KR100253420B1 - Diagnostic method using purified protein expressed in E. coli as diagnostic antigen - Google Patents

Abstract

PURPOSE: Provided is a diagnosis method using a protein expressed in and purified from E. coli as a diagnosis antigen by reacting the antigen with human serum having antibodies from E. coli. CONSTITUTION: A diagnosis method using protein expressed in and purified from E. coli as an antigen for diagnosis is characterized by the next steps of: (i) obtaining extract of E. coli producing an antigen for diagnosis but having no vector that expresses a protein; (ii) mixing the extract with serum obtained from a patient to eliminate antibodies in the serum through antigen-antibody reaction; and (iii) reacting the serum with purified antigen as a recombinant protein manufactured in E. coli.

Description

Diagnostic method using purified protein expressed in E. coli as diagnostic antigen

Figure 1 shows the base sequence of the primers for the synthesis and amplification of nucleocapsid gene from Seoul virus R22.

2 is a schematic diagram illustrating the preparation of pET-sNP, an expression plasmid containing the nucleocapsid gene of Seoul virus R22.

3 shows a method for purifying nucleocapsid proteins expressed from E. coli containing pET-sNP, respectively.

4 is a polyacrylamide gel electrophoresis (a) and a Westin blotting photograph showing the purity of the transformants of each nucleoside seed protein expressed from E. coli BL (pET-sNT). b). The numbers on these pictures indicate the following:

1: molecular weight marker

2: solution suspended after precipitation with ammonium sulfate

3: the supernatant after centrifuging the crushed cells

4: flow through the column with phenyl thetarose

5: eluent of phenyl thetarose column

FIG. 5 confirmed the reactivity with sera of patients, normal subjects, and vaccinates via Western blot to confirm the possibility of use as a diagnostic antigen for nucleocapsid proteins produced and purified from Escherichia coli containing pET-sNP.

1. Size marker

2. Normal Serum

3. Patient Serum

4.-9. Vaccine Serum

Figure 6 is a result of Western blot to examine the reactivity with the nucleocapsid protein expressed and purified in E. coli after the antibody to E. coli-derived protein in human serum is removed by a pretreatment method using E. coli extract.

1. Size marker

2. Normal Serum Not Pretreated

3. Pretreated Serum

4. Pretreated Patient Serum

7 is an example of the results of a diagnostic test using real patient serum and normal serum using a diagnostic stick sample.

1. Patient Serum

2. Normal Serum

[Purpose of invention]

An object of the present invention is to eliminate inaccurate diagnosis by E. coli-derived impurity proteins in using antigens expressed in E. coli for the purpose of diagnosis through gene synthesis method.

[Technical Field of the Invention and Prior Art in the Field]

When the protein produced in Escherichia coli is used as a diagnostic antigen by using genetic recombination technology, the present invention is included in the purified diagnostic antigen and reacts with an antibody against E. coli-derived protein contained in human serum. The present invention relates to a method for removing an incorrect response by the present invention, and thus, due to such an incorrect reaction, the protein expressed in Escherichia coli is not suitable for use as a diagnostic antigen, and a high-purity purification should be preceded even if used. According to the present invention, the protein expressed in E. coli can be used as a diagnostic antigen without undergoing high purity purification.

[Technical Challenges to Invent]

Advances in genetic engineering have made it possible to produce certain proteins in several microorganisms, including E. coli. Among many microorganisms, Escherichia coli has been studied so far and is easy to cultivate, and many expression vectors have been developed and used for producing a large number of proteins including pharmaceuticals. Medicines administered to the human body require high-purity tablets because of their safety, including problems such as side effects when they contain impurities. In the case of a diagnostic antigen, it is not necessary to administer it directly to the human body, so safety is not taken into consideration. Purity is required beyond the drug administered. Therefore, it has been known that it is inappropriate to use a protein expressed in E. coli as a diagnostic antigen. Most people have indirect contact with E. coli in their daily lives, and therefore many people have antibodies to some proteins produced by E. coli. In general, an antigen antibody response may well occur even when a small amount of antigen is present when the specificity is high. Therefore, the production of specific proteins in E. coli through genetic engineering methods is very advantageous in terms of cost, yield, or ease of operation, but is not suitable for use as diagnostic antigen due to the presence of antibodies to E. coli-derived proteins in human serum. It was inappropriate. Of course, it is possible to purify the antigen with high purity to remove the antigen-antibody reaction of E. coli-derived protein, but this requires very high purification costs, lowers the purification yield, and it is almost impossible to completely remove E. coli-derived protein.

