WO2017095135A1 - Igm for early diagnosis of tsutsugamushi disease, igg antibody detection kit, and method for preparing same - Google Patents

Abstract

The present invention relates to a diagnostic kit for tsutsugamushi disease and a diagnostic method using the same. More particularly, the present invention can rapidly and accurately diagnose a tsutsugamushi disease patient by using a diagnostic kit for tsutsugamushi disease comprising a first kit and a second kit, wherein the first kit and the second kit each comprises mixed antigens in which 56kDa fusion protein (cr56) of Orientia tsutsugamusi Gilliam, Karp, and Kato and 56 kDa protein (kr56) of Orientia tsutsugamushi Kanwon are mixed at a volume ratio of 5:3.

Description

IGM, IGG Antibody Detection Diagnostic Kit for Early Diagnosis of Tsutsugamushi Disease and Method for Preparing the Same

The present invention relates to a Tsutsugamus disease diagnosis kit and a Tsutsugamus disease diagnosis method comprising a first kit and a second kit.

Tsutsugamus disease is caused by Orientia tsutsugamushi belonging to the Scrub typhus group of the genus Rickettsia, and is an acute febrile disease transmitted by tick larvae. About 10 days (1 to 3 weeks) after the bite of the tick larva, chills, fever, headache appear, about 1 week before the onset of the body and the rash spreading to the limbs appear. The bite of the mite larvae causes blisters, forming ulcers of the size of 0.5 to 0.8 cm, which are covered with black scab, which is called eschar. Some patients complain of respiratory symptoms and chest X-rays show about half the lung infiltration. In severe cases, the central nervous system may be invaded, leading to loss of consciousness or death. Tsutsugamushi disease occurs frequently in Asia, including Japan, China, Malaysia, Thailand, and Vietnam, and frequently occurs in people who work outdoors in the fields such as work or camping. The pathogenicity of Orientia tsutsugamushi is due to systemic microvasculitis due to invasion of vascular endothelial cells. Orientia Tsutsugamu, which enters the blood, proliferates in vascular endothelial cells and destroys vascular endothelial cells, or proliferates to cause microthrombosis and inflammation around blood vessels. Therefore, histopathologic features of blood vessel damage of the general organs, resulting in skin necrosis and rash, encephalitis and hearing loss, myotitis and heart failure. In addition, hypofibrinosis caused by vascular coagulation has been reported. In each group (serum type) of Orientia Tsutsugamushi, the symptoms and epidemiologic symptoms of the disease differ from case to case. There is a serological relationship between the bacteria in the same group, but the serological cross-reactivity with other bacteria is weak or absent. Orientia Tsutsugamu is classified as one species, but there are many serotypes with different antigenic structures, and according to the difference of these antigens, they are classified into serotypes such as Giliam, Kaf, and Kato. In Korea, Kangwon and Boryong have been separated from Giliam and Kaf. Species-specific antigens and serotype-specific antigens are present in the cell membrane protein antigens of Orientia Tsutsugashimi, which determine such a variety of serotypes. Purification of Orientia Tsutsugamushi and the analysis of antigens by polyacrylamide gel electrophoresis and immunoblotting revealed that major antigens of 70, 60, 54-56 and 46-47 kDa were present (Takahashi K et al., Immunol. 29: 475, 1985), of which 46-47 kDa, 54-56 kDa and 70 kDa proteins are antigenic, and 70 kDa and 46-47 kDa proteins are known as strain specific antigens (Tamura A et al., Microbiol. Immunol. 26: 321, 1982). In general, Tsutsugamus disease is recognized as a disease with mild clinical symptoms and easy clinical diagnosis. However, due to many cases of atypical clinical symptoms, the characteristic findings of Tsutsugamus disease, such as generalized lymphadenopathy or skin formation, If not, differential diagnosis with acute febrile diseases such as leptospirosis, rash and renal hemorrhagic fever is difficult. Tsutsugamu's disease is diagnosed by a method of isolating pathogens or using immunofluorescent antibodies to prove the antibody. However, the above-mentioned diagnosis can be performed only in a limited number of laboratories because cell culture must be performed to obtain antigens of Orientia Tsutsugamu, and long-term storage of the produced antigens is difficult, and a fluorescent microscope is required for the test. Therefore, it is difficult to carry out immunofluorescence antibody in the first-line hospitals located in rural areas where many patients actually occur.

It is an object of the present invention to provide a Tsutsugamu disease diagnosis kit and a diagnostic method using the same, and more specifically, the first kit and the second kit is composed of the first kit and the second kit comprises a mixed antigen, respectively , The mixed antigen is a Tsutsugamushi disease diagnostic kit, which is a mixture of 56kDa fusion protein (cr56) of Orientia Tsutsugamu Giliam, Kaf, Kato and 56kDa protein (kr56) of Orientia Tsutsugamu Gangwon at a volume ratio of 5: 3, and It is to provide a diagnostic method used.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

A first aspect of the present invention comprises a first kit and a second kit, wherein the first kit and the second kit each include a mixed antigen, wherein the mixed antigen is Orientia Tsutsugashimi Giliam, Kaf, Kato Tsutsugamushi disease diagnostic kit, wherein the 56kDa fusion protein (cr56) and 56kDa protein (kr56) from Kangwon, Orientia Tsutsugamushi are mixed in a volume ratio of 5: 3.

