KR20170023589A

KR20170023589A - A Method and Kit for simultaneously detecting multiple diseases based on 3D protein nanoparticle probes

Info

Publication number: KR20170023589A
Application number: KR1020150118939A
Authority: KR
Inventors: 이지원; 이종환; 서혁성
Original assignee: 고려대학교 산학협력단
Priority date: 2015-08-24
Filing date: 2015-08-24
Publication date: 2017-03-06

Abstract

TECHNICAL FIELD The present invention relates to a simultaneous detection method for multiple diseases based on highly sensitive three-dimensional protein nanoparticle probes and a kit for simultaneous detection of multiple diseases using highly sensitive three-dimensional protein nanoparticle probes. More particularly, A conjugation pad in which signal molecules are planted; A membrane comprising a protein nanoparticle probe in which a disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; And an absorbent pad, and a method for simultaneous detection of multiple diseases using the kit. The protein nanoparticle probe according to the present invention is characterized in that disease marker specific epitopes on the surface have proper orientation and are highly integrated And can detect various disease marker specific epitopes at various ratios by controlling the mixing ratios of these marker markers, and a method of detecting disease markers using the method can detect various disease marker specific epitopes on the surface of protein nanoparticles And is excellent in sensitivity and accuracy.

Description

TECHNICAL FIELD [0001] The present invention relates to a method and kit for simultaneously detecting three-dimensional protein nanoparticle probe-

TECHNICAL FIELD The present invention relates to a simultaneous detection method for multiple diseases based on highly sensitive three-dimensional protein nanoparticle probes and a kit for simultaneous detection of multiple diseases using highly sensitive three-dimensional protein nanoparticle probes. More particularly, A conjugation pad in which signal molecules are planted; A membrane comprising a protein nanoparticle probe in which a disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; And a kit for the simultaneous detection of multiple diseases. The method for simultaneous detection of multiple diseases using a highly sensitive three-dimensional protein nanoparticle probe according to the present invention is a method for simultaneous detection of multiple diseases, By using protein nanoparticles containing two or more epitopes each, the sensitivity and accuracy of simultaneous detection of multiple diseases are excellent.

Infectious diseases such as AIDS, hepatitis, pneumonia, tuberculosis and malaria are responsible for 15 million deaths annually, which is about 25% of the world's population deaths. Deaths due to infectious diseases are mostly occurring in developing countries with poor health care facilities and services, thereby creating the need for affordable, convenient, and patient-friendly medical services. Enzyme-linked immunosorbent assay (ELISA), proteome chips, polymerase chain reaction (PCR), and microfluidic system-based assays. Although it has been developed, expensive equipment and skilled medical personnel are required, which limits its use in developing countries. Lateral Flow Assay (LFA) technique is simple, economical, and patient-friendly method that can confirm the results in a short time (about 30 minutes). However, LFA Technique has a high false positive rate due to non-specific binding between the probe and the target, and has a limitation that the sensitivity is low due to a short reaction time. Also, in the simultaneous diagnosis of various diseases, optimal conditions for diagnosis of different diseases are different, and there is a cross-reactivity between diseases, so that simultaneous diagnosis is difficult.

Blood transfusions are an important pathway of viral infectious disease, and although many advanced diagnostic techniques have been developed, serious social problems are still occurring. When the disease is first infected, there is a 'window period', which is a non-diagnostic area due to technical limitations. At this time, the concentration of the disease marker in the sample is very low. This is a frequent occurrence. Therefore, the economic and psychological suffering of the patients is inevitably increased, and the patient is exposed to the secondary infection to the other person without knowing the fact of the infection of the patient. Diagnostic techniques with sensitive sensitivity such as nucleic acid testing (NAT) have been developed, however, in the case of nucleic acid diagnostic tests, additional pretreatment of the sample is indispensable and analysis of the sample requires expensive equipment , It is difficult to apply it to emergencies such as the examination of samples of patients whose identity and unconsciousness are uncertain, and there is a limit to the universal utilization in areas where the medical facilities are weak.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are known to have the same route of infection. Most are known to be infected through blood, through sexual intercourse, the use of the same syringe, and blood transfusions. Because both of these viruses have the same route of infection, there are many cases of simultaneous infection in both viruses and it is known that about 500 to 10 million patients are simultaneously infected. When hepatitis C patients are coinfected with HIV, the progression of hepatocellular fibrosis is accelerated and leads to rapid cirrhosis or liver cancer. It is known that AIDS patients reach a faster immune deficiency state when they are co-infected with HCV. Different viral infectious diseases should be treated differently, and corresponding treatments should be provided for simultaneous infection. Therefore, it is necessary to develop a simultaneous diagnosis system for simultaneous diagnosis of two diseases. In the case of hepatitis A, an effective vaccine has been developed, but its pathway can also be infected by food or simple contact. In the case of hepatitis A virus (HAV), even in non-biological conditions, It is possible to survive and survive extreme temperatures and pHs, and 1.5 million new patients are emerging each year. When the HAV virus is infected, the IgM type antibody is formed in the body after 5-10 days, and when the symptoms are alleviated, the IgG type antibody is generated and remains in the body for a lifetime. IgG antibodies have been used to detect both types of antibodies.

In vitro diagnostics has been developing technologies that are more accurate, faster, and easier to measure / analyze. Currently, artificial synthetic nanomaterials (gold nanoparticles, silicon nanowires, magnetic nanoparticles , Silica nanoparticles, etc.) are difficult to mass-produce / mass-produce nanomaterials / particles having a uniform size / particle size distribution because the synthesis process is very complicated and coherent reaction control is not easy, and biological functions are imparted to the surface of particles , Surface distribution of peptides, proteins, etc.), chemical modification of the surface is indispensably required. Therefore, the sensitivity, specificity and reproducibility of the diagnosis remain low due to the degradation of the probe activity on the artificial synthetic nanomaterial surface, the orientation of the probe and the difficulty in controlling the degree of integration. Due to these technical limitations, Sensitivity and specificity.

Ferritin is composed of 24 identical protein subunits composed of a heavy chain and a light chain and forms a hollow shell in vivo. The protein binding to iron has an iron storage function and functions as an iron detoxification (Harrison et al., Biochim Biophys Acta., 1275 (3): 161-163, 1996). The protein functions as a cytoprotective protein that maintains iron balance in the cell for the growth and survival of most tissues and minimizes oxygen-free radical formation due to the binding of iron in the cell (Lawson et al., Nature, 349: 541-544, 1991). Ferritin has a molecular weight of about 500.000 Da and is composed of a heavy chain and a light chain. It has a self-assembly ability and exhibits unique characteristics to form spherical particles.

Human heavy chain ferritin (21 kDa) is a protein nanoparticle formed by self-assembly of 24 monomers in a 4-3-2 symmetry phase in a cell. Human heavy chain ferritin (hFTH) monomer Are highly biodegradable and expressible in E. coli cells, while they form nanoparticles of about 12 nm in diameter by self-assembling properties. In addition, it is excellent in stability and can be freely imparted to the surface function / characteristics of the particle by a relatively simple genetic manipulation, and when a different foreign protein is fused to a monomer, a single protein nano- It is possible to manufacture particles. Therefore, by using protein nanoparticles as a base material, it is possible to fundamentally solve problems of probe activity, orientation, and synthesis efficiency, and by using a self-assembly process of protein nanoparticles, a three-dimensional detection probe can be manufactured, Can also be implemented.

Chronic diseases and viral infectious diseases, in which early diagnosis is important, include various disease markers even in the same disease, and there is a difficulty in accurate diagnosis because the distribution of disease markers in individual patients is different. In addition, if disease markers with high sensitivity and specificity are not found in the vast majority of patients, the disease may be diagnosed according to the frequency of detection of various disease-related markers. Therefore, it is required to develop a simultaneous multi-disease marker detection system capable of simultaneously detecting various disease markers in the body for accurate early diagnosis.

The present inventors have developed a system capable of detecting a high sensitivity of a disease marker using a hydrogel immobilized with protein nanoparticles expressing a probe for detecting a disease marker in Korean Patent No. 10-2012-0126843. As a model disease, AIDS However, there is a disadvantage that only one disease marker can be detected at a time.

As a result of intensive efforts to develop a simultaneous detection method for multiple diseases with high sensitivity and accuracy, the present inventors have designed and produced expression vectors encoding human ferritin protein monomers fused with different epitopes for the same diseases Protein nanoparticle probes expressing multiple sensitive antigenic epitopes on the surface of protein nanoparticles, which are highly sensitive to disease markers when co-expressed in E. coli, can be used to detect multiple disease markers at the same time, Protein nanoparticle probes that can control the expression frequency of antigenic epitopes have been developed. Further, as a result of using the high-function protein nanoparticle probe capable of simultaneously detecting multiple disease markers of the present invention for simultaneous detection of AIDS, hepatitis C and hepatitis A, it was confirmed that the sensitivity and accuracy were remarkably superior to those of the peptide probe Thus completing the present invention.

It is an object of the present invention to provide a lateral flow assay kit comprising a protein nanoparticle probe containing various disease marker specific epitopes on its surface.

