KR102293984B1 - 3-dimensional immunodiagnostics based on detection signal control - Google Patents

Abstract

The present invention realizes self-amplification of detection signals by simultaneously linking immunodiagnosis using a protein nanoparticle-based three-dimensional detection probe and gold particle formation by gold ion reduction in liquid conditions, and at the same time, controls the viscosity control factor of liquid phase. A kit for detecting disease-specific markers, which detects disease-specific markers with an accurate immunodiagnostic method that can reduce the generation of false-positive signals by utilizing metal ions that control the detection signal generation time and do not cause interference with the detection signal The present invention relates to a method for detecting a disease-specific marker used, and more specifically, by controlling the content of a viscosity control factor in a diagnostic solution, it is possible to derive consistent diagnostic results without being affected by differences in the viscosity of samples according to individual differences, as well as In addition to gold ions, metal ions that do not generate optical signals when reduced to metal particles are additionally utilized to solve the problem of false positive noise while dramatically reducing the amount of detection probes (protein nanoparticle-based three-dimensional antibody probes) It relates to a kit for detecting a disease-specific marker characterized by high accuracy and economic feasibility, which can significantly reduce manufacturing costs, and a method for detecting a disease-specific marker using the same.

Description

Accurate three-dimensional immunodiagnostics based on detection signal control

The present invention realizes self-amplification of detection signals by simultaneously linking immunodiagnosis using a protein nanoparticle-based three-dimensional detection probe and gold particle formation by gold ion reduction in liquid conditions, and at the same time, controls the viscosity control factor of liquid phase. A kit for detecting disease-specific markers, which detects disease-specific markers with an accurate immunodiagnostic method that can reduce the generation of false-positive signals by utilizing metal ions that control the detection signal generation time and do not cause interference with the detection signal The present invention relates to a method for detecting a disease-specific marker used, and more specifically, by controlling the content of a viscosity control factor in a diagnostic solution, it is possible to derive consistent diagnostic results without being affected by differences in the viscosity of samples according to individual differences, as well as In addition to gold ions, metal ions that do not generate optical signals when reduced to metal particles are additionally utilized to solve the problem of false positive noise while dramatically reducing the amount of detection probes (protein nanoparticle-based three-dimensional antibody probes) It relates to a kit for detecting a disease-specific marker characterized by high accuracy and economic feasibility, which can significantly reduce manufacturing costs, and a method for detecting a disease-specific marker using the same.

In modern disease diagnosis methods, the most ideal method is a diagnosis method that is fast, accurate, and has a simple procedure. And this rapid diagnosis is more important in the case of patients in emergency situations such as acute myocardial infarction. In case of emergency patients, appropriate treatment according to such a fast and accurate diagnosis is directly related to survival rate, so it is more important than non-emergency patients.

In the field of clinical examination, diagnosis of various diseases is performed using biological samples (blood, urine, etc.). As such a diagnosis method, various measurement methods have been developed and used. As a representative method of such a measurement method, a biochemical measurement method using an enzyme reaction or an immunoassay method using an antigen-antibody reaction may be mentioned. In recent years, it is required to precisely measure components in biological samples, and immunoassay methods using antigen-antibody reactions with high specificity have been widely used.

The immunoassay method is a method that utilizes the antigen-antibody binding ability. Primarily, immunoassays are used to assay for the presence of a specific antigen (or antibody)-specific antibody (or antigen) in a sample. This is done by washing the sample over a specific antigen (or antibody) immobilized on a solid support, followed by visualization of the bound antibody (or antigen) using a variety of techniques.

In general, immunoassay methods require the use of a calibrator to assign a concentration to an unknown sample. In a classical immunoassay, a set of calibrators is run, a calibration curve of signal versus concentration is plotted and the concentration of an unknown sample is determined by interpolation (Ibrahim A. Darwish, International Journal of Biomedical Science, Vol. 2 pp. 217-235, 2006).

Immunoassays include radioimmunoassay (RIA) that detects signals using radioactive isotopes according to the detection principle and method, enzyme-linked immunosorbent assay (ELISA) that uses signal amplification by enzymes; Alternatively, it can be divided into enzyme immunoassay (EIA), fluorescence antibody technique (FA: fluorescence antibody technique) that detects using fluorescence, and chemiluminescence immunoassay (CLIA: chemiluminescence immunoassay) using chemiluminescence. Various classifications are possible depending on the method of use or the type of substrate.

This ELIA method has the advantages of high sensitivity, fast analysis speed, and easy automation, but it has to go through several steps, and the production cost for a labeled antibody or antigen is expensive. In the case of ruthenium pyridine, which is mainly used, It has the disadvantages of being greatly affected by the present invention, having limited luminous efficiency, and being expensive.

In order to overcome this disadvantage, the present inventors disclosed in Korean Patent Application Laid-Open No. 10-2018-0115480, when gold particle formation using gold ion reduction reaction and antigen-antibody immune response are simultaneously induced, gold particle aggregates A method has been developed that can rapidly detect the presence or absence of disease-specific markers by self-amplification of the detection signal according to the formation. This method can diagnose the disease in a single step without the process of immobilization and washing normally involved in diagnosis such as the conventional ELISA, and the diagnostic test time that takes several hours or several days is about 10 minutes. In particular, it can be effectively used for diagnosis of diseases such as patients in emergency situations, and has the advantage of being able to visually check the test results without a separate analysis device.

The present inventors, based on the diagnostic platform, utilize a material capable of adjusting the viscosity of a sample to be diagnosed, thereby not only being able to derive consistent diagnostic results without being affected by differences in serum viscosity according to individual differences, but also A novel disease-specific marker detection kit with improved sensitivity and economic feasibility, which can solve the signal noise problem and significantly reduce manufacturing costs by dramatically reducing the amount of protein nanoparticles and antibodies used compared to the phosphorus liquid immunodiagnostic method, and By developing a method for detecting a disease-specific marker using the same, the present invention was completed.

It is an object of the present invention to provide a composition and/or kit for detecting a disease-specific marker that has high accuracy and economic efficiency by controlling the self-amplification of a detection signal in a rapid and convenient single-step liquid immunodiagnostic method.

Another object of the present invention is to provide a method for detecting a disease-specific marker using the aforementioned kit.

In order to solve the above problems, the present invention provides a composition for detecting a disease marker comprising:

a pre-assay solution in which an antibody for detecting disease-specific markers or protein nanoparticles with antigen and tags for adsorption of gold ions, free gold ions and adsorbed gold ions are present;

reducing agent;

metal ions showing no color change before and after reduction; and

A substance capable of adjusting the viscosity of a diagnostic solution.

According to a preferred embodiment of the present invention, the disease-specific marker may be a protein marker including troponin I for acute myocardial infarction.

According to another preferred embodiment of the present invention, the protein particles include ferritin, ferritin-like protein, magnetosome component protein, virus component protein, DNA binding protein (DPS), and It may be at least one selected from the group consisting of proteasome.

According to another preferred embodiment of the present invention, the virus component protein may be a component protein of HBV core protein (hepatitis B virus core protein) or tobacco mosaic virus (tobacco mosaic virus).

According to another preferred embodiment of the present invention, the gold ion adsorption tag may be one or more amino acids selected from the group consisting of histidine, lysine and arginine.

According to another preferred embodiment of the present invention, the reducing agent may be at least one selected from the group consisting of ascorbic acid, imidazole, pyrazole, histamine, hydroxylamine, citric acid and sodium borohydride. .

According to another preferred embodiment of the present invention, the metal ion showing no color change before and after the reduction is selected from the group consisting ^{of Ni 2+} , Zn ²⁺ , Fe ³⁺ , Mg ²⁺ , Ca ²⁺ and Na ⁺ There may be more than one type.

According to another preferred embodiment of the present invention, the substance capable of adjusting the viscosity of the diagnostic solution may include bovine serum albumin (BSA).

The present invention also provides a kit for diagnosing a disease comprising the above-described composition for detecting a disease marker.

According to a preferred embodiment of the present invention, the disease may be selected from the group consisting of acute myocardial infarction, human immunodeficiency syndrome, hepatitis C, Sjogren's syndrome, multiple sclerosis syndrome, hepatitis A, stroke, cerebral hemorrhage and cancer. .

The present invention also provides a method for detecting a disease-specific marker using self-amplification of a detection signal, comprising the steps of:

(a) preparing a pre-assay solution in which an antibody for detecting a disease-specific marker or an antigen and a tag for adsorption of an antigen and a tag for adsorption of gold ions are expressed, and a pre-assay solution in which free gold ions and adsorbed gold ions are present at the same time;

(b) generating a sample mixture by adding a material capable of controlling viscosity and a metal ion showing no color change before and after reduction to a sample to be diagnosed;

(c) gold by antigen-antibody immune reaction and reduction of gold ions in an assay solution prepared by adding the sample mixture and reducing agent of step (b) to the pre-diagnosis solution of step (a) simultaneously inducing a particle formation reaction; and

(d) confirming the presence or absence of a disease-specific marker through a color reaction caused by the formation of gold particles.