According to one embodiment, the first kit and the second kit may each further comprise a goat anti-chicken IgY antibody.

According to one embodiment, the first kit comprises a first gold conjugate pad comprising anti-human IgM conjugate gold and chicken IgY conjugate gold, and the second kit comprises an anti-human IgG conjugate And a second gold conjugate pad comprising gold and chicken IgY conjugate gold.

According to one embodiment, the anti-human IgM conjugate gold is OD 1 to 1.5 concentration, the anti-human IgG conjugate gold is OD 2 to 4 concentration, the chicken IgY conjugate gold is OD 0.1 to 0.5 concentration Can be.

According to one embodiment, the mixed antigen may be included in 0.4 to 0.8 mg / ml.

According to one embodiment, the first kit and the second kit, respectively, may comprise an entire reaction line region containing 1.5 to 3 mg / ml E. coli extract.

A second aspect of the present invention provides a method for providing information for diagnosing Tsutsugamosis, comprising simultaneously detecting Tsutsugamosis IgM and IgG antibodies in a patient's sample using the Tsutsugamosis disease diagnostic kit.

According to one embodiment, the information for diagnosing Tsutsugashimu is information necessary for diagnosing Tsutsugamushi disease patients whose Ab titers (Ab titers) measured by IFA are IgM 1: 5 to 1:20 and IgG 1:50 to 1: 100. Can be.

According to the present invention, the Tsutsugamosis disease kit comprising the first kit and the second kit is capable of quickly and accurately diagnosing Tsutsugamosis disease, and has high sensitivity and excellent specificity by eliminating non-specific reactions. It is kept constant so that the confidence in the evaluation results can be secured.

Figure 1 shows a strip-type Tsutsugashi disease diagnosis kit according to an embodiment of the present invention.

2 shows the kit reaction according to the antigen concentration.

Figure 3 shows the kit reaction according to the mixed antigen composition.

Figure 4 shows the kit response according to the protein of the control line.

Figure 5 shows the kit reaction according to the E. coli extract concentration of the entire reaction line.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

As used herein, the term 'diagnosis' means identifying the presence or characteristic of a pathological condition. In addition, for the purposes of the present invention, the diagnosis is to identify Tsutsuma's disease.

The term 'tsutsugamushi disease' used in the present invention refers to a disease caused by the pathogenicity of Orientia tsutsugamushi.

A first aspect of the present invention comprises a first kit and a second kit, wherein the first kit and the second kit each include a mixed antigen, wherein the mixed antigen is Orientia Tsutsugashimi Giliam, Kaf, Kato Tsutsugamushi disease diagnostic kit, which is a mixture of 56kDa fusion protein (cr56) and 56kDa protein (kr56) from Kangwon, Orientia, at a volume ratio of 5: 3.

More specifically, the first kit and the second kit constituting the diagnostic kit of the present invention, each of (a) a sample pad (sample pad) is absorbed; (b) a gold conjugation pad that binds to human antibodies in the sample; (c) a reaction membrane (or test membrane) to which a test line comprising a Tsutsugamu mixed antigen and a control line comprising a control protein are treated; And (d) an absorption pad on which the remaining sample is absorbed.

The diagnostic kit of the present invention comprises a Tsutsugashi mixed antigen. Tsutsugamushi mixed antigens that can be used in the present invention are recombinant antigen proteins and Kangwon derived from Orientia tsutsugamushi Gilliam, Karp and Kato that cause Tsutsugamushi disease. The antigenic protein derived from is a mixed protein. That is, the Tsutsugamus mixed antigen of the present invention is an antigen of a recombinant protein derived from a recombinant gene serially connecting a gene encoding a protein constituting an antigenic determinant of Orientia Tsutsugamus Gilioma, Kaf and Kato and an Orientia Tsutsugamu Kangwonju antigen Refers to a mixture of recombinant proteins derived from genes having activity. Specifically, in the present invention, based on the fact that the 56kDa protein of Orientia Tsutsugamu has antigenicity and is of diagnostic value, the standard serotypes Gilliam and Karp are used. And amplifying a gene encoding some fragments of the 56 kDa protein of Kato strain by Polymerase Chain Reaction (PCR), connecting the amplified DNA fragments in series, and cloning them in a protein expression vector in E. coli. The fusion protein antigen, which can be expressed in and separated and purified, can be produced and separated and purified simultaneously in a single process. In addition, in the present invention, in order to improve the sensitivity of diagnosis of Tsugamus disease in addition to Orientia Tsutsugamus Giliam, Kaf and Kato, the gene encoding the 56kDa protein having the antigenic activity of Orientia Tsutsugamu Kangwon (Orientia tsutsugamushi Kangwon) Amplified by reaction, these amplified DNA fragments were cloned into a protein expression vector, expressed in Escherichia coli, and isolated and purified to obtain Tsutsugamushi disease together with the 56kDa protein recombinant protein derived from the Orientia Tsutsugamus Gilioma, Kaf and Kato. Used for diagnosis.