It is another object of the present invention to provide a method for simultaneous detection of multiple diseases using the lateral flow assay kit.

In order to achieve the above object, the present invention provides a semiconductor device comprising: a sample pad; A conjugation pad in which signal molecules are planted; A membrane comprising a protein nanoparticle probe having a first disease marker specific epitope fused and expressed on a surface of a self-assembly protein; And an absorbent pad.

The present invention also provides a semiconductor device comprising: a sample pad; A conjugation pad in which signal molecules are planted; A protein nanoparticle probe in which a first disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A membrane comprising a protein nanoparticle probe having a second disease marker specific epitope fused and expressed on a surface of a self-assembly protein; And an absorbent pad.

The present invention also provides a semiconductor device comprising: a sample pad; A conjugation pad in which signal molecules are planted; A protein nanoparticle probe in which a first disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A protein nanoparticle probe in which a second disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A membrane comprising a protein nanoparticle probe having a third disease marker specific epitope fused and expressed on a self-assembly protein surface; And an absorbent pad.

The present invention also provides a semiconductor device comprising: (a) a sample pad; A conjugation pad in which signal molecules are planted; A protein nanoparticle probe in which a first disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A protein nanoparticle probe in which a second disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; And a step of reacting a detection target sample with a side flow assay kit comprising a membrane and an absorbent pad including a protein nanoparticle probe having a third disease marker specific epitope fused and expressed on the surface of a self-assembly protein ; And (b) measuring fluorescence with a naked eye or a densitometer. The present invention also provides a method for simultaneous detection of multiple diseases using a lateral flow assay.

The protein nanoparticle probe according to the present invention is a three-dimensional nanostructure that is naturally biosynthesized in a cell, and is always synthesized while maintaining an accurate quadratic structure and a particle topology, dramatically increasing the problem of probe activity, orientation, and synthesis efficiency , And is a safe three-dimensional bio-nanoparticle free from the problem of nano-toxicity, and has the advantage of being able to diagnose with high sensitivity and high accuracy of many viral infectious diseases.

The simultaneous detection of multiple diseases using the protein nanoparticle probe according to the present invention is remarkably improved in sensitivity and specificity at the time of simultaneous detection of multiple diseases and is useful for early diagnosis of infectious diseases.

FIG. 1 is a schematic diagram of an expression vector for developing a three-dimensional protein nanoparticle probe for the diagnosis of AIDS, hepatitis C and hepatitis A, a schematic diagram of a protein nanoparticle probe monomer and a three-dimensional structure of the expressed protein nanoparticle, will showing the nanoparticle probes of _HIV P _-1 and P _-2 _HIV, (B) will showing a hepatitis C diagnostic protein nanoparticle probe of _HCV P _-1 and P _-2 _HCV, (C) a hepatitis diagnosis Protein nanoparticle probes P _HAV _-1 , P _HAV _-2 and P _HAV _-2 are shown.
Figure 2 shows the results of SDS-PAGE of purified protein nanoparticle probes via Ni2 + -NTA affinity chromatography (M: size molecular marker, S: water soluble protein, IS: insoluble protein).
FIG. 3 is a transmission electron microscope (TEM) photograph of a purified protein nanoparticle probe, wherein (A) shows a protein nanoparticle probe for AIDS diagnosis, (B) shows a protein nanoparticle probe for diagnosing hepatitis C, C) shows a protein nanoparticle probe for hepatitis A diagnosis.
FIG. 4 is a schematic diagram of a protein nanoparticle probe-based lateral flow assay (LFA) system. The LFA system includes a sample pad, a conjugation pad, a nitrocellulose membrane, absorbent pad. On the membrane, protein nanoparticle probes for the diagnosis of AIDS, hepatitis C and hepatitis A are immobilized on a test line, and anti-human secondary antibodies are immobilized on a control line.
FIG. 5 shows (A) transmission electron microscope (TEM) photographs and (B) dynamic light scattering (DLS) analysis results of colloidal gold nanoparticles operating as signal molecules.
Figure 6 shows the results of a protein A-optimal wreath binding to colloidal gold utilizing ultraviolet and visible absorption spectroscopy (ultimately 40 nM protein A is used).
FIG. 7 compares the detection limits of protein nanoparticle probe-based LFA and peptide probe-based LFA with (A) AIDS, (B) hepatitis C and (C) hepatitis A.
FIG. 8 shows the results of verifying the sensitivity of the protein nanoparticle probe-based LFA system to serum samples of 20 AIDS patients.
FIG. 9 shows the results of verifying the sensitivity of the protein nanoparticle probe-based LFA system to serum samples of 20 patients with hepatitis C virus.
FIG. 10 shows the results of verifying the sensitivity of the protein nanoparticle probe-based LFA system to serum samples of 20 hepatitis A patients.
FIG. 11 shows the results of verifying the sensitivity of a peptide probe-based LFA system to serum samples of 20 AIDS patients.
FIG. 12 shows the results of a test for the sensitivity of a peptide probe-based LFA system to serum samples of 20 patients with hepatitis C virus.
FIG. 13 shows the results of a test for the sensitivity of a peptide probe-based LFA system to serum samples of 20 hepatitis A patients.
FIG. 14 shows the results of verifying the specificity of the protein nanoparticle probe-based LFA system in 20 normal serum samples.
FIG. 15 is a result of a Duplex assay (concurrent diagnosis of two types of viral infectious diseases) of a protein nanoparticle probe-based LFA system and verifying AIDS and hepatitis C. FIG.
FIG. 16 is a result of verifying AIDS and hepatitis A as a Duplex assay (simultaneous diagnosis of two types of viral infectious diseases) of a protein nanoparticle probe-based LFA system.
17 is a Duplex assay (simultaneous diagnosis of two types of viral infectious diseases) of a protein nanoparticle probe-based LFA system, which is a result of verifying hepatitis C and hepatitis A.
18 shows the result of triple assay (simultaneous diagnosis of three kinds of virus infectious diseases) of the protein nanoparticle probe-based LFA system.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Human ferritin is a protein nanoparticle produced by self-assembly of 24 heavy chain (21 kDa) and light chain (19 kDa) ferritin monomers in a 4-3-2 symmetry phase in a cell, The human heavy chain ferritin monomer forms nanoparticles of about 12 nm in diameter by self-assembling properties while being biosynthesized in a high expression rate and acceptability in E. coli cells. The active terminal heavy chain ferritin forming the nanoparticles has an amino terminal (N-term.) Exposed to the outside of the particle and a carboxyl terminal (C-term.) When the foreign protein or peptide is fused. It is possible to perform surface property modification of ferritin nanoparticles by fusing the peptide or protein of the detection probe function to the amino- or carboxyl terminus using a gene recombination technique.

In the present invention, synthetic peptides for specific amino acid sequences including immunodominant epitopes of HIV, HCV and HAV among various disease-specific markers are fused and expressed at the carboxyl terminal of human heavy chain ferritin, And to confirm the ease of mass production, uniform particle size distribution, ease of epitope density / structure / orientation control, and stability of protein nanoparticles in which hepatitis C and hepatitis A specific epitopes are fused and expressed on the surface of nanoparticles.

In the present invention, gp41 (I580 S613) peptide (HIV1) and HIV24 gag protein p24 (P166 G354) protein (HIV2) in HIV envelope glycoprotein gp160, which are two HIV critical antigenic epitopes, are determined as disease marker specific epitopes for AIDS diagnosis Derived from the core portion c22p (K10 to S53) of the four HCV important antigenic epitopes, c33c (A1192 to C1457) of the NS3 portion, 511p (I1694 to L1735) and c100p (I1920 to V1935) portions of the NS4 portion Considering the size and structure of the peptides / proteins, it is possible to use the epitope A [c33p (A1192 to C1457), HCV1], the epitope B [511p (I1694 to L1735) -c100p (I1920 to V1935) ]. In order to diagnose hepatitis A, eleven peptide epitopes derived from HAV surface antigens 3A, 3B, 3C, 2B and 2C were selected and constituted three types of epitopes which are combinations of these peptides:

Epitope 1 (E1): [(E64-F83) - (G289-Q308) - (D572-F591) - (V779-

Epitope 2 (E2): [(K961-Q980) - (S1421-K1449) - (H1500-Q1519) - (V1719-Q1738)

Epitope 3 (E3): [(G119-G130) - (F355-V366) - (T502-I516)].

Next, an expression system in Escherichia coli was constructed using a vector in which each epitope was fused with a ferritin protein using a gene recombination technique (Fig. 1). Using these, protein nanoparticles expressing important antigenic epitopes capable of binding anit-HIV, anti-HCV, and anti-HAV antibodies to the surface of protein nanoparticles were prepared, respectively. As a result, it was confirmed that the protein nanoparticles were expressed in a water-soluble state in E. coli and purified by Ni2 + -NTA affinity chromatography (FIG. 2). Further, the purified protein nanoparticles were photographed using a transmission electron microscope (TEM), and it was confirmed that spherical protein nanoparticles were formed (FIG. 3).