According to a preferred embodiment of the present invention, the protein particles are ferritin, ferritin-like protein, magnetosome component protein, virus component protein (eg, HBV core protein or tobacco). It may be one or more proteins selected from the group consisting of mosaic virus constituent proteins), DNA binding protein (DPS), and proteasome.

According to another preferred embodiment of the present invention, the gold ion adsorption tag in step (a) may be one or more amino acids selected from the group consisting of histidine, lysine and arginine.

According to another preferred embodiment of the present invention, the step (b) may be to mix the material capable of controlling the viscosity and the metal ion showing no color change before and after reduction until combined.

According to another preferred embodiment of the present invention, the reducing agent of step (b) is 1 selected from the group consisting of ascorbic acid, imidazole, pyrazole, histamine, hydroxylamine, citric acid and sodium borohydride. may be more than one species.

According to another preferred embodiment of the present invention, the metal ions showing no color change before and after the reduction in step (b) are Ni ²⁺ , Zn ²⁺ , Fe ³⁺ , Mg ²⁺ , Ca ²⁺ and Na ^It may be at least one selected from the group consisting of +.

According to another preferred embodiment of the present invention, the substance capable of adjusting the viscosity of step (b) may include bovine serum albumin (BSA).

According to another preferred embodiment of the present invention, the sample of step (b) may be at least one selected from the group consisting of blood, plasma, serum, urine, saliva, oral mucosa and saliva.

According to another preferred embodiment of the present invention, when the sample is serum, the amount of serum may be 1.2% (v/v) to 12.5% (v/v) of the total diagnostic solution.

According to another preferred embodiment of the present invention, the amount of the protein particles in step (c) may be 1.5 nM to 25 nM.

According to another preferred embodiment of the present invention, the concentration of the substance capable of controlling the viscosity in the mixture of step (c) may be more than 135 μM and less than 293 μM.

According to another preferred embodiment of the present invention, the concentration of the metal ion showing no color change before and after the reduction in the mixture of step (c) may be 33 mM to 66 mM in the case ^{of Ni 2+ .}

According to another preferred embodiment of the present invention, the mixture of step (b), including a form in which a metal ion showing no color change before and after reduction is bound to a substance capable of controlling the viscosity, is 5% ( v/v) to 25% (v/v).

According to another preferred embodiment of the present invention, the color reaction in step (d) can be confirmed by measuring the absorbance at 500 nm to 600 nm.

According to another preferred embodiment of the present invention, the color reaction in step (d) may appear within 10 to 40 minutes.

According to another preferred embodiment of the present invention, the disease may be selected from the group consisting of acute myocardial infarction, human immunodeficiency, hepatitis C, Sjogren's syndrome, multiple sclerosis syndrome, hepatitis A, stroke, cerebral hemorrhage and cancer. have.

The kit for detecting a disease-specific marker according to the present invention can derive consistent diagnostic results without being affected by differences in sample viscosity according to individual differences by controlling the content of the viscosity control factor in the diagnostic solution, and, in addition to gold ions, By additionally utilizing metal ions that do not generate optical signals when reduced to metal particles, the problem of false positive noise is solved, while the use of detection probes (protein nanoparticle-based three-dimensional antibody probes) is dramatically reduced to reduce manufacturing costs. It can significantly reduce the accuracy and economic efficiency.

1 is a schematic diagram of protein particles for diagnosing acute myocardial infarction by the method of the present invention and results of confirming the produced protein particles with an electron microscope.
2 is a schematic diagram of the principle of the method for detecting disease-specific markers of the present invention, illustrating a process in which the formation of gold particles due to the reduction reaction and the antigen-antibody reaction occur simultaneously to form clusters, thereby generating rapid self-signal amplification. will be.
3 is a single-step immunoassay using BSA, a substance that can control the viscosity of a diagnostic sample, at a concentration of 181 μM. The results of detecting nin I (troponin I) are shown as an absorbance graph.
4 shows Acute myocardial infarction from serum of 15 healthy subjects (H) and patients with acute myocardial infarction (P), respectively, in a single-step immunoassay using BSA, a substance that can control the viscosity of a diagnostic sample, at a concentration of 181 μM. The result of detecting the marker troponin I (troponin I) is shown as an absorbance graph, (A) ^{shows the result when Ni 2+} ions were used, (B) shows the results when Ni ²⁺ ions were not used. show the results when
Figure 5 shows tropho, an acute myocardial infarction marker, from serum of healthy normal people (H) and acute myocardial infarction patients (P) in a single-step immunoassay using BSA, a substance that can control the viscosity of a diagnostic sample, at a concentration of 248 μM. The results of detecting nin I (troponin I) are shown as an absorbance graph.
6 shows Acute myocardial infarction from serum of 15 healthy subjects (H) and patients with acute myocardial infarction (P), respectively, in a single-step immunoassay using BSA, a substance that can control the viscosity of a diagnostic sample, at a concentration of 248 μM. The result of detecting the marker troponin I (troponin I) is shown as an absorbance graph, (A) ^{shows the result when Ni 2+} ions were used, (B) shows the results when Ni ²⁺ ions were not used. show the results when
Figure 7 shows tropho, an acute myocardial infarction marker, from serum of healthy normal people (H) and acute myocardial infarction patients (P) in a single-step immunoassay using BSA, a substance that can control the viscosity of a diagnostic sample, at a concentration of 293 μM. The results of detecting nin I (troponin I) are shown as an absorbance graph.
8 is a single-step immunoassay using BSA, a substance that can control the viscosity of a diagnostic sample, at a concentration of 293 μM, and acute myocardial infarction from the serum of 15 healthy subjects (H) and acute myocardial infarction patients (P), respectively. The result of detecting the marker troponin I (troponin I) is shown as an absorbance graph, (A) ^{shows the result when Ni 2+} ions were used, (B) shows the results when Ni ²⁺ ions were not used. show the results when
9 is a photograph showing the color change before and after reduction of various metal ions (Ni ²⁺ , Zn ²⁺ , Cu ²⁺ , Fe ²⁺ and Fe ^{3+ ).}
10 is a single-step immunoassay using Tween 20 at various concentrations (0.5%, 1%, 5%, 10% and 20% (w/v), respectively) as a substance capable of controlling the viscosity of a diagnostic sample. The result of detecting troponin I, an acute myocardial infarction marker, from the serum of healthy normal people (H) and patients with acute myocardial infarction (P) was confirmed by changing the color of the diagnostic solution.
11 is a single-step immunoassay using Tween 20 at various concentrations (0.5%, 1%, 5%, 10% and 20% (w/v), respectively) as a substance capable of controlling the viscosity of a diagnostic sample. The results of the detection of troponin I, an acute myocardial infarction marker, from the serum of healthy normal people (H) and patients with acute myocardial infarction (P) were confirmed by an absorbance graph.
12 is a graph quantitatively confirming the sensitivity of the diagnostic platform to the detection amount of the disease marker (troponin I) according to the BSA concentration through the absorbance graph.
13 is an absorbance graph confirming the effect of reducing signal generation noise in a diagnostic platform for a normal person according to whether ^{Ni 2+ ions are used or not.}

As described above, the present inventors utilize a material that can control the viscosity of a sample to be diagnosed, so that consistent diagnostic results can be derived without being affected by differences in sample viscosity according to individual differences, and metals other than gold ions By additionally utilizing metal ions that do not generate optical signals when reduced to particles, the problem of false positive noise is solved, while the use of detection probes (protein nanoparticle-based three-dimensional antibody probes) is dramatically reduced, greatly reducing manufacturing costs. By developing a disease-specific marker detection kit with improved accuracy and economic feasibility, and a method for detecting disease-specific markers using the same, a novel immunodiagnostic technology platform based on viscosity modulating factors was completed.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for detecting a disease marker comprising:

a pre-assay solution in which an antibody for detecting a disease-specific marker or a protein particle labeled with an antigen and tag for adsorption of gold ions, free gold ions and adsorbed gold ions exist;

reducing agent;

metal ions showing no color change before and after reduction; and

A substance capable of adjusting the viscosity of a diagnostic solution.

In the composition of the present invention, the disease-specific marker is a specific marker capable of confirming whether a specific disease has occurred, and upon contact with a sample to be diagnosed, the presence or absence of a specific disease-specific marker in the sample can be confirmed. The present antibody or antigen is expressed on the surface of the protein particle, so that the presence of the antibody or antigen or antibody that specifically reacts with the antibody or antigen in the sample can be checked.

In the composition of the present invention, when the disease is acute myocardial infarction, an antibody that specifically binds to troponin I (troponin I) may be expressed on the surface of a protein particle and used.