The present inventors focused on eliminating the reaction of a small amount of E. coli-derived protein contained in the purified antigen with an antibody against E. coli-derived protein contained in human serum in using a protein expressed in E. coli as a diagnostic antigen. As a result, the human serum to be diagnosed was solved through a kind of pretreatment. That is, a method for removing antibodies against E. coli-derived proteins present in the serum of a person to be diagnosed in advance, which is the same as the strain producing the antigen for diagnosis, but the extract solution of E. coli without the vector expressing the antigen for diagnosis and Antibodies to E. coli-derived proteins present in human serum were previously removed by antigen antibody reaction by mixing human serum to be diagnosed before the diagnostic test. The present invention has been completed by discovering that inaccurate diagnosis can be dramatically reduced or eliminated by reacting such pretreated human serum with purified diagnostic antigen.

[Configuration and Function of Invention]

Accordingly, the present invention is directed to an E. coli-derived antigen protein present in the serum of a patient by mixing an extract solution of E. coli that contains the same but not expressing a vector for expressing the diagnostic antigen protein. After the antibody is removed by an antigen-antibody reaction, the serum of the pretreated patient is reacted with a diagnostic antigen purified as a recombinant protein prepared in Escherichia coli.

In addition, the present invention is an E. coli-derived antigen protein present in the inoculator's serum by mixing E. coli's extract solution, which is the same as E. coli producing a diagnostic antigen, but does not contain a vector expressing the diagnostic antigen protein, and serum collected from the vaccine. The present invention provides a method for diagnosing the antibody formation of an inoculator by removing the antibody against the antibody by an antigen-antibody reaction and then reacting the serum of the pretreated inoculator with a diagnostic antigen purified as a recombinant protein prepared in Escherichia coli.

The present invention provides a method for removing inaccurate responses caused by E. coli-derived impurity proteins in using a protein expressed in E. coli as a diagnostic antigen. More specifically, the present invention purifies the use of nucleocapsid proteins of Seoul virus, Hantan virus, Fumala virus, or sinnobrivirus belonging to Hantavirus genus expressed in E. coli for the purpose of diagnosing nephrotic bleeding. Antibodies to E. coli-derived proteins contained in nucleocapsid proteins and to E. coli-derived proteins contained in human serum may actually be mistaken as patients even though they do not have antibodies to nucleocapsid proteins. It provides a method to exclude the possibility through the pretreatment of patient serum.

In addition, the present invention provides a diagnostic formulation for use in the above-mentioned diagnostic method of the present invention by adsorbing an antigenic protein and human immunoglobulin G on a nitrocellulose membrane. More specifically, the present invention provides a method for preparing a nephrotic bleeding hemorrhagic fever diagnostic agent in the form of a stick and applying the pretreatment method of the patient's serum to the actual diagnosis.

The present invention is a strain for the production of antigen for diagnosis of nephrotic bleeding hemorrhagic fever with the applicant Applicant deposited on September 9, 1996 with the Korean spawn association was granted accession number KFCC-10914 on September 16, 1996. E. coli transformant BL (pET-sNP) was used.

Three primers used for the synthesis of the nucleoside seed gene according to the present invention are shown in FIG. 1 and the cloning process of the nucleocapsid gene is shown in FIG. First, by carrying out two polymerase chain reactions using the three primers shown in FIG. 1, a sufficient amount of nucleocapsid genes for cloning into the plasmid was obtained, and these genes were assigned to plasmid pET-3a. Appropriate restriction enzyme recognition sites are added for cloning. That is, in the first polymerase chain reaction, the same amplification using two primers of NP1 and NP3, and in the second polymerase chain reaction, the gene for insertion into pET-3A contains NPg and EcORI recognition sites. Amplify using NP3.