That is, in one embodiment of the present invention, the ease of mass production of protein nanoparticles in which the AIDS, hepatitis C and hepatitis A specific epitopes are fused and expressed on the surface of the nanoparticles, uniform particle size distribution, epitope density / The ease and stability of structural / directional control were confirmed.

In addition, when the protein nanoparticle probe prepared in the present invention is immobilized on a lateral flow assay (LFA) strip, since the epitope of specific disease markers is expressed while maintaining a perfect three-dimensional morphology, The sensitivity and the specificity of the simultaneous detection of the hyperglycemia can be realized.

That is, in another embodiment of the present invention, a sample pad; Conjugation pads; Membrane; Transmission electron microscopy and dynamic light scattering analysis of staphylococcus aureus- derived protein A and colloidal gold nanoparticles were performed to calculate the optimum signal molecular weight for the lateral flow assay strip (FIG. 4) (Fig. 5 and Fig. 6) were immobilized on a conjugation pad. Each of the disease-specific protein nanoparticle probes was immobilized on the membrane by forming test lines, and immobilized on all human antibodies (Control line) with an anti-human secondary antibody derived from goat. It was confirmed that the detection sensitivity of AIDS, hepatitis C and hepatitis A markers of the LFA strip prepared in this manner was drastically increased as compared with that of the peptide probe (FIG. 7), and serum samples of 20 patients with disease- It was confirmed that the accuracy of the LFA strip was superior to that of the LFA strip containing the peptide probe (FIG. 8 to FIG. 14). It was confirmed by using one LFA strip that two diseases or three diseases were simultaneously detected with high accuracy and sensitivity (Fig. 15 to Fig. 18).

Thus, in one aspect, the present invention provides a semiconductor device comprising: a sample pad; A conjugation pad in which signal molecules are planted; A membrane comprising a protein nanoparticle probe having a first disease marker specific epitope fused and expressed on a surface of a self-assembly protein; And an absorbent pad.

In the present invention, the disease can be any viral disease regardless of the type, but is preferably selected from the group consisting of AIDS, hepatitis C and hepatitis A, and the first disease marker and second disease marker and 3 disease markers are different from each other.

In the present invention, the disease of the first disease marker-specific epitope may be AIDS, and the AIDS marker-specific epitope may be any epitope recognized by the AIDS antibody, preferably gp41 and p24 And the like.

In the present invention, the disease of the second disease marker-specific epitope may be hepatitis C, and the hepatitis C marker-specific epitope may be any epitope recognized by the hepatitis C antibody, And preferably selected from the group consisting of c22p, c33p, 511p, c100p and an epitope in which they are fused two or more times.

In the present invention, the two or more fused epitopes are selected from the group comprising c22p-c33p, c22p-511p, c22p-c100p, c33p-511p, c33p-c100p, 511p-c100p and c22p-511p-c100p .

In the present invention, the disease of the third disease marker-specific epitope may be hepatitis A, and the hepatitis A marker-specific epitope may be any epitope recognized by the hepatitis A antibody, Preferably, E1 [(E64-F83) - (G289-Q308) - (D572-F591) - (V779-K829)], E2 [(K961-Q980) - (S1421- (V1719-Q1738)] and E3 [(G119-G130) - (F355-V366) - (T502-I516)].

In the present invention, the self-assembly protein can be used without limitation as long as it can self-assemble in a cell. Preferably, the self-assembly protein is a human ferritin heavy chain protein. have.

In the present invention, the protein nanoparticles may have a diameter of 1-20 nm.

In the present invention, the protein nanoparticle probe in which the first disease marker-specific epitope is fused and expressed on the surface of the self-assembly protein is characterized in that the first disease marker-specific epitope is expressed by the fusion protein nano- And a particle probe. That is, when the disease of the first disease marker-specific epitope of the present invention is AIDS according to an embodiment of the present invention, the protein nanoparticle probe of the present invention is a protein nanoparticle probe in which gp41 expressed protein nanoparticle and p24 expressed protein nanoparticle May be mixed in a ratio of 5: 5.

In the present invention, the protein nanoparticle probe in which the second disease marker-specific epitope is fused and expressed on the surface of the self-assembly protein is characterized in that the second disease marker-specific epitope is expressed by fusion protein nano- And a particle probe. That is, when the disease of the second disease marker-specific epitope of the present invention is hepatitis C according to an embodiment of the present invention, the protein nanoparticle probe of the present invention is a protein nanoparticle probe in which HCV1 expressed protein nanoparticle and HCV2 expressed protein Nanoparticles may be mixed in a ratio of 7: 3.

In the present invention, the protein nanoparticle probe in which the third disease marker-specific epitope is fused and expressed on the surface of the self-assembly protein includes a protein nano-protein having different third disease marker-specific epitopes, And a particle probe. That is, when the disease of the third disease marker-specific epitope of the present invention is hepatitis A according to an embodiment of the present invention, the protein nanoparticle probe of the present invention is a protein nanoparticle probe comprising E1-labeled protein nanoparticles and E2- Nanoparticles and protein nanoparticles to which E3 has been expressed in a ratio of 3: 3: 3.

In the present invention, the protein nanoparticle probe may be characterized in that different protein nanoparticles having a disease marker-specific epitope fused and expressed on the surface thereof are mixed at a ratio of 1: 9 to 9: 1.

In the present invention, the membrane may further include not only three kinds of protein nanoparticle probes in which a disease marker specific epitope is fused and expressed on the surface of a self-assembly protein, but also four or more kinds thereof .

In the present invention, the signal molecule may be protein A-colloidal gold nanoparticle or protein G-colloidal gold nanoparticle.

In the present invention, the protein nanoparticle probe may be immobilized on the membrane to form a test line, and may further include a control line on which the control antibody is immobilized. can do.

The present invention also provides a semiconductor device comprising: (a) a sample pad; A conjugation pad in which signal molecules are planted; A protein nanoparticle probe in which a first disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A protein nanoparticle probe in which a second disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A membrane comprising a protein nanoparticle probe having a third disease marker specific epitope fused and expressed on a self-assembly protein surface; Reacting a sample fluid to be detected with a side flow assay kit comprising an adsorption pad and an adsorption pad; And (b) measuring chromogenic light of the signal molecule using a visual or densitometer. The present invention also relates to a method for simultaneous detection of multiple diseases using a lateral flow assay.

In the present invention, when a disease-specific antibody marker is present in the sample to be detected, the antibody binds to a signal molecule, protein A-colloidal gold, and is detected on a protein nanoparticle probe immobilized on a test line of a nitrocellulose membrane And red color appears by the signal molecule. On the other hand, since the signal molecule binds not only to the disease-specific antibody but also to other antibodies present in the sample, it also binds to the control line on which the anti-human secondary antibody is immobilized, resulting in red color, When no specific antibody is present, no red line appears on the test line, and only a red line appears on the test line. In the present invention, the presence or absence of a disease marker-specific antibody was determined by the presence or absence of a red line appearing on the test line and the control line 30 minutes after the sample to be detected was introduced into the sample pad and the intensity of the red line was quantified (GS-800 Calibrated Densitometer, BIO-RAD Inc., Hercules, California, USA) analyzer and image analysis software (Quantity One, BIO-RAD Inc., Hercules, California) , USA) was used to convert the red line intensity to a digitized signal intensity.

In the present invention, the disease marker-specific epitope of step (a) may be any viral disease regardless of the type thereof, but is preferably selected from the group including AIDS, hepatitis C and hepatitis A .

As described above, in the present invention, a protein nanoparticle probe in which a sensitive antigenic epitope is expressed on the surface of protein nanoparticles based on human ferritin heavy chain, which is useful for antibody measurement, is prepared, and it is concluded to simply detect various kinds of antigenic epitopes However, a probe capable of effectively controlling the expression frequency according to the importance of antigenicity was prepared by varying the mixing ratio. In the present invention, the inventors have developed a method for simultaneously detecting highly sensitive multiple disease markers by effectively fusing the above-described protein nanoparticle probes with the LFA system and utilizing the same for simultaneous detection of multiple diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 : Diagnosis of virus infectious diseases Epitope Production of expression vector for biosynthesis of expressed protein nanoparticles

1-1. For the diagnosis of viral infectious diseases Epitope selection

A variety of literature studies and previous studies have identified gp41 (I580 S613) peptide in HIV envelope glycoprotein gp160 and p24 (P166 G354) protein in HIV gag protein as epitopes for AIDS diagnosis. For the diagnosis of hepatitis C, (I1694-L1735) and c100p (I1920 V1935) peptides or proteins in NS4, c22c (K10-S53), non-structural proteins in NS3 As an epitope. Finally, peptide epitopes derived from HAV surface antigens 3A, 3B, 3C, 2B and 2C were selected for the diagnosis of hepatitis A, and three types of probes, which are combinations of these peptides, were selected (epitope 1 (E1) : [(E64-F83) - (G289-Q308) - (D572-F591) - (V779-K829)], epitope 2 (E2): [(K961-Q980) - (S1421- ) - (V1719-Q1738), epitope 3 (E3): [(G119-G130) - (F355-V366) - (T502-I516)].

