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

Igm for early diagnosis of tsutsugamushi disease, igg antibody detection kit, and method for preparing same Download PDF

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WO2017095135A1
WO2017095135A1 PCT/KR2016/013954 KR2016013954W WO2017095135A1 WO 2017095135 A1 WO2017095135 A1 WO 2017095135A1 KR 2016013954 W KR2016013954 W KR 2016013954W WO 2017095135 A1 WO2017095135 A1 WO 2017095135A1
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Prior art keywords
kit
gold
disease
protein
conjugate
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PCT/KR2016/013954
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French (fr)
Korean (ko)
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김윤원
김영진
박상진
김동환
김소라
최은지
김은예
강희근
유정하
김태용
이남택
권순환
서원준
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주식회사 이뮨메드
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Priority to CN201680001830.3A priority Critical patent/CN107209185A/en
Publication of WO2017095135A1 publication Critical patent/WO2017095135A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54386Analytical elements
    • G01N33/54387Immunochromatographic test strips
    • G01N33/54388Immunochromatographic test strips based on lateral flow
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first kit and the second kit may each further comprise a goat anti-chicken IgY antibody.
  • the first kit comprises a first gold conjugate pad comprising anti-human IgM conjugate gold and chicken IgY conjugate gold
  • the second kit comprises an anti-human IgG conjugate And a second gold conjugate pad comprising gold and chicken IgY conjugate gold.
  • 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.
  • the mixed antigen may be included in 0.4 to 0.8 mg / ml.
  • the first kit and the second kit 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.
  • 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.
  • 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.
  • 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.
  • the term 'diagnosis' means identifying the presence or characteristic of a pathological condition.
  • the diagnosis is to identify Tsutsuma's disease.
  • '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.
  • 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.
  • 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.
  • 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
  • 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.
  • PCR Polymerase Chain Reaction
  • 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 Republic 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.
  • 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.
  • a pretest line comprising an E. coli extract that does not express the Tsutsugamu antigen protein.
  • 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 TM XA3J072100) of a standard size (10 ⁇ 500 nm).
  • a suitable material such as a nitrocellulose membrane (Milipore TM 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.
  • 0.2M sodum borate buffer pH 8.6
  • 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.
  • 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
  • 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.
  • OD optical density
  • 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.
  • 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.
  • the first kit may be for detecting human IgM antibodies against Tsutsugamus antigen protein
  • the second kit may be for detecting human IgG antibodies to Tsutsugamus antigen protein, and their order may be reversed. have.
  • 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.
  • 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
  • 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.
  • 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.
  • the antibody in the biological sample binds to the gold-labeled marker to form a complex, and the complex migrates along the membrane.
  • 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.
  • 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.
  • the reaction line on the membrane because it does not bind with the Tsutsugamus mixed antigen 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.
  • Tsutsugamushi 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.
  • the method of diagnosing Tsutsugamushi disease mainly uses immunofluorescent antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the diagnostic kit of the present invention can show a negative response can be diagnosed as not Tsutsugamushi disease.
  • 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
  • the result of the negative response to Tsutsuga musi disease is the case where only the control line develops color.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ligase buffer 250 mM Tris-HCl pH 7.8, 100 mM MgCl 2, 20 mM DTT, 4 mM ATP
  • the reaction was carried out for a time.
  • 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.
  • the ligation product prepared in (3) and the 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ Ultra 30K and quantified using BCA protein assay.
  • 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.
  • Table 2 below shows the kit reaction according to the antigen concentration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Tsutsugashim diagnostic kit of the present invention 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.
  • IFA immunofluorescent antibody method
  • the method using the Tsutsugamu diagnostic kit of the present invention is as follows.
  • Tsutsugamushi diagnostic kits of other companies were used in accordance with the instructions attached to the diagnostic kits.
  • 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.

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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

쯔쯔가무시병 조기진단을 위한 IGM, IGG 항체 검출 진단키트 및 이의 제조방법IGM, IGG Antibody Detection Diagnostic Kit for Early Diagnosis of Tsutsugamushi Disease and Method for Preparing the Same
본 발명은 제1 키트 및 제2 키트로 구성된 쯔쯔가무시병 진단 키트 및 쯔쯔가무시병 진단 방법에 관한 것이다.The present invention relates to a Tsutsugamus disease diagnosis kit and a Tsutsugamus disease diagnosis method comprising a first kit and a second kit.
쯔쯔가무시병은 리케치아 속의 스크럽 티푸스(Scrub typhus)군에 속하는 오리엔티아 쯔쯔가무시(Orientia tsutsugamushi)에 의한 것으로, 진드기유충에 의해 전파되는 급성열성질환이다. 진드기유충에 물린 뒤 약 10일 (1~3주)이 지나면 오한, 발열, 두통이 나타나며, 발병 1주 전후하여 몸통에서 시작하여 사지로 퍼져나가는 발진이 나타난다. 진드기의 유충에 물린 자리에는 수포가 생기고, 0.5 내지 0.8cm 크기의 궤양을 형성하며, 이것이 검은 딱지로 덮이게 되는데 이를 가피(eschar)라고 한다. 일부 환자에서 호흡기 증상을 호소하며, 흉부 X-선 촬영을 하면 약 반수에서 폐침윤을 볼 수 있다. 심한 경우에는 중추신경계를 침범하여 의식을 잃거나 사망하는 경우도 있다. 쯔쯔가무시병은, 주로 일본, 중국, 말레이시아, 태국, 베트남 등을 포함하는 아시아 지역에서 자주 발병하며, 주로 들판에서 작업이나 캠핑 등의 야외 활동을 하는 사람들에게서 빈번하게 발병한다. 오리엔티아 쯔쯔가무시(Orientia tsutsugamushi)의 병원성은 혈관 내피 세포의 침입으로 인한 전신 미세혈관염에 기인한다. 혈중으로 들어온 오리엔티아 쯔쯔가무시는 혈관 내피세포에 증식하여 혈관 내피 세포를 파괴하거나, 증식이 촉발되어 미세혈전과 혈관 주위의 염증을 유발한다. 따라서 병리조직학적으로 전신장기의 혈관손상이 특징이며, 이로 인한 피부괴사와 발진, 뇌염과 청력상실, 신근염과 심부전을 일으키기도 한다. 뿐만 아니라 혈관 내 응고증에 의한 저 피브린혈증도 보고 되고 있다. 오리엔티아 쯔쯔가무시의 각 군(혈청형)에 따라서 병의 증상이나 역학적인 양상은 경우에 따라 상이한 차이를 보이고 있다. 같은 군 속의 균 상호간에는 혈청학적으로 연관이 있으나, 다른 군의 균과는 혈청학적 교차반응이 약하게 있거나 없다. 오리엔티아 쯔쯔가무시는 한 개의 종으로 분류되고 있으나, 항원구조가 다른 혈청형이 많이 존재하며, 이러한 항원의 차이에 따라 길리암, 카프, 카토(Gilliam, Karp, Kato) 등의 혈청형으로 분류된다. 국내에서는 길리암, 카프 외에 기존에 알려진 원형균주와는 혈청학적 반응 양상이 다른 강원주(Kanwon)과 보령주(Boryong)가 분리된 바 있다. 이와 같은 혈청형의 다양성을 결정짓는 오리엔티아 쯔쯔가무시의 세포막 단백 항원에는 종특이적 항원과 혈청형 특이적 항원 등이 존재한다. 오리엔티아 쯔쯔가무시를 정제하여 항원을 폴리아크릴아미드 젤 전기영동과 면역블로팅(immunoblotting)으로 분석한 결과 70, 60, 54~56, 46~47kDa의 주요 항원이 존재하는 것으로 밝혀졌는데 (Takahashi K 등, Microbiol. Immunol. 29: 475, 1985), 이들 중 46~47kDa, 54~56kDa 및 70kDa의 단백질이 항원성을 가지며, 70kDa과 46~47kDa 단백질은 균주 특이 항원으로 알려져 있다(Tamura A 등, Microbiol. Immunol. 26: 321,1982). 일반적으로 쯔쯔가무시병은 임상 증상이 가벼우며 증상에 의한 임상 진단이 용이한 질환으로 인식되고 있지만, 비전형적인 임상 증상을 나타내는 경우가 많기 때문에, 전신 임파선 종창이나 가피 형성 등과 같은 쯔쯔가무시병의 특징적인 소견이 없는 경우에는 렙토스피라증, 발진열 및 신증후출혈열 등의 급성열성질환과의 감별 진단이 어렵다. 쯔쯔가무시병의 진단법은 병원균을 분리하거나 면역형광항체법으로 항체를 증명하는 방법에 의한 정밀진단이 있다. 그러나, 상기 진단법은 오리엔티아 쯔쯔가무시의 항원을 얻기 위하여 세포 배양을 실시해야 하며 생산된 항원의 장기 보관이 어려울 뿐 아니라 검사에 있어 형광현미경을 필요로 하므로 제한된 일부 검사실에서만 시행할 수가 있다. 따라서 실제로 환자가 많이 발생하는 농촌지역에 위치한 일선 병원에서는 면역형광항체법은 실시하기엔 어려운 점이 있다.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.