In the composition of the present invention, when the disease is a disease related to viral infection, all kinds of amino acids translated by viral genes, such as viral proteins, viral peptides, and epitopes, that specifically bind to an antibody capable of detecting a virus It can be used by expressing it on the surface of a protein particle, or by using the virus-derived protein as an antigen and expressing an antibody capable of binding thereto.

In the present invention, the term "epitope" refers to a specific site that binds to an antigenic molecular antibody or T cell receptor (TCR) and the major histocompatibility complex (MHC), and is used in the same sense as an antigenic determinant.

For example, hepatitis C virus (HCV) is a virus belonging to the Flaviviridae family that causes non-A and non-B hepatitis, and the HCV genome is a single-stranded RNA with about 3,010 amino acids. It expresses a polyprotein consisting of (Choo et al., Science, 244:359-362, 1989). The single complex protein expressed by HCV is cleaved back into 10 different proteins by the host cell and viral proteases.

The genetic makeup of HCV is NH ₂ -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Steven Rosenberg, J. Mol. Biol., 313:451-464, 2001). The protein can be largely divided into structural proteins comprising C (Core), E1, E2, and p7 and non-structural proteins comprising NS2, NS3, NS4A, NS4B, NS5A, and NS5B.

The C (core) protein of HCV is believed to play a role in the development of liver cancer by regulating gene transcription, growth and proliferation of host cells, as well as playing a structural role in encapsidation of HCV genomic RNA, E1 and E2 proteins is a type 1 transmembrane protein and is known to play an important role in cell infection as a viral envelope protein. In the case of E1 protein, it has not received much attention due to its inability to induce neutralizing antibodies. Phase clinical trials are in progress, and in particular, it is evaluated as very encouraging as it shows good effects on type 1b HCV infection, which is not particularly effective due to the use of alpha interferon.

In addition to its structural role, the E2 protein is a major viral envelope protein and binds to a putative cell receptor, CD81, in the host immune system and interferon-mediated anti-viral response and escapes from oncogenesis or autoimmune liver disease ( It is known as a multifunctional protein causing autoimmnune liver disease), so it is recognized as a main antigen for the development of an effective HCV vaccine as well as a main target for the development of anti-HCV drugs. The function of the P7 protein is unknown, NS2 is part of the metallo-protease and NS3 has the viral serine protease domain at the N-terminus and the RNA helicase domain at the C-terminus. have on

NS4A is a cofactor of viral protease, and NS4B has been reported to have potential oncogenic potential. NS5A has been reported to have a function that makes HCV resistant to interferon and has an antiapoptotic function, and NS5B is known to function as an RNA-dependent RNA polymerase of a virus. It will be apparent to those skilled in the art that it can be used as a virus-specific epitope of

Specifically, when the virus infection-related disease is hepatitis C, the disease-specific antibody may be an antibody that specifically binds to HCV, and when the antigen is expressed on the surface of a protein particle, HCV c22p, c33c, 5-1-1p, c100p, and those selected from the group consisting of epitopes fused to two or more thereof may be used, and in the form of two or more fused epitopes, c22p-c33c, c22p-5-1-1p, c22p-c100p, One selected from the group consisting of c33c-5-5-1p, c33c-c100p, 5-1-1p-c100p and c22p-5-1-1p-c100p may be used, but the present invention is not limited thereto.

The term "virus" used in the present invention refers to an obligatory intracellular parasite that has DNA or RNA as a nucleic acid, proliferation begins with nucleic acid, does not proliferate by mitosis, and does not have an enzyme system necessary for ATP production. it means.

The virus of the present invention includes a naked virus and an enveloped virus, and specifically, the virus according to an embodiment of the present invention may be an enveloped virus.

In the present invention, the envelope virus is specifically a DNA virus including a herpesvirus, a poxvirus, and a hepadnavirus, a flavivirus, a togavirus, and a corona virus. Viruses (coronavirus), hepatitis C (hepatitis C), orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, human immunodeficiency As long as it belongs to RNA viruses including viruses (HIV) and retroviruses (retrovirus), they may be included.

The orthomyxovirus includes all viruses included in the genera of influenza virus A, influenza virus B, influenza virus C, isavirus, thogotovirus and quaranjavirus.

The coronavirus includes all viruses included in the genus alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus.

The paramyxovirus includes all viruses included in the genus Paramyxovirus, rubulavirus, mobilivirus and pneumovirus.

The human immunodeficiency virus (HIV) genome consists of two positive single-stranded RNAs that encode nine viral genes. RNA is surrounded by a conical capsid composed of about 2,000 viral proteins p24. The inside of the virus contains single-stranded RNA, nuclear capsid protein p7, and enzymes essential for the formation of virion protein (reverse transcriptase, protease, ribonuclease, and integrase).

A substrate composed of the viral protein p17 again surrounds the capsid for virion integrity. The substrate is again surrounded by a viral membrane composed of a phospholipid bilayer. The virus membrane is first formed when a new virus emerges from the host cell. The virus membrane being created is embedded in the cell membrane of the host cell and is composed of the host cell protein and 70 complex HIV proteins that protrude from the surface of the virus particle.

The complex HIV protein known as Env consists of a cap composed of 3 molecules of glycoprotein 120 and a stem composed of 3 molecules of gp41 (which serves to anchor the protein structure to the viral membrane). Glycoprotein conjugates are important in initiating the infection cycle, allowing the virus to attach to and fuse to target cells. Two surface glycoproteins of viruses, especially gp120, are known as targets for future therapeutics or vaccines.

Two TAT proteins (p14 and p16) are activators for the LTR promoter, which act by binding to TAR RNA. TAR can also be processed by microRNAs that regulate apoptosis genes (ERCC1 and IER3). Rev protein (p19) is involved in binding RNA to PRE RNA molecules and transporting them from the nucleus to the cytoplasm. Vif protein (p23) inhibits the function of APOBEC3G (a cellular protein that removes amines from DNA:RNA hybrids or interferes with Pol protein), and Vpr protein (p14) binds host cells at the G2/M stage and prevents cell division. Nef protein (p27) inhibits MHC class I and MHC class II molecules as well as CD4 (major viral receptor) of T cells.

Nef also interacts with the SH3 domain. The Vpu protein (p16) affects the release of new viral particles from infected cells. The GAG gene is a gene that expresses the matrix proteins (MA, GAG p17), capsid proteins (CA, GAG p24), and nucleic acid capsids (NC, GAG p7), which are central to making virus particles, and the Pol gene is an enzyme related to virus proliferation. It contains genes, and is composed of the RT part involved in the expression of the reverse transcriptase of viral RNA, the PR part responsible for the proteolytic enzyme, and the IN part related to the integrase.

In addition, the TAT gene is known to express a regulator that activates the proliferation of viruses during the infection process, and the Nef gene is known to play a role in inhibiting the proliferation of viruses together with the LTR region on the contrary. LTR (Long Term Repeat), which repeats the same nucleotide sequence, is attached to both ends of the total HIV RNA. The LTR region can act as a switch that regulates the production of new viruses, or it can be triggered by HIV or host cell proteins. The retroviral Psi element is involved in packaging the viral genome and being recognized by the GAG and Rev proteins. It is known to those skilled in the art that the SLIP element (TTTTTT) is related to the conformational shift in the GAG-Pol translation structure necessary for making functional Pol, and it will be apparent to those skilled in the art that this epitope of HIV can be used for the detection of the virus of the present invention.

Specifically, when the viral infection-related disease is acquired immunodeficiency syndrome, the disease-specific antibody may be an antibody that specifically binds to HIV, and when the antigen is expressed on the surface of a protein particle, HIV gp41, p24 And gp100 may be used, but is not limited thereto.

Human papillomavirus (HPV) DNA contains 8000 base pairs and is surrounded by a pentameric capsid protein rather than a lipid membrane. The capsid protein consists of two structural proteins, L1 and L2, which are expressed later in the viral replication cycle. There are eight ORFs in the genome of all human papillomavirus (HPV), and each ORF is divided into three functional regions. It is composed of E1-E7, a gene necessary for virus replication, L1-L2, a gene expressing structural proteins constituting virion, and LCR, which regulates virus replication and transcription.

E6 binds to p53 and promotes ubiquitination of p53, thereby inhibiting the function of p53 as a tumor suppressor gene. It also induces degradation of BAK, a pro-apoptotic protein. Through activation of telomerase, the cell cycle of the host cell is activated, and E7 interacts with RB (retinoblastoma) to degrade RB. Through this, E2F, a transcriptional promoter that was inhibited by RB, is released. In addition, it activates the cell cycle of the host cell by activating cyclin E and cyclin A, which act in the S phase of the cell cycle. can be caused by However, the role of other genes such as E1, E2, and E4 in HPV in the development of cancer is still unknown.

L1 assembles themselves to form pentameric capsomers. These capsomers form capsids through disulfide bonds with adjacent L1 molecules to package human papillomavirus DNA, and L2 is present in a smaller amount than L1, and facilitates the packaging of the viral genome. In addition, it is known that human papillomavirus plays an important function when infiltrating new host cells.