Epitope for AIDS diagnosis:

gp41 (I580 S613), p24 (P166 G354)

Epitope for the diagnosis of hepatitis C:

C22p (K10 S53), C33c (A1192 C1457), 5-1-1p (I1694 L1735), C100p (I1920 V1935)

Epitope for the diagnosis of hepatitis A:

E1: [(E64-F83) - (G289-Q308) - (D572-F591) - (V779-K829)

E2: [(K961-Q980) - (S1421-K1449) - (H1500-Q1519) - (V1719-Q1738)

E3: [(G119-G130) - (F355-V366) - (T502-I516)]

1-2. Production of clones for diagnosis of viral infectious diseases

By using PCR or assembly PCR using the primers shown in Table 1 below, the following gene clones for diagnosis of diseases were obtained,

- human heavy chain ferritin (hFTH) monomer Gene clone: NH ₂ -NdeI- (hFTH) -G ₃ SG ₃ TG ₃ SG ₃ (linker) -XhoI-COOH,

Epitope gene clones for AIDS diagnosis:

_{NH 2 -XhoI- (gp41) 3 -H} 6 -ClaI-COOH ( hereinafter _{HIV1), NH 2 -XhoI-p24} -H 6 -ClaI-COOH ( hereinafter HIV2)

Epitope clone for the diagnosis of hepatitis C:

_{NH 2 -XhoI-c33c-H 6} -HindIII-COOH ( hereinafter HCV1),

NH ₂ -XhoI-511p-G ₃ SG ₃ TG ₃ SG ₃ -c100p-c22p-H ₆ -ClaI-COOH (hereinafter referred to as HCV2)

Epitope gene clone for the diagnosis of hepatitis A:

_{NH 2 -XhoI- (E1) 2 -H} 6 -ClaI-COOH ( hereinafter HAV1),

NH ₂ -Xhol- (E ₂ ) ₂ -H ₆ -ClaI-COOH (hereinafter referred to as HAV 2),

NH ₂ -XhoI- (E 3) ₂ -H ₆ -ClaI-COOH (hereinafter HAV3).

The gene clone was synthesized through a series of ligation in plasmid pT7-7 to produce a fusion protein in which carboxyl terminus of human heavy chain ferritin fused an epitope for diagnosis of each viral infectious disease. The plasmid expression (Fig. 1).

Protein nanoparticle probe expression vectors for AIDS diagnosis: pT7-hFTH-HIV1, pT7-hFTH-HIV2,

Protein nanoparticle probe expression vector for diagnosis of hepatitis C virus: pT7-hFTH-HCV1, pT7-hFTH-HCV2,

Protein nanoparticle probe expression vector for hepatitis A diagnosis: pT7-hFTH-HAV1, pT7-hFTH-HAV2, pT7-hFTH-HAV3,

All plasmid expression vectors were sequenced by complete DNA sequencing after gel - purification.

1-3. human Ferritin Heavy chain Production of Expression Vector for Protein Nanoparticle Synthesis

(Primer sequence 1 containing restriction enzyme NdeI) and XhoI and linker sequence (amino acid sequence G3SG3TG3SG3) using the human ferritin heavy chain gene sequence (NCBI Nucleotide accession number: 130-681 sequence in M97164 sequence) PCR was carried out using SEQ ID NOs: 2 and 3. As a result, a PCR product composed of 5'-NdeI- (hFTH) -G ₃ SG ₃ TG ₃ SG ₃ (linker) -XhoI-3 'was obtained. The amplified PCR product was digested with restriction enzymes NdeI and XhoI and inserted into the restriction enzymes NdeI and XhoI of pT7-7 (Novagen, USA) and named pT7-hFTH.

1-4. Production of Expression Vector for Protein Nanoparticle Synthesis for AIDS Diagnosis

1) gp41 (I580 S613) (NCBI nucleotide accession number: 7507-7609 sequence in NC 001802 sequence) site, the XhoI restriction enzyme cleavage site (CTCGAG) at the amine terminus and the EcoRI restriction enzyme cleavage site at the carboxyl terminus Gp41 epitope with GAATTC was synthesized by assembly PCR. A total of four primers (Primers SEQ ID NO: 4, 5, 6, 7) a respective buffer for DNA polymerase, including reaction by 50 pmol (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH 4) 2 SO 4; 20 mM Tris -HCl (pH8.8); 2 mM MgSO 4; 0.1% Triton X-100) in the Taq DNA polymerase for 94 ℃ / 1 minute 30 seconds given put (denaturation), 54 ℃ / 2 minutes (annealing), 72 ℃ / 3 min (elongation), and PCR was performed using primers 8 and 9 as a template for the assembly PCR product to finally obtain a PCR product of XhoI-gp41-EcoRI. In the present invention, in order to continuously position the gp41 epitope, PCR was performed using primers 10 and 11. As a result, the terminal of the amine was cleaved with hexahistidine (H6), termination codon (Stop) and ClaI restriction The gene containing the enzyme cleavage site was synthesized (EcoRI-gp41-H6-stop-ClaI). The two clones thus synthesized were sequentially ligated to an expression vector containing pT7-hFTH synthesized in advance. As a result, an expression vector of pT7-hFTH-XhoI-gp41-EcoRI-gp41-H6-stop- . In the present invention, in order to maximize the detection effect of the disease marker, the EcoRI site of pT7-hFTH-XhoI-gp41-EcoRI-gp41-H6-stop-ClaI was transformed to insert one gp41 epitope, GGAGGC-gp41-H6-stop-ClaI gene clone was obtained by replacing the restriction enzyme cleavage site with another nucleotide sequence (GGAGGC). This gene clone was subjected to PCR using primers 10 and 11 having an EcoRI restriction site at the amine terminus and a ClaI restriction enzyme cleavage site at the carboxyl terminus. EcoRI-gp41-GGAGGC-gp41-H6- gp41-H6-stop-ClaI gene was obtained by treating pT7-hFTH-XhoI-gp41-EcoRI-gp41-H6-stop-ClaI expression vector with EcoRI and ClaI. Finally, EcoRI-gp41-GGAGGC- And finally, pT7-hFTH-XhoI-gp41-EcoRI-gp41-GGAGGC-gp41-H6-stop-ClaI expression vector was obtained.

2) p16 (P166 G354) (NCBI nucleotide accession number: 831-1397 sequence of NC 001802) as a template, primer sequence 12 containing XhoI restriction enzyme cleavage site, hexahistidine (H6), termination codon stop and ClaI restriction enzyme cleavage site to obtain a gene clone of XhoI-p24-H6-stop-ClaI. This was ligated to an expression vector containing pT7-hFTH, resulting in the expression vector of pT7-hFTH-XhoI-p24-H6-stop-ClaI.

1-5. Production of expression vector for protein nanoparticle synthesis for hepatitis C diagnosis

1), primer sequence 14 containing an XhoI restriction enzyme cleavage site, hexahistidine (H6), stop codon stop (SEQ ID NO: 1) containing the XhoI restriction enzyme site using the c33c (A1192 C1457) (NCBI nucleotide accession number: 3915-4712 sequence in M62321 sequence) ) And HindIII restriction enzyme cleavage site, and a clone of XhoI-c33c-H6-stop-ClaI gene was obtained. This was ligated to an expression vector containing pT7-hFTH, resulting in the expression vector pT7-hFTH-XhoI-c33c-H6-stop-ClaI.

2) NH3-511p-G ₃ SG ₃ TG ₃ SG ₃ -c100p-c22p-H ₆ -ClaI-COOH, 511p-G ₃ SG ₃ TG ₃ SG ₃ -c100p and c22p-H6 Were synthesized by assembly PCR. First, to synthesize 511p-G ₃ SG ₃ TG ₃ SG ₃ -c100p, assembly PCR was carried out using primer sequence 16-21, followed by primer sequence 22 containing XhoI restriction enzyme cleavage site, using the primer sequence 23, which contains the EcoRI restriction enzyme cleavage site, it was synthesized _{XhoI-511p-G 3 SG 3} TG 3 SG 3 -c100p-EcoRI. In order to synthesize c22p-H6, assembly PCR was performed using primers 24-27, totaling four primers. Primer sequence 28 containing an EcoRI restriction site, hexahistidine (H6), and termination codon stop) and a ClaI restriction enzyme cleavage site to obtain EcoRI-c22p-H6-stop-ClaI clone. In the two kinds of expression vectors that include a gene clone _{(XhoI-511p-G 3 SG} 3 TG 3 SG 3 -c100p-EcoRI, EcoRI-c22p-H6-stop-ClaI) to pT7-hFTH obtained above was sequentially ligated, finally, to prepare a pT7-hFTH-XhoI-511p- G 3 SG 3 TG 3 SG 3 -c100p-c22p-H6- stop-ClaI expression vector.

1-5. Production of expression vector for protein nanoparticle synthesis for hepatitis A diagnosis

In order to synthesize epitope 1 (E1) [(E64-F83) - (G289-Q308) - (D572-F591) - (V779-K829)], epitope sequences were encoded, , And the assembly PCR product was subjected to PCR using the primer sequences 38 and 39 to obtain the XhoI-E1-EcoRI gene clone. Respectively. PCR was carried out using primers 40 and 41 using this as a template to obtain EcoRI-E1-H6-stop-ClaI gene. The obtained two gene clones (XhoI-E1-EcoRI and EcoRI-E1-H6-stop-ClaI) were sequentially ligated to an expression vector containing pT7-hFTH and finally pT7-hFTH-XhoI-E1- H6-stop-ClaI expression vector.

