CN107167606B - More diagnostic kits - Google Patents
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- CN107167606B CN107167606B CN201610825982.7A CN201610825982A CN107167606B CN 107167606 B CN107167606 B CN 107167606B CN 201610825982 A CN201610825982 A CN 201610825982A CN 107167606 B CN107167606 B CN 107167606B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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Abstract
Disclose more diagnostic kits, it is made of test strips, the test strips include tsutsugamushi antigen albumen, Leptospira antigen and HFRS Antigen albumen, and the tsutsugamushi antigen albumen contains the 56kDa fusion protein (cr56) of Orientia Tsutsugamushi Gilliam, Karp and Kato and the 56kDa albumen (kr56) of Orientia Tsutsugamushi Kangwon.
Description
Technical field
The present invention relates to more diagnostic kits.
Background technique
Be commonly used for diagnosing the illness as yochubio, leptospirosis and hemorrhagic fever with renal syndrome technology mainly with
Under type carries out: biological sample (blood or urine of the patient such as under a cloud for having infected the above disease) is coated onto diagnosis test paper
It is upper and check its reacting with the fixture in the test strips, thereby determine that whether patient has infected the above disease.
However, this kind of technology is only available for diagnosing single disease, in order to check the infection of a variety of diseases, it is necessary to independently into
Row is each to test and must inject multiple biological samples, therefore it is extremely complex to diagnose process.
Summary of the invention
Accordingly, it is considered to the present invention is made that the problem of encountering in the related technology, the present invention is intended to provide more diagnosis examinations
Agent box, can quickly and easily diagnosis of tsutsugamushi disease, leptospirosis and hemorrhagic fever with renal syndrome infection.
By following description, in conjunction with attached drawing, it will be clear that ground understands the present invention.
Embodiment of the present invention provides more diagnostic kits including test strips, and the test strips include tsutsugamushi antigen egg
White, Leptospira antigen and HFRS Antigen albumen, the tsutsugamushi antigen albumen contain Orientia Tsutsugamushi
The 56kDa fusion protein (cr56) and Orientia Tsutsugamushi of (Orientia tsutsugamushi) Gilliam, Karp and Kato
The 56kDa albumen (kr56) of Kangwon.
The Leptospira antigen may include the core polysaccharide and O- prepared from the lipopolysaccharides (LPS) of Leptospira
Specific side chain.
The HFRS Antigen albumen may include nuclear shell protein, and the nuclear shell protein is from Soochong
Amino acid sequence prepared by virus and with the SEQ ID NO:2 or 3 from eukaryocyte.
According to an embodiment of the invention, more diagnostic kits can detect yochubio, leptospirosis and kidney syndrome
The infection of Hemorrhagic fever.More diagnostic kits can diagnose simultaneously the infection of a variety of diseases by bolus injection biological sample.
In addition, more diagnostic kits do not include any non-specific antibody, so that it is guaranteed that the diagnostic result of high precision.
Detailed description of the invention
From detail specifications below and in conjunction with attached drawing, will be more clearly understood above-mentioned and other feature of the invention and
Advantage, in which:
Fig. 1 shows more diagnostic kits of embodiment of the present invention；
Fig. 2 shows the test strips of more diagnostic kits of embodiment of the present invention；
Fig. 3 shows the reaction of the kit of the dependence tsutsugamushi antigen protein concentration of embodiment of the present invention；
Fig. 4 shows the reaction of the kit for relying on tsutsugamushi antigen albumen composition of embodiment of the present invention；
Fig. 5 shows the antigenicity of the polysaccharide of purifying, is embodiment according to the present invention from patoc type and lai type
The SDS-PAGE (A) of middle isolated polysaccharide and the result of immunoblotting (B)；
Fig. 6 shows the nuclear shell protein (NP), complete NP (CNP) and truncated NP of embodiment of the present invention
(CNP) in the gene structure of the S section of Soochong virus；
Fig. 7 shows the DNA band of the TNP after the polymerase chain reaction (PCR) of embodiment of the present invention (about
370bp) and the DNA band (about 1300bp) of CNP；
Fig. 8 shows embodiment according to the present invention for the DNA clone of the DNA of TNP and CNP to pGem-T-Easy
In PCR cloning vector；
Fig. 9 shows embodiment according to the present invention for the PCR product of pGT-SooV2-TNP and pGT-SooV2-CNP
It is cloned into baculovirus plasmid carrier of the end N- with His-Taq；
Figure 10 shows embodiment according to the present invention and uses rod string design from pBac-SooV2-
TNP and pBac-SooV2-CNP forms Bac-SooV2-TNP and Bac-SooV2-CNP；
Figure 11 is shown embodiment according to the present invention and is printed using the protein of patients serum and normal serum detection TNP
The result of mark method；And
Figure 12 shows the protein of the pellets (pellet) of the purifying of the TNP and CNP of embodiment of the present invention
The result of blotting.