Since the starting plasmid pET-3a used for recombination with the nucleocapsid gene comprises part of the s10 derived amino acid sequence, the nucleocapsid protein cloned and expressed in this plasmid is a fusion protein containing 14 more amino acids at the amino terminus. Expressed in form.

Cloning the nucleocapsid gene in plasmid pET-3a to produce the expression plasmid pET-sNP desired in the present invention is shown in FIG. In addition, each expression plasmid prepared according to this procedure was inserted into the host cell Escherichia coli BL21 (DE3) using an electric field shock, thereby transforming the E. coli to produce a large amount of the desired nucleocapsid protein. E. coli BL (pET-sNP) is obtained.

According to a conventional method used in the field of genetic engineering, the E. coli transformant is cultured in a suitable medium to express a large amount of protein, and the expressed protein is purified by performing the procedure shown in FIG. That is, a protein is obtained by a purification method using an ammonium, sulfate precipitation method, or a phenyl sepharose resin column, and polyacrylamide gel electrophoresis and western blotting are performed on the purified protein to nucleoside of the desired Seoul virus. Confirm that it is a capsid protein. 4 shows the results of electrophoresis and western blotting according to the present invention. Purity of the purified protein determined from the result of electrophoresis according to the present invention is 90% or more.

The molecular weight of the nucleocapsid protein obtained from the expression plasmid pET-sNP is about 49.5 kDa, which, as already mentioned above, is present in front of the BamHI site, the plasmid cloning site of pET-3a used in the construction of pET-sNP. This is due to the sequence. That is, the nucleocapsid protein produced from pET-sNP contains 14 more amino acids than the protein in its natural state.

Western blotting is performed using the serum of the patient and the vaccinates to investigate the diagnostic efficacy of the nucleocapsid protein of approximately 90% pure Seoul virus produced by the method described above. In addition to nucleocapsid protein positions of molecular weight 50,000, unwanted reactions are observed at positions of about 90,000 molecular weight. Inaccurate reactions are observed when such antigens are used as diagnostic antigens. In order to eliminate the reaction causing the inaccurate diagnosis, human blood is pretreated with an extract of Escherichia coli which is identical to the strain used for the production of the diagnostic antigen but does not contain an expression vector. The use of proteins can dramatically reduce this inaccurate response.

A sample of nephrotic bleeding hemorrhagic fever was prepared using the purified nucleocapsid protein and pre-treated with Escherichia coli extract in 70 human serum, and the diagnostic test was carried out in Vero E6 cells. 93% concordance with the indirect Immunofluorescent Antibody Assay (ELISA) and the Enzyme Linked Immuno Sorbent Assay (ELISA) method was used.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, this is intended to illustrate the invention and should not be understood as limiting the scope of the invention to these examples.

EXAMPLE

Example 1

Purification of RNA Genome and Synthesis of cDNA from Seoul Virus R22

Seoul virus R22 strain [Song Gan, etc., The Journal of Infectious Diseases Vol. 150, p889-894, 1984] was taken from solution A (4.2M guanidine isothiocyanate, 25 mM Tris-HC1 (pH 8.0), 0.5% Sarkosy1, 0.7% β-mercaptoethanol) Mix well with 400 μl. 50 μl of solution B (1M Tris-HC1 (pH 8.0), 0.1 EDTA, 10% SDS) was added thereto, mixed well, and dissolved at 65 ° C. for 5 minutes. The same volume of phenol / chloroform (1: 1) mixture was added to the mixed solution, and the mixture was left at 65 ° C. for 30 minutes, followed by centrifugation to take only the supernatant, and 300 µl of solution A was added to the remaining solution. Then centrifuged. The supernatant taken here was combined with the supernatant taken first, and then extracted once with phenol / chloroform (1: 1) mixture and once with chloroform. 1/10 volume of 3M sodium acetate and 2 volumes of isopropyl alcohol were added to the extracted solution, mixed well, and then stored at −20 ° C. for 16 hours.

It was precipitated by centrifugation at 12,000 rpm for 15 minutes in a microcentrifuge and the precipitate was washed twice with 70% ethyl alcohol and dried. The dried precipitate was dissolved in 10 μl of ribonuclease (RNase) in sterile water and then stored at 4 ° C. 5 μl of ribonucleic acid was synthesized and cDNA was synthesized in a total volume of 20 μl. The reaction conditions are as shown in Table 1 below and reacted at 37 ° C. for 1 hour.