In order to synthesize epitope 2 (E2) [(K961-Q980) - (S1421-K1449) - (H1500-Q1519) - (V1719-Q1738)], epitope sequences were coded, And the assembly PCR product was subjected to PCR using the primer sequences 48 and 49 to obtain XhoI-E2-EcoRI gene clones. Respectively. PCR was carried out using the primers 50 and 51 as a template, and the EcoRI-E2-H6-stop-ClaI gene was also obtained. The obtained two gene clones (XhoI-E2-EcoRI and EcoRI-E2-H6-stop-ClaI) were sequentially ligated into an expression vector containing pT7-hFTH, and finally pT7-hFTH-XhoI-E2- H6-stop-ClaI expression vector.

In order to synthesize epitope 3 (E3) [(G119-G130) - (F355-V366) - (T502-I516)], the epitope sequence is encoded, and in consideration of Codon usage, The PCR product was then subjected to PCR using the primer sequences 56 and 57 to obtain an XhoI-E3-EcoRI gene clone. PCR was carried out using primers 58 and 59 as a template to obtain EcoRI-E3-H6-stop-ClaI gene. The obtained two gene clones (XhoI-E3-EcoRI and EcoRI-E3-H6-stop-ClaI) were sequentially ligated to an expression vector containing pT7-hFTH and finally pT7-hFTH-XhoI-E3- H6-stop-ClaI expression vector.

Primer sequence SEQ ID NO: Base sequence SEQ ID NO: 1 5'-CATATGATGACGACCGCGTCCACCTCG-3 ' SEQ ID NO: 2 5'-CTCGAGAGTGCCTCCCCCACTTCCGCCACCGCTTTCATTATC-3 ' SEQ ID NO: 3 5'-CTCGAGCCCACCGCCGCTGCCACCTCCAGTGCCTCCCCCACT -3 ' SEQ ID NO: 4 5'-CTCGAGATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA-3 ' SEQ ID NO: 5 5'-GAGCAACCCCAAATCCCCAGGAGCTGTTGATCCTTTAGGTATCTTTCCAC-3 ' SEQ ID NO: 6 5'-GGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGC-3 ' SEQ ID NO: 7 5'-GAATTCACTAGCATTCCAAGGCACAGCAGTGGTGCAAAT-3 ' SEQ ID NO: 8 5'-CTCGAGATCCTGGCTGTGG-3 ' SEQ ID NO: 9 5'-GAATTCACTAGCATTCCAAGGCA-3 ' SEQ ID NO: 10 5'-GAATTCATCCTGGCTGTGG-3 ' SEQ ID NO: 11 5'-ATCGATTTAGTGATGGTGATGGTGATGACTAGCATTCCAAGGCA-3 ' SEQ ID NO: 12 5'-CTCGAGCCAGAAGTGATACCC-3 ' SEQ ID NO: 13 5'-ATCGATTTAGTGATGGTGATGGTGATGTCCTACTCCCTGACAT-3 ' SEQ ID NO: 14 5'-CTCGAGGCGGTGGACTTTATCCCT-3 ' SEQ ID NO: 15 5'-AAGCTTTTAGTGATGGTGATGGTGATGACACGTATTGCAGTCTATCACCGAGTC-3 ' SEQ ID NO: 16 5'-CTCGAGATCATCCCCGATAGGGAAGTTCTCTACCAGGAGTTCGACGAG-3 ' SEQ ID NO: 17 5'-TGTTCGATGTAAGGGAGGTGTTGGGAACACTCCTCCATCTCGTCGAACTCCTGGTAG-3 ' SEQ ID NO: 18 5'-CACCTCCCTTACATCGAACAGGGAATGATGCTCGCCGAGCAATTCAAACAGAAGGCGC-3 ' SEQ ID NO: 19 5'-CTCCAGTGCCTCCCCCACTTCCGCCACCCAACCCGAGCGCCTTCTGTTTGAATTGC-3 ' SEQ ID NO: 20 5'-GGGGGAGGCACTGGAGGTGGCAGCGGCGGTGGGATAGCGTTCGCCTCGCGGGG-3 ' SEQ ID NO: 21 5'-GAATTCCACATAGTGCGTGGGGGAGACGTGGTTACCCCGCGAGGCGA-3 ' SEQ ID NO: 22 5'-CTCGAGATCATCCCCGATAGG-3 ' SEQ ID NO: 23 5'-GAATTCCACATAGTGCGTGGG-3 ' SEQ ID NO: 24 5'-AAAAACAAACGTAACACCAACCGCCGCCCACAGGATATTAAGTT-3 ' SEQ ID NO: 25 5'-TAAACTCCACCAACGATCTGACCACCGCCCGGGAACTTAATATCCTGTGGGCGGC-3 ' SEQ ID NO: 26 5'-GTCAGATCGTTGGTGGAGTTTACTTGTTGCCGCGCAGGGGCCCCAGGTTGG-3 ' SEQ ID NO: 27 5'-GGAAGTCTTCCTAGTCGCGCGCACACCCAACCTGGGGCCC-3 ' SEQ ID NO: 28 5'-GAATTCAAAAACAAACGTAACACCAACCG-3 ' SEQ ID NO: 29 5'-ATCGATTTAGTGATGGTGATGGTGATGGGAAGTCTTCCTAGTCGCG-3 ' SEQ ID NO: 30 5'-CTCGAGGAACCTTTGAGAACCTCTGTTGATAAACCTGGTTCTAAGAAGACTCAG-3 ' SEQ ID NO: 31 5'-TGTCCAAGTAGTAAAATGAGTAATTTTAATTCCACCGAACTTCTCCCCCTGAGTCTTCTTAGAACCAGGT-3 ' SEQ ID NO: 32 5'-GAATTAAAATTACTCATTTACTACTTGGACATCCATTCCAACCTTGGCTGCTCAGGATCACATGTCTATT-3 ' SEQ ID NO: 33 5'-GAACGTAAAAGTACACAGAAAATGAGACCTTCCCATGAATTTATAAATAGACATGTGATCCTGAGCAGC-3 ' SEQ ID NO: 34 5'-TCTCATTTTCTGTGTACTTTTACGTTCGTGGATGATCCTAGATCAGAGGAGGACAAAAGATTTGAGAG-3 ' SEQ ID NO: 35 5'-CCTCCAATCTCAATTCTTTGTATGGTTTCCTACATTCTATATGACTCTCAAATCTTTTGTCCTCCTC-3 ' SEQ ID NO: 36 5'-CCATACAAAGAATTGAGATTGGAGGTTGGGAAACAAAGACTCAAATATGCTCAGGAAGAGTTGTCAAAT-3 ' SEQ ID NO: 37 5'-GAATTCTTTCCTAGGAGGTGGAAGCACTTCATTTGACAACTCTTCCTGAGCATATTTG-3 ' SEQ ID NO: 38 5'-CTCGAGGAACCTTTGAGAACCTCTG-3 ' SEQ ID NO: 39 5'-GAATTCTTTCCTAGGAGGTGGAAG-3 ' SEQ ID NO: 40 5'-GAATTCGAACCTTTGAGAACCTCTG-3 ' SEQ ID NO: 41 5'-ATCGATTTAGTGATGGTGATGGTGATGTTTCCTAGGAGGTGGAAG-3 ' SEQ ID NO: 42 5'-CTCGAGAAAATCAATTTGGCAGATAGAATGCTTGGATTGTCTGGAGTTCAGGAAATTAA-3 ' SEQ ID NO: 43 5'-CAGCAGAGTCTCATTATCGTCATCTGAAATTCCCTGAGATTGTTCTTTAATTTCCTGAACTCCAGACAATCC-3 ' SEQ ID NO: 44 5'-GATGACGATAATGACTCTGCTGTGGCTGAGTTTTTCCAGTCTTTTCCATCTGGTGAACCATCAAATTCT-3 ' SEQ ID NO: 45 5'-ATCTGCATCCAATTTAATCACCTGTTTGGGCTTAGTCACGCCATGTTTAGAATTTGATGGTTCACCAGATGG-3 ' SEQ ID NO: 46 5'-CAAACAGGTGATTAAATTGGATGCAGATCCAGTAGAGTCTCAGGTAGCAAAGTTGGTTACTCAAGAAATGTTTC-3 ' SEQ ID NO: 47 5'-GAATTCCTGACTTTCAATTTTCTTATCAATATTTTGAAACATTTCTTGAGTAACCAACTTTG-3 ' SEQ ID NO: 48 5'-CTCGAGAAAATCAATTTGGCAGATAG-3 ' SEQ ID NO: 49 5'-GAATTCCTGACTTTCAATTTTCTTATCAATATTTT-3 ' SEQ ID NO: 50 5'-GAATTCAAAATCAATTTGGCAGATAG-3 ' SEQ ID NO: 51 5'-ATCGATTTAGTGATGGTGATGGTGATGCTGACTTTCAATTTTCTTATCAATA-3 ' SEQ ID NO: 52 5'-CTCGAGGGTTTGTTGAGATACCATACGTATGCAAGATTTGGCTT-3 ' SEQ ID NO: 53 5'-CCTGGAAATCGAAGACAAGATCTCCCCTCCAGAAGCCAAATCTTGCATACGTATGG-3 ' SEQ ID NO: 54 5'-AGATCTTGTCTTCGATTTCCAGGTTACAGTTTCTACAGAGCAGAATGTTCCTGAT-3 ' SEQ ID NO: 55 5'-GAATTCTATGCCGACTTGGGGATCAGGAACATTCTGCTCTGTAG-3 ' SEQ ID NO: 56 5'-CTCGAGGGTTTGTTGAGATACC-3 ' SEQ ID NO: 57 5'-GAATTCTATGCCGACTTGGGG-3 ' SEQ ID NO: 58 5'-GAATTCGGTTTGTTGAGATACC-3 ' SEQ ID NO: 59 5'-ATCGATTTAGTGATGGTGATGGTGATGTATGCCGACTTGGGG-3 '

