, enzyme proteins such as HRP) used as a labeling substance for the first antibody used in the immunoassay.

 In the present invention, the binding substance is any substance as long as it has a configuration in which any substance capable of binding directly or indirectly specifically with the first antibody is labeled with an oligonucleic acid chain. For example, an antibody labeled with an oligonucleic acid chain (second antibody) is preferable. In this case, the first antibody or the other substance bound to the first antibody becomes an antigen for the second antibody, and examples of the second antibody include an anti-Ig antibody and an anti-HRP antibody. The

[0019] As the oligonucleic acid chain used for the labeling treatment of the binding substance, an oligonucleic acid chain having a binding region with the primer used in the nucleic acid amplification method is used. Nucleic acid amplification methods include, for example, a nucleic acid amplification method (for example, PCR method) in which a reaction is carried out under a plurality of temperature controlled conditions using a thermal cycler, etc. Law) There is no limit. The amplification region by the nucleic acid amplification method may be a part or all of the oligonucleic acid chain.

 Here, the region that binds to the PCR primer is a region that includes two regions that serve as a basis for designing a primer set composed of an F primer and an R primer, and can be amplified using this primer set. It means a region, and the specific nucleic acid sequence is not limited. The PCR method is preferably a real-time PCR method from the viewpoint of ease of detection and quantification described later. In particular, in the case of the TaqMan method, the oligonucleic acid chain having a binding region with a TaqMan probe is used.

[0020] The region binding to the primer for LAMP method is FIP, BIP, F3 primer, B3 primer

 Means a region that contains 6 regions that can be used as a basis for designing a primer set composed of (Prime F and / or Loop Primer B if necessary) and that can be amplified using this primer set. However, the specific nucleic acid sequence is not limited.

 The region that binds to the ICAN primer is a region that includes two regions that are the basis for designing a primer set composed of two chimeric primers (one of the F and R primers). It means a region that can be amplified using, and the specific nucleic acid sequence is not limited.

 The oligonucleic acid chain to be labeled is, for example, one having a region cleavable by a restriction enzyme, one having a region cleavable by light irradiation, or one having a region cleavable by active oxygen. May be. In this case, before the amplification reaction, the oligonucleic acid chain containing the amplification region can be separated and isolated from the complex with the binding substance to form a cage, and a more efficient amplification reaction can be performed.

In the present invention, the oligonucleic acid chain is an oligonucleotide chain (oligo DNA chain, oligo RNA chain (especially oligo DNA chain)), oligopeptide nucleic acid chain (oligo PNA chain), or a mixture thereof. Preferred is a strand, and more preferred is an oligonucleotide chain. In addition, oligonucleic acid chains include those containing oligopeptide chains in part. When the oligopeptide chain is contained at one end of the oligonucleic acid chain, for example, in order to facilitate the labeling of the binding substance, or to cleave the oligopeptide chain later when it is separated from the binding substance, Peptide chains can be used. Oligonucleic acid strands are natural products. However, it is usually preferable to be a synthetic product. The length of the oligonucleic acid chain used as a label is not particularly limited. For example, it is preferably 100-5, OOOmer, more preferably 100-1,000 mer, and still more preferably 100-50 Omer. When the length of the oligonucleic acid chain satisfies the above range, it is easy to conjugate (label) to the binding substance, stabilize the state after complexing, improve detection sensitivity, and shorten the detection time. Can do.

[0022] The binding substance labeled with the oligonucleic acid chain can be prepared, for example, by covalently binding one end of the oligonucleic acid chain to the binding substance body. In this case, for example, one or two or more thiol groups, amino groups (substituents), biotin (or avidin), etc. are introduced into the oligonucleic acid chain by chemical or enzymatic treatment (preferably chemical treatment). May be. This facilitates complexation with a binding substance, further stabilizes the state after complexation, improves the yield, and increases the detection sensitivity and detection effect. Specifically, the method of labeling the oligonucleic acid chain on the binding substance is as follows: (a) Fixing the oligonucleic acid chain with an amino group or thiol group at the 5 'end to the binding substance using a divalent cross-linking agent (See E. Hendrickson et al., Nucl. Acids Res., Vol.23 (3), p.52 2-529 (1995)) and (b) Preferred is a method of immobilizing the oligonucleic acid chain to the binding substance via avidin by mixing the binding substance and the oligonucleic acid chain and adding avidin.