The diagnostic kit of the present invention comprises a Tsutsugamushi mixed antigen obtained by mixing a recombinant 56kDa protein of Orientia Tsutsugamus Gilioma, Cap and Kato strains, and 56kDa protein of Orientia Tsuzumushi Kangwon Province in a volume ratio of 5: 3. The mixed antigen may be included at 0.4 to 0.8 mg / ml, preferably 0.5 to 0.6 mg / ml, most preferably 0.533 mg / ml. More specifically, the Tsutsugamu mixed antigen may be deposited on a test line in the kit.

Diagnostic kits of the invention include control proteins capable of binding gold-labeled proteins. Control proteins that can be used in the present invention include, for example, goat anti-chicken IgY antibodies, rabbit anti-goat IgG polyclonal antibodies, goat anti-human IgG polyclonal antibodies, rabbit anti At least one selected from the group consisting of a goat IgM polyclonal antibody and a goat anti-human IgM polyclonal antibody, preferably, a goat anti-chicken IgY antibody. In case of using goat anti-chicken IgY antibody, the control line is uniformly developed regardless of the response level of the reaction line, thereby ensuring the reliability of diagnostic kit operability. The control protein may be included at a concentration of 0.1 mg / ml to 2 mg / ml, preferably at a concentration of 1 mg / ml.

The diagnostic kit of the present invention may further comprise a pretest line comprising an E. coli extract that does not express the Tsutsugamu antigen protein. In the whole reaction line, the E. coli-derived protein contained in the purified antigen protein and the E. coli-derived protein contained in human serum are used for the purpose of diagnosing Tsutsugamushi disease. This is to exclude the possibility that the antibody against may actually be mistaken as a patient even though it does not actually have an antibody against the Orientia Tsutsugamu antigen protein. The E. coli extract may be included in the whole reaction line at a concentration of 1.5 mg / ml to 3 mg / ml, preferably 2 mg / ml.

The reaction membrane included in the diagnostic kit of the present invention may include a suitable material such as a nitrocellulose membrane (Milipore ™ XA3J072100) of a standard size (10 × 500 nm). The reaction membrane is preferably arranged in the order of the pre-reaction line, the reaction line and the control line, these pre-reaction line, the reaction line and the control line may be arranged in an interval of 2.0 to 4.5mm, preferably 2.5 to 3.0mm.

The sample pad included in the diagnostic kit of the present invention was immersed in a solution containing 0.2M sodum borate buffer (pH 8.6) 750ml, 20% Tween 20 75ml, 10% BSA 30ml, 10% NaN3 1.5ml, DW 630ml. After that, it was prepared by drying.

Gold conjugate pads included in the diagnostic kits of the present invention may include suitable markers, for example anti-human IgM conjugated gold, anti-human IgG conjugated gold ( Anti-human IgG conjugated gold, chicken IgY conjugated gold, and the like. More specifically, the first kit constituting the diagnostic kit of the present invention comprises a first gold conjugate pad comprising anti-human IgM conjugate gold and chicken IgY conjugate gold, and the second kit is an anti-human A second gold conjugate pad comprising IgG conjugate gold and chicken IgY conjugate gold. The gold conjugate pad is preferably located adjacent to the sample pad.

The marker included in the gold conjugate pad used in the present invention may have an optical density (OD, measured at 530 nm) at a concentration of 0.1 to 6, preferably 0.1 to 3. When the amount of the marker is excessive, the Tsutsugamus antibody to be analyzed may be expressed as a non-specific signal even in a relatively low region, resulting in false positive results. For example, the anti-human IgM conjugate gold may be included at a concentration of OD 1 to 1.5, preferably OD 1.25, and the anti-human IgG conjugate gold may be at a concentration of OD 2 to 4, preferably OD 3. Chicken IgY conjugate gold may be included at a concentration of OD 0.1 to 0.5, preferably OD 0.3. Absorption pads are located at opposite ends of the membrane and absorb biological samples such as serum, plasma, etc. that have migrated along the membrane by capillary action.