TABLE 1

Example 2

Amplification of Nucleocapsid Gene by Polymerase Chain Reaction

Nucleocapsid genes for cloning to known plasmid pET-3a according to the present invention were amplified by polymerase chain reaction, respectively, as described below using the primers shown in FIG.

To amplify the nucleocapsid gene for cloning into plasmid pET-3a, first, 5 µl of the solution containing the cDNA of the nucleocapsid synthesized in Example 1 was taken and first amplified using primers NP1 and NP3. Since the desired nucleocapsid gene was not sufficient in the first reaction solution, 5 µl of the first reaction solution was taken and a second polymerase chain reaction was performed using primers NP2 and NP3.

At this time, the 5 'end of the primers NP2 and NP3 used contained restriction enzyme recognition sites of Bg1II (AGATCT) and EcoRI (GAATTC), respectively.

The device used for the polymerase chain reaction was a deoxyribonucleic acid temperature cycler (DNA thermal cycler 480) of PERKIN ELMER (PERKIN ELMER), the conditions of the polymerase chain reaction is shown in Table 2 below.

TABLE 2

Polymer Element Chain Reaction Solution Preparation and Procedure

Example 3

Construction of the Expression Plasmid pET-sNP

The cloning process of the nucleocapsid gene is shown in FIG. After confirming the size of the two-stranded DNA amplified according to the method of Example 2 in agarose gel electrophoresis, DNA fragments containing a 1.3kb nucleocapsid gene were recovered using a Ginclean kit. . The recovered DNA fragments were cloned into plasmid pET-3a, each containing a portion of the s10 derived amino acid sequence. Genes for cloning pET-3a in the recovered DNA fragments were double digested with restriction enzymes Bg1II and EcRI, extracted once with phenol / chloroform mixture (1: 1), and dissolved in final 20 μl of sterile distilled water. Meanwhile, the gene carrier pET-3a was double-digested with EcoRI and BamHI, and then heat-treated at 70 ° C. for 10 minutes in the presence of 5 mM EDTA, extracted once with a phenol / chloroform mixture (1: 1), and then in 20 μl of sterile distilled water. Dissolved. The construction of the plasmid pET-sNP according to the present invention was completed by mixing 5 µl of each of the truncated gene carriers and 10 µl of nucleocapsid DNA and conjugating by reacting for 3 hours at 25 ° C using T4 DNA ligase.

Example 4

Preparation of Transformant

E. coli transformation was performed using electric field bombardment, and pET-sNP was used as E. coli BL21 (DE3) as a host cell.

E. coli BL21 (DE3) was inoculated from agar plate medium in 20 ml of LBQOWL in order to obtain a large amount of transformed host cells and shaken for 18 hours at 37 ℃. This was inoculated in 1 liter of fresh LB medium and shaken at 37 ° C. until absorbance reached 0.5 to 0.8 at a wavelength of 600 nm. In order to recover the cells, the culture solution was left at 0 ° C. for 20 minutes and the cells were recovered by centrifugation. The recovered cells were washed once with 1 liter of cold sterile distilled water, and then again with 0.5 liter and finally with 20% glycerol. This was resuspended in 3 ml of 10% glycerol solution, aliquoted, stored at -70 ° C, and used one by one for each transformation. The instrument used in the electric field impact method was a Gene-Pulser manufactured by Bio-Rad, the transformation method is as follows. 40μl of cells stored at -70 ℃ and 2μl of deoxyribonucleic acid were mixed and placed in a cuvette with an electrode spacing of 0.2cm, and the Gene-Rulser was fixed at a capacitance of 25㎌, a resistance of 200Ω and an electric field of 12.5㎸ / ㎝. One electric field shock was shocked and immediately 1 ml of SOC medium (2% bactotriptone, 0.5% yeast extract, 10 mM NaC1, 2.5 mM MgC1 ₂ , 10 mM MgSO ₄ , 20 mM glucose) was added. After shaking for 1 hour at 37 ° C., 0.1 ml and 0.9 ml of each plate were added to two LB agar plates containing 50 μg / ml of ampicillin, followed by incubation for 12 to 18 hours in a 37 ° C. incubator. Screening of the recombinant plasmids used a cracking method of single colonies.