본 발명의 목적은, 쯔쯔가무시병 진단 키트 및 이를 이용한 진단 방법을 제공하는 것으로, 보다 구체적으로는, 제1 키트 및 제2 키트로 구성되며 상기 제1 키트 및 제2 키트는 각각 혼합항원을 포함하고, 상기 혼합항원은 오리엔티아 쯔쯔가무시 길리암, 카프, 카토의 56kDa의 융합 단백질(cr56)과 오리엔티아 쯔쯔가무시 강원의 56kDa 단백질(kr56)을 5 : 3의 부피비로 혼합한 것인 쯔쯔가무시병 진단 키트 및 이를 이용한 진단 방법을 제공하는 것이다.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.
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.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.
본 발명의 제1 측면은, 제1 키트 및 제2 키트로 구성되며, 상기 제1 키트 및 제2 키트는 각각, 혼합항원을 포함하고, 상기 혼합항원은, 오리엔티아 쯔쯔가무시 길리암, 카프, 카토의 56kDa의 융합 단백질(cr56)과 오리엔티아 쯔쯔가무시 강원의 56kDa 단백질(kr56)이 5 : 3의 부피비로 혼합된 것인 쯔쯔가무시병 진단 키트를 제공한다.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.
일 실시예에 따르면, 상기 제1 키트 및 제2 키트는 각각, 산양 항-닭 IgY 항체를 더 포함할 수 있다.According to one embodiment, the first kit and the second kit may each further comprise a goat anti-chicken IgY antibody.
일 실시예에 따르면, 상기 제1 키트는, 항-인간 IgM 컨쥬게이트 골드 및 닭 IgY 컨쥬게이트 골드를 포함하는 제1 골드 컨쥬게이트 패드를 포함하고, 상기 제2 키트는, 항-인간 IgG 컨쥬게이트 골드 및 닭 IgY 컨쥬게이트 골드를 포함하는 제2 골드 컨쥬게이트 패드를 포함할 수 있다.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.
일 실시예에 따르면, 상기 항-인간 IgM 컨쥬게이트 골드는 OD 1 내지 1.5 농도이고, 상기 항-인간 IgG 컨쥬게이트 골드는 OD 2 내지 4 농도이며, 상기 닭 IgY 컨쥬게이트 골드는 OD 0.1 내지 0.5 농도일 수 있다.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.
일 실시예에 따르면, 상기 혼합 항원은 0.4 내지 0.8 mg/ml로 포함될 수 있다.According to one embodiment, the mixed antigen may be included in 0.4 to 0.8 mg / ml.
일 실시예에 따르면, 상기 제1 키트 및 제2 키트는 각각, 대장균 추출물을 1.5 내지 3 mg/ml 포함하는 전반응선 영역을 포함할 수 있다.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.
본 발명의 제2 측면은, 상기 쯔쯔가무시병 진단 키트를 사용하여 환자의 시료 내 쯔쯔가무시병 IgM 및 IgG 항체를 동시에 검출하는 단계를 포함하는 쯔쯔가무시병 진단을 위한 정보를 제공하는 방법을 제공한다.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.
일 실시예에 따르면, 상기 쯔쯔가무시 진단을 위한 정보는, IFA로 측정되는 Ab 역가 (Ab titer)가 IgM 1:5 내지 1:20 및 IgG 1:50 내지 1:100인 쯔쯔가무시병 환자 진단에 필요한 정보일 수 있다.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.
본 발명에 따른, 제1 키트 및 제2 키트로 구성된 쯔쯔가무시병 진단 키트는, 빠르고 정확하게 쯔쯔가무시병을 진단할 수 있는 것으로서, 민감도가 높고 비특이 반응을 제거하여 특이도가 우수하며, 대조선의 발색이 일정하게 유지되므로 평가 결과에 대한 신뢰도를 확보할 수 있다.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.
도 1은, 본 발명의 일 실시예에 따른 스트립 타입의 쯔쯔가무시병 진단 키트를 나타낸 것이다.Figure 1 shows a strip-type Tsutsugashi disease diagnosis kit according to an embodiment of the present invention.
도 2는, 항원 농도에 따른 키트 반응을 나타낸 것이다.2 shows the kit reaction according to the antigen concentration.
도 3은, 혼합 항원 조성에 따른 키트 반응을 나타낸 것이다.Figure 3 shows the kit reaction according to the mixed antigen composition.
도 4는, 대조선의 단백질에 따른 키트 반응을 나타낸 것이다.Figure 4 shows the kit response according to the protein of the control line.
도 5는, 전반응선의 대장균 추출물 농도에 따른 키트 반응을 나타낸 것이다.Figure 5 shows the kit reaction according to the E. coli extract concentration of the entire reaction line.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 각 도면에 제시된 동일한 참조 부호는 동일한 부재를 나타낸다.Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.
아래 설명하는 실시예들에는 다양한 변경이 가해질 수 있다. 아래 설명하는 실시예들은 실시 형태에 대해 한정하려는 것이 아니며, 이들에 대한 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다.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.
실시예에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 실시예를 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성 요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성 요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.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.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.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.
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조 부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.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.
본 발명에서 사용된 용어, '진단'은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 또한, 본 발명의 목적상, 진단은 쯔쯔가무시병을 확인하는 것이다. 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.
본 발명에서 사용된 용어, '쯔쯔가무시병'은 오리엔티아 쯔쯔가무시(Orientia tsutsugamushi)의 병원성이 일으키는 질환을 말한다.The term 'tsutsugamushi disease' used in the present invention refers to a disease caused by the pathogenicity of Orientia tsutsugamushi.
본 발명의 제1 측면은, 제1 키트 및 제2 키트로 구성되며, 상기 제1 키트 및 제2 키트는 각각, 혼합항원을 포함하고, 상기 혼합항원은, 오리엔티아 쯔쯔가무시 길리암, 카프, 카토의 56kDa의 융합 단백질(cr56)과 오리엔티아 쯔쯔가무시 강원의 56kDa 단백질(kr56)을 5 : 3의 부피비로 혼합한 것인 쯔쯔가무시병 진단 키트를 제공한다. 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.