Yes

[0023] In addition, according to the present invention, an oligonucleic acid chain that becomes a labeling moiety can be complexed with a binding substance via at least a part of the adapter. By fixing the oligonucleic acid chain to the binding substance via a part of the adapter, the structural stability after complexation can be further enhanced, resulting in a higher yield and higher detection sensitivity and detection effect. . Here, as the adapter portion, for example, any protein selected from protein G, protein A, and protein L may be used, and it may be a fusion protein with at least two proteins selected from protein G, protein A, and protein. It may be a fusion protein of at least one protein selected from protein G, protein A and protein L and another protein, or any combination thereof. these This adapter protein is preferable because it can be easily and stably bound to the antibody, particularly when the binding substance is an antibody molecule. Examples of proteins other than protein G, protein A, and protein L include anti-IgG antibodies.

[0024] The method of complexing the oligonucleic acid chain and the binding substance via the adapter part is, for example, (0 First, the oligonucleic acid chain is bound to the adapter part, and (ii) The adapter part is then bound to the binding substance. Specifically, in (i), the adapter part is avidin-modified, the oligonucleic acid chain is biotinized, and the both are mixed to bind the oligonucleic acid chain to the adapter part. The adapter part and the oligonucleic acid chain are vitinized in advance / !, and the adapter part and the oligonucleic acid chain are mixed and added with avidin to convert the oligonucleic acid chain into the adapter part via avidin. In the case of the former method, the avidin modification of the adapter part starts with the linker compound as the adapter part. After the binding reaction, avidin may be bound to the compound.If the adapter part used here is protein A, G or L, etc., as a linker compound, for example, “Sulfosuccinimidyl 4- (N- maleimidomethyl) cyclohexane-l-carboxylate (Sulfo_SMCC) ”etc. can be preferably used.Next, in (ii), when the adapter part and the binding substance have binding reactivity, the key obtained in (i) can be used. A complex can be obtained by mixing the conjugate part of the doveter / label part and the binding substance, and if the adapter part and the binding substance do not have binding reactivity originally, for example, both of them are biotin. The oligonucleic acid chain and the binding substance can be complexed using a method similar to the method that can be used in (i), such as mixing in the presence of avidin.

[0025] In this step, a reaction in which the first antibody bound to the antigen or another substance bound to the first antibody is brought into contact with the above-described binding substance (including a solution state), that is, The reaction for binding the first antibody or other substance to the binding substance is not limited in its method and conditions, and can be set as appropriate. For example, the reaction is preferably performed by stirring at room temperature for 30 to 60 minutes. Before the contact, it is desirable to perform a blocking treatment with a known blocking solution in advance. After the above reaction, the substance is bound to the first antibody or other substance by washing with a known washing solution such as PBS. Remove unnecessary components other than.

[0026] (3) Amplification process

 In this step, the oligonucleic acid chain in the binding substance after the binding step is amplified by the various amplification methods described above (for example, PCR method, LAMP method, ICAN method). Specifically, oligonucleic acid is obtained by mixing a predetermined primer set, DNA synthase, substrate, etc. (including solution state) in the system after the binding step and reacting under a predetermined temperature control condition. Amplifies a given region of the chain. Further, as the nucleic acid amplification method, it is preferable to use a real-time PCR method (for example, intercalator method, TaqMan method, cyclin probe method) so that amplification products can be easily detected in the detection step described later. The reaction solution composition and reaction conditions (temperature, time, etc.) in the nucleic acid amplification method can be appropriately set by conventional methods.

 In this step, when the oligonucleic acid chain has a region that can be cleaved by a restriction enzyme as described above, an appropriate restriction enzyme is added to the system obtained in the binding step in advance. By reacting, an oligonucleic acid chain can be isolated and amplified as a cage. If the oligonucleic acid chain to be labeled has a region that can be cleaved by light irradiation, the oligonucleic acid chain is isolated in advance by irradiation with light of a predetermined wavelength, and this is amplified as a cage. That power S. In addition, if the oligonucleic acid chain to be labeled has a region that can be cleaved by active oxygen, a reagent that produces and releases free radicals such as HRP (horseradish peroxidase) and Fe complexes is added in advance to activate. By generating oxygen, an oligonucleic acid chain can be isolated and amplified as a cage.