The diagnostic kit of the present invention consists of a first kit and a second kit branched from one sample pad. For example, the first kit may be for detecting human IgM antibodies against Tsutsugamus antigen protein, and the second kit may be for detecting human IgG antibodies to Tsutsugamus antigen protein, and their order may be reversed. have. According to an embodiment of the invention, the first kit comprises a first gold conjugate pad comprising anti-human IgM conjugate gold and chicken IgY conjugate gold and a control line comprising a goat anti-chicken IgY antibody, The second kit may comprise a second gold conjugate pad comprising anti-human IgG conjugate gold and chicken IgY conjugate gold and a control line comprising goat anti-chicken IgY antibodies. According to an embodiment of the present invention, the first kit and the second kit may be arranged in parallel, but the present invention is not limited thereto, and the first kit and the second kit may be arranged in series with each other in opposite directions or various Can be arranged in a direction. The diagnostic kit of the present invention can be individually packaged in a case surrounding the test strip to ensure safety. The diagnostic kit can be stored for a long time (at least 18 months) at room temperature without loss of diagnostic activity.

The first kit and the second kit constituting the Tsutsugamus disease diagnosis kit of the present invention include a sample pad, a gold conjugate pad, a reaction membrane, and an absorption pad, respectively. The sample pad is overlaid on the gold conjugate pad to form a first overlapping portion, and the gold conjugate pad is overlaid on the film to form a second overlapping portion. The membrane and absorbent pad also form a third overlapping portion. When the biological sample is deposited on the sample pad, capillary action causes the biological sample to pass through the gold conjugate pad. The gold particles forming the gold-label preferably have a diameter size of 20 to 55 nm, more preferably a diameter size of 20 to 40 nm, and serve as an indicator dye. As it passes through the gold conjugate pad, the antibody in the biological sample binds to the gold-labeled marker to form a complex, and the complex migrates along the membrane.

As used herein, the term biological sample refers to serum, plasma, and the like of mammals including humans suspected of having Tsutsugamus disease. Biological samples can be used with or without dilution prior to dropping onto the sample pad of the diagnostic kit of the present invention. If the biological sample contains Tsutsugamu antibody, the antibody passes through the reaction line containing the Tsutsuga mu mixed antigen in the diagnostic kit of the present invention, and the antibody binds to the Tsutsuga mu mixed antigen to allow color to be visually identified in the reaction line on the membrane. Change happens. That is, the formation of an antigen-antibody complex results in a reddish purple band that can be detected while the indicator dye is precipitated by the gold complex, thereby showing a positive reaction in which the Tsutsugamus antibody is present in the biological sample. However, if there is no Tsutsugamus antibody in the biological sample, no change occurs in the reaction line on the membrane because it does not bind with the Tsutsugamus mixed antigen of the present invention.

According to a second aspect of the present invention, the Tsutsugamus disease IgM and IgG antibodies are simultaneously detected in a patient's sample using the Tsutsuga Mus disease disease kit, thereby providing information for diagnosing Tsutsuga Mus disease.

In general, in order to diagnose whether or not a patient suspected of Tsutsugamushi disease is infected with Orientia Tsutsugamushi, it is necessary to confirm the presence of Orientia Tsutsugamushi in the human body. Currently, the method of diagnosing Tsutsugamushi disease mainly uses immunofluorescent antibody. However, this method has high sensitivity and specificity, and can distinguish between IgM and IgG, so that it can distinguish between initial infection and re-infection. However, cell culture facilities are required because the cell culture must be performed. It is complicated and can only be performed by a specialist, and there is a problem that it takes a lot of time and maintenance costs. On the contrary, since the diagnostic method of the present invention uses an immunochromatography method by antigen-antibody reaction, it is possible to diagnose Tsutsugamushi disease easily and accurately within a quick time of about 10 minutes, and thus has excellent efficacy compared to the conventional diagnostic method. Have.

The Tsutsugamus disease antibody detection method using the Tsutsugamus disease diagnostic kit of the present invention is as follows. First, when a biological sample to be sampled is dropped on a sample pad which is a site where a sample of the diagnostic kit is absorbed, the biological sample reaches the gold conjugate pad by capillary action so that the antibody in the sample is a marker in the gold conjugate pad, eg For example, anti-human IgM conjugated gold, anti-human IgG conjugated gold, anti-human IgG conjugated gold, chicken IgY conjugated gold, etc. Will form. The biological sample continues to move without being immobilized on the gold conjugate pad to reach a test line in which the Tsutsugamu mixed antigen is immobilized. In the case of a biological sample of a patient infected with Tsugasuga disease, an antigen-antibody reaction occurs with the Tsutsugamu mixed antigen, that is, the Tsutsugamus antibody bound to a specific marker in the patient's gold conjugate pad binds to the Tsutsugamus mixed antigen fixed to the reaction line. By forming a red purple band can be observed with the naked eye. In addition, the markers in the remaining gold conjugate pads that did not react with the antibodies in the biological sample reached the control line, reacting with the control protein to form a red purple band, indicating the suitability of the test. As described above, the diagnostic kit of the present invention is a method capable of visually confirming test results without special equipment by using an immunochromatography method by an antigen-antibody reaction.