Specifically, when the disease is cervical cancer, the disease-specific antibody may be an antibody that specifically binds to HPV, and when the antigen is expressed on the surface of a protein particle, L1 or L2 may be used, but limited thereto it's not going to be

Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a type of coronavirus, was developed from a sample collected from the lungs of a 60-year-old man with acute pneumonia and acute renal failure in Jeddah, Saudi Arabia. As a virus, it is known to cause Middle East Respiratory Syndrome.

Therefore, in the present invention, the antigen for detecting the antiviral (MERS-CoV) antibody present in the pre-assay solution may be a MERS-CoV surface protein, but is not limited thereto.

Hepatitis A Virus (HAV) is classified into the genus Picornaviridae and Hepatovirus, has no capsule and is icosahedral, and has a particle size of 27-32 nm. The virion has three major structural polypeptides (VP1, VP2 and VP3) ) is composed of

Specifically, when the disease is hepatitis A, the disease-specific antibody may be an antibody that specifically binds to HAV, and when an antigen is expressed on the surface of a protein particle, VP1, VP2, VP3 or a portion thereof One or more epitopes selected from the group consisting of epitopes prepared by extraction or linking may be used, but the present invention is not limited thereto.

In the present invention, the disease may be cancer, and the cancer is lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, thyroid cancer, esophageal cancer, uterine cancer, liver cancer, kidney cancer , may be selected from the group consisting of biliary tract cancer, glioblastoma and testicular cancer, but is not limited thereto.

In the present invention, when the disease is cancer, the disease-specific antibody may be an antibody that specifically binds to a tumor marker, but is not limited thereto.

In the present invention, the tumor markers may be as shown in Table 1 below for each cancer type, but is not limited thereto.

Tumor markers by cancer type carcinoma tumor markers brain tumor SCC lung cancer SLX, NSE, SCO, ADH, ACTH liver cancer AFP, PIVKA-II, ALP stomach, duodenal cancer CA19-9, CEA, AFP prostate cancer PSA, PA testicular cancer AFP, HCO, NSE thyroid cancer CEA, Calcitonin, Thyroglobulin breast cancer CA 15-3, CEA, CA549, TPA pancreatic cancer CA19-9, Elatase I, CA50, Du-Pan-2, SPAN-1, KMO1, POA, PST colorectal cancer CEA, NCC-ST-439 uterine cancer SCC, CEA, hCG ovarian cancer CA125, CA72-4 blood cancer β-2-Microglobulin

In the present invention, when the disease is lymphoma or leukemia, the disease-specific antibody may be an antibody that specifically binds to CD20, CD30, CD33 or CD52, but is not limited thereto.

In the present invention, when the disease is breast cancer, colon cancer, lung cancer, or ovarian cancer, the disease-specific antibody is EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, FAP, tenasin, It may be an antibody that specifically binds to EpCAM, CEA, gpA33, Mucin, TAG-72, CAIX, PSMA or Folate-binding protein, but is not limited thereto.

In the present invention, the disease may be an infectious disease, and the infectious disease is influenza (influenza), smallpox, polio, foot-and-mouth disease, Ebola, measles, yellow fever, dengue fever, SARS, pneumonia, tuberculosis, cholera, typhoid, dysentery, diphtheria. And it may be selected from the group consisting of Lyme disease, but is not limited thereto.

In the present invention, when the disease is influenza, the disease-specific antibody may be an antibody that specifically binds to an influenza virus, but is not limited thereto.

Influenza virus is a single-stranded RNA virus belonging to the Orthomyxoviridae family. It has two glycoprotein surface antigens, hemagglutinin and neuraminidase, and depending on antigenicity, influenza A, B, classified as type C. Types A and B mainly cause epidemics. Type A is classified into subtypes according to the characteristics of HA (H1-H15) and NA (N1-N9), and type B has no subtypes. Therefore, when diagnosing influenza by the method of the present invention, the antibody used in the present invention may be an antibody specific to each influenza virus subtype, but is not limited thereto.

In the present invention, when the disease is polio, the disease-specific antibody may be an antibody that specifically binds to a poliovirus, but is not limited thereto.

In the present invention, when the disease is Ebola, the disease-specific antibody may be an antibody that specifically binds to the Ebola virus, but is not limited thereto. When the disease is measles, the disease-specific antibody is It may be an antibody that specifically binds to a paramyxovirus, but is not limited thereto, and when the disease is yellow fever, the disease-specific antibody is a yellow fever virus belonging to the flavivirus family. ), but is not limited thereto, and when the disease is dengue fever, the disease-specific antibody may be an antibody that specifically binds to dengue virus, but is not limited thereto. it is not

In the present invention, the disease may be an autoimmune disease, and the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's disease, lupus, scleroderma, psoriasis and vitiligo. However, the present invention is not limited thereto.

In the present invention, when the disease is Sjogren's syndrome, the disease-specific antibody may be an anti-Ro (Sjogren's syndrome A, SSA) antibody or an anti-La (Sjogren's syndrome B, SSB) antibody, but is not limited thereto no.

In the present invention, when the disease is multiple sclerosis, the disease-specific antibody may be an anti-MOG antibody, an anti-myelin antibody, or an anti-KIR4.1 antibody, but is not limited thereto. .

In the present invention, when the disease is stroke, the disease-specific antibodies are anti-NR2A/2B and metalloproteinases (MMPs) antibodies, antinuclear antibodies (ANA), antiphospholipid antibodies (APL), anti-D -It may be a dimer antibody, an anti-S100β antibody, an anti-B-type natriuretic peptide (BNP) antibody, or an anti-cardiolipin antibody (ACL), but is not limited thereto.

In the present invention, when the disease is cerebral hemorrhage, the disease-specific antibody may be an anti-glial fiber nitrate protein (GFAP) antibody, an anti-symmetric dimethylarginine (ADMA) antibody, or an anti-D-dimer antibody, but is not limited thereto. it is not

In the composition of the present invention, the protein particles are ferritin, ferritin-like protein, magnetosome component protein, virus component protein, DPS (DNA binding protein), and proteasome ( proteasome), but may be at least one selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the virus constituent protein may be a constituent protein selected from the group consisting of capsid proteins of human hepatitis B virus or a constituent protein of tobacco mosaic virus.

Preferably, the capsid protein of the human hepatitis B virus may be an HBV core protein.

In the composition of the present invention, the size of the protein particles may be 10nm-50nm, but is not limited thereto.

In the composition of the present invention, in the antibody for detecting a disease-specific marker expressed on the surface of the protein particle, a domain capable of binding to the Fc domain of the antibody is expressed on the surface of the protein particle, so that the domain and the disease-specific marker A detection antibody may bind.

In the composition of the present invention, the domain capable of binding to the Fc domain of the antibody may be selected from the group consisting of domain B and protein G of staphylococcal protein A (SPA), but is not limited thereto.

According to an embodiment of the present invention, the protein particle is a hepatitis B virus core protein 1 to 79 amino acid sequence portion, a plurality of SPA domain B (SPA _B ) portion and the 81 to 149 amino acid sequence portion of the core protein wherein the SPA _B domain portion may be located between the 1 to 78 amino acid sequence portion and the 81 to 149 amino acid sequence portion of the hepatitis B virus core protein.

In the composition of the present invention, the domain capable of binding to the Fc domain of the antibody is expressed on the surface of the protein particle, and such expression may be more effectively expressed due to the linker protein.

In the composition of the present invention, the linker protein may be any amino acid capable of effectively expressing the domain capable of binding to the Fc domain of the antibody on the surface of the protein particle, preferably G4SG4T or G4SG4.

In the composition of the present invention, the gold ion adsorption tag may be used without limitation as long as it is a material capable of adsorbing gold ions, and preferably may be an amino acid selected from the group consisting of histidine, lysine and arginine, but limited thereto it's not going to be

In the composition of the present invention, the reducing agent may be any material capable of reducing gold ions to gold particles under the reaction conditions of the present invention, preferably ascorbic acid, imidazole, pyrazole, histamine, and hydride. and may be selected from the group consisting of hydroxylamine, citric acid and sodium borohydride.

In the composition of the present invention, the metal ion showing no color change before and after the reduction is at least one selected from the group consisting ^{of Ni 2+} , Zn ²⁺ , Fe ³⁺ , Mg ²⁺ , Ca ²⁺ and Na ⁺ can, but is not limited thereto.

In the composition of the present invention, the substance capable of adjusting the viscosity of the diagnostic solution may include bovine serum albumin (BSA).

According to a preferred embodiment of the present invention, the metal ion that does not show a color change before and after the reduction is Ni ²⁺ , and ^{the use of Ni 2+} is interference caused by the combination of ^{BSA with Au 3+} added for viscosity control. to prevent this from happening. This effect is achieved ^{by pre-binding Ni 2+} with BSA and administering it to the pre-diagnosis solution. Therefore, the metal ion binding to BSA does not affect the color reaction that occurs following the formation of gold particles by the reduction reaction of the gold ion. In order to avoid this, the color change should not be induced by the reduction reaction after binding to BSA.