 [0028] (4) Detection process

 By detecting the amplification product obtained in the amplification step, the presence or absence of the antigen in the test sample can be confirmed. Detection of the amplification product can be performed by appropriately selecting from known detection means used in various nucleic acid amplification methods. For example, it can be easily performed by various electrophoresis methods, turbidity measurement, and visual observation, and when a real-time PCR method is used, it can be performed by detecting a predetermined fluorescence wavelength with a spectrofluorometer.

 [0029] (5) Quantitative process

In the present invention, by using the result obtained in the detection step as an index, The antigen of can also be quantified.

 The quantification can be performed by appropriately selecting from known quantification methods used in combination with various nucleic acid amplification methods. Specifically, for example, measurement of band concentration after electrophoresis using a densitometer, turbidity measurement (monitoring) of an amplification product using an absorptiometer or spectrophotometer, fluorescence intensity measurement (monitoring) using a spectrofluorometer, etc. Using the measurement results obtained by the above method as an index, a method of quantifying by comparison conversion with the measurement results (calibration curve, etc.) of controls prepared in advance can be mentioned.

 [0030] 2. Detection method of virus, cell and microorganism

 The following describes in detail how to detect viruses, cells, and microorganisms without destroying or extracting them. In particular, the force S, which will be described in detail using hepatitis C virus as an example, can be similarly applied to cases where detection is performed on other viruses, cells, and microorganisms using ordinary technical common knowledge of those skilled in the art. And

 [0031] As described above, the method for detecting hepatitis C virus of the present invention includes the following steps (i) to (v).

 (Step of binding hepatitis c virus in the test sample to the immobilized primary antibody (primary reaction step)

 (ii) A step of binding a secondary antibody (corresponding to the first antibody) to the virus bound to the primary antibody (secondary reaction step)

 (iii) A step of binding a binding substance (for example, a tertiary antibody (corresponding to the second antibody)) labeled with an oligonucleic acid chain to a secondary antibody bound to the virus (binding substance binding step)

 (iv) Amplifying the oligonucleic acid strand (Amplification step)

 (V) Process for detecting amplification products (Detection process)

[0032] The detection method of the present invention may include other steps in addition to the above steps, and is not limited to, for example, (vi) in the test sample using the detection result of the amplification product as an index. Of quantifying hepatitis C virus (quantitative process), (vii) comparing the amount of virus quantified with other factors (for example, severity of inflammation caused by cytodynamic force-in and liver condition as clinical findings) The process of diagnosing the current medical condition is preferred. Below, the detail about each said process is demonstrated.

[0033] (1) Primary reaction process

 The test sample containing hepatitis C virus is usually a component collected from a living body (for example, blood, serum, plasma, saliva, urine, tissue fragment), but it is not limited and collected. A biological component may be appropriately subjected to a known treatment in the field of various detection methods. Since the detection method of the present invention is extremely sensitive, hepatitis C, which is included in a trace amount of saliva and urine, which are not normally used as test samples in the detection of hepatitis C virus, among the above biological components. Viruses can be easily detected. Thus, by detecting the hepatitis c virus using the patient's blood, plasma, saliva and urine as the test sample, the presence or absence of hepatitis C virus infection is non-invasively detected in the patient. That's the power S.

 [0034] In the present invention, the amount of hepatitis C virus in the test sample is not limited. For example, even if the amount of virus per mL of the test sample is less than ng in terms of weight, It can be clearly detected, and may be below the pg order, and may be below the fg order. In the previous report, it was reported that all patients treated with interferon showed complete efficacy when the hepatitis C virus level in hepatitis C patients was 330 fg / mL (15 fmol / L) or less. (J. New Rem. & Clin., Vol. 50, 8, p.865-875, 2001). Since the detection method of the present invention is extremely sensitive, it can be sufficiently detected even if the amount of hepatitis C virus in the test sample is not more than the above virus amount (330 fg / mL), and not more than 20 fg / mL. Even the amount of virus can be detected.

 [0035] The primary antibody used in this step is not particularly limited as long as it is an antibody that can specifically bind to hepatitis C virus. For example, antibodies against HCV envelope, HCV core protein and the like are preferable.