The diagnostic kit of the present invention includes a pretest line, which eliminates the possibility of being mistaken for having an antibody against an Orientia Tsutsugamus antigen protein, thereby eliminating the accuracy of Tsutsugamus disease diagnosis. Improved.

According to one embodiment of the present invention, using the Tsutsugashi disease diagnosis kit of the present invention, the time taken to confirm the diagnosis result is about 5 to 10 minutes, which is within 15 minutes. The results of the diagnosis can be used to diagnose the condition of Tsutsugamushi infection in the patient. For example, if the biological sample is collected from a patient infected with Tsutsugamu, the diagnostic kit of the present invention may be positive for Tsutsugamushi antibody and diagnosed with Tsutsugamu disease, and if the biological sample is collected from a patient not infected with Tsutsugamu In the case of the diagnostic kit of the present invention can show a negative response can be diagnosed as not Tsutsugamushi disease. More specifically, the result of the positive reaction of the present invention is the case where the reaction line containing the Tsutsugashi mixed antigen in addition to the control line develops color, and the result of the negative response to Tsutsuga musi disease is the case where only the control line develops color.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, this is only added to help the understanding of the present invention, the scope of the present invention is not limited by these examples.

Example  1. Antigen Protein Expression and Isolation

1-1. Chimeric  Recombination 56 kDa (cr56)  Protein expression

The chimeric recombinant protein (cr56) comprising a major antigenic site of Orientia tsutsugamushi Gilliam, Karp, Kato was prepared by the same method as described in the examples of Korean Patent Publication No. 2002-0020281. Expression and isolation were purified.

That is, Gilien, Cap and Kato of Orientia Tsutsugamu were cultured in mouse L-929 cells, harvested and purified. After enzymatic digestion of the purified strain, DNA was purified by phenol extraction and ethanol precipitation. In addition, based on the nucleotide sequence of the 56kDa protein gene of Orientia, a pair of oligonucleotides were selected in Gilium, Kape, and Kato, respectively, by selecting a protein region showing 30% or more homology between amino acids of Gilium, Kafe, and Kato serotypes. Prepare nucleotide primers. With each of these primer pairs, a PCR reaction is carried out using the Orientia Tsutsugamus DNA extracted above as a template to amplify the DNA fragments of Gillium, Cap and Kato. The PCR product is purified and cloned to link to the corresponding restriction enzyme cleavage buoy of the pTYB12 vector. First, a vector pTG3 is prepared by introducing a Gilt DNA fragment into a pTYB12 vector, a DNA fragment of a cap is introduced into pTG3 to prepare a vector pTGP1, and a pTGPT2 is prepared by introducing a Kato DNA fragment into pTGP1. In addition, a series linkage of DNA fragments of Gillium, Cap and Kato was cut from the vector pTGPT2 and introduced into the pET22b (+) vector to prepare a vector pETb7. E. coli is transformed with the produced expression vector, and the transformed E. coli is cultured to induce fusion protein expression. After confirmation by electrophoresis and Western blot, the expressed fusion protein antigen was isolated and purified.

1-2. Recombination Kangwon 56kDa  Protein ( kr56 Expression of

Phylogenic analysis results (FEMA Microbiology Letters, 1999, 180: 163-169) show that the global prototypes of Karp, Giliam, Kato and Yeoncheon ( Considering that they can be largely classified as Yonchon (most similar to Kangwon), it was considered very appropriate to use these four strains as antigens. Karp, Giliam and Kato are shown as cr56. Yeonchon is also presented as kr56 of Kangwon. Therefore, the present inventors decided to use Kangwon 56kDa recombinant protein (kr56) as an auxiliary antigen of chimeric recombinant protein (cr56).

(1) Overview of Kangwon 56kDa protein (kr56) expression

A pair of primers were prepared by modifying the amino acids 84 (250bp) to 445 (1,335bp), known as the major antigenic domain of Kangwon 87-61, to contain Nco I and Sal I recognition sites (Table 1). ), A PCR reaction was performed. The PCR product and the expression vector pET30a were digested with Nco I and Sal I restriction enzymes and then ligated with each other. This plasmid was transformed into Escherichia coli BL21 and the proteins were extracted.

Plasmid pET30a expresses His-Tagged fusion protein.