Example 2: human Ferritin Heavy chain Based three-dimensional protein nanoparticle Probe Biosynthesis

In order to prepare protein nanoparticles having respective epitopes for diagnosis of viral infectious diseases, Escherichia coli strain BL21 (DE3) [F-ompThsdSB (rB-mB-)] was transformed into the expression vector pT7-hFTH-HIV1, pT7- The method described by Hanahan (Hanahan D, DNA Cloning vol. 1) was performed by using pT7-hFTH-HAV2, pT7-hFTH-HCV1, pT7-hFTH-HCV2, pT7-hFTH-HAV1, pT7- .1 109-135, IRS press 1985). More specifically, calcium chloride (CaCl ₂₎ was transformed with each of the thermal shock method of the vector into E. coli BL21 (DE3) treatment, the ampicillin (ampicillin) is that the expression vector is transformed by culturing in medium containing ampicillin resistance , And then the colonies were inoculated in LB (Luria-Bertani) medium containing 100 mg / ml ampicillin, and then cultured in LB medium overnight at 37 ° C at 130 rpm Cultured. When the culture turbidity (OD ₆₀₀₎ of the culture reached 0.5 to 0.6, the expression of the recombinant gene was induced by adding IPTG (Isopropyl-β-D-thiogalactopyranoside) (1 mM). After incubation at 20 ° C for 16-18 hours, E. coli cultured was centrifuged at 4,500 rpm for 10 minutes to collect the cell lysate. Then, 7 ml of a lysis solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA ) And disrupted using an ultrasonic wave crusher (Branson Ultrasonics Corp., Danbury, CT, USA). After crushing, the mixture was centrifuged at 13,000 rpm for 10 minutes. Then, the supernatant and the insoluble aggregate were separated. The separated supernatant was used for purification according to Example 3.

Example 3: human Ferritin Heavy chain Based three-dimensional protein nanoparticle Probe refine

Among the recombinant proteins expressed in Example 2, self-assembled antigen epitope fusion protein nanoparticles were purified through two-step purification.

1) After performing Ni2 + -NTA affinity chromatography using histidine-nickel bond fusion with the recombinant protein, 2) using an ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, Mass.) Was used to change the aqueous solution containing the recombinant protein into PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4) Lt; / RTI > Details of each step are as follows.

1) Ni2 + -NTA affinity chromatography

E. coli cultured by the method of Example 2 was recovered and the cell pellet was resuspended in 7 mL of a lysis buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole) to purify the recombinant protein, After the cells were disrupted using a crusher, the crushed cell lysate was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was separated, and each recombinant protein was separated using Ni2 + -NTA column (Qiagen, Hilden, Germany) (Washing buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 80 mM imidazole / elution buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 200 mM imidazole).

2) buffer change and concentration

(Amicon Ultra 100K, Millipore, Billerica, Mass.) And centrifuged at 5,000 g for 10 minutes. The column top was washed with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4) and centrifuged at 5,000 g until 500 μl of solution remained in the column. This was repeated three times, then the final volume was adjusted to 1 ml, and the concentrated solution was analyzed by SDS-PAGE. A portion of the concentrated solution was mixed with 1: 4 with 5 x SDS (0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) and boiled for 10 minutes at 100 ° C. Well and developed for 2 hours at 125 V. The gel was stained with Coomassie staining method and then decolorized to confirm the purified protein nanoparticles as shown in FIG.

Implementation 4: Human Ferritin Heavy chain Based three-dimensional protein nanoparticle Of the probe Expression pattern and structure analysis

For the structural analysis of the recombinant protein nanoparticles purified in Example 3, recombinant protein nanoparticles were photographed using a transmission electron microscope (TEM). First, the unfiltered protein samples were naturally dried after carbon-coated copper electron microscopy grids and the electron microscope grids containing the naturally dried samples were incubated with 2% ( w / v) aqueous uranyl acetate solution for 10 minutes at room temperature, and washed 3-4 times with distilled water. The protein nanoparticle image was observed using a Philips Technai 120 kV electron microscope and it was confirmed that spherical nanoparticles were formed as shown in FIG.

Example 5: Protein A fusion Colloidal Gold nanoparticle (signal molecule) synthesis

Add 1.98 L of distilled water to 20 mL of hydrogen tetrachloraurate (III) trihydrate (HAuCl ₄ .3H ₂ O) (1% w / v) and heat to 100 ° C. Add 36 mL of sodium citrate solution The color changes from yellow to dark red. The color-changed solution was further boiled for 40-45 minutes, cooled to below 30 ° C until the temperature of the solution was lowered, and 100 mL of protein A (0.05 mg / mL / repligen, Waltham, USA ) Protein was added and mixed. Then 100 mL of a solution of bovine serum albumin (BSA) (10% w / v / Equitech-Bio, Inc., Texas, USA) and 100 mL of sodium dodecyl sulfate (SDS) (0.4% w / v / Sigma Aldrich Co. , MO, USA). The prepared protein A binding-colloidal gold synthesis solution was concentrated and applied to a conjugate pad, followed by drying at 50 ° C for 1 hour. The colloidal gold used in the invention has a diameter of 25 nm and was analyzed using a transmission electron microscope (TEM) and a dynamic light scattering (DLS) method. As a result, In order to optimize the amount of protein A bound to colloidal gold, UV-vis absorption spectra analysis was used. As a result, as shown in FIG. 6, The optimal amount was finally found to be 40 nM (Fig. 6).

Example 6: Protein nanoparticles Probe Produce

To construct the LFA system as shown in FIG. 4, a protein nanoparticle probe in which disease marker specific protein nanoparticles were mixed was prepared. That is, AIDS-specific protein nanoparticle probes were prepared by mixing 0.06 mM of protein nanoparticles expressing HIV-1 epitope on the surface and 0.06 mM of protein nanoparticles expressing HIV-2 on the surface. The hepatitis C specific protein The nanoparticle probes were prepared by mixing 0.015 mM of protein nanoparticles expressing the HCV-1 epitope on the surface and 0.06 mM of protein nanoparticles expressing the HCV-2 epitope on the surface, and the hepatitis A-specific protein nanoparticle probe 0.04 mM of protein nanoparticles expressing the HAV-1 epitope on the surface, 0.04 mM of protein nanoparticles expressing the HAV-2 epitope on the surface, and 0.04 mM of protein nanoparticles expressing the HAV-3 epitope on the surface.

Example 7: Side Fluid Assay (Lateral Flow Assay LFA )) Strip configuration and judgment

To construct the LFA system as shown in Figure 4, a sample pad (Cat. No. 8115 2250, Whatman, UK), a conjugation pad (Cat 8131 1250, Whatman, UK), a nitrocellulose membrane nitrocellulose membrane, Cat. G344469, Whatman, UK) and absorbent pad (Cat 8115 1250, Whatman, UK).

7-1. LFA Strip configuration

The sample pad was blocked with a blocking solution (5% w / v casein, 2% w / v BSA, and 0.1% w / v SDS) to reduce the nonspecific binding between the diagnostic probe and various molecules in the sample. Respectively. The sample to be detected is transferred from the sample pad to the conjugation pad and then to the colloidal gold, a signal molecule that was dried on the conjugation pad. In order to immobilize a wide variety of human-derived antibody, that is an Fc domain and a strong affinity of the antibody was immobilized a known Staphylococcus aureus-derived Protein A protein (staphylococcus aureus) in the colloidal gold surface, increasing the non-specific binding and dispersing Bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS) were also immobilized. Test lines and control lines for the detection of disease markers were prepared in nitrocellulose membranes. The test line was a line for confirming the presence or absence of disease in the actual patient, and the protein nanoparticles prepared in Example 6 (Goat anti-human secondary antibodies, Cat. 201-1006, KPL, Maryland, USA) were used to confirm the normal operation of the system. Was used as 0.5 μL / cm. Protein nanoparticle probes and anti-human secondary antibodies were immobilized on nitrocellulose membranes using BIODOT dispensers (XYZ platform, AirJet Quanti 3000 Kit, BioJet 3000 kit, BioDot, Inc., CA, USA) Lt; 0 > C for 4 hours. The absorptive pad is located at the end of the strip and absorbs the sample to be detected so that it can be moved by a capillary phenomenon.