In other words, add 1 loop of cells from agar plate medium to 80 µl of cracking buffer (0.05M Tris, pH 6.8, 1% SDS, 2mM EDTA, 0.4M Scroose, 0.01% Bromophenol Blue) and suspend with a vortex mixer. After centrifugation at 12,000rpm for 15 minutes using a microcentrifuge, the supernatant was taken to identify pET-sNP plasmid which is a desired nucleocapsid gene carrier by agarose gel electrophoresis. In addition, screening was carried out by cutting and confirming the finally isolated recombinant plasmid with restriction enzymes.

Example 5

Culture of transformants

For the cultivation of Escherichia coli, glucose and empicillin were added to LB medium (0.5% yeast extract, 1% bactotrypton, 1% NaC1, pH 7.0) at 0.5% and 100µg / ml, respectively. Was inoculated at 5% in 1 liter of fresh medium and shaken at 37 ° C at 200 rpm. When the absorbance (A ₆₀₀ ) of the culture reached 0.5 to 0.8, IPTG was added to 0.1 to 2 mM and further cultured for 4 to 8 hours. The culture solution was centrifuged to recover the cells and washed once with 0.8% NaC1 solution.

Example 6

Isolation and Purification of Nucleocapsid Proteins

The method for purifying nucleocapsid protein expressed from Escherichia coli including pET-sNPFMF is shown briefly in FIG. The recovered cells were suspended in 50 to 100ml of TE buffer solution (50mM Tris, 1mM DETA, pH 8.0) and the cells were crushed using a sonicator. The degree of crushing was determined using the Bradford assay method. Was performed until the protein concentration of the lysate no longer increased. The supernatant was taken by centrifuging the crushed solution at 8,000 ⅹg for 1 hour. Ammonium sulfate was added to the supernatant at 25 to 505 in saturation concentration to dissolve, and the mixture was left at room temperature for 1 hour and then centrifuged at 8,000 8,000g for 30 minutes to discard the supernatant, and the precipitate was resuspended in 20 ml of TE buffer solution. . Dialysis was performed twice in 2 liters of buffer solution for 2 hours to remove ammonium sulfate in the resuspended solution. The dialyzed solution was first subjected to gel filtration using Sephacryl S200, manufactured by Pharmacia, and secondly purified using phenyl sepharose CL-4B resin. The loading of the sample was performed by adding ammonium sulfate to 0.6M in a solution of 40 mM KH ₂ PO ₄ / NA ₂ HPO ₄ , (pH 8.0), and the elution was 40 mM KH ₂ PO ₄ / Na ₂ HPO ₄ (PH 8.0). A solution of was used. At this time, most of the nucleocapsid protein flowed out through the flow (flow), and most of the other proteins derived from E. coli. This was dialyzed again with PBS buffer and centricon or centriprep manufactured by Amicon was used to concentrate the protein to the desired concentration. Purified protein at each step was confirmed by polyacrylamide gel electrophoresis and western blotting (see FIG. 4). In electrophoresis, the purity of the finally purified protein was determined to be greater than 90%. As the primary antibody of the western blot, a single group antibody ht9040 against nucleocapsid protein was used. At this time, PBS solution containing 5% of the skim milk (skim milk) for adhesion of the specific protein only (8g NaC1, 0.2g KC1, 1.44g Na ₂ HPO ₄ , 0.24g KH ₂ PO ₄ , pH per liter 7.4) and then reacted for 30 minutes with the above-mentioned primary antibody at room temperature for at least 1 hour. Membranes were washed three times for 5 minutes each with PBST solution (PBS + 0.5% Tween20) to remove unattached antibodies. The washed membrane was shaken for 1 hour with a mixture of PBS solution containing an appropriately diluted anti-mouse immunoglobulin G and 5% skim milk with peroxidase enzyme. Again, the membranes were washed three times with PBST solution for 5 minutes each, followed by a color developing reagent [25 ml of 0.1 M Tris (pH 7.4), 4-chloro-1-nathtol 25 mg / methanol), 3 μl of H ₂ O _2. 50 ml] was used for color development. When proper color appeared, the reaction was stopped and washed with distilled water. The color reaction was carried out to identify the desired nucleocapsid protein specific band (about 50 kD) at the expected position.