Specific embodiment
Hereinafter, embodiment of the present invention will be described in detail.The present invention is not limited to realities disclosed herein
Scheme is applied, but different form can be modified to.There is provided these embodiments is at large explain present disclosure simultaneously
Spirit of the invention is fully conveyed to those skilled in the art.Therefore, propose that these embodiments are understood not to
The limitation present invention.
Although term such as " first ", " second " are for describing various elements, the element should not be by such term institute
Limitation.These terms are only used to element is distinguished from each other.In addition, when the first element is described as being arranged in the second element,
Mean that the first element can be formed directly in the second element, or can be inserted into third element between the first and second elements.
In all the appended drawings, the size of element or the relative size of element may be described large to provide to of the invention
Readily comprehensible description.Further, since the variation of manufacturing method, the shape of element shown in the drawings can slightly change.Therefore,
Unless otherwise indicated, it should be appreciated that embodiment of the present invention is not limited in shape shown in the drawings, and some modifications may be incorporated into
Wherein.
As used herein, term " yochubio " is the disease as caused by Orientia Tsutsugamushi.
As used herein, term " leptospirosis " is the pathogenic spiral mattress by Leptospira (Leptospira)
Caused disease.
As used herein, term " hemorrhagic fever with renal syndrome " is viral by the Chinese his (Hantaan) and Seoul (Seoul) is viral
Caused disease.
As used herein, term " diagnosis " refers to the presence of pathologic state or the determination of feature, and in the present invention, examines
It is disconnected to be used to identify yochubio, leptospirosis and hemorrhagic fever with renal syndrome.
As used herein, term " sample ", which refers to, under a cloud has infected yochubio, leptospirosis and hemorrhagic fever renal syndrome
Blood, serum and the blood plasma of the mammal --- including people --- of heat.
Fig. 1 shows more diagnostic kits of embodiment of the present invention, and Fig. 2 shows the more of embodiment of the present invention
The test strips of diagnostic kit.
Referring to Fig.1 with 2, more diagnostic kits 10 may include test strips 100, and test strips 100 may include the first test strips
100a and the second test strips 100b.
First test strips 100a and the second test strips 100b can detect tsutsugamushi mite antibody, antibodies against Leptospira from sample
And nephrotic syndrome hemorrhagic fever antibody.It is comprehensive that first test strips 100a can detect tsutsugamushi mite IgM antibody, Leptospira IgM antibody and kidney
Simulator sickness Hemorrhagic fever IgM antibody, the second test strips 100b can detect tsutsugamushi mite IgG antibody, Leptospira IgG antibody and kidney syndrome
Hemorrhagic fever IgG antibody.
Each of first test strips 100a and the second test strips 100b may include supporting element 110, reaction film 120, sample
Product pad 130, gold-labelled pad (gold conjugate pad) 140, pre-reaction line 150, response line 160, control line 170 and suction
Receive pad 180.
Supporting element 110 is the base of test strips 100, and on supporting element 110 settable reaction film 120, sample pad 130,
Gold-labelled pad 140 and absorption pad 180.