The present invention also provides a kit for diagnosing a disease comprising the above-described composition for detecting a disease-specific marker.

In the kit of the present invention, the disease may be selected from the group consisting of acute myocardial infarction, human immunodeficiency syndrome, hepatitis C, Sjogren's syndrome, multiple sclerosis syndrome, hepatitis A, stroke, cerebral hemorrhage, and cancer.

In the kit of the present invention, an antibody for detecting a disease-specific marker or a protein particle labeled with an antigen and a tag for adsorption of gold ions, a pre-assay solution in which free gold ions and adsorbed gold ions are present, a reducing agent, It is preferable that metal ions that do not change color before and after reduction and substances capable of adjusting the viscosity of the diagnostic solution are separately contained in different containers.

In the kit of the present invention, each component included in the kit is the same as the above-described composition for detecting a disease-specific marker, and thus description thereof will be omitted.

The kit of the present invention may further include an external package, and the external package may include instructions for use of the components.

In one embodiment of the present invention, a protein particle in which a tag for adsorption of gold ions and a tag for binding to an antibody Fc domain are expressed on the surface of a core protein derived from recombinant B-type mesenchymal virus capsid (HBV-capsid) is prepared, After attaching an antibody capable of detecting an acute myocardial infarction marker to the surface of the protein particle, an aqueous solution of protein particle adsorbed with gold ions was prepared.

More specifically, in one embodiment of the present invention, six histidines capable of adsorbing gold ions are expressed at the N-terminus of the HBV core protein, and linker amino acids and Fc of the antibody in the loop portion. Recombinant HBV capsid protein particles having a surface modified by binding to the B domain of SPA ( Staphylococuccal Protein A) capable of binding to the domain were prepared and used in the composition and kit of the present invention (FIG. 1).

The present invention also provides a method for detecting a disease-specific marker using self-amplification of a detection signal, comprising the steps of:

(a) preparing a pre-assay solution in which an antibody for detecting a disease-specific marker or an antigen and a tag for adsorption of an antigen and a tag for adsorption of gold ions are expressed, and a pre-assay solution in which free gold ions and adsorbed gold ions are present at the same time;

(b) generating a sample mixture by adding a material capable of controlling viscosity and a metal ion showing no color change before and after reduction to a sample to be diagnosed;

(c) gold by antigen-antibody immune reaction and reduction of gold ions in an assay solution prepared by adding the sample mixture and reducing agent of step (b) to the pre-diagnosis solution of step (a) simultaneously inducing a particle formation reaction; and

(d) confirming the presence or absence of a disease-specific marker through a color reaction caused by the formation of gold particles.

The method for detecting a disease-specific marker of the present invention may be implemented using the above-described composition for detecting a disease-specific marker and/or a kit for diagnosing a disease comprising the same, and the composition used for implementing the detection method; / or each configuration of the kit is the same as described above, and thus description thereof is omitted.

As used herein, the term “pre-assay solution” refers to adsorbed gold ions, free gold ions, and an antigen or antigen to be detected or an antibody or antigen that specifically binds, preferably the antibody or antigen is It means a solution containing the expressed protein particles, and other buffers for reaction conditions may be additionally included. More preferably, an amino acid that adsorbs gold ions may be additionally expressed on the antibody or antigen-expressed protein particle.

The term "assay solution" used in the present invention refers to the pre-diagnostic solution, a metal ion that does not change color before and after reduction, a diagnostic sample containing a disease marker to be diagnosed, a substance capable of controlling viscosity, and a reducing agent. means a mixed solution, and the disease marker to be detected in the sample may include an antigen or an antibody.

As used herein, the term "antibody" is an immunoglobulin selected from the group consisting of IgA, IgE, IgM, IgD, IgY and IgG, and can specifically bind to a target antigen. A light chain and a heavy chain are formed by gathering two each, and each chain consists of a variable domain having a variable amino acid sequence and a constant domain having a constant sequence. An antigen-binding site is located at the end of the three-dimensional structure of the variable region, and this region is formed by aggregation of three complementarity determining regions present in each of the light and heavy chains. The complementarity determining region is a region with particularly high amino acid sequence variability among variable regions, and specific antibodies to various antigens can be found by such high variability. The scope of the present invention includes not only complete antibody forms, but also antigen-binding fragments of the antibody molecules.

As used herein, the term "ScFv (single-chain Fv, single-chain fragment antibody or antibody fragment)" is an antibody in which the variable regions of the light and heavy chains are linked. In some cases, it may include a linker consisting of a peptide chain in which about 15 or more amino acids are linked, in this case, the ScFv is a light chain variable region-linking region-heavy chain variable region, or heavy chain variable region-linking It may have a structure of a region-light chain variable region, and has the same or similar antigenic specificity as the original antibody.

) 및 람다(

) 타입을 가진다.A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain is kappa (

) and lambda(

) type.

Antigen-binding fragment or antibody fragment of an antibody refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab') ₂ and Fv. Among the antibody fragments, Fab has a structure having variable regions of light and heavy chains, a constant region of a light chain and a first constant region (CH1) of a heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombinant technology for generating Fv fragments is described in PCT International Patent Applications WO88/10649, WO88/106630, WO88/07085, WO88/07086 and WO88/09344. has been disclosed. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and single-chain Fv (scFv) is generally a heavy chain variable region and a light chain variable region through a peptide linker. This covalent bond or directly linked at the C-terminus can form a dimer-like structure like a double-stranded Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, by restriction digestion of the whole antibody with papain, Fab can be obtained, and F(ab') ₂ fragment can be obtained by digestion with pepsin), gene It can also be produced through recombinant technology.

Antibodies of the present invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of such antibodies, and the like.

In the method of the present invention, in the step (b), when the material capable of controlling the viscosity is mixed with the material capable of adjusting the viscosity until the metal ion showing no color change before and after the reduction is combined, when the material capable of controlling the viscosity is injected, in advance It is possible to prevent binding to ^{Au 3+ in the} diagnostic solution.

In the method of the present invention, it is possible to derive consistent diagnostic results that are not affected by individual differences in serum viscosity, which is commonly used as a diagnostic sample, by using a material that can control the viscosity of the sample.

However, when adding a material capable of controlling the viscosity, interference phenomenon due to non-specific binding to ^{Au 3+ contained in the pre-diagnosis solution may be induced.} By mixing the additional metal ion solution to induce bonding between the material capable of controlling the viscosity and the additional metal ion, it is preferable to block the bonding with ^{Au 3+ in advance.}

At this time, so that the metal ions binding to the material capable of controlling the viscosity do not affect the generation of the detection signal according to the reduction reaction by the reducing agent used for the reduction reaction of the gold ions, the color change due to the reduction reaction is induced after binding to the BSA. shouldn't be

The method of the present invention uses a substance that can control the viscosity, and as compared to the conventional liquid immunodiagnostic method, the amount of serum is greatly reduced from 20% to 5% of the total assay solution, so that the serum viscosity A diagnostic platform that is not affected by individual differences has been established, and the method of the present invention enables diagnosis using blood (currently, a serum sample with hemolysis can be newly diagnosed).

In addition, as the amount of serum is reduced, the amount of protein nanoparticles and antibodies is also greatly reduced, so that the diagnostic method can be implemented with only 1/20 to 1/500 used compared to the conventional liquid immunodiagnostic method. Reduction in the amount of protein nanoparticles and antibodies not only solves the problem of non-specific signal noise caused by the presence of excessive amounts of three-dimensional probes, but also improves the economic feasibility of producing diagnostic kits by dramatically reducing manufacturing costs.

Therefore, the method of the present invention suggests a new technology for immunodiagnostic based on a substance that can control viscosity, that is, BSA.

In the method of the present invention, the sample of step (b) may be selected from the group consisting of blood, plasma, serum, urine, saliva, oral mucosa and saliva, but is not limited thereto.

In the method of the present invention, when the sample is serum, the amount of serum may be used in an amount of 1.2% (v/v) to 12.5% (v/v), which is the amount of serum used compared to the total diagnostic solution in the existing method (20 % (v/v)), it can be seen that the sensitivity of the method of the present invention is improved compared to the existing method.

In the method of the present invention, the amount of protein nanoparticles in step (c) (concentration in 40 μl of diagnostic solution 1.5 nM to 25 nM) is the amount of protein nanoparticles used in conventional methods (concentration in 100 μl of diagnostic solution) 50 nM), which can solve the problem of signal noise caused by the use of an excessive amount of three-dimensional detection probe in a conventional method, and significantly reduce kit manufacturing cost.