보다 구체적으로, 본 발명의 진단 키트를 구성하는 제1 키트 및 제2 키트는 각각, (a) 시료가 흡수되는 샘플패드(sample pad); (b) 시료 내의 인간의 항체와 결합하는 골드 컨쥬게이트 패드(gold conjugation pad); (c) 쯔쯔가무시 혼합 항원을 포함하는 반응선(test line)과 대조군 단백질을 포함하는 대조선(control line)이 처리되어 있는 반응막 (또는 테스트 막); 및 (d) 잔량의 시료가 흡수되는 흡수패드(absorption pad) 를 포함한다.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.
본 발명의 진단 키트는 쯔쯔가무시 혼합 항원을 포함한다. 본 발명에서 사용될 수 있는 쯔쯔가무시 혼합 항원은, 쯔쯔가무시병의 원인이 되는 오리엔티아 쯔쯔가무시(Orientia tsutsugamushi) 길리암(Gilliam), 카프(Karp) 및 카토(Kato)로부터 유래한 재조합 항원 단백질 및 강원(Kangwon)으로부터 유래한 항원 단백질이 혼합된 단백질이다. 즉, 본 발명의 쯔쯔가무시 혼합 항원은, 오리엔티아 쯔쯔가무시 길리암, 카프 및 카토의 항원 결정기를 구성하는 단백질을 코딩하는 유전자를 직렬로 연결한 재조합 유전자로부터 유래한 유전자 재조합 단백질과 오리엔티아 쯔쯔가무시 강원주의 항원 활성을 갖는 유전자로부터 유래한 유전자 재조합 단백질이 혼합되어 있는 것을 말한다. 구체적으로, 본 발명에서는 오리엔티아 쯔쯔가무시의 56kDa 단백질이 항원성을 가져 진단적 가치가 있다는 대한민국 공개특허 제2002-0020281호 등에 기재된 사실에 근거하여, 표준 혈청형인 길리암(Gilliam), 카프(Karp) 및 카토(kato)주의 56kDa 단백의 일부 절편을 코딩하는 유전자를 중합효소연쇄반응 (Polymerase Chain Reaction; PCR)으로 증폭하고, 이들 증폭된 DNA 절편을 직렬 연결한 후 단백질 발현용 벡터에 클로닝하여 대장균 내에서 발현시키고, 이를 분리 정제함으로써 쯔쯔가무시병 진단에 이용할 수 있는 융합 단백질 항원을 단일 공정으로 동시에 생산, 분리 정제할 수 있도록 하였다. 또한, 본 발명에서는 오리엔티아 쯔쯔가무시 길리암, 카프 및 카토주 외에 쯔쯔가무시병 진단의 민감도를 향상시키기 위하여 오리엔티아 쯔쯔가무시 강원주(Orientia tsutsugamushi Kangwon)의 항원 활성을 갖는 56kDa 단백질을 코딩하는 유전자를 중합효소연쇄반응으로 증폭하고, 이들 증폭된 DNA 절편을 단백 발현용 벡터에 클로닝하여 대장균 내에서 발현시키고 이를 분리 정제하여 상기 오리엔티아 쯔쯔가무시 길리암, 카프 및 카토주로부터 유래한 56kDa 단백 유전자 재조합 단백질과 함께 쯔쯔가무시병 진단을 위하여 사용하였다.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.
본 발명의 진단 키트는 오리엔티아 쯔쯔가무시 길리암, 카프 및 카토주의 재조합 56kDa 단백질 및 오리엔티아 쯔즈가무시 강원주의 56kDa 단백질을 5 : 3의 부피비로 섞은 쯔쯔가무시 혼합 항원을 포함한다. 상기 혼합 항원은 0.4 내지 0.8 mg/ml, 바람직하게는 0.5 내지 0.6 mg/ml, 가장 바람직하게는 0.533 mg/ml로 포함될 수 있다. 보다 구체적으로, 상기 쯔쯔가무시 혼합 항원은 키트 내 반응선 (test line)에 점적될 수 있다.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.
본 발명의 진단 키트는 골드-표지된 단백질과 결합할 수 있는 대조군 단백질을 포함한다. 본 발명에서 사용될 수 있는 대조군 단백질은, 예를 들면, 산양 항-닭 IgY 다클론 항체 (Goat anti-chicken IgY), 토끼 항-산양 IgG 다클론 항체, 산양 항-인간 IgG 다클론 항체, 토끼 항-산양 IgM 다클론 항체 및 산양 항-인간 IgM 다클론 항체로 이루어진 군으로부터 선택되는 적어도 어느 하나일 수 있으며, 바람직하게는, 산양 항-닭 IgY 항체일 수 있다. 산양 항-닭 IgY 항체를 사용하는 경우, 반응선의 반응 정도과 관계없이 대조선이 일정하게 발색되어, 진단키트 작동성에 관한 신뢰도를 확보할 수 있다는 이점이 있다. 상기 대조군 단백질은 0.1mg/ml 내지 2mg/ml의 농도로, 바람직하게는 1mg/ml의 농도로 포함될 수 있다.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.
본 발명의 진단 키트는, 쯔쯔가무시 항원 단백질을 발현하지 않는 대장균 추출물을 포함하는 전반응선(pretest line)을 더 포함할 수 있다. 상기 전반응선은 본 발명의 대장균에서 발현된 오리엔티아 쯔쯔가무시의 항원 단백질을 쯔쯔가무시병 진단 목적으로 사용하는데 있어 정제된 항원 단백질에 포함되어 있는 대장균 유래의 단백질과 사람 혈청 중에 포함되어 있는 대장균 유래의 단백질에 대한 항체에 의해 실제로 오리엔티아 쯔쯔가무시 항원 단백질에 대한 항체를 보유하고 있지 않음에도 환자인 것으로 오판될 수 있는 가능성을 배제하기 위함이다. 이 대장균 추출물은 1.5mg/ml 내지 3mg/ml, 바람직하게는 2mg/ml의 농도로 전반응선에 포함될 수 있다.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.
본 발명의 진단 키트에 포함되는 반응막은 규격화된 크기(10 × 500nm)의 니트로셀룰로즈막 (MiliporeTM XA3J072100) 등의 적합한 물질을 포함할 수 있다. 반응막은 상기 전반응선, 반응선 및 대조선이 순서대로 배열되는 것이 바람직하며, 이들 전반응선, 반응선 및 대조선은 2.0 내지 4.5mm의 간격, 바람직하게는 2.5 내지 3.0mm로 배열될 수 있다. 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.
본 발명의 진단 키트에 포함되는 샘플패드는, 0.2M sodum borate buffer (pH 8.6) 750ml, 20% Tween 20 75ml, 10% BSA 30ml, 10% NaN3 1.5ml, D.W 630ml를 포함하는 용액에 담가 흡수시킨 후에, 건조하여 제조되었다. 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.
본 발명의 진단 키트에 포함되는 골드 컨쥬게이트 패드는, 적합한 마커를 포함할 수 있으며, 예를 들면, 항-인간 IgM 컨쥬게이트 골드 (Anti-human IgM conjugated gold), 항-인간 IgG 컨쥬게이트 골드(Anti-human IgG conjugated gold) 및 닭 IgY 컨쥬게이트 골드 (Chicken IgY conjugated gold) 등을 포함할 수 있다. 보다 구체적으로는, 본 발명의 진단 키트를 구성하는 제1 키트는 항-인간 IgM 컨쥬게이트 골드 및 닭 IgY 컨쥬게이트 골드를 포함하는 제1 골드 컨쥬게이트 패드를 포함하고, 제2 키트는 항-인간 IgG 컨쥬게이트 골드 및 닭 IgY 컨쥬게이트 골드를 포함하는 제2 골드 컨쥬게이트 패드를 포함할 수 있다. 상기 골드 컨쥬게이트 패드는 샘플 패드에 인접하여 위치하는 것이 바람직하다. 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.