Reaction film 120 can be nitrocellulose filter.Settable pre-reaction line 150,160 and of response line on reaction film 120
Control line 170.Pre-reaction line 150, response line 160 and control line 170 can be arranged with 2.0 to 4.5mm interval.
The absorbable sample injected via sample injection port 200 of sample pad 130.The sample pad 130 of first test strips 100a
It can be manufactured by following steps: immerse and contain 50mM Tris (pH8.0), 1% polysorbas20,0.5M NaCl and 0.1%NaN3's
In solution, then dry.The sample pad 130 of second test strips 100b can be manufactured by following steps: being immersed and contained 50mM Tris
(pH 8.0), 1% polysorbas20,0.2%BSA and 0.1% NaN3Solution, then dry.
Gold-labelled pad 140 can be configured to Chong Die with sample pad 130.The sample for being absorbed into sample pad 130 can pass through capillary
Effect is moved in gold-labelled pad 140.
Gold-labelled pad 140 may include tag antibody (marker antibody).The tag antibody may include that gold mark is anti-human
IgM, gold mark anti-human igg and gold mark chicken IgY.The concentration that the gold for being included marks anti-human IgM can be OD 1 to 3, the gold mark for being included
The concentration of anti-human igg can be OD2 to 4, and the concentration for the gold mark chicken IgY for being included can be OD 0.1 to 1.
First test strips 100a may include that gold marks anti-human IgM and gold marks chicken IgY, and the second test strips 100b may include that gold mark is anti-
Human IgG and gold mark chicken IgY.
The tag antibody can be with the tsutsugamushi mite antibody, antibodies against Leptospira and nephrotic syndrome hemorrhagic fever antibody knot
It closes, compound is consequently formed.Gold-labelled pad 140 can be configured to Chong Die with reaction film 120, thus the compound can be made to reaction
Film 120 is mobile.It can make the residue not in conjunction with the tsutsugamushi mite antibody, antibodies against Leptospira and nephrotic syndrome hemorrhagic fever antibody
Antibody is mobile to reaction film 120.
The tag antibody may include coloured particle.The coloured particle can be colloid gold particle, coloured glass or plastics
Pearl.
Response line 160 may include the first response line 161, the second response line 162 and third response line 163.
First response line 161 may include Leptospira antigen.The Leptospira antigen may include being originated from hook end spiral shell
Revolve the core polysaccharide and O- specific side chain of the lipopolysaccharides of body.The concentration for the Leptospira antigen for being included can be 2 to 4mg/ml.
Sample include antibodies against Leptospira in the case where, the antibodies against Leptospira can in gold-labelled pad 140
Tag antibody combines, and compound is consequently formed.When the compound passes through the first response line 161, the Leptospira is anti-
Body can be in conjunction with the Leptospira antigen of the first response line 161.This combination enables the coloured particle of the tag antibody
Enough Precipitations in the first response line 161, to form aubergine band.
Second response line 162 may include tsutsugamushi antigen albumen.The tsutsugamushi antigen albumen may include mixed with 5: 3 volume ratio
The 56kDa gene recombinant protein of Orientia Tsutsugamushi Gilliam, Karp and Kato of conjunction and Orientia Tsutsugamushi Kangwon's
56kDa albumen.The concentration for the tsutsugamushi antigen albumen for being included can be 0.4 to 0.8mg/ml.
Sample include tsutsugamushi mite antibody in the case where, the tsutsugamushi mite antibody can in conjunction with the tag antibody in gold-labelled pad 140,
Compound is consequently formed.When the compound passes through the second response line 162, the tsutsugamushi mite antibody can be with the second response line 162
Tsutsugamushi antigen protein binding.This combination enables the coloured particle of the tag antibody to sink in the second response line 162
Precipitation goes out, to form aubergine band.