Example 7

Diagnostics using Western Blot of Purified Nucleocapsid Protein

Diagnosis of patients with renal hemorrhagic fever by antigen-antibody reaction with antibody to nucleocapsid protein in sera of renal hemorrhagic fever using Hantavirus nucleocapsid protein expressed in E. coli An experiment was conducted to investigate the possibility that the inoculator of the hemorrhagic fever vaccine could be used to determine whether the antibody had been properly formed. Vaccine sera were tested using samples taken one month after two doses of a commercial vaccine at monthly intervals. Purified nucleocapsid protein was subjected to electrophoresis on polyacrylamide gel and transferred to nitrocellulose membrane. Here, the primary antibody was used by diluting 100 times the serum of normal, patient or vaccinated serum, and the secondary antibody used peroxidase-attached goat's antihuman antibody G (peroxidase conjugate of goat anti-human IgG). Other conditions were carried out in the same manner as in Example 6. The results are shown in FIG. 5, and the reaction was clearly confirmed at the molecular weight of 5., 000 positions with the patient serum (No. 3). On the other hand, in normal subjects (No. 2), no response was observed at this position, so the possibility of use as an antigen for diagnosing nephrotic bleeding fever was confirmed. In this result, two other important facts can be observed. One of the six vaccinates (Nos. 4-9) formed two antibodies (Nos. 4 and 9) against the nucleocapsid protein, indicating that the antibody against hantavirus was normally formed after vaccination. It can be seen that no antibody is yet formed. Of course, the time required for vaccination antibody formation to be different may vary depending on the person, but at least, it is proved that the nucleocapsid protein expressed in Escherichia coli can be used as a material to determine whether the vaccine is formed. It is. Another fact found here is that antigen-antibody reactions between E. coli-derived proteins and human sera occur at a position of about 90,000 molecular weights in FIG. 1 (4 out of 8 sera). The need to remove this protein has emerged as it has the potential to be negative.

Example 8

Elimination of the reaction of E. coli-derived proteins contained in the purified antigen with antibodies in human serum

In order to remove the E. coli-derived protein using a variety of purification methods in order to remove the reaction with the popular bacteria-derived protein observed in Example 7, purification can completely remove the E. coli-derived protein that causes half the human serum There was no. Therefore, it is impossible to remove such E. coli-derived protein by purification, so that only E. coli host cells that do not express nucleocapsid protein are cultured to make an extract, and then reacted with patient serum beforehand. Antibodies that cause antigen-antibody reactions were previously removed. This method was so efficient that even if the purity of the nucleocapsid protein used for diagnosis was not high, the reaction with E. coli-derived protein was eliminated. Therefore, the nucleocapsid protein used herein is a nucleocapsid protein having a purity of less than about 50% performed only until the precipitation process using ammonium sulfate in the purification process of Example 6. Preparation of E. coli extract was also carried out until the precipitation using the same ammonium sulfate as the purification process of the nucleocapsid protein expressed in E. coli. The nucleocapsid protein precipitated with ammonium sulfate and E. coli extract were both resuspended in TE buffer, dialyzed twice with the same buffer, and concentrated to an appropriate concentration using an ultrafilter. The pretreated patient serum was reacted with the purified nucleocapsid protein to prevent the possibility of reaction with trace amounts of E. coli derived protein contained in the purified nucleocapsid protein. In the method, when Western serum was added to human serum as the primary antibody, 300 ug of E. coli extract was added to 10 ml PBS reaction solution containing 5% skim milk, and 10 μl of human serum was added simultaneously. The subsequent procedure was performed in the same manner as in Example 7. The results are shown in FIG. 6. In case 2, the normal serum was not pretreated with E. coli extract, but the reaction with E. coli-derived protein was confirmed at the molecular weight of 90,000, but the same serum was pretreated with E. coli extract (time 3). It was confirmed that this reaction was reduced. In addition, even when the patient serum was pretreated (No. 4), the reaction was confirmed at the molecular weight of 50,000, so that pretreatment of the serum of the serum can effectively remove antibodies against E. coli-derived proteins without significantly affecting the reactivity with the nucleocapsid protein. I could see that.