본 발명에서 사용되는 골드 컨쥬게이트 패드에 포함되는 마커는, 흡광도(optical density; OD, 530nm에서 측정된)가 0.1 내지 6, 바람직하게는 0.1 내지 3의 농도로 포함될 수 있다. 상기 마커의 양이 과량인 경우에는 분석하고자 하는 쯔쯔가무시 항체가 비교적 낮은 영역에서도 비특정 신호로 발현되어 위양성 결과를 초래할 수 있다. 예를 들면, 항-인간 IgM 컨쥬게이트 골드는 OD 1 내지 1.5, 바람직하게는 OD 1.25의 농도로 포함될 수 있으며, 항-인간 IgG 컨쥬게이트 골드는 OD 2 내지 4, 바람직하게는 OD 3의 농도로 포함될 수 있으며, 닭 IgY 컨쥬게이트 골드는 OD 0.1 내지 0.5, 바람직하게는 OD 0.3의 농도로 포함될 수 있다. 흡수패드는 막의 반대편 말단에 위치하며 모세관 현상에 의해 막을 따라 이동한 생물학적 시료, 예를 들면 혈청, 혈장 등을 흡수한다.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.
본 발명의 진단 키트는, 하나의 샘플패드로부터 분지되는 제1 키트 및 제2 키트로 구성된다. 예를 들면, 제1 키트는 쯔쯔가무시 항원 단백질에 대한 인간 IgM 항체를 검출하기 위한 것일 수 있고, 제2 키트는 쯔쯔가무시 항원 단백질에 대한 인간 IgG 항체를 검출하기 위한 것일 수 있으며, 이들의 순서는 바뀔 수 있다. 본 발명의 일 실시예에 따르면, 제1 키트는 항-인간 IgM 컨쥬게이트 골드와 닭 IgY 컨쥬게이트 골드를 포함하는 제1 골드 컨쥬게이트 패드 및 산양 항-닭 IgY 항체를 포함하는 대조선을 포함하며, 제2 키트는 항-인간 IgG 컨쥬게이트 골드와 닭 IgY 컨쥬게이트 골드를 포함하는 제2 골드 컨쥬게이트 패드 및 산양 항-닭 IgY 항체를 포함하는 대조선을 포함할 수 있다. 본 발명의 일 실시예에 따르면, 상기 제1 키트와 제2 키트는 병렬로 배열될 수 있으나, 본 발명은 이에 제한되지 않고, 제1 키트와 제2 키트가 반대방향으로 서로 직렬 배치되거나 또는 다양한 방향으로 배열될 수 있다. 본 발명의 진단 키트는 개별적으로 안전성을 확보하기 위하여 테스트 스트립을 둘러싸는 케이스로 패키징될 수 있다. 이 진단 키트는 진단 활성의 손실 없이 실온에서 오랜 기간(적어도 18개월 이상) 보관될 수 있다.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.
본 발명의 쯔쯔가무시병 진단 키트를 구성하는 제1 키트 및 제2 키트는 각각, 샘플패드, 골드 컨쥬게이트 패드, 반응막 및 흡수패드를 포함한다. 샘플 패드는 골드 컨쥬게이트 패드 위에 겹쳐져 있어 제1 중첩 부분을 형성하고, 골드 컨쥬게이트 패드는 막 위에 겹쳐져 있어 제2 중첩 부분을 형성한다. 또한 막과 흡수패드는 제3 중첩 부분을 형성한다. 생물학적 시료가 샘플패드 상에 점적되면 모세관 현상에 의하여 생물학적 시료는 골드 컨쥬게이트 패드를 통과하게 된다. 골드-표지를 형성하는 골드 입자는 바람직하게 20 내지 55nm의 지름 크기, 보다 바람직하게는 20 내지 40nm의 지름 크기를 가지며, 염색 지시자(indicator dye)로서 작용한다. 상기 골드 컨쥬게이트 패드를 통과함에 따라, 생물학적 시료 내의 항체는 골드-라벨된 마커와 결합하여 복합체를 형성하고, 상기 복합체는 막을 따라 이동한다.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.
본 발명에서 사용된, 용어 생물학적 시료는 쯔쯔가무시병이 의심되는 인간을 포함한 포유동물의 혈청, 혈장 등을 말한다. 생물학적 시료는 본 발명의 진단 키트의 샘플 패드에 점적하기 전에 희석하여 사용하거나 또는 희석하지 않고 사용할 수 있다. 생물학적 시료에 쯔쯔가무시 항체가 있는 경우에는 본 발명의 상기 진단키트 내 쯔쯔가무시 혼합 항원을 포함하는 반응선을 통과함에 따라 상기 항체가 쯔쯔가무시 혼합 항원과 결합하여 육안으로 식별할 수 있도록 막 위의 반응선에서 색 변화가 일어난다. 즉, 항원-항체 복합체의 형성으로 상기 골드 복합체에 의한 염색 지시자(indicator dye)가 침전하면서 검출할 수 있는 붉은 보라색 밴드를 나타내고, 이에 따라 생물학적 시료 내에 쯔쯔가무시 항체가 존재하는 양성 반응을 나타내게 된다. 그러나 생물학적 시료에 쯔쯔가무시 항체가 없는 경우에는 본 발명의 쯔쯔가무시 혼합 항원과 결합하지 않아 막 위의 반응선에서 아무런 변화가 일어나지 않는다.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.
본 발명의 제2 측면은, 상기 쯔쯔가무시병 진단 키트를 사용하여 환자의 시료 내 쯔쯔가무시병 IgM 및 IgG 항체를 동시에 검출하여, 쯔쯔가무시병 진단을 위한 정보를 제공한다. 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.
일반적으로, 쯔쯔가무시병이 의심되는 환자가 오리엔티아 쯔쯔가무시에 감염되었는지를 진단하기 위해서는 인체 내 오리엔티아 쯔쯔가무시의 존재 유무를 확인하여야 하는데, 현재 쯔쯔가무시병 진단방법은 주로 면역형광항체법을 이용하고 있다. 그러나, 이 방법은 감수성과 특이도가 높고, IgM과 IgG를 감별하여 측정할 수 있어 초기 감염과 재감염을 어느 정도 구분할 수 있다는 장점이 있으나, 균체 배양을 하여야 하기 때문에 세포배양시설이 필요하고 방법이 복잡하여 전문가에 의해서만 수행될 수 있으며, 시간과 유지비용 등이 많이 소요된다는 문제점이 있다. 이에 반하여, 본 발명의 진단 방법은 항원-항체반응에 의한 면역크로마토그래피 방법을 이용하기 때문에, 10분 내외의 빠른 시간 내에 간편하고 정확하게 쯔쯔가무시병을 진단할 수 있어 종래의 진단 방법에 비하여 우수한 효능을 가지고 있다.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.