 (2) Cloning and Expression of Genes

DNA amplified by polymerase chain reaction (amino acids 84 (250bp) to 445 (1,335bp) 362 aa (total 1,086bp)) was electrophoresed on 1.2% agarose gel and then QIAGEN gel elution kit (QIAGEN Inc., USA). Bands amplified to the expected size on a UV transilluminator were cut out and transferred to microtubes. Three times the volume of NaI solution of agarose gel was added and then left at 60 ° C. for 5 minutes to completely dissolve the gel. Glass milk was added thereto and stirred for 10 seconds. This was centrifuged at 17,000 g (Hanil, AI.5S-24, 12,000 rpm) for 1 minute at room temperature, and then the supernatant was removed. The microtubes were allowed to stand at room temperature for 5 minutes to dry and then stirred by addition of elution buffer, followed by centrifugation at 17,000 x g (12,000 rpm) to transfer the supernatant to a new microtube. The purified PCR product and pET30a vector were completely cleaved with restriction enzymes Nco I and Sal I, and then recovered using Geneclean kit II. 100 ng of the cleaved vector and 100 ng of the cleaved PCR product are mixed, and a 10-fold concentration of ligase buffer (250 mM Tris-HCl pH 7.8, 100 mM MgCl 2, 20 mM DTT, 4 mM ATP) is mixed with 18 at 4 ° C. The reaction was carried out for a time.

 (3) Preparation and transformation of competent cells

Escherichia coli BL21 incubated in LB medium for 18 hours was diluted 100-fold in LB medium and cultured at 600 nm until the absorbance became 0.3. After standing in ice for 30 minutes, the supernatant of the medium was discarded, and E. coli was suspended by adding 0.1 M CaCl2 to 1/2 the volume of the medium. It was left for 1 hour in ice, centrifuged at 2,000 g (4,100 rpm) for 10 minutes, and then suspended in 1/10 volume of 0.1 M CaCl 2 of the culture medium, which was used for transformation.

 (4) transformation

The ligation product prepared in (3) and the competent cells (competent cells) were mixed and allowed to stand in ice and then subjected to thermal shock at 42 ° C. for 1 minute 30 seconds. After adding LB medium, the cells were incubated at 37 ° C. for 1 hour. Cultures were inoculated in LB agar plates and incubated at 37 ° C.

 (5) Isolation of plasmid DNA of transformed Escherichia coli

The transformed E. coli colonies were inoculated in LB medium containing Kanamycin and shaken at 37 ° C. Plasmid DNA extraction was performed using AccuPrep Plasmid Extraction kit. The culture was centrifuged at 750 g (2,500 rpm) for 10 minutes at room temperature, and then only cell precipitates were recovered. Resuspenstion buffer was added thereto, followed by stirring to add lysis buffer and allowed to stand at room temperature for 5 minutes. Neutralization buffer was added and left at room temperature, followed by centrifugation at 17,000xg (12,000 rpm) to transfer the supernatant to a binding column tube. This was centrifuged at 17,000xg (12,000rpm) at room temperature and then the extract was removed. 80% ethanol was aliquoted and centrifuged, and only the binding column was transferred to a new tube. Elution buffer was dispensed and left at room temperature, followed by centrifugation to transfer the supernatant to a new microtube.

 (6) Expression and Isolation of Proteins

The culture medium was inoculated with the culture solution of the seed culture step. The inoculation was at 37 ° C. and stirred at 220 rpm for 1 hour 45 minutes after inoculation. When the O.D value was in the range of 0.5 to 0.6, IPTG was inoculated with 0.5 mM and incubated for 3 hours. Thereafter, centrifugation at 3,000 rpm for 30 minutes at 4 ℃, 1xbinding buffer (6 M Urea, 500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM Imidazole) in the pellet and using a Sonicator ( pulse: 10 sec on, 20 sec off, time: 6 min X 2 times (inverting once in the middle), Amp .: 45%) The cells were disrupted. Kr56 was centrifuged at 14,000 rpm for 15 minutes at 4 ° C. (rotor 7 in autoclaved ultra centrifuge bottle), then the supernatant was refrigerated and the pellet resuspended in 1 × Binding buffer (50 ml based on 1 L Erlenmeyer flask culture). It was. After leaving for 1 hour in ice (after 30 minutes, inverting), and centrifuged at 14,000 rpm for 30 minutes at 4 ℃ (rotor 7 in autoclaved ultra centrifuge bottle) to obtain a supernatant. The supernatant was diluted twice with 1 × Binding buffer (6M urea), filtered and stored at 4 ° C. (0.45 μm syringe filter).

 (7) protein purification

Hisbind resin (Novagen) was charged to the column to 2ml, the column was prepared using distilled water, 1 X charge buffer, 1 X binding buffer. Antigen proteins were passed through the column and washed with 1 X washing buffer. 1 ml of protein was recovered by passing through 1 × Elution buffer (6 M Urea, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0), 500 mM Imidazole. Protein concentrations in each fraction were quantified using BCA protein assay, concentrated with Amicon ㄾ Ultra 30K and quantified using BCA protein assay.

For the specific amino acid sequence of the Orientia Tsutsugamu Gangwon 56kDa protein included in the Tsutsugamu mixed antigen of the present invention, reference may be made to the amino acid sequences disclosed in Patent No. 10-0796772.