Third response line 163 may include HFRS Antigen albumen.The HFRS Antigen albumen
May include from Soochong virus the amino acid sequence with SEQ ID NO:2 complete nuclear shell protein (CNP) or
The truncated nuclear shell protein (TNP) of the amino acid sequence with SEQ ID NO:3 from Soochong virus.Included
The concentration of HFRS Antigen albumen can be 0.1 to 1.0mg/ml.
In the case where sample includes nephrotic syndrome hemorrhagic fever antibody, the nephrotic syndrome hemorrhagic fever antibody can be with gold-labelled pad
Tag antibody in 140 combines, and compound is consequently formed.When the compound passes through third response line 163, the kidney is comprehensive
Levying hemorrhagic fever antibody can be with the HFRS Antigen protein binding of third response line 163.This combination makes the mark
The coloured particle of will antibody Precipitation in third response line 163, to form aubergine band.
It can make the remaining mark not in conjunction with the tsutsugamushi mite antibody, antibodies against Leptospira and nephrotic syndrome hemorrhagic fever antibody
Will antibody is mobile to control line 170 by response line 160.
Pre-reaction line 150 may include the first pre-reaction line 151 and the second pre-reaction line 152.
First pre-reaction line 151 may include Escherichia coli (E.coli) extract.The E. coli extract can be with
One non-specific antibody combines.First non-specific antibody can be and be originated from Escherichia coli protein bound antibody.
First pre-reaction line 151 can exclude following situations: although not actually existing the antibody for tsutsugamushi antigen albumen,
But due to above-mentioned non-specific antibody and include the albumen from Escherichia coli in tsutsugamushi antigen albumen combination, it is described anti-
Body not actually exists but is mistaken for existing.
Second pre-reaction line 152 may include that insect cell Sf (Spodopterafrugiperda (Spodoptera frugiperda)) is mentioned
Take object.The insect cell Sf extract can be in conjunction with the second non-specific antibody.Second non-specific antibody can for
Protein bound antibody from insect cell Sf.
Second pre-reaction line 152 can exclude following situations: since above-mentioned non-specific antibody goes out with included in kidney syndrome
The combination of the albumen from insect cell Sr21 in blood-head antigen protein, for the antibody of HFRS Antigen albumen
It not actually exists but is mistaken for existing.
Control line 170 may include reference protein.The reference protein can be in conjunction with the tag antibody.This combination makes
The coloured particle of the tag antibody can in control line 170 Precipitation, to form aubergine band.
The reference protein can be more selected from the anti-chicken IgY polyclonal antibody of goat (the anti-chicken IgY of goat), rabbit-anti goat IgG
At least one of clonal antibody and rabbit-anti goat IgM polyclonal antibody.The concentration for the reference protein for being included can be 0.1 to 2mg/
ml。
The absorbable remaining sample moved along reaction film 120 of absorption pad 180.Absorption pad 180 can be configured to and react
Film 120 is overlapped.
For the embodiment of tsutsugamushi antigen albumen
The expression and separation of 1. antigen protein of embodiment
1.1. it is fitted into the expression of recombination 56kDa (cr56) albumen
Orientia Tsutsugamushi with Korean patent application publication the 2002-0020281st disclosed in embodiment method
The chimeric weight of the Primary epitope comprising Orientia Tsutsugamushi Gilliam, Karp and Kato is expressed, separates and purified to identical mode
Histone (cr56).