본 발명의 쯔쯔가무시병 진단 키트를 이용하여 쯔쯔가무시병 항체를 검출하는 방법은 다음과 같다. 우선, 검체하고자 하는 생물학적 시료를 상기 진단 키트의 시료가 흡수되는 부위인 샘플패드에 점적하면, 생물학적 시료는 모세관 현상에 의해 골드 컨쥬게이트 패드에 도달하여 시료 내의 항체가 골드 컨쥬게이트 패드 내의 마커, 예를 들면, 항-인간 IgM 컨쥬게이트 골드 (Anti-human IgM conjugated gold), 항-인간 IgG 컨쥬게이트 골드(Anti-human IgG conjugated gold) 및 닭 IgY 컨쥬게이트 골드 (Chicken IgY conjugated gold) 등에 결합하여 콜로이드를 형성하게 된다. 생물학적 시료는 상기 골드 컨쥬게이트 패드에 고정화되지 않고 계속 이동하여 쯔쯔가무시 혼합 항원이 고정화되어 있는 반응선(test line)에 도달하게 된다. 쯔쯔가무시병에 감염된 환자의 생물학적 시료인 경우에는 상기 쯔쯔가무시 혼합 항원과 항원-항체반응을 일으키게 되는데, 즉 환자의 골드 컨쥬게이트 패드 내의 특정 마커에 결합된 쯔쯔가무시 항체는 반응선에 고정된 쯔쯔가무시 혼합 항원에 결합하여 붉은 보라색 밴드를 형성함으로써 육안으로 관찰할 수 있게 된다. 그리고, 생물학적 시료 내 항체와 반응하지 않은 나머지 골드 컨쥬게이트 패드 내의 마커는 대조선에 도달하여 대조군 단백질과 반응하여 붉은 보라색 밴드를 형성함으로써 검사의 적합성을 나타내게 된다. 이와 같이 본 발명의 진단 키트는 항원-항체 반응에 의한 면역크로마토그래피 방법을 이용함으로써 특별한 장비 없이 육안으로 검사결과를 확인할 수 있는 방법이다.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.
본 발명의 진단 키트는 전반응선(pretest line)을 포함함으로써, 실제로 오리엔티아 쯔쯔가무시 항원 단백질에 대한 항체를 보유하고 있지 않음에도 보유하고 있는 것으로 오판될 수 있는 가능성을 제거함으로써 쯔쯔가무시병 진단의 정확성을 향상시켰다.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.
본 발명의 일 실시예에 따르면, 본 발명의 쯔쯔가무시병 진단 키트를 사용하여, 진단 결과를 확인하기까지 걸리는 시간은 약 5 내지 10분으로서, 15분 이내이다. 진단 결과는 환자의 쯔쯔가무시 감염 상태를 진단하는데 사용할 수 있다. 예를 들면, 만일 쯔쯔가무시에 감염된 환자로부터 수집된 생물학적 시료라면, 본 발명의 진단 키트는 쯔쯔가무시 항체에 대해 양성 반응을 나타내어 쯔쯔가무시병으로 진단할 수 있으며, 만일 쯔쯔가무시에 감염되지 않은 환자로부터 수집된 생물학적 시료인 경우 본 발명의 진단 키트는 음성 반응을 나타내어 쯔쯔가무시병이 아닌 것으로 진단할 수 있다. 보다 구체적으로는, 본 발명의 대한 양성 반응 결과는 대조선 외에 쯔쯔가무시 혼합 항원을 포함하고 있는 반응선이 발색하는 경우이고, 쯔쯔가무시병에 대한 음성 반응 결과는 대조선만이 발색하는 경우이다. 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.
이하, 본 발명을 하기 실시예에 의해 보다 구체적으로 설명한다. 그러나 이는 본 발명의 이해를 돕기 위해 추가된 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.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.
실시예Example 1. 항원 단백질 발현 및 분리 1. Antigen Protein Expression and Isolation
1-1. 1-1. 카이메릭Chimeric 재조합  Recombination 56kDa(cr56)의56 kDa (cr56) 단백질 발현 Protein expression
오리엔티아 쯔쯔가무시 길리암, 카프, 카토(Orientia tsutsugamushi Gilliam, Karp, Kato)의 주요 항원 부위를 포함하는 카이메릭 재조합 단백질(cr56)은 대한민국 특허공개 제 2002-0020281호의 실시예에 기재된 방법과 동일한 방법으로 발현 및 분리 정제하였다.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.
즉, 오리엔티아 쯔쯔가무시의 길리엄, 카프, 카토를 마우스 L-929 세포에서 배양하여 수확한 후 정제한다. 정제된 균주를 효소 분해시킨 후 페놀 추출과 에탄올 침전으로 DNA를 정제한다. 또한, 오리엔티아 쯔쯔가무시 56kDa 단백 유전자의 염기서열을 근거로 하여, 길리엄, 카프, 카토 혈청형간 아미노산 서열이 30% 이상 상동성을 보이는 단백 부위를 선택하여, 길리엄, 카프, 카토 주에서 각각 한쌍의 올리고뉴클레오티드 프라이머를 제조한다. 이들 각각의 프라이머쌍을 가지고 상기에서 추출한 오리엔티아 쯔쯔가무시 DNA를 주형으로 하여 PCR 반응을 실시하여 길리엄, 카프 및 카토의 DNA 절편을 증폭시킨다. PCR 산물을 정제하여 pTYB12 벡터의 해당 제한효소 절단 부이에 연결되도록 클로닝한다. 먼저 pTYB12 벡터에 길리엄의 DNA 절편을 도입하여 벡터 pTG3를 제작하고, pTG3에 카프의 DNA 절편을 도입하여 벡터 pTGP1을 제작한 후, pTGP1에 카토의 DNA 절편을 도입하여 pTGPT2를 제작한다. 또한, 벡터 pTGPT2로부터 길리엄, 카프 및 카토의 DNA 절편의 직렬 연결체를 절단하여 pET22b(+) 벡터에 도입하여 벡터 pETb7을 제작한다. 제작한 발현 벡터로 대장균을 형질전환하고, 형질전환된 대장균을 배양하여 융합 단백질 발현을 유도한다. 전기영동 및 웨스턴 블롯으로 확인한 후 발현된 융합 단백 항원을 분리 정제하였다.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. 재조합 1-2. Recombination KangwonKangwon 56kDa56kDa 단백( Protein ( kr56kr56 )의 발현Expression of
계통발생 분석(Phylogenic analysis) 결과(FEMA Microbiology Letters, 1999, 180:163-169)를 참고하면 전세계적 주혈청형(prototype)인 카프(Karp), 길리엄(Gilliam), 카토(Kato)와 연천(Yonchon) (Kangwon과 가장 유사)으로 크게 분류될 수 있음을 고려할 때 이들 4가지 균주를 항원으로 이용하는 것이 매우 적절할 것으로 여겨졌다. 카프(Karp), 길리엄(Gilliam) 및 카토(Kato)는 cr56으로 제시된다. 또한 연천(Yonchon)은 유사한 강원(Kangwon)의 kr56으로 제시된다. 이에 본발명자들은 강원 56kDa 재조합단백질(kr56)을 카이메릭 재조합 단백(cr56)의 보조항원으로 사용하기로 하였다.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) Kangwon 56kDa protein (kr56) 발현의 개요(1) Overview of Kangwon 56kDa protein (kr56) expression
강원(Kangwon) 87-61의 주 항원 도메인(major antigenic domain)으로 알려진 부분 아미노산 84(250bp)부터 445(1,335bp)까지 NcoⅠ과 SalⅠ인식부위를 함유하도록 변형하여 프라이머 한 쌍을 제작하였으며(표 1), PCR 반응을 수행하였다. PCR 산물과 발현벡터 pET30a를 NcoⅠ과 SalⅠ 제한효소를 처리하여 절단한 다음, 이들을 서로 라이게이션 시켰다. 이 플라스미드를 대장균주 BL21에 형질전환 시킨 후 단백을 추출하였다.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.