7-2. Judgment of experimental results

If a disease-specific antibody marker is present in the sample to be detected, the antibody binds to the signaling molecule protein A-colloidal gold and then to the nitrocellulose membrane. The disease-specific antibody marker associated with such a signal molecule is detected in a protein nanoparticle probe immobilized on a test line of a nitrocellulose membrane and a red line is generated by a signal molecule (colloidal gold) . In addition, the anti-human secondary antibody, which binds to all human-derived antibodies, is immobilized on the control line, and a red line appears on the control line regardless of the presence or absence of the disease-specific antibody marker. In the present invention, the disease was judged by the presence of the red line appearing on the test line and the control line at the point of 30 minutes after the sample to be detected was introduced into the sample pad, and more accurate analysis and numerical value of the intensity of the red line Using a GS-800 Calibrated Densitometer (BIO-RAD, Inc., Hercules, Calif., USA) analyzer and analysis software (Quantity One, BIO-RAD Inc., Hercules, California, USA) , And the red line strength was converted into a numerical signal intensity. In addition, since the detection signal (intensity of red line) of the LFA system is dependent on the amount of signal molecules actually participated in the experiment, the relative intensity defined by the intensity ratio of the detection signal of the test line and the reference line (test line detection signal intensity / More accurate analysis was performed by introducing relative peak intensity (RPI).

Example 8: Peptibe Probe And three-dimensional protein nanoparticles Of the probe Comparison of detection limits

For comparison of the detection limit and sensitivity of the protein nanoparticle probe and the peptide probe, the same molar concentration of the peptide probe expressed in the protein nanoparticle probe (AIDS diagnostic peptide probe: (gp41) 3 (0.06 mM) + p24 (E1) 2 (0.04 mM), hepatitis A diagnostic peptide probe: 511p (0.015 mM) + c100p (0.015 mM) + c22p (0.015 mM) + c33c (0.06 mM) (E2) 2 (0.04 mM) + (E3) 2 (0.04 mM), the LFA system was constructed in the same manner as in Example 7, and used as a control group for detection limit and sensitivity analysis. Detection limits were analyzed by serial dilution, and 50 μL of patient serum samples were diluted by mixing with 50 μL of standard serum samples (Cat. H4522, Sigma-Aldrich Co., MO, USA) , A diluted sample of 2 to 128 times was prepared, 10 μL of a diluted serum sample was used for the experiment. The determination of the experimental results was carried out using a GS-800 Calibrated Densitometer analyzer and analysis software (Quantity One) as described in Example 7. As a result, The detection limit of the particle probe was found to be much better in all three disease models (AIDS, hepatitis C, hepatitis A) than the detection limit of the peptide probe (FIG. 7).

In other words, when the protein nanoparticle probe was used, the detection signal (RPI signal) gradually decreased as the dilution rate of the serum sample to be detected increased, while the detection signal was detected up to 64 times diluted serum sample in all three disease models, In the case of using a peptide probe, the detection signal was found to be 8 times that of the AIDS serum sample and 16 times of the dilution sample of the hepatitis C and hepatitis A sera samples. By utilizing protein nanoparticle probes, the inactivation, denaturation, and aggregation problems of probes, which have been pointed out as disadvantages of existing peptide probes, are solved. The probes are three-dimensionally expressed on the surface of protein nanoparticles uniformly while maintaining proper structure, And the detection limit of the existing peptide probe can be improved by maximizing the accessibility with the peptide probe.

Example 9: Single disease object Peptides Probe And three-dimensional protein nanoparticles Of the probe Sensitivity / Specificity Verification

First, the specificity of the LFA system constructed in the present invention was verified against 20 normal human serum samples before applying the serum samples of various viral infectious diseases. It was confirmed that no false-positive results were obtained by non-specific binding at all three kinds of detection lines (AIDS, hepatitis C and hepatitis A) (Fig. 14).

The sensitivity of the protein nanoparticle probe-based LFA system and the peptide probe-based LFA system were compared in serum samples from 20 patients with three types of viral infection diseases (AIDS, hepatitis C, and hepatitis A). In the experiment, 10 μL of the serum samples of each viral infectious disease confirmed patient were used, and the result was judged after 30 minutes. As a result, in the case of the protein nanoparticle probe-based LFA, all of the detection lines in all the patient samples of the three types of viral infections (AIDS, hepatitis C, and hepatitis A) were red and the sensitivity was 100% (Figs. 8 to 10).

On the other hand, in the case of peptide probe-based LFA, four patients (P1, P5, P12, P18) (sensitivity: 80%, Fig. 11) in serum samples of AIDS patients and two patients P5, P15) (sensitivity: 90%, Fig. 12) and 7 patients ((P1, P4, P7, P10, P11, P16, P18) It is confirmed that a false negative result is obtained in which no detection signal appears on the detection line.

Thus, protein-based nanoparticle probe-based LFA not only detected disease markers in all patients, but also showed that the respective detection signals (RPI signals) were also higher overall than peptide probe-based LFAs, It is a result of dramatically improving the directionality, density and accessibility of the probe, overcoming limitations such as inactivation, denaturation and coagulation phenomenon in the peptide of the existing peptide probe.

Example 10: Multiple diseases Target three-dimensional protein nanoparticle Of the probe Simultaneous diagnosis possibility verification

10-1. Duplex assay

To confirm the possibility of simultaneous diagnosis of multiple viral infectious diseases, sera from two or three kinds of AIDS, hepatitis C and hepatitis A patients were mixed in the same volume, and finally 10 μL of mixed sample was applied to the experiment . In order to confirm the possibility of simultaneous diagnosis of two diseases, three types of experiments were performed: AIDS-C hepatitis, AIDS-A hepatitis, Hepatitis C-Hepatitis A. The same volume of 20 serum samples of two types of diseases was mixed and applied to the protein nanoparticle probe-based LFA. As shown in FIGS. 15 to 17, the 20 mixed serum samples , The detection signal was only found in the test line corresponding to each disease, and it was confirmed that no false positive signal was detected for the disease not applied to the experiment. That is, no detection signal was shown in the hepatitis A test line of FIG. 15, the hepatitis C test line of FIG. 16, and the AIDS hepatitis test line of FIG. This indicates that the protein nanoparticle probe for disease diagnosis selectively detects only the target marker for the disease in question and has high specificity without cross-reactivity (sensitivity: 100%, specificity: 100%).