Specifically, Gilliam, Karp and Kato plants of Orientia Tsutsugamushi are cultivated in mouse L-929 cell, receive
Collection, then purifies.By the strain enzymic digestion of purifying, DNA purifying is then carried out by phenol extraction and ethanol precipitation.Based on tsutsugamushi mite
The base sequence of the 56kDa protein gene of sick Orientia, in Gilliam, Karp and Kato serotype selection have 30% or
The more protein loci of homoamino acid sequence homology, and for a pair of few core of Gilliam, Karp and Kato plants of each strain preparation
Thuja acid primer.Use the DNA of extracted Orientia Tsutsugamushi as template, PCR is carried out using each primer pair, with amplification
Gilliam, Karp and Kato plants of DNA fragmentation.PCR product is purified and cloned, to be connected to the corresponding limit of pTYB12 carrier
Enzymic digestion site processed.Firstly, Gilliam plants of DNA fragmentation is introduced into pTYB12 carrier to prepare carrier pTG3, and will
Karp plants of DNA fragmentation is introduced into pTG3 carrier to prepare carrier pTGP1.Kato plants of DNA fragmentation is introduced into pTGP1 to carry
To prepare carrier pTGPT2 in body.Further by with the concatenated connector of Gilliam, Karp and Kato plants of DNA fragmentation from
It is cut on pTGPT2 carrier, and the connector is introduced into pET22b (+) carrier to prepare carrier pETb7.Using prepared
Expression vector convert Escherichia coli, cultivate the transformed Escherichia coli then with the expression of induced fusion albumen.It uses
Electrophoresis and immunoblotting identify the fusion protein, separate later and purify expressed fusion protein antigen.
1-2. recombinates the expression of Kangwon 56kDa albumen (kr56)
In view of strain is divided into global prototype-strain Karp, Gilliam and Kato and Yonchon (with Kangwon most phase
Like), based on Phylogenetic Analysis (phylogenic analysis) result (FEMA Microbiology Letters,
1999,180:163-169), above-mentioned four kinds of strains are considered being most suitable for being used as antigen.Karp, Gilliam and Kato are by cr56 table
Show.Equally, it is similar to be represented as kr56, Kangwon and the Yonchon of Kangwon by Yonchon.The present inventor uses Kangwon
Helper antigen of the 56kDa recombinant protein (kr56) as chimeric recombinant protein (cr56).
(1) general introduction of Kangwon 56kDa albumen (kr56) expression
One long to 84 (250bp) to 445 (1335bp) of the referred to as major antigen structural domain of Kangwon 87-61
Amino acid sequence is divided to be modified to prepare pair of primers and carry out PCR comprising Nco I and Sal I recognition site.Using limitation
Enzyme Nco I and Sal I digests PCR product and expression vector pET30a, and then they are connected to each other.Using acquired
Plasmid convert e. coli bl21, then extract albumen from the e. coli bl21.Above-mentioned primer is shown in the following table 1.
[table 1]
* plasmid pET30a expresses the fusion protein of His- label.
(2) gene cloning and expression
By by the DNA of PCR amplification, (362 amino acid of 84 (250bp) to 445 (1335bp) are (altogether
Electrophoresis 1086bp)) is carried out in 1.2% Ago-Gel, then using QIAGEN gel elution kit (QIAGEN Inc.,
The U.S.) recycling.It will be cut on ultraviolet transilluminator through expanding with the DNA band for being suitable for required size, be transferred to micro tube, with
It is equivalent to 3 times of Ago-Gel volume of amount and NaI solution is added, and stand 5 minutes at 60 DEG C, so that gel is completely dissolved.
It is newborn (glass milk) that glass is further added into gel solution, stirs 10 seconds, and at room temperature 17000g (Hanil,
AI.5S-24,12000rpm) under be centrifuged 1 minute, then remove supernatant.So that micro tube is stood 5 minutes at room temperature, then does
It is dry.Elution buffer is added thereto, stirs micro tube later, is then centrifuged at 17000g (12000rpm), and by supernatant
Liquid is transferred to new micro tube.The PCR product of purifying and pET30a carrier are digested completely with restriction enzyme Nco I and Sal I, so
It is recycled afterwards using Geneclean kit II by identical method.Carrier and 100ng warp by 100ng through restriction enzyme digestion
The PCR product of restriction enzyme digestion mutually mixes, with 10 × ligase buffer solution (250mM Tris-HCl pH 7.8,100mM
MgCl2, 20mM DTT and 4mM ATP) and ligase combine, and reacted 18 hours at 4 DEG C.