Figure PCTKR2016013954-appb-I000001
Figure PCTKR2016013954-appb-I000001
*플라스미드 pET30a는 His-Tagged 융합 단백질을 발현함.Plasmid pET30a expresses His-Tagged fusion protein.
(2) 유전자의 클로닝 및 발현 (2) Cloning and Expression of Genes
중합효소 연쇄반응으로 증폭한 DNA (amino acid 84(250bp)부터 445 (1,335bp)까지 362 a.a. (total 1,086bp))를 1.2% 아가로즈 겔에서 전기영동한 다음 QIAGEN gel elution kit (QIAGEN Inc., USA)를 사용하여 회수하였다. UV 투과조명기(transilluminator)상에서 예상크기에 맞게 증폭된 밴드를 잘라내어 마이크로튜브로 옮겼다. 아가로즈 겔 부피의 3배량의 NaI 용액을 첨가한 다음 60℃에서 5분간 방치하여 겔을 완전히 녹였다. 여기에 글라스 밀크(glass milk)를 첨가하여 10초간 교반하였다. 이를 상온에서 17,000 g(Hanil, AI.5S-24,12,000rpm)로 1분간 원심분리한 다음 상층액을 제거하였다. 마이크로튜브를 상온에서 5분간 방치하여 건조시킨 다음 elution buffer를 첨가하여 교반한 후, 17,000 x g(12,000rpm)로 원심분리하여 상층액을 새로운 마이크로튜브로 옮겼다. 이렇게 정제된 PCR 산물과 pET30a 백터를 제한효소 NcoⅠ과 SalⅠ으로 완전절단한 후, Geneclean kit Ⅱ를 이용하여 동일한 방법으로 회수하였다. 절단된 백터 100ng과 절단된 PCR 산물 100ng을 혼합하고, 여기에 10배 농도의 리가제 완충액 (250mM Tris-HCl pH 7.8, 100mM MgCl2, 20mM DTT, 4mM ATP)과 리가제를 혼합하여 4℃에서 18시간 동안 반응시켰다.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) 적격세포(competent cell) 제작 및 형질전환 (3) Preparation and transformation of competent cells
LB 배지에서 18시간 배양한 대장균 BL21을, LB 배지에 100배 희석하여 600nm에서 흡광도가 0.3이 될 때까지 재배양하였다. 얼음 속에서 30분간 방치한 후 배지 상층액을 버리고, 0.1M CaCl2를 배지의 1/2 부피가 되도록 첨가하여 대장균을 부유시켰다. 얼음 속에서 1시간 동안 방치하고, 10분 동안 2,000 g(4,100rpm)에서 원심분리한 후, 배양한 배지의 1/10부피의 0.1M CaCl2로 부유하여 이를 형질전환에 사용하였다.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) 형질전환 (4) transformation
상기 (3)에서 제작한 리게이션 산물과 적격세포(competent cell)를 혼합하여 얼음 속에서 방치한 후 42℃에서 1분 30초간 열충격을 주었다. LB 배지를 첨가한 후 37℃에서 1시간 동안 배양하였다. 배양액을 LB 한천 평판배지에 접종한 후 37℃에서 배양하였다.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) 형질전환된 대장균의 plasmid DNA 분리 (5) Isolation of plasmid DNA of transformed Escherichia coli
형질전환된 대장균 단일집락을 카나마이신(Kanamycin)이 함유된 LB배지에 접종하여 37℃에서 진탕배양하였다. 플라스미드 DNA 추출은 AccuPrep Plasmid Extraction kit를 이용하였다. 배양액을 상온에서 750g (2,500rpm)로 10분간 원심분리한 다음 세포침전물만 회수하였다. 여기에 resuspenstion buffer를 첨가한 다음 교반하여 lysis buffer를 첨가하고 상온에서 5분간 방치하였다. Neutralization buffer를 첨가하여 상온에서 방치한 후 17,000xg (12,000rpm)로 원심분리하여 상층액을 바인딩 컬럼튜브(binding column tube)로 옮겼다. 이를 상온에서 17,000xg (12,000rpm)로 원심분리한 다음 추출액을 제거하였다. 여기에 80% 에탄올을 분주하여 원심분리한 다음 바인딩 컬럼만을 새로운 튜브로 옮겼다. 여기에 Elution buffer를 분주하여 상온에서 방치한 후 원심분리하여 상층액을 새로운 마이크로튜브로 옮겼다.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) 단백질의 발현 및 분리 (6) Expression and Isolation of Proteins
본배양배지에 종배양공정의 배양액을 접종하였다. 상기 접종은 37℃에서 이루어졌으며, 접종 후 1시간 45분동안 220 rpm 으로 교반되었다. O.D 값이 0.5 내지 0.6 의 값을 나타낼 때, IPTG를 0.5mM 접종하여 3시간 동안 배양하였다. 이 후, 4℃에서 30분 동안 3,000rpm으로 원심분리하여, 펠렛에 1xbinding Buffer(6 M Urea, 500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM Imidazole)를 넣고 Sonicator를 사용하여 (pulse: 10 sec on, 20 sec off, time: 6 min X 2회 (중간에 한 번 inverting), Amp.: 45%) 세포를 파쇄하였다. Kr56은 4 ℃에서 15분 동안 14,000 rpm으로 원심분리 (rotor 7 in autoclaved ultra centrifuge bottle)한 후, 상층액은 냉장 보관하고, 펠렛은 1X Binding buffer (1L Erlenmeyer flask 배양액 기준으로 50 ml)로 재현탁 하였다. Ice에서 1시간 동안 (30분 방치 후, inverting)놓아둔 후, 4 ℃에서 30분 동안 14,000 rpm으로 원심분리 (rotor 7 in autoclaved ultra centrifuge bottle)한 후, 상층액을 얻었다. 상층액은 1X Binding buffer (6M urea)로 2배 희석 후, 여과하여 (0.45 ㎛ syringe filter) 4℃에 보관하였다.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) 단백질 정제 (7) protein purification
Hisbind 레진 (Novagen)을 컬럼에 2ml이 되도록 채운 후, 증류수와 1 X charge buffer, 1 X binding buffer를 사용해 컬럼을 준비하였다. 항원단백을 컬럼에 통과시킨 후, 1 X washing buffer로 세척하였다. 1xElution 버퍼 (6 M Urea, 500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 500 mM Imidazole)를 통과시켜 단백질을 1 ml씩 회수하였다. 각각 분획내의 단백질 농도는 BCA protein assay 을 이용하여 정량하였으며, Amiconㄾ Ultra 30K로 농축한 후에 BCA protein assay를 이용하여 정량하였다. 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.
본 발명의 쯔쯔가무시 혼합 항원에 포함되는 오리엔티아 쯔쯔가무시 강원 56kDa 단백질의 구체적인 아미노산 서열은 등록특허 제10-0796772호에 개시된 아미노산 서열을 참고할 수 있다.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.
실시예Example 2. 항원 농도에 따른  2. Depending on antigen concentration 키트의Of kit 반응 (위양성 유무 판단) Reaction (determination of false positives)
상기 실시예 1에 따라 제조되는 본 발명의 혼합 항원을 각각 0.6 mg/ml, 0.9 mg/ml, 1.2 mg/ml 및 1.5 mg/ml 농도로 준비하였다. 각 농도의 항원을 포함하는 키트를 4개씩 준비하였다. 쯔쯔가무시병을 진단하는 표준진단시험법의 일종인 면역형광항체법을 이용하여 환자의 시료를 평가한 후, 음성 혈청 1개 (DK13)와, 양성 혈청 3개 (90-202, 00-350, 89-129)를 준비하여, 이를 서로 다른 항원 농도를 갖는 진단 키트에 각각에 적용하여 반응을 측정하였다. 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.