10-2. Triplex assay

To confirm the possibility of simultaneous diagnosis of three viral infectious diseases, a mixed serum sample of patients with three diseases was applied to protein nanoparticle probe-based LFA. Ten μL of mixed serum samples containing the patient's serum of the three diseases were mixed at the same volume. As a result of applying 20 mixed serum samples to the LFA, it was confirmed that detection signals were generated in all the test lines, thereby obtaining a sensitivity of 100% (FIG. 18). Thus, it was confirmed that the sensitivity, specificity, and simultaneous diagnosis of three viral infectious diseases can be diagnosed through the application of LFA to protein nanoparticle probes.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> A Method and Kit for simultaneous detection of multiple diseases based on 3D protein nanoparticle probes <130> P15-B250 <160> 59 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 1 <400> 1 catatgatga cgaccgcgtc cacctcg 27 <210> 2 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 2 <400> 2 ctcgagagtg cctcccccac ttccgccacc gctttcatta tc 42 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 3 <400> 3 ctcgagccca ccgccgctgc cacctccagt gcctccccca ct 42 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> 4 <400> 4 ctcgagatcc tggctgtgga aagataccta aaggatcaac a 41 <210> 5 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> 5 <400> 5 gagcaacccc aaatccccag gagctgttga tcctttaggt atctttccac 50 <210> 6 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> 6 <400> 6 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgc 47 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> 7 <400> 7 gaattcacta gcattccaag gcacagcagt ggtgcaaat 39 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 8 <400> 8 ctcgagatcc tggctgtgg 19 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 9 <400> 9 gaattcacta gcattccaag gca 23 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 10 <400> 10 gaattcatcc tggctgtgg 19 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 11 <400> 11 atcgatttag tgatggtgat ggtgatgact agcattccaa ggca 44 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 12 <400> 12 ctcgagccag aagtgatacc c 21 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> 13 <400> 13 atcgatttag tgatggtgat ggtgatgtcc tactccctga cat 43 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 14 <400> 14 ctcgaggcgg tggactttat ccct 24 <210> 15 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> 15 <400> 15 aagcttttag tgatggtgat ggtgatgaca cgtattgcag tctatcaccg agtc 54 <210> 16 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 16 <400> 16 ctcgagatca tccccgatag ggaagttctc taccaggagt tcgacgag 48 <210> 17 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> 17 <400> 17 tgttcgatgt aagggaggtg ttgggaacac tcctccatct cgtcgaactc ctggtag 57 <210> 18 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> 18 <400> 18 cacctccctt acatcgaaca gggaatgatg ctcgccgagc aattcaaaca gaaggcgc 58 <210> 19 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> 19 <400> 19 ctccagtgcc tcccccactt ccgccaccca acccgagcgc cttctgtttg aattgc 56 <210> 20 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> 20 <400> 20 gggggaggca ctggaggtgg cagcggcggt gggatagcgt tcgcctcgcg ggg 53 <210> 21 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> 21 <400> 21 gaattccaca tagtgcgtgg gggagacgtg gttaccccgc gaggcga 47 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 22 <400> 22 ctcgagatca tccccgatag g 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 23 <400> 23 gaattccaca tagtgcgtgg g 21 <210> 24 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 24 <400> 24 aaaaacaaac gtaacaccaa ccgccgccca caggatatta agtt 44 <210> 25 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 25 <400> 25 taaactccac caacgatctg accaccgccc gggaacttaa tatcctgtgg gcggc 55 <210> 26 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> 26 <400> 26 gtcagatcgt tggtggagtt tacttgttgc cgcgcagggg ccccaggttg g 51 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> 27 <400> 27 ggaagtcttc ctagtcgcgc gcacacccaa cctggggccc 40 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> 28 <400> 28 gaattcaaaa acaaacgtaa caccaaccg 29 <210> 29 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> 29 <400> 29 atcgatttag tgatggtgat ggtgatggga agtcttccta gtcgcg 46 <210> 30 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> 30 <400> 30 ctcgaggaac ctttgagaac ctctgttgat aaacctggtt ctaagaagac tcag 54 <210> 31 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> 31 <400> 31 tgtccaagta gtaaaatgag taattttaat tccaccgaac ttctccccct gagtcttctt 60 agaaccaggt 70 <210> 32 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> 32 <400> 32 gaattaaaat tactcatttt actacttgga catccattcc aaccttggct gctcaggatc 60 acatgtctat t 71 <210> 33 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> 33 <400> 33 gaacgtaaaa gtacacagaa aatgagacct tcccatgaat ttataaatag acatgtgatc 60 ctgagcagc 69 <210> 34 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> 34 <400> 34 tctcattttc tgtgtacttt tacgttcgtg gatgatccta gatcagagga ggacaaaaga 60 tttgagag 68 <210> 35 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> 35 <400> 35 cctccaatct caattctttg tatggtttcc tacattctat atgactctca aatcttttgt 60 cctcctc 67 <210> 36 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> 36 <400> 36 ccatacaaag aattgagatt ggaggttggg aaacaaagac tcaaatatgc tcaggaagag 60 ttgtcaaat 69 <210> 37 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> 37 <400> 37 gaattctttc ctaggaggtg gaagcacttc atttgacaac tcttcctgag catatttg 58 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 38 <400> 38 ctcgaggaac ctttgagaac ctctg 25 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 39 <400> 39 gaattctttc ctaggaggtg gaag 24 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 40 <400> 40 gaattcgaac ctttgagaac ctctg 25 <210> 41 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 41 <400> 41 atcgatttag tgatggtgat ggtgatgttt cctaggaggt ggaag 45 <210> 42 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> 42 <400> 42 ctcgagaaaa tcaatttggc agatagaatg cttggattgt ctggagttca ggaaattaa 59 <210> 43 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> 43 <400> 43 cagcagagtc attatcgtca tctgaaattc cctgagattg ttctttaatt tcctgaactc 60 cagacaatcc 70 <210> 44 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> 44 <400> 44 gatgacgata atgactctgc tgtggctgag tttttccagt cttttccatc tggtgaacca 60 tcaaattct 69 <210> 45 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> 45 <400> 45 atctgcatcc aatttaatca cctgtttggg cttagtcacg ccatgtttag aatttgatgg 60 ttcaccagat gg 72 <210> 46 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> 46 <400> 46 caaacaggtg attaaattgg atgcagatcc agtagagtct caggtagcaa agttggttac 60 tcaagaaatg tttc 74 <210> 47 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> 47 <400> 47 gaattcctga ctttcaattt tcttatcaat attttgaaac atttcttgag taaccaactt 60 tg 62 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 48 <400> 48 ctcgagaaaa tcaatttggc agatag 26 <210> 49 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 49 <400> 49 gaattcctga ctttcaattt tcttatcaat atttt 35 <210> 50 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 50 <400> 50 gaattcaaaa tcaatttggc agatag 26 <210> 51 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> 51 <400> 51 atcgatttag tgatggtgat ggtgatgctg actttcaatt ttcttatcaa ta 52 <210> 52 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 52 <400> 52 ctcgagggtt tgttgagata ccatacgtat gcaagatttg gctt 44 <210> 53 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> 53 <400> 53 cctggaaatc gaagacaaga tctcccctcc agaagccaaa tcttgcatac gtatgg 56 <210> 54 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 54 <400> 54 agatcttgtc ttcgatttcc aggttacagt ttctacagag cagaatgttc ctgat 55 <210> 55 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 55 <400> 55 gaattctatg ccgacttggg gatcaggaac attctgctct gtag 44 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 56 <400> 56 ctcgagggtt tgttgagata cc 22 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 57 <400> 57 gaattctatg ccgacttggg g 21 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 58 <400> 58 gaattcggtt tgttgagata cc 22 <210> 59 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 59 <400> 59 atcgatttag tgatggtgat ggtgatgtat gccgacttgg gg 42

Claims

Sample pad; A conjugation pad in which signal molecules are planted; A membrane comprising a protein nanoparticle probe having a first disease marker specific epitope fused and expressed on a surface of a self-assembly protein; And an absorbent pad.

Sample pad; A conjugation pad in which signal molecules are planted; A protein nanoparticle probe in which a first disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A membrane comprising a protein nanoparticle probe having a second disease marker specific epitope fused and expressed on a surface of a self-assembly protein; And an absorbent pad.

Sample pad; A conjugation pad in which signal molecules are planted; A protein nanoparticle probe in which a first disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A protein nanoparticle probe in which a second disease marker-specific epitope is fused and expressed on the surface of a self-assembly protein; A membrane comprising a protein nanoparticle probe having a third disease marker specific epitope fused and expressed on a self-assembly protein surface; And an absorbent pad.

4. The method according to any one of claims 1 to 3, wherein the disease is selected from the group comprising AIDS, hepatitis C and hepatitis A, and wherein the first disease marker, the second disease marker and the third disease marker are different Side Flow Assay Kit.

5. The lateral flow assay kit of claim 4, wherein the marker specific epitope of AIDS is selected from the group comprising gp41 and p24.

5. The side flow assay kit of claim 4, wherein the marker-specific epitope of hepatitis C is selected from the group consisting of c22p, c33p, 511p, c100p, and epitopes fused therewith.

The method of claim 6, wherein the two or more fused epitopes are selected from the group consisting of c22p-c33p, c22p-511p, c22p-c100p, c33p-511p, c33p-c100p, 511p-c100p and c22p-511p-c100p Characterized by a lateral flow assay kit.

5. The side flow assay kit of claim 4, wherein the marker-specific epitope of hepatitis A is selected from the group comprising E1, E2, and E3.

4. The lateral flow assay kit according to any one of claims 1 to 3, wherein the self-assembly protein comprises a human ferritin heavy chain protein.

The protein nanoparticle probe according to claim 1, wherein the first disease marker-specific epitope is fused and expressed on the surface of the self-assembly protein, wherein the first disease marker-specific epitope is fusion protein Lt; RTI ID = 0.0 > nanoparticle < / RTI > probes.

The protein nanoparticle probe according to claim 2, wherein the second disease marker-specific epitope is fused and expressed on the surface of the self-assembly protein, wherein the second disease marker-specific epitope is fusion protein Lt; RTI ID = 0.0 > nanoparticle < / RTI > probe.

The protein nanoparticle probe according to claim 3, wherein the third disease marker-specific epitope is fused and expressed on the surface of the self-assembly protein is a protein nano-particle probe in which different third disease marker- specific epitopes are fusion- Lt; RTI ID = 0.0 > nanoparticle < / RTI > probe.

The protein nanoparticle probe according to any one of claims 10 to 12, wherein the protein nanoparticle probe is a mixture of different protein nanoparticles in which a disease marker specific epitope is fused and expressed on the surface in a ratio of 1: 9 to 9: 1 Wherein the lateral flow estimation kit comprises:

The lateral flow assay kit according to any one of claims 1 to 3, wherein the signal molecule is protein A-colloidal gold nanoparticle or protein G-colloidal gold nanoparticle.

The method according to any one of claims 1 to 3, wherein the protein nanoparticle probe is immobilized on the membrane in a form of forming a test line, and further comprises a control line on which the control antibody is immobilized Wherein the first and second fluid flow passages are spaced apart from each other.

A method for simultaneous detection of multiple diseases using a lateral flow assay comprising the steps of:
(a) reacting the side flow assay kit of claims 1 to 3 with a sample to be detected; And
(b) measuring the chromogenic light of the signal molecule using the naked eye or a densitometer.

17. The method according to claim 16, wherein the chromogenic light is present on a test line by a signal molecule when the antibody against the disease marker is present in the sample and protein nanoparticles are bound thereto.