Further, the primary antibody used in this step is preferably a solid phase immobilized antibody. Specifically, the primary antibody is preferably fixed to an arbitrary support. By using a solid-phased primary antibody, substances other than hepatitis C virus in the test sample can be efficiently excluded, and the selectivity of the virus can be increased. Reduce power by S. As a support for immobilizing the primary antibody, an antibody molecule can be immobilized, and the test sample, a secondary antibody and a binding substance (including a solution state) described later are added to the antibody molecule. There is no limitation as long as it can be brought into contact with each other. As such a support, insoluble materials and shapes are usually used. For example, known supports that can be used in the assembly system by antigen-antibody reaction are preferred, such as multi-plastic wellore plates, plastic beads, latex beads, magnetic dejugate beads, plastic tubes, nylon membranes, and A nitrocellulose membrane is preferred.

[0036] Immobilization (immobilization) of the primary antibody to the support can be performed by a known method in immunoassay, and is not limited. In addition, a commercially available support on which a primary antibody is immobilized in advance may be used.

 The binding reaction (antigen antibody reaction) between the hepatitis C virus in the test sample and the primary antibody immobilized on the support is performed by bringing the test sample into contact with the support. Various reaction conditions such as reaction temperature and reaction time can be appropriately set according to conventional methods of immunoassay. In addition, before bringing the test sample into contact with the support, it is desirable to perform a blocking treatment with a known blocking solution in advance. After the above reaction, unnecessary components other than the hepatitis C virus bound to the primary antibody are removed by washing with a known washing solution such as PBS.

[0037] (2) Secondary reaction step

 This step is a step of binding the secondary antibody to the hepatitis C virus bound to the primary antibody.

 The secondary antibody is preferably an antibody that can specifically bind to the hepatitis C virus and has a different epitope than the primary antibody, but is not limited thereto. As the secondary antibody, for example, antibodies against core protein, envelope protein El, envelope protein E2 and the like are preferable, and antibodies against core protein are preferable.

The binding reaction (antigen-antibody reaction) between hepatitis C virus and the secondary antibody is carried out by bringing the secondary antibody solution into contact with the support. Various reaction conditions such as reaction temperature and reaction time can be appropriately set according to conventional methods of immunoassay. In addition, it is desirable to perform a blocking treatment with a known blocking solution in advance before bringing the secondary antibody solution into contact with the support. Good. After the above reaction, unnecessary components other than the secondary antibody bound to hepatitis C virus are removed by washing with a known washing solution such as PBS.

[0038] In the present invention, as the secondary antibody used in this step, an antibody that has been previously labeled with an oligonucleic acid chain described later (that is, an oligonucleic acid chain labeled with a binding substance described later) may be used. it can. In this case, the joining process described later can be simplified and more rapid detection is possible.

[0039] (3) Subsequent steps

 Hereinafter, the binding substance binding step, the amplification step, and the detection step may be performed in the same manner as in the case of antigen detection.

 In this way, the presence or absence of hepatitis C virus in the test sample can be confirmed. In addition, hepatitis c virus in the test sample can be quantified by using the result obtained in the detection step as an index. The quantification method may be performed in the same manner as the antigen detection.

 [0040] 2. Detection kit

 As described above, the kit for detecting hepatitis c virus of the present invention comprises: (0) a support on which a primary antibody against hepatitis c virus is immobilized, (ii) a secondary antibody against hepatitis C virus,

(iii) a binding substance labeled with an oligonucleic acid chain, which can be directly or indirectly bound to the secondary antibody, and (iv) a primer used for amplification of the oligonucleic acid chain. It is what.

 The kit of the present invention can be used effectively for carrying out the above-described detection method of the present invention, and is extremely useful.

The kit of the present invention may contain other components in addition to the components (i) to (iv) listed above. Other components include, for example, dNTP, DNA polymerase, probes (TaqMan probe, cyclin probe, etc.), fluorescent reagents (DNA intercalators such as SYBR Green I, etc.), RNase H, restriction enzymes, various buffers, sterile water , Various reaction vessels (Eppendorf tubes, etc.), blocking agents (serum components such as Bovine Serum Albumin (BSA), Skim milk, Goat serum), and preservatives such as detergents, surfactants, sodium azide, etc. In addition to the experimental operation manual (instructions), etc., if necessary, a thermal cycler, a thermostatic chamber (any liquid, gas or solid medium), turbidity measurement device (minute Control or measurement equipment such as a photometer, etc.) and a spectrofluorometer.