Example  2. Depending on antigen concentration Of kit  Reaction (determination of false positives)

The mixed antigen of the present invention prepared according to Example 1 was prepared at concentrations of 0.6 mg / ml, 0.9 mg / ml, 1.2 mg / ml and 1.5 mg / ml, respectively. Four kits containing antigens of each concentration were prepared. After evaluating the patient's sample using immunofluorescent antibody, a standard diagnostic test for diagnosing Tsutsugamushi disease, 1 negative serum (DK13) and 3 positive serum (90-202, 00-350, 89) -129) were prepared and applied to each of the diagnostic kits having different antigen concentrations to measure the response.

2 shows the kit reaction according to the antigen concentration.

Table 2 below shows the kit reaction according to the antigen concentration.

As shown in Table 2 and FIG. 2, false positives were shown in a diagnostic kit with antigen concentrations of 0.9, 1.2, and 1.5 mg / mL, but no false positives were shown in a diagnostic kit with an antigen concentration of 0.6 mg / mL.

Example  3. According to the mixed antigen composition Kit  reaction

The mixed antigen of the present invention prepared according to Example 1 was prepared at 0.6 mg / ml, and the conventional mixed antigen was prepared at the same concentration. The conventional mixed antigen is a mixture of cr56, kr56, and r21 proteins in a 5: 2: 1 ratio, respectively. The diagnostic kit containing the mixed antigen of the present invention and the conventional diagnostic kit containing the mixed antigen were prepared, and the reactivity of negative serum and positive serum was measured. As a control, each negative and positive serum was measured for Ab titer according to IFA.

Figure 3 shows the kit reaction according to the mixed antigen composition.

Table 3 below shows the kit reaction according to the mixed antigen composition.

As shown in Table 3 and FIG. 3, when the ratio of kr56 was increased without including r21 in the mixed antigen, response sensitivity to the antibody was improved.

Example  4. According to the protein of the control line Kit  reaction

The diagnostic kit of the present invention comprising 0.6 mg / ml of the mixed antigen of the present invention prepared according to Example 1, and 1.0 mg / ml of goat anti-chicken IgY as a protein instilled in the control line was prepared. A diagnostic kit was prepared in which all other conditions were the same, but the protein deposited on the control line was anti-protein A. After evaluating the patient's samples using the immunofluorescent antibody method, a standard diagnostic test for diagnosing Tsutsugamushi disease, negative and positive serum were applied to each diagnostic kit to measure the response.

Figure 4 shows the kit response according to the protein of the control line.

Table 4 below shows the kit response according to the control protein.

As shown in Table 4 and FIG. 4, there was a difference in the color development of the control line when anti-protein A was used as the control protein. However, when goat anti-chicken IgY was used as the control protein, there was no difference in control color development. Level was maintained.

Example  5. contained in purified antigen E. coli derived  Study on elimination of reaction between protein and antibody in human serum

Antigen was purified using His-bind affinity chromatography, but E. coli-derived proteins could not be completely removed. Therefore, it is difficult to completely remove these E. coli-derived proteins. Purification of the E. coli host cells that do not express the T. TSUGAMUSHI antigen protein makes extracts, and then by dropping them as pretest lines in the front of the test line. Included to remove antigens that cause antigen-antibody reactions with E. coli-derived proteins.

Example  6. Full response  E. coli extract concentration Kit  reaction

The diagnostic kit of the present invention comprising 0.6 mg / ml of the mixed antigen of the present invention prepared according to Example 1, and 1.0 mg / ml of goat anti-chicken IgY as a protein instilled in the control line was prepared. E. coli extracts in the diagnostic kits of the present invention were instilled in the entire reaction line at 0.5 mg / ml, 1 mg / ml or 2 mg / ml, respectively. Negative serum was prepared and applied to each diagnostic kit to measure response.

Figure 5 shows the kit reaction according to the E. coli extract concentration of the entire reaction line.

Table 5 below shows the kit reaction according to the E. coli extract concentration of the entire reaction line.

As shown in Table 5 and Figure 5, the concentration of E. coli extract of the whole reaction line was 0.5 mg / ml and 1 mg / ml showed false positive, but the concentration of E. coli extract of the entire reaction line is 2 mg / ml There was no false positive.