Example 9

Preparation and diagnosis of diagnostic sample in the form of stick

The efficacy of the Seoul virus nucleoside seed protein expressed in Escherichia coli as a diagnostic agent for nephrotic bleeding fever and the pretreatment of human serum by the use of E. coli extracts are effective in eliminating inaccurate reactions. Diagnostic stick samples were prepared using a device called Bio-Jet Dispensor from Dot (17781, Sky Park Circle, Irine, California 92714, USA). The material of the stick was a nitrocellulose membrane and the samples were coated with two proteins. One is a nucleocapsid protein of about 50% purity obtained from E. coli and obtained using ammonium sulfate to detect antibodies to the virus present in the serum of the patient, and the other is a normal reaction of the color system. Purified human immunoglobulin G was used to determine. 1 μl of nucleocapsid protein per stick was used and 0.1 μl of purified human immunoglobulin G. Diagnosis was performed by adding 50ul (3mg / ml) and 1ul of E. coli extracted protein to 1ml PBST solution containing 5% of skim milk, shaking at room temperature for 20 minutes, and then again using 1ml PBST solution. 2 min of peroxidase conjugated goat anti-human IgG with peroxidase attached to 1 ml PBST containing 5% skim milk It was added as an antibody and reacted again for 20 minutes. After washing again with 1 ml of PBST for 2-3 minutes, a color developing reagent [25 ml of 0.1M Tris (pH 7.4), 4-chloro-1-natol 25 mg / methanol), 3 µl / 50 ml of H ₂ O _{2 was} added. ] 1 ml was used for color reaction. When proper color appeared, the reaction was stopped and washed with distilled water. A photograph of the results of diagnosis using normal and patient serum using an actual diagnostic sample is shown in FIG. Two lines appear in the patient's serum, whereas only one line appears in the upper part of the serum of a normal person. In order to confirm the efficacy of this diagnostic agent, 70 human sera including normal subjects of the patient were tested and indirect immunofluorescent using Hantan virus as antigen for culture in Vero E6 cells. Antibody assay) and ELISA were performed to compare the results. Indirect immunofluorescent antibody was positive when the maximum dilution factor of fluorescent serum was 512 times or more and negative when it was 32 times or less. Elyza was diluted 50-fold with serum, and the serum showed higher absorbance than the average value obtained by adding absorbance at 450 nm using five sera determined as negative control by indirect immunofluorescence antibody method as a negative control. For positive, negative was determined. The results are shown in Table 3. Comparing the results, the results of the stick sample, except for serum 48 and 49, were almost 97% accurate, almost in agreement with the results of indirect immunofluorescent antibody / ELISA analysis.

TABLE 3

Diagnostic Results Using Diagnostic Stick Samples

Stick sample; +: Positive,-: negative, ±: uncertain

IFA / ELISA; +: All positive,-: all negative, x: uncertain

Claims (4)

Antigen-antibodies for E. coli-derived antigen proteins present in the serum of a patient are mixed with an E. coli extract solution that is the same as E. coli producing a diagnostic antigen but does not contain a vector expressing the diagnostic antigen protein. A method for diagnosing a human disease, characterized in that the serum of the pretreated patient after removal by the reaction is reacted with a diagnostic antigen purified as a recombinant protein prepared in Escherichia coli. The method of claim 1, wherein the recombinant protein is a viral recombinant protein of the Hantavirus genus. The method of claim 1, wherein the recombinant protein is a recombinant protein of Seoulvirus. Antibodies against E. coli-derived antigen proteins present in the inoculator's serum are mixed by mixing E. coli's extract solution, which is the same as E. coli that produces the diagnostic antigen, but does not contain a vector that expresses the antigenic protein. A method for diagnosing antibody formation of an inoculator, characterized in that the serum of the inoculated pretreatment after removal by the antibody reaction is reacted with a diagnostic antigen purified as a recombinant protein prepared in Escherichia coli.

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