In the method of the present invention, the concentration of the gold ions (free gold ions + adsorbed gold ions) present in the prognostic solution may be 1 mM to 10 mM, but is not limited thereto.

In the method of the present invention, the concentration of the reducing agent present in the diagnostic solution may be characterized in that it is 0.001 M to 0.02 M, but is not limited thereto, and the amount of the reducing agent used in the method of the present invention is the amount used in the existing method. It is greatly reduced compared to the volume of the reducing agent (20 μl), and can be used in 5 μl to 10 μl.

In the method of the present invention, when the concentration of the gold ion, the concentration of the reducing agent, and the amount of the sample are less than the reference values, effective disease marker detection is not performed, and when the concentration is greater than the reference concentration, negative effects such as false positives may appear.

In the method of the present invention, the immune reaction of the antigen or antibody for detecting a disease-specific marker may occur on the surface of a protein particle, and the tag adsorbs gold ions to generate and aggregate gold particles in the presence of a reducing agent. can induce

In the method of the present invention, the concentration of the substance capable of controlling the viscosity in the mixture of step (c) is preferably more than 135 μM and less than 293 μM, and if it is out of the above range, effective diagnostic results cannot be obtained.

In the method of the present invention, the concentration of metal ions showing no color change before and after reduction in the mixture of step (c) is preferably 33 mM to 66 mM, and when it is out of the above range, effective diagnostic results are obtained. can't For example, when the concentration is less than the above range, the BSA adsorption of free gold ions increases, which causes interference to the detection signal generation. An interference phenomenon also occurs in the gold ion reduction reaction.

In the method of the present invention, the mixture of step (b), including a form in which a metal ion showing no color change before and after reduction is bound to a substance capable of controlling the viscosity, is 5% (v/v) in the total diagnostic solution to 25% (v/v).

In the method of the present invention, the color reaction in step (d) can be used without limitation as long as it is a method capable of detecting a change in visible light, and preferably can be detected with the naked eye or a spectrophotometer, 500 nm It can be confirmed by measuring the absorbance of to 600 nm, but is not limited thereto.

In the method of the present invention, when the patient's sample is injected, the color reaction may appear faster than when the normal sample is injected, and in the case of the patient, it may appear within 10 to 40 minutes, but the color reaction time is It may vary depending on the conditions.

In the present invention, self-amplification of the detection signal detects a small amount of a disease marker and displays the result as a color change that can be visually confirmed. The self-amplification of this signal is caused by a phenomenon in which gold ions dissolved in a diagnostic solution are reduced to gold particles by a reducing agent. will appear

In the protein particles used in the present invention, hexa-histidine is fused to their monomers. Since gold ions and histidine amino acids have a binding property, gold ions bound to aggregates made by antigen-antibody reaction are combined with a reducing agent. The reaction is reduced to gold particles, and as other gold ions are continuously reduced, they increase in size to form large gold particles and have color due to these aggregates.

In the case of normal human serum, since antigen-antibody reaction does not occur, the time at which the same color appears due to the slow rate of formation of aggregates by gold particles is significantly delayed.

In Example 1 of the present invention, a protein particle was prepared by expressing the expression vector of the _{H 6} -SPA _{B -HBVC capsid structure in E. coli.} The protein particles prepared in this way are hexa-histidine and SPA _B are expressed on the surface, and function to bind to gold ions and the Fc domain of the antibody.

If gold ions are adsorbed to an aqueous solution mixed with protein particles and then injected into a patient's blood sample or a normal person's blood sample, clusters of protein particles and gold particles are formed only in the patient's blood sample, resulting in a blue color reaction. .

In the method of the present invention, the disease may be selected from the group consisting of acute myocardial infarction, human immunodeficiency syndrome, hepatitis C, Sjogren's syndrome, multiple sclerosis syndrome, hepatitis A, stroke, cerebral hemorrhage, and cancer, but is limited thereto it is not

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Preparation of expression vector for synthesis of HBV capsid protein particles

_{An expression vector for preparing protein particles H 6} -SPAB-capsid was prepared through PCR according to the vector schematic shown in Table 2 below.

The prepared plasmid expression vector was subjected to agarose gel-purification and the entire DNA sequence was confirmed.

Specifically, PCR products required for each expression vector were prepared using the primer sets in Table 3, and then the PCR products were sequentially inserted into the pT7-vector to construct an expression vector capable of expressing protein particles. The vector for expression of protein particles is _{pT7-H 6} -SPAB-capsid.

Expression vector construction for protein particle construction protein particles expression vector H ₆ -SPA _B -capsid ₂ NH - Nde I-H ₆ -HBVC- (SPA _{B) 2-COOH} -HBVC- Cla I

Construction of primers for PCR for expression vector production protein particles SEQ ID NO: primer H ₆ -SPA _B -capsid One
2 5'primer: CTC GAG GCA CCG AAA GCT GAT AAC
3'primer: GGA TCC GTC AGC TTT TAG TGC TTG

As shown in Table 2, HBV capsid protein nanoparticles are largely divided into the 1-78th amino acid sequence part of the capsid protein, the part containing two _{SPA B consecutively, and the 81-149th amino acid sequence part of the capsid protein.} (The 1-78th sequence of the capsid protein is positions 1901-2134 (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) encoded thereby in the nucleotide sequence of NCBI Nucleotide accession No. AF286594, and 81- of the capsid protein The 149th sequence is positions 2141-2347 (SEQ ID NO: 5) in the nucleotide sequence of AF286594 and the amino acid sequence encoded by it (SEQ ID NO: 6), and the sequence of SPA is positions 625-813 in the nucleotide sequence of NCBI Nucleotide accession No. M18264 (SEQ ID NO: 7) and the amino acid sequence encoded thereby (SEQ ID NO: 8)).

Biosynthesis and separation and purification of HBV capsid protein particles

2-1. biosynthesis of protein particles

E. coli strain BL21(DE3)[F-ompThsdSB(rB-mB-)] was transformed through the expression vector prepared in Example 1, respectively, and ampicillin-resistant transformants were selected. Transformed E. coli was cultured in flasks (250 mL Erlenmeyer flasks, 37°C, 150 rpm) containing 50 mL of Luria-Bertani (LB) medium (containing 100 mg L-1 ampicillin).

When the turbidity (OD 600) of the medium reached about 0.4-0.6, Isopropyl-β-D-thiogalactopyanosid (IPTG) (1.0 mM) was injected to induce expression of the recombinant gene. After culturing at 20°C for 12 to 16 hours, the cultured E. coli was centrifuged at 4,500 rpm for 10 minutes to obtain a cell precipitate, and then suspended in 5 ml of a disruption solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA). After crushing using an ultrasonic crusher (Branson Ultrasonics Corp., Danbury, CT, USA), centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and insoluble aggregates. Purification was performed using the separated supernatant.

2-2. Protein Particle Purification

In order to purify the fusion protein particles recombined by self-assembly expressed in Example 2-1, the following three-step purification process was performed. ^{First, 1) Ni 2+} -NTA affinity chromatography using the binding of histidine and nickel fused to the recombinant protein was performed, and then 2) the self-recovery of the monomers was performed by concentrating the recombinant protein and exchanging the buffer of the concentrated protein. Assembly was performed, and 3) sucrose gradient ultracentifugation was performed to separate only self-assembled protein particles. The detailed description of each step is as follows.

1) Ni ²⁺ -NTA affinity chromatography

In order to purify the recombinant protein, E. coli cultured by the above-mentioned method was recovered, the cell pellet was resuspended in 5 mL of a disintegration solution (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole), and the cell pellet was resuspended using a sonicator. Cells were disrupted. The lysed cell solution was centrifuged at 13,000 rpm for 10 minutes to separate only the supernatant, and then each recombinant protein was separated using a Ni ²⁺ -NTA column (Qiagen, Hilden, Germany) (washing buffer: pH 8.0, 50 mM). sodium phosphate, 300 mM NaCl, 50 mM imidazole / elution buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 100 mM imidazole).

2) Concentration and buffer exchange

Put 3 ml of recombinant protein eluted through Ni ²⁺ -NTA affinity chromatography in an ultracentrifuge filter (Amicon Ultra 10K, Millipore, Billerica, MA) and centrifuge at 5,000 rpm until 1 ml of solution remains on the column. did. The self-assembly of the monomers was carried out by exchanging the protein particles with a buffer of Tris-HCl (50 mM Tris-HCl, 500 mM NaCl, pH 7.0).

3) sucrose gradient ultrafast centrifugation

Tris-HCl (50 mM Tris-HCl, 500 mM NaCl, pH 7.0) was added to each concentration of sucrose in buffer to prepare solutions containing 60%, 50%, 40%, 30%, and 20% sucrose, respectively. Put 2 ml of each concentration (60-20%) sucrose solution from the highest concentration to the ultraclear 13.2ml tube (Beckman). After filling the solution, 1 ml of the recombinant protein solution was finally filled, followed by ultra-high speed centrifugation at 24,000 rpm at 4° C. for 16 hours (Ultracentrifuge L-90k, Beckman). After centrifugation, a portion of the 40-50% sucrose solution was carefully removed using a pipette, and the recombinant protein buffer was replaced using an ultracentrifuge filter and Tris-HCl buffer as described in 2).