도 2는, 항원 농도에 따른 키트 반응을 나타낸 것이다.2 shows the kit reaction according to the antigen concentration.
아래의 표 2는, 항원 농도에 따른 키트 반응을 나타낸 것이다.Table 2 below shows the kit reaction according to the antigen concentration.
Figure PCTKR2016013954-appb-I000002
Figure PCTKR2016013954-appb-I000002
표 2 및 도 2에 나타난 바와 같이, 항원 농도가 0.9, 1.2, 1.5 mg/mL 인 진단 키트에서는 위양성이 나타났으나, 항원 농도가 0.6 mg/mL인 진단 키트에서는 위양성이 나타나지 않았다.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.
실시예Example 3. 혼합 항원 조성에 따른  3. According to the mixed antigen composition 키트Kit 반응 reaction
상기 실시예 1에 따라 제조되는 본 발명의 혼합 항원을 0.6 mg/ml로 준비하고, 종래의 혼합 항원을 동일한 농도로 준비하였다. 종래의 혼합 항원은, cr56, kr56, 및 r21 단백질을 각각 5 : 2: 1 비율로 혼합된 것이다. 본 발명의 혼합 항원을 포함하는 진단 키트와, 종래의 혼합 항원을 포함하는 진단 키트를 제조하여, 음성 혈청과 양성 혈청에 대한 반응도를 측정하였다. 대조군으로서, 각각의 음성 혈청과 양성 혈청은 IFA에 따른 Ab 역가가 측정되었다. 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.
도 3은, 혼합 항원 조성에 따른 키트 반응을 나타낸 것이다.Figure 3 shows the kit reaction according to the mixed antigen composition.
아래의 표 3은, 혼합 항원 조성에 따른 키트 반응을 나타낸 것이다.Table 3 below shows the kit reaction according to the mixed antigen composition.
Figure PCTKR2016013954-appb-I000003
Figure PCTKR2016013954-appb-I000003
표 3 및 도 3에 나타난 바와 같이, 혼합 항원에 r21이 포함되지 않고 kr56의 비율을 증가시킨 경우, 항체에 대한 반응 민감도가 향상되었다.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.
실시예Example 4. 대조선의 단백질에 따른  4. According to the protein of the control line 키트Kit 반응 reaction
상기 실시예 1에 따라 제조되는 본 발명의 혼합 항원 0.6 mg/ml, 및 대조선에 점적되는 단백질로 산양 항-닭 IgY를 1.0 mg/ml 포함하는, 본 발명의 진단 키트를 준비하였다. 다른 조건은 모두 동일하게 포함하지만, 대조선에 점적되는 단백질이 항-프로틴 A인 진단 키트를 준비하였다. 쯔쯔가무시병을 진단하는 표준진단시험법인 면역형광항체법을 이용하여 환자의 시료를 평가한 후, 음성 혈청과 양성 혈청을 각각의 진단 키트에 적용하여 반응을 측정하였다. 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.
도 4는, 대조선의 단백질에 따른 키트 반응을 나타낸 것이다.Figure 4 shows the kit response according to the protein of the control line.
아래의 표 4는, 대조선의 단백질에 따른 키트 반응을 나타낸 것이다.Table 4 below shows the kit response according to the control protein.
Figure PCTKR2016013954-appb-I000004
Figure PCTKR2016013954-appb-I000004
표 4 및 도 4에 나타난 바와 같이, 항-프로틴 A를 대조선의 단백질로 사용하였을 때는 대조선의 발색이 차이가 있었으나, 산양 항-닭 IgY를 대조선의 단백질로 사용한 경우에는 대조선 발색의 차이가 없이 일정한 수준을 유지하였다.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.
실시예Example 5. 정제된 항원 중에 포함된  5. contained in purified antigen 대장균유래E. coli derived 단백질과 사람 혈청 중의 항체와의 반응 제거 연구 Study on elimination of reaction between protein and antibody in human serum
His-bind 친화 크로마토그래피를 이용하여 항원을 정제하였으나 대장균유래 단백질을 완전히는 제거할 수 없었다. 따라서 정제로는 이러한 대장균유래 단백질을 완전히 제거하는 것이 힘들다고 판단하여 쯔쯔가무시 항원 단백질을 발현하지 않는 대장균 숙주세포만을 배양하여 추출액을 만든 후 테스트라인 앞 부분에 전반응선(pretestline)으로 점적함으로써 사람 혈청중에 포함되어 대장균유래의 단백질과 항원-항체 반응을 일으키는 항체를 제거하였다.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.
실시예Example 6.  6. 전반응선의Full response 대장균 추출물 농도에 따른  E. coli extract concentration 키트Kit 반응 reaction
상기 실시예 1에 따라 제조되는 본 발명의 혼합 항원 0.6 mg/ml, 및 대조선에 점적되는 단백질로 산양 항-닭 IgY를 1.0 mg/ml 포함하는 본 발명의 진단 키트를 준비하였다. 본 발명의 진단 키트 내 대장균 추출물은 각각 0.5mg/ml, 1mg/ml 또는 2mg/ml로 전반응선에 점적되었다. 음성 혈청을 준비하여 각각의 진단 키트에 적용하여 반응을 측정하였다. 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.
도 5는, 전반응선의 대장균 추출물 농도에 따른 키트 반응을 나타낸 것이다.Figure 5 shows the kit reaction according to the E. coli extract concentration of the entire reaction line.
아래의 표 5는, 전반응선의 대장균 추출물 농도에 따른 키트 반응을 나타낸 것이다.Table 5 below shows the kit reaction according to the E. coli extract concentration of the entire reaction line.
Figure PCTKR2016013954-appb-I000005
Figure PCTKR2016013954-appb-I000005
표 5 및 도 5에 나타난 바와 같이, 전반응선의 대장균 추출물의 농도가 0.5 mg/ml 및 1 mg/ml인 경우에는 위양성이 나타났으나, 전반응선의 대장균 추출물의 농도가 2 mg/ml인 경우에는 위양성이 나타나지 않았다.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.