 Example

 [0041] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

 [0042] [Example 1]

 1. Preparation of standard dilution system 歹 IJ (test sample) for HCV antigen

 Obtain a “soso HCV antigen ELISA test” (manufactured by soso 'clinical' Diagnostic), a kit for quantitative detection of hepatitis C virus, and use the HCV recombinant antigen standard solution (HCV concentration: 3600 fmol / L = 72 pg / mL) and a standard diluent were used to prepare a dilution series of HCV recombinant antigen solution.

 Specifically, as shown in Table 1 below, first, an antigen standard solution (72 pg / mL) was added to a standard dilution solution (208 mL) and diluted by a factor of 3.6 to prepare a 20 pg / mL antigen standard solution. Next, this 20 pg / mL antigen standard solution is diluted 5-fold with a standard dilution solution to prepare a 4 pg / mL antigen standard solution, and then the 5-fold dilution operation is repeated four times in the same manner to obtain 0.0064 pg A dilution series up to an antigen standard solution of / mL (6.4 fg / mL) was prepared.

 [0043] [Table 1]

[0044] 2. Primary reaction process (HCV antigen sensitization)

First, 100 L / well of a reaction solution (supplied with the kit) was added to each well of a multi-wall plate (supplied with the kit) on which a primary antibody against HCV was immobilized. Next, 100 L / well of each dilution series of the antigen standard solution prepared in 1. was added to each well to which the reaction solution was added. After the addition, pipetting was performed in each well, and the reaction solution and the antigen standard solution were mixed uniformly. The mixed plate was agitated with a plate mixer for 60 minutes at room temperature (20 30 ° C.) to carry out a reaction between the HCV recombinant antigen and the primary antibody. After completion of the reaction, the liquid in each well was removed with an aspirator.

 Thereafter, to each well, a cleaning solution (attached to the kit; 10-fold diluted with MilliQ was used) was added ^ OL / well, allowed to stand for 20 seconds, and then the cleaning solution was removed with an aspirator. The same washing operation was repeated 5 more times (6 times in total).

[0045] 3. Secondary reaction step (sensitization of HCV secondary antibody)

 To 50 L of HRP-labeled secondary antibody solution against HCV (supplied with the kit), 5 mL of an antibody diluent (supplied with the kit) was added and diluted approximately 100 times.

 200 L / well of the diluted secondary antibody solution was added to each well after washing in 2. above. After the addition, the mixture was allowed to stand at room temperature (20 30 ° C) for 30 minutes to react with the HCV recombinant antigen and the secondary antibody. After completion of the reaction, the liquid in each well was removed with an aspirator.

 Thereafter, the same washing operation as in 2. above was performed for each well (six times in total).

[0046] 4. Binding step (sensitization of oligonucleic acid chain-labeled antibody)

 (1) Preparation of oligonucleotide-labeled antibody

 First, an anti-HRP antibody labeled with a predetermined oligonucleotide is prepared by the following procedure.

; ^^

 [0047] (1-1) Preparation of oligonucleotide chain

 A 550-mer oligonucleotide was prepared by PCR using pcDNA3.1 (manufactured by In vitrogen) with Pinl inserted as a truncated DNA and using the following primers (5_MUSTagBio, 3-MUsag515). 5-MUSTagBio had biotin bound to the 3 ′ end and was used as the 5 ′ primer. 3_MUSTag515 was used as the 3 ′ primer.

 [0048] 5-MUSTagBio: SEQ ID NO: 1)

 3-MUSTag515:

 5'— AGCTTGACGGGGAAAGCCGG— 3 '(IJ number 2)

[0049] The PCR was performed under the following reaction solution composition and reaction conditions. <Reaction solution composition>

 Taq polymerase: 2.5unit

 5 'primer (20 M): 2 β L

 3 'primer (20 M): 2 β L

 dNTP (2.5mM each): 8 L

 10 X Buffer: lO ^ L

 Sterilized water: appropriate amount (approx. 77 L)

 Total: 100

 [0050] <Reaction conditions>

 A total of 35 cycles under the cycle condition of “thermal denaturation at 95 ° C for 60 seconds. Dissociation → annealing at 55 ° C for 60 seconds → synthesis at 72 ° C for 30 seconds' extension”.