Assembly verification of protein particles

For structural analysis of the recombinant protein particles purified in Example 2, the recombinant protein particles were photographed with a transmission electron microscope (TEM). First, unstained, purified protein samples were placed on carbon-coated copper electron microscope grids and then air-dried. To obtain stained images of protein particles, electron microscope grids containing naturally dried samples were incubated with 2% (w/v) aqueous uranyl acetate solution for 10 min at room temperature, washed 3-4 times with distilled water. did.

In the case of obtaining an image of an aggregate in which gold particles are gathered, a sample of the assay solution, which has changed color used for diagnosis, was placed on a grid without a separate dyeing process, and then dried naturally.

The images of protein particles and gold particle aggregates were observed using a Philips Technai 120 kV electron microscope, and as shown in FIG. 1 , it was confirmed that each protein particle formed a spherical shape and the gold particle aggregate formed a radial shape particle. .

BSA concentration and Ni using signal self-amplification ²⁺ Check single-step diagnosis results with or without presence

4-1. Ni in BSA 181 μM ²⁺ Diagnosis result with or without ionic solution

In the case of acute myocardial infarction (AMI), the H ₆ -SPA _B -capsid protein particles prepared in Example 2 were 6.25 nM, goat anti-TnI polyclonal IgG (cat. no. 70-XG82, Fitzgerald, Acton, MA). , USA) antibody was mixed with 1 ml of a solution having a concentration of 66.7 nM at 4° C. for 12 to 16 hours.

^{500 μL of 14.7 mM Au 3+} ion solution (HAuCl ₄ (cat. no. 254169, Sigma Aldrich, St. Louis, MO, USA)) was added to the antibody-immobilized protein particles and mixed and combined at 4°C for 12 to 16 hours. .

Put 72 μL of the protein particle solution (pre-assay solution) immobilized with the antibody and gold ions in a glass vial, and then add 1.2 mM BSA and 245 mM Ni ²⁺ ions to it. Add 18 μL of the mixed solution and mix. Immediately after, 6 μL of serum samples from normal or AMI patients mixed with 38.5 mM Ni ²⁺ ions were added and mixed well, followed by 0.05 M L-ascorbic acid (cat. no. A7506, Sigma Aldrich, St. Louis, MO, USA). ) Add 24 μL of reducing agent. 40 μL of the complete diagnostic solution was added to each well of a 384-well plate (cat. no. 3640, Corning, NY, USA), and the color change caused by self-signal amplification was confirmed. In addition, it was confirmed that this color change is due to high absorbance in the 576 nm wavelength band, and the difference in absorbance between the serum of a patient and a normal person in this wavelength band was confirmed with a microplate reader (Infinite M200 Pro, TECAN, Zurich, Switzerland). As a result, as shown in FIG. 3 , a color reaction was confirmed within 14 minutes of the reaction, and it was confirmed that there was a clear difference in absorbance between the patient and the normal person.

4(A) is the result of performing the same single-stage immunoassay as above on serum samples from 15 healthy and acute myocardial infarction patients, respectively. Similarly, the difference between patients and normal people can be clearly distinguished through the difference in absorbance. It was confirmed that it is possible.

However, if under the same conditions as in the Ni ²⁺ ion solution are not added, the degree woke the patient is part of the normal absorbance curves overlapping patterns appear as will be obtained from the 4 (B), BSA, which is Au ³⁺ It was confirmed that the interference phenomenon caused by the combination with

4-2. Ni in BSA 248 μM ²⁺ Diagnosis result with or without ionic solution

A single-stage immunoassay was performed under the same conditions as in Example 4-1, except that the BSA concentration was changed to 248 μM.

As a result, as shown in FIG. 5 , a color reaction was confirmed within 25 minutes of the reaction, and it was confirmed that there was a clear difference in absorbance between the patient and the normal person. In addition, as a result of performing a single-step immunoassay on serum samples from 15 healthy individuals and acute myocardial infarction patients, respectively, a color reaction was confirmed in 25 minutes as shown in FIG. but can be clearly distinguish between the patients and normal persons of difference, Ni ²⁺ ion when the solution is not added, the more interference is caused by a combination of BSA and Au ³⁺ as shown in (B) of Figure 6 significantly over It was confirmed that the absorbance curve of the patient group spreads.

4-3. Ni in BSA 293 μM ²⁺ Diagnosis result with or without ionic solution

A single-step immunoassay was performed under the same conditions as in Example 4-1, except that the BSA concentration was changed to 293 μM.

As a result, as shown in FIG. 7 , a color reaction was confirmed within 40 minutes of the reaction, and it was confirmed that there was a clear difference in absorbance between the patient and the normal person. In addition, in the result of performing a single-step immunoassay on serum samples from 15 healthy individuals and acute myocardial infarction patients, respectively, a color reaction was confirmed in 40 minutes as shown in FIG. 8A , and the difference in absorbance was able to clearly distinguish between the patients and normal persons of difference over, Ni ²⁺ ion when the solution is not added, the more interference is caused by a combination of BSA and Au ³⁺ as shown in (B) of Figure 8 set off It was confirmed that the absorbance curve of the patient group spreads.

Ni ²⁺ Check the availability of metal ions other than

^{The purpose of Ni 2+} used in the single-step immunoassay of the present invention is to prevent interference caused by binding of BSA to Au ³⁺ for viscosity control, which is pre-bonded with ^{Ni 2+ and BSA.} The effect is exerted by administration to the prognostic solution. Therefore, the color change should not be induced by the reduction reaction after binding to the BSA so that the metal ion binding to BSA does not affect the color reaction that occurs according to the formation of gold particles by the reduction reaction of the gold ion.

In this embodiment, as well as Ni ²⁺ to determine the metal ion which satisfies the condition as described above in addition to ^{Ni 2+, Zn 2+, Cu 2+} , Fe 2+ and Fe ³⁺ reduction before and after the change in color of the metal ion Confirmed.

As a result, as confirmed in FIG. 9 , it was found that Zn ²⁺ and Fe ³⁺ metal ions did not show color change before and after reduction, so that they were suitable for use in preventing the bonding ^{between BSA and Au 3+ . In addition, Mg +2} , Ca ⁺² and Na ⁺ , which are metal ions satisfying the above conditions, may be used in the single-step immunoassay of the present invention according to the properties of various known metal ions.

Identification of substances for viscosity control of diagnostic solutions other than BSA

In this example, in order to identify a substance capable of adjusting the viscosity of a diagnostic solution other than BSA, a single-step immunoassay was performed using Tween 20 instead of BSA, using sera from normal healthy people and acute myocardial infarction patients as samples.

Since Tween 20 has a blocking effect that reduces non-specific binding in an immunodiagnostic system using an antigen-antibody reaction such as ELISA, it is often used as a blocking agent like BSA. Since Tween 20 or BSA serves to improve sensitivity by reducing noise or background signal of the diagnostic platform as a result by using it, in this embodiment, Tween 20 is selected as a control to exhibit the same effect as BSA. I wanted to check whether

Except for using 0.5%, 1%, 5%, 10% and 20% of Tween 20 instead of BSA, respectively, the remaining experimental conditions are the same as in Example 4-1.

As a result, as shown in FIG. 10, there was no clear difference in color development that could distinguish a patient from a normal person at all concentrations of Tween 20, and the graph of a patient and a normal person was not clearly differentiated in the absorbance graph of FIG. It was confirmed that it is unsuitable as a viscosity modifier.

Through the above results, the unique functionality of BSA in the disease-specific marker detection method of the present invention was verified.

Identify the optimal effective concentration range for BSA

In this example, the critical concentration range of BSA used to control the viscosity of the diagnostic solution was confirmed. Sensitivity analysis was performed after mixing by adjusting the concentration of BSA to 135 μM, 248 μM, 271 μM, and 293 μM, respectively, in samples containing 1 ng/ml, 2.5 ng/ml and 5 ng/ml of troponin I, respectively. and the remaining experimental conditions were the same as in Example 4-1.

As a result, as shown in FIG. 12 , at the concentration of 135 μM BSA, the viscosity was not controlled, so the reduction reaction of gold ions occurred very quickly, and gold particle aggregates were generated regardless of the presence or absence of the disease marker, and the detection signal according to the concentration of the disease marker occurred indiscriminately, and at 293 μM BSA concentration, no color change due to reduction reaction was observed in all experimental groups. Confirmed.