실시예Example 6. 스틱 형태의  6. Stick form 진단제Diagnostics 샘플의 제조 Manufacture of samples
대장균에서 발현된 쯔쯔가무시 항원 (cr56, kr56) 단백질에 대한 쯔쯔가무시병 진단제로서의 효능을 확인하고 Kinematic automation(M1600)사의 디스펜서(dispenser_관리번호 IMM-E-P-005)라는 기기를 이용하여 진단용 테스트 스트립 샘플을 제작하였다. 테스트 스트립의 구조는 제3에서 보는 바와 같이 니트로셀룰로즈 막, 샘플패드, 흡수패드, 금결합패드(유리섬유)로 구성되어 있다. 니트로셀룰로즈 막은 밀리포아사에서 제작한 것을 사용하였는데 막이 쉽게 손상되는 것을 방지하기 위해 한쪽에 투명한 플라스틱을 부착하였다. 니트로셀룰로즈 막에는 3종의 단백질을 스프레이하였다. 1종은 환자의 혈청중에 존재하는 쯔쯔가무시에 대한 항체를 검출하기 위해 대장균에서 발현된cr56, kr56 단백항원을 적당한 양으로 혼합한 단백질로서 반응선(test line)이며, 다른 1종은 발색시스템의 정상적인 반응 여부를 판단하기 위한 대조선(control line)이다. 또 다른 1종은 사람 혈청중에 포함되어 대장균유래의 단백질과 항원-항체 반응을 일으키는 항체를 제거하기 위해 쯔쯔가무시 항원 단백질을 발현하지 않은 대장균 배양 추출액으로서 전반응선(pretest line)이다. 디스펜서를 사용하여 적정량의 단백질을 전반응선, 반응선, 대조선에 스프레이한 후 25℃에서 건조시켰다. 유리섬유에 5% 트레할로이즈에 적당량 콜로이달 골드 콘주게이트를 희석하여 흡수시킨 후 25℃에서 24시간 동안 건조시켰다. 샘플패드와 흡수패드는 각각 원하는 크기로 절단한 후 샘플패드에는 다시 혈청이나 혈장을 잘 흡수할 수 있도록 0.1M sodium borate buffer에 트윈 20을 0.5%가 되게 만든 용액에 담가 흡수시킨 후 상온에서 건조될 때까지 완전 건조시켰다. 다음에는 니트로셀룰로즈 막 아래쪽에 콜로이달 골드 콘주게이트가 스프레이된 금결합패드(유리섬유)를 접착하고, 그 아래에 다시 샘플패드를 부착하였다. 니트로셀룰로즈 막 위쪽에는 흡수패드를 부착하였다. 최종 진단제 스틱의 크기는 너비 4mm 높이 60mm되게 절단하였다. 본 발명에서는 두개의 스트립을 한 플라스틱에 넣어 동시에 진단할 수 있도록 제작하였다. 이에 따라 제작된 진단제 샘플에 대해 환자 혈청과 정상인 혈청을 사용하여 효능 시험을 수행해 본 결과, 신속, 정확, 간편한 쯔쯔가무시병 진단제로서의 효능이 입증되었다. 또한 다른 급성열성질환(신증후출혈열, 발진열, 렙토스피라증) 환자 혈청으로 교차 실험한 결과 반응이 일어나지 않은 것으로 확인되었다.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).
실시예Example 7.  7. 진단키트의Diagnostic Kit 성능 시험Performance test
본 발명의 쯔쯔가무시 진단키트의 민감도와 특이도를 비교 측정하기 위하여, 타사 (S사)의 쯔쯔가무시 진단키트와 비교 시험하였다. 쯔쯔가무시병을 진단하는 표준진단시험법인 면역형광항체법 (IFA)을 이용하여 평가된, 양성검체 60개와 음성검체 100개를 준비하였다. 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.
본 발명의 쯔쯔가무시 진단키트를 사용한 방법은 아래와 같다.The method using the Tsutsugamu diagnostic kit of the present invention is as follows.
1) 혈청, 혈장의 경우 3 ㎕, 또는 전혈의 경우 6 ㎕를 마이크로피펫으로 취하여 검체주입부 바닥의 샘플패드에 흡수되도록 주입하고, 즉시 검체희석액을 7방울을 검체주입부에 떨어뜨린다. 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) 대조선(C) 지역에 붉은 띠가 나타날 때까지 기다린다. 검사 개시 후 15분에 결과를 판독하며, 15분 이후에 새롭게 나타나는 붉은 띠는 결과판정에 포함시키지 않는다.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.
타사의 쯔쯔가무시 진단키트는, 해당 진단키트에 첨부된 설명서에 따라 사용되었다. Tsutsugamushi diagnostic kits of other companies were used in accordance with the instructions attached to the diagnostic kits.
진단키트의 성능을 시험한 결과, 본 발명의 쯔쯔가무시 진단키트는 민감도 99%, 특이도 100%로 평가된 반면, S사의 민감도 83%, 특이도 100%로 평가되어, 본 발명의 쯔쯔가무시 진단키트의 민감도가 더 높은 것으로 나타났다.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.
이상과 같이 실시예들이 비록 한정된 실시예와 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기의 기재로부터 다양한 수정 및 변형이 가능하다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다.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.
그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 특허청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the claims that follow.

Claims (8)

  1. 제1 키트 및 제2 키트로 구성되며,Consisting of a first kit and a second kit,
    상기 제1 키트 및 제2 키트는 각각, 혼합항원을 포함하고,The first kit and the second kit each include a mixed antigen,
    상기 혼합항원은, 오리엔티아 쯔쯔가무시 길리암, 카프, 카토의 56kDa의 융합 단백질(cr56)과 오리엔티아 쯔쯔가무시 강원의 56kDa 단백질(kr56)이 5 : 3의 부피비로 혼합된 것인, 쯔쯔가무시병 진단 키트.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. 제1항에 있어서,The method of claim 1,
    상기 제1 키트 및 제2 키트는 각각, 산양 항-닭 IgY 항체를 더 포함하는 것인, 쯔쯔가무시병 진단 키트.The first kit and the second kit, respectively, Tsutsugamushi disease diagnostic kit that further comprises a goat anti-chicken IgY antibody.
  3. 제1항에 있어서,The method of claim 1,
    상기 제1 키트는, 항-인간 IgM 컨쥬게이트 골드 및 닭 IgY 컨쥬게이트 골드를 포함하는 제1 골드 컨쥬게이트 패드를 포함하고, The first kit comprises a first gold conjugate pad comprising anti-human IgM conjugate gold and chicken IgY conjugate gold,
    상기 제2 키트는, 항-인간 IgG 컨쥬게이트 골드 및 닭 IgY 컨쥬게이트 골드를 포함하는 제2 골드 컨쥬게이트 패드를 포함하는 것인, 쯔쯔가무시병 진단 키트.Wherein said second kit comprises a second gold conjugate pad comprising anti-human IgG conjugate gold and chicken IgY conjugate gold.
  4. 제3항에 있어서,The method of claim 3,
    상기 항-인간 IgM 컨쥬게이트 골드는 OD 1 내지 1.5 농도이고, The anti-human IgM conjugate gold is in an OD 1-1.5 concentration,
    상기 항-인간 IgG 컨쥬게이트 골드는 OD 2 내지 4 농도이며, The anti-human IgG conjugate gold is at an OD 2-4 concentration,
    상기 닭 IgY 컨쥬게이트 골드는 OD 0.1 내지 0.5 농도인 것인, 쯔쯔가무시병 진단 키트.The chicken IgY conjugate gold is an OD of 0.1 to 0.5 concentration, Tsutsugamus disease diagnostic kit.
  5. 제1항에 있어서,The method of claim 1,
    상기 혼합 항원은 0.4 내지 0.8 mg/ml로 포함되는 것인, 쯔쯔가무시병 진단 키트.Wherein the mixed antigen is contained in 0.4 to 0.8 mg / ml, Tsutsugamus disease diagnostic kit.
  6. 제1항에 있어서,The method of claim 1,
    상기 제1 키트 및 제2 키트는 각각, 대장균 추출물을 1.5 내지 3 mg/ml 포함하는 전반응선 영역을 포함하는 것인, 쯔쯔가무시병 진단 키트.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. 제1항의 쯔쯔가무시병 진단 키트를 사용하여 환자의 시료 내 쯔쯔가무시병 IgM 및 IgG 항체를 동시에 검출하는 단계를 포함하는, 쯔쯔가무시병 진단을 위한 정보를 제공하는 방법.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. 제7항에 있어서,The method of claim 7, wherein
    상기 쯔쯔가무시병 진단을 위한 정보는, IFA로 측정되는 Ab 역가 (Ab titer)가 IgM 1:5 내지 1:20 및 IgG 1:50 내지 1:100인 쯔쯔가무시병 환자 진단에 필요한 정보인, 쯔쯔가무시병 진단을 위한 정보를 제공하는 방법.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.
PCT/KR2016/013954 2015-11-30 2016-11-30 Igm for early diagnosis of tsutsugamushi disease, igg antibody detection kit, and method for preparing same WO2017095135A1 (en)

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