[0051] The amplification product after each PCR was purified to a single oligonucleotide by subjecting the supernatant obtained by centrifugation after PCR to filter purification using a MinElute PCR Purification spin column (Qiagen).

[0052] (1-2) Preparation of biotinylated antibody

 Usagi-derived anti-HRP monoclonal antibodies (hereinafter, anti-HRP antibodies) were prepared by a conventional method for producing monoclonal antibodies.

Next, an anti-HRP antibody and Sulfo-NHS-LC_Biotin (Pierce Co.), the molar ratio of 1: ² becomes zero by Uni were mixed and reacted for 30 minutes at room temperature (20 to 30 ° C). The reaction solution was passed through a 5 mL desalting column, and the target antibody fraction was recovered to obtain a biotinylated anti-HRP antibody.

[0053] (1-3) Preparation of oligonucleotide-labeled antibody

 Biotinylated anti-HRP antibody and 550mer oligonucleotide were mixed in a 1: 1 molar ratio. Next, NeutrAvidin (Pierce) was added in an equimolar amount to the biotinylated anti-HRP antibody and allowed to react at room temperature (20-30 ° C.) for 15 minutes. The reaction solution was passed through a 5 mL desalting column, and the target fraction was collected to obtain a solution of anti-HRP antibody (oligonucleotide-labeled antibody) labeled with a 550-mer oligonucleotide chain.

[0054] (2) Sensitization of oligonucleotide-labeled antibody To 211 L of the oligonucleotide-labeled antibody solution (stock solution) obtained in (1) above, 1 mL of PBS was added and diluted about 500 times.

 50 L / well of the diluted oligonucleotide-labeled antibody solution was added to each well after washing in 3. above. After the addition, the mixture was allowed to stand at room temperature (20 to 30 ° C.) for 60 minutes, and the HRP labeled with the secondary antibody was reacted with the oligonucleotide-labeled antibody obtained in (1) above. After completion of the reaction, the liquid in each well was removed with an aspirator.

 Thereafter, PBS was added to each well at 400 L / well and allowed to stand for 20 seconds, and then the washing solution was removed with an aspirator. The same washing operation was repeated two more times (3 times in total).

[0055] 5. Amplification step (Amplification of oligonucleic acid strand)

 (l) EcoRI treatment

 An EcoRI enzyme solution was added to each of the tools after the washing in 4. to cleave the oligonucleotide chain in the oligonucleotide-labeled antibody.

 Specifically, an EcoRI enzyme solution was first prepared at the following volume ratio.

[0056] EcoRI (20unit / μL): 0.4

 10 X Buffer: 4

 Sterile water: 35.6

 Total: 40

[0057] To each well, 40 µL / well of the prepared EcoRI enzyme solution was added. The plate after addition was allowed to react at 30 ° C. for 30 minutes.

 After the reaction, the liquid in each well was collected and centrifuged to obtain a supernatant (including the oligonucleotide fragment after EcoRI treatment).

[0058] (2) Ryanoletime PCR

 The supernatant after centrifugation from each well is used as a vertical DNA solution, and real-time PCR is performed using the following primers (5- MUSTag-Forw3, 3-MUSTag-GEX) and TaqMan probe (# 7_cy5). went. 5-MUSTag_Forw3 was used as a 5 ′ primer and 3-MUST ag-GEX was used as a 3 ′ primer.

[0059] 5-MUSTag-Forw3:

5'- TGCATCTAGAGGGCCCTATTCTATA-3 '(SEQ ID NO: 3) 3-MUSTag-GEX:

 5'-GGCAAGCCACGTTTGGTG-3 '(SEQ ID NO: 4)

 # 7-cy5:

 5 '-[Cy5] -ACCGACAATTGCATGAAGAACTC- [BHQ] -3' (SEQ ID NO: 5)

[0060] The above real-time PCR was performed using MX-3005p real-time PCR apparatus (Stratagene) under the following reaction solution composition and reaction conditions.