Ni ²⁺ Investigation of additional functionalities of ions

^{In this Example, the function of Ni 2+} ions was additionally confirmed in normal serum serum in which troponin I, a disease marker, does not exist. Since normal human sera lacks disease markers, diagnostic signals are significantly delayed compared to patient sera. However, even when diagnosing normal serum, non-specific signals may occur due to different serum-specific conditions. In order to control this subtle difference, an experiment was conducted to prevent non-specific signal generation due to adsorption of gold ions with other proteins in serum by additionally binding ^{Ni 2+ ions to normal serum in advance.}

As a result, when the Ni ²⁺ ion was not attached, it was confirmed that there was also a serum showing a subtle non-specific signal for each normal serum, but ^{by binding Ni 2+} ion to each serum in advance, it was confirmed that this non-specific signal disappeared.

SEQUENCE LISTING <110> CELLEMEDY CO.,LTD CELLEGAIN Co., Ltd. <120> Liquid immunodiagnostics based on detection signal control <130> 1066886 <160> 8 <170> PatentIn version 3.2 <210> 1 <211> 24 <212> DNA <213> <220> <223> H6-SPAB-capsid_F <400> 1 ctcgaggcac cgaaagctga taac 24 <210> 2 <211> 24 <212> DNA <213> <220> <223> H6-SPAB-capsid_R <400> 2 ggatccgtca gcttttagtg cttg 24 <210> 3 <211> 234 <212> DNA <213> <220> <223> HBV capsid protein 1-78 <400> 3 atggacattg acccgtataa agaatttgga gcttctgtgg agttactctc ttttttgcct 60 tctgacttct ttccttctat tcgagatctc ctcgacaccg cctctgctct gtatcgggag 120 gccttagagt ctccggaaca ttgttcacct caccatacag cactcaggca agctattctg 180 tgttggggtg agttgatgaa tctggccacc tgggtgggaa gtaatttgga aagac 234 <210> 4 <211> 78 <212> PRT <213> <220> <223> HBV capsid protein 1-78 <400> 4 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp 65 70 75 <210> 5 <211> 207 <212> DNA <213> <220> <223> HBV capsid protein 81-149 <400> 5 tccagggaat tagtagtcag ctatgtcaac gttaatatgg gcctaaaaat cagacaacta 60 ttgtggtttc acatttcctg tcttactttt ggaagagaaa ctgttcttga gtatttggtg 120 tcttttggag tgtggattcg cactcctccc gcttacagac caccaaatgc ccctatctta 180 tcaacacttc cggaaactac tgttgtt 207 <210> 6 <211> 69 <212> PRT <213> <220> <223> HBV capsid protein 81-149 <400> 6 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 1 5 10 15 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 20 25 30 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 35 40 45 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 50 55 60 Glu Thr Thr Val Val 65 <210> 7 <211> 189 <212> DNA <213> <220> <223> SPAB <400> 7 gcaccgaaag ctgataacaa attcaacaaa gaacaacaaa atgctttcta tgaaatctta 60 catttaccta acttaaatga agaacaacgc aatggtttca tccaaagctt aaaagatgac 120 ccaagccaaa gcgctaacct tttagcagaa gctaaaaagc taaatgatgc acaagcacca 180 aaagctgac 189 <210> 8 <211> 63 <212> PRT <213> <220> <223> Protein A <400> 8 Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 1 5 10 15 Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly 20 25 30 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 35 40 45 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp 50 55 60

Claims (26)

a pre-assay solution in which an antibody for detecting disease-specific markers or protein particles with antigen and tags for adsorption of gold ions, free gold ions and adsorbed gold ions exist;
reducing agent;
metal ions showing no color change before and after reduction; and
a substance capable of adjusting the viscosity of the diagnostic solution;
including,
A composition for detecting a disease marker, wherein the metal ion showing no color change before and after the reduction is used by binding to a substance capable of adjusting the viscosity of the diagnostic solution. The composition for detecting a disease marker according to claim 1, wherein the disease-specific marker is a protein marker including troponin I for acute myocardial infarction. According to claim 1, wherein the protein particle is ferritin (ferritin), ferritin-like protein (ferritin-like protein), magnetosome (magnetosome) component protein, virus component protein, DPS (DNA binding protein), and proteasome (proteasome) ) at least one selected from the group consisting of, a composition for detecting a disease marker. According to claim 3, wherein the viral constituent protein is HBV core protein (hepatitis B virus core protein) or tobacco mosaic virus (tobacco mosaic virus) constituent protein, the composition for detecting a disease marker. The composition for detecting a disease marker according to claim 1, wherein the gold ion adsorption tag is at least one amino acid selected from the group consisting of histidine, lysine, and arginine. According to claim 1, wherein the reducing agent is at least one selected from the group consisting of ascorbic acid, imidazole, pyrazole, histamine, hydroxylamine, citric acid and sodium borohydride, the composition for detecting a disease marker. According to claim 1, wherein the metal ion does not show a color change before and after the reduction is Ni ²⁺ , Zn ²⁺ , Fe ³⁺ , Mg ²⁺ , Ca ²⁺ and at least one selected from the group consisting of ^{Na +,} A composition for detecting a disease marker. The composition for detecting a disease marker according to claim 1, wherein the substance capable of adjusting the viscosity of the diagnostic solution comprises bovine serum albumin (BSA). A kit for diagnosing a disease comprising the composition for detecting any one of claims 1 to 8 for a disease marker. The kit of claim 9, wherein the disease is selected from the group consisting of acute myocardial infarction, human immunodeficiency syndrome, hepatitis C, Sjogren's syndrome, multiple sclerosis syndrome, hepatitis A, stroke, cerebral hemorrhage, and cancer. A method for detecting a disease-specific marker using self-amplification of a detection signal, comprising the steps of:
(a) preparing a pre-assay solution in which an antibody for detecting a disease-specific marker or an antigen and a tag for adsorption of an antigen and a tag for adsorption of gold ions are expressed, and a pre-assay solution in which free gold ions and adsorbed gold ions are present at the same time;
(b) generating a sample mixture by adding a material capable of controlling viscosity and a metal ion showing no color change before and after reduction to a sample to be diagnosed;
(c) gold by antigen-antibody immune reaction and reduction of gold ions in an assay solution prepared by adding the sample mixture and reducing agent of step (b) to the pre-diagnosis solution of step (a) simultaneously inducing a particle formation reaction; and
(d) confirming the presence or absence of a disease-specific marker through a color reaction caused by the formation of gold particles. 12. The method of claim 11, wherein the protein particles are ferritin, ferritin-like protein, magnetosome component protein, virus component protein, DNA binding protein (DPS), and proteasome. ), which is one or more proteins selected from the group consisting of, a method for detecting a disease-specific marker. The method of claim 11, wherein the gold ion adsorption tag in step (a) is one or more amino acids selected from the group consisting of histidine, lysine and arginine. The method of claim 11 , wherein in step (b), a material capable of adjusting the viscosity and a metal ion showing no color change before and after reduction are mixed until combined. According to claim 11, wherein the reducing agent of step (b) is at least one selected from the group consisting of ascorbic acid, imidazole, pyrazole, histamine, hydroxylamine, citric acid and sodium borohydride, disease-specific A method for detecting enemy markers. According to claim 11, wherein the metal ions that do not show a color change before and after the reduction in step (b) is Ni ²⁺ , Zn ²⁺ , Fe ³⁺ , Mg ²⁺ , Ca ²⁺ and selected from the group consisting of ^{Na +} A method for detecting one or more disease-specific markers. The method of claim 11 , wherein the substance capable of controlling the viscosity of step (b) includes BSA (Bovine serum albumin). The method of claim 11, wherein the sample of step (b) is at least one selected from the group consisting of blood, plasma, serum, urine, saliva, oral mucosa and saliva. The method of claim 11 , wherein when the sample is serum, the amount of serum is 1.2% (v/v) to 12.5% (v/v). The method of claim 11, wherein the amount of the protein particles in step (c) is 1.5 nM to 25 nM. The method of claim 11, wherein the concentration of the substance capable of controlling the viscosity in the mixture of step (c) is greater than 135 μM and less than 293 μM. The method of claim 11, wherein the concentration of metal ions showing no color change before and after reduction in the mixture of step (c) is 33 mM to 66 mM. 15. The method of claim 14, wherein the mixture of step (b) containing a metal ion showing no color change before and after reduction to a substance capable of controlling viscosity is combined with 5% (v/v) to 5% (v/v) in the total diagnostic solution. A method for detecting a disease-specific marker, present in a proportion of 25% (v/v). The method of claim 11, wherein the color reaction in step (d) is confirmed by measuring the absorbance at 500 nm to 600 nm. The method of claim 11, wherein the color reaction in step (d) appears within 10 to 40 minutes. 12. The method of claim 11, wherein the disease is selected from the group consisting of acute myocardial infarction, human immunodeficiency syndrome, hepatitis C, Sjogren's syndrome, multiple sclerosis syndrome, hepatitis A, stroke, cerebral hemorrhage, and cancer. Detection of a disease-specific marker Way.

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