Example  6. Stick form Diagnostics  Manufacture of samples

Test efficacy of Tsutsugamus disease (cr56, kr56) protein expressed in Escherichia coli as a Tsutsugamus disease diagnostic agent and a test strip sample for diagnosis using a device called a dispenser (control dispenser_control number IMM-EP-005) from Kinematic automation (M1600) Was produced. The structure of the test strip is composed of a nitrocellulose membrane, a sample pad, an absorption pad, and a gold bonding pad (glass fiber), as shown in the third. Nitrocellulose membrane was manufactured by Millipore Inc., and a transparent plastic was attached to one side to prevent the membrane from being easily damaged. Nitrocellulose membranes were sprayed with three proteins. One type is a test line which is a protein containing a proper amount of cr56 and kr56 protein antigens expressed in Escherichia coli to detect an antibody against Tsutsugashimu in the serum of a patient. The other type is a test line. It is a control line to judge the response. The other is E. coli culture extract that does not express the Tsutsugamus antigen protein in order to remove antibodies that are contained in human serum and cause antigen-antibody reactions with E. coli-derived proteins, and is a pretest line. Using a dispenser, the appropriate amount of protein was sprayed on the whole reaction line, the reaction line, and the control line and dried at 25 ° C. The glass fiber was diluted by absorbing an appropriate amount of colloidal gold conjugate in 5% trehalose and dried at 25 ° C. for 24 hours. The sample pad and the absorbent pad are cut into the desired size, and the sample pad is soaked in a solution made of 0.5% of Tween 20 in 0.1M sodium borate buffer to absorb the serum or plasma again, and then dried at room temperature. Dry thoroughly until. Next, a gold bond pad (glass fiber) sprayed with a colloidal gold conjugate was attached to the bottom of the nitrocellulose membrane, and a sample pad was attached again below. An absorbent pad was attached on top of the nitrocellulose membrane. The final diagnostic stick was cut to a width of 4 mm and a height of 60 mm. In the present invention, two strips were put in one plastic and manufactured to diagnose at the same time. As a result of the efficacy test using the patient serum and the normal serum on the prepared diagnostic sample, the efficacy as a rapid, accurate and convenient Tsutsugamus disease diagnosis was demonstrated. In addition, it was confirmed that the reaction did not occur after cross-testing with serum of patients with other acute febrile diseases (nephrotic hemorrhage, rash, leptospirosis).

Example  7. Diagnostic Kit Performance test

In order to compare and measure the sensitivity and specificity of the Tsutsugashim diagnostic kit of the present invention, it was compared with the Tsutsugashimu diagnostic kit of other company (S). Sixty positive samples and 100 negative samples, which were evaluated using the immunofluorescent antibody method (IFA), a standard diagnostic test for diagnosing Tsutsugamushi disease, were prepared.

The method using the Tsutsugamu diagnostic kit of the present invention is as follows.

1) Take 3 μl of serum, plasma, or 6 μl of whole blood with a micropipette and inject it to be absorbed into the sample pad at the bottom of the sample injection section. Immediately, 7 drops of sample diluent is dropped into the sample injection section.

2) Wait until the red band appears in the control line (C) area. Results are read 15 minutes after the start of the test, and any new red bands appearing after 15 minutes are not included in the results.

Tsutsugamushi diagnostic kits of other companies were used in accordance with the instructions attached to the diagnostic kits.

As a result of testing the performance of the diagnostic kit, the Tsutsugamushi diagnostic kit of the present invention was evaluated to have a sensitivity of 99% and a specificity of 100%, while it was evaluated to have a sensitivity of 83% and a specificity of 100% of the S company. The sensitivity was found to be higher.

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the claims that follow.

Claims (8)

1. Consisting of a first kit and a second kit,

   The first kit and the second kit each include a mixed antigen,

   Said mixed antigen, Tsutsugamushi disease diagnostic kit is a mixture of 56kDa fusion protein (cr56) of Orientia Tsutsugamu Giliam, Kafe, Kato and 56kDa protein (kr56) of Orientia Tsutsugamu Gangwon at 5: 3 volume ratio.
2. The method of claim 1,

   The first kit and the second kit, respectively, Tsutsugamushi disease diagnostic kit that further comprises a goat anti-chicken IgY antibody.
3. The method of claim 1,

   The first kit comprises a first gold conjugate pad comprising anti-human IgM conjugate gold and chicken IgY conjugate gold,

   Wherein said second kit comprises a second gold conjugate pad comprising anti-human IgG conjugate gold and chicken IgY conjugate gold.
4. The method of claim 3,

   The anti-human IgM conjugate gold is in an OD 1-1.5 concentration,

   The anti-human IgG conjugate gold is at an OD 2-4 concentration,

   The chicken IgY conjugate gold is an OD of 0.1 to 0.5 concentration, Tsutsugamus disease diagnostic kit.
5. The method of claim 1,

   Wherein the mixed antigen is contained in 0.4 to 0.8 mg / ml, Tsutsugamus disease diagnostic kit.
6. The method of claim 1,

   Wherein the first kit and the second kit, each of which comprises a whole reaction line region containing 1.5 to 3 mg / ml E. coli, Tsutsugamus disease diagnostic kit.
7. A method of providing information for diagnosing Tsutsugamus disease, comprising simultaneously detecting Tsutsugamus disease IgM and IgG antibodies in a patient's sample using the Tsutsugamus disease diagnostic kit of claim 1.
8. The method of claim 7, wherein

   The information for diagnosing Tsutsugamushi disease is Tsutsugamushi disease diagnosis, which is information necessary for diagnosing Tsutsugamushi disease patients whose Ab titers (Ab titer) measured by IFA are IgM 1: 5 to 1:20 and IgG 1:50 to 1: 100. How to provide information for.

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