 <Reaction solution composition>

 Vertical DNA (Supernatant after centrifugation): 3 L

 2 X Buffer: lO ^ L

 (TaqMan Universal PCR master mix; Applied Biosystems)

 5 'primer

 5- MUSTag-Forw3 (20 μM): 0Λ β L

 3 'primer

 3-MUSTag-GEX (20 μ Μ): 0Λ β L

 TaqMan probe

 # 7- cy5 (2.5 \): 1 ^ L

 Reference Dye (Rox II): 0.4 μL

 Sterilized water: appropriate amount (about 4.8 ^ L)

 Total: 20 L

 [0061] <Reaction conditions>

 First, heat denaturation at 95 ° C for 10 minutes, then "heat denaturation at 98 ° C for 15 seconds 'dissociation → annealing at 60 ° C for 60 seconds · synthesis' extension (so-called two-step method)" 40 cycles in total.

[0062] 6. Detection step (Detection of amplification product)

 By measuring the amount of fluorescence that fluctuates with the synthesis reaction of the DNA strand by real-time PCR every minute after the start of the reaction, it is possible to detect and amplify the amplified fragment for each wall of each dilution series of HCV recombinant antigen. The change in quantity was observed.

[0063] 7.Result As described above, according to the detection method of the present invention, as a result of attempting to detect the antigen using a dilution series of HCV recombinant antigen (72 pg / ml to 0.9 fg / ml) as a test sample, an antigen concentration of 0.02 pg / ml (20 fg / ml) was able to be detected (see Fig. 1 (a)). Therefore, in order to confirm the reliability of the detected value in this detectable range, an approximate curve was created based on the least square approximation method, and the R ² value was 0.9315, sufficiently high! (See Figure 1 (b)).

 [0064] On the other hand, as a comparative example, the same antigen dilution series and antibody (primary antibody and secondary antibody) as in the detection method of the present invention were used, and as a result of trying to detect the antigen by ELISA, the highest detection sensitivity was obtained. Was 2.67 pg / ml (see Figure 2).

 That is, the detection method of the present invention was able to achieve a sensitivity improvement of about 134 times compared to the conventional ELISA method (see FIG. 3).

 Industrial applicability

 [0065] According to the present invention, a novel method for detecting HCV is a simple and low-cost method that can easily detect even a trace amount of viruses and microorganisms that could not be detected in the past. You can power to provide. In particular, it is possible to provide a method capable of detecting a trace level of HCV at which a therapeutic effect by interleukin administration can be sufficiently exerted. In addition, a detection kit for carrying out the detection method can be provided.

 [0066] The detection method of the present invention cannot be found by a known immunoassay method such as the HCV core protein quantification method! /, HCV-infected persons, that is, "the amount of HCV infection is extremely small and sufficient treatment is achieved by administration of interferon. The ability to easily find “potentially infected people with HCV” (including complete cure). As a result, it is possible to provide appropriate and sufficient treatment for the above-mentioned infected people, which can contribute to improving the patient's QOL and prevent the spread of HCV infected people. I will be given strength. The present invention is extremely useful in the above points.

Claims

The scope of the claims  [1] a step of binding a first antibody that binds to an antigen to the antigen;  Binding a second antibody that binds to the first antibody labeled with an oligonucleic acid chain to the first antibody;  Amplifying the oligonucleic acid strand; and  Process for detecting amplification products  A method for detecting an antigen, comprising: [2] The detection method according to [1], wherein the antigen is immobilized on a support.  [3] The step of binding the virus and / or microorganism in the test sample to the immobilized primary antibody,  Binding a secondary antibody to the virus and / or microorganism bound to the primary antibody;  A tertiary antibody that binds to the secondary antibody labeled with an oligonucleic acid chain is bound to the wine and / or microorganism.  Binding to the prepared secondary antibody,  Amplifying the oligonucleic acid strand; and  Process for detecting amplification products  A method for detecting viruses and / or microorganisms. [4] Furthermore, the virus and / or microorganism in the test sample is determined using the detection result of the amplification product as an index.  4. The method of claim 3, comprising the step of measuring. [5] The method according to any one of claims 1 to 4, wherein the amplification method is a real-time PCR method. [6] The method according to any one of claims 3 to 5, wherein the virus is hepatitis C virus. [7] A support on which a primary antibody against a virus and / or microorganism is immobilized,  A secondary antibody against the virus and / or microorganism, A tertiary antibody labeled with an oligonucleic acid chain and bound to the secondary antibody, and a primer used for amplification of the oligonucleic acid chain A kit for detecting viruses and / or microorganisms.  The kit according to claim 7, wherein the virus is hepatitis C virus.