(3) preparation and conversion of competent cell
The large intestine bar mattress BL21 cell LB culture medium cultivated in LB culture medium 18 hours is diluted 100 times, then
It is further cultured for reaching 0.3 to the absorbance at 600nm.So that cell is stood 30 minutes in ice, falls off on culture medium later
Clear liquid, and 0.1M CaCl is added to be equal to the amount of culture volume half2, with the Bacillus coli cells that suspend.By the large intestine of suspension
Bacilli-cell stands 1 hour in ice, is centrifuged 10 minutes, is then suspended in as culture volume ten at 2000g (4100rpm)
/ mono- 0.1M CaCl2In, it is subsequently used for converting.
(4) it converts
The competent cell prepared in above-mentioned (3) is mixed with connection product, is placed in ice, the then heat shock 1 at 42 DEG C
Divide 30 seconds.LB culture medium is added into gained mixture, and is cultivated 1 hour at 37 DEG C.Meat soup is inoculated into LB agar plate
In culture medium, then cultivated at 37 DEG C.
(5) isolated plasmid dna from the large intestine bar mattress of conversion
The single mattress of the large intestine bar mattress of conversion is fallen and is inoculated into LB culture medium containing kanamycin, then at 37 DEG C
Shake culture.Plasmid is carried out using AccuPrep plasmid extraction kit (AccuPrep Plasmid Extraction kit)
The extraction of DNA.Meat soup is centrifuged at 750g (2500rpm) at room temperature 10 minutes, thus only recycling cell granular precipitates, so
Settling flux buffer is added in the backward cell granular precipitating, stirring is added lysis buffer, then stands 5 at room temperature
Minute.Neutralization buffer is added into products therefrom, stands at room temperature, is then centrifuged at 17000 × g (12000rpm),
Supernatant is transferred in conjunction in column tube later, is then centrifuged at 17000 × g (12000rpm) at room temperature, to remove warp
The solution of extraction.Then, 80% ethyl alcohol equal part (aliquoting) and centrifugation are carried out, column is only placed on new test tube later
In.Elution buffer is added into column, stands it at room temperature, and is centrifuged, is then transferred to supernatant new micro
Guan Zhong.
(6) protein expression and separation
Current culture medium is inoculated at 37 DEG C using the meat soup of seed culture method, stirs 1 at 220rpm
45 minutes hours were inoculated with when OD is 0.5 to 0.6 using 0.5mM IPTG, and were cultivated 3 hours.Thereafter, at 4 DEG C
It is centrifuged 30 minutes under 3000rpm, 1 × combination buffer (6M urea, 500mM NaCl, 20mM is added into pellets
Tris-HCl (pH 8.0), 5mM imidazoles), and by clasmatosis, (pulse: opening for 10 seconds, closes within 20 seconds, the time: 6 using ultrasonoscope
Minute × 2 times (once being commutated during this period), Amp.:45%).Thereafter, by kr56 at 4 DEG C in 14000rpm (high pressure
Rotor 7 in the ultracentrifugation engine room of sterilizing) under be centrifuged 15 minutes, later, supernatant is freezed, and by pellets settling flux
In 1 × combination buffer (50ml, based on the meat soup in 1L conical flask).Pellets are placed 1 hour in ice and (stands 30
Minute, then it is inverted), 30 points are centrifuged under 14000rpm (rotor 7 in autoclaved ultracentrifugation engine room) at 4 DEG C
Clock, to obtain supernatant.Supernatant is diluted 2 times with 1 × combination buffer (6M urea), filtering (0.45nm injection filtering
Device) and stored at 4 DEG C.
(7) protein purification
Using 2ml Hisbind resin (Novagen) packed column, distilled water, 1 × loading buffer are then used
(charge buffer) and 1 × combination buffer are prepared.It is slow using 1 × washing after so that antigen protein is passed through the column
Fliud flushing washs the column.Make 1 × elution buffer (6M urea, 500mM NaCl, 20mM Tris-HCl (pH 8.0), 500mM
Imidazoles) column is passed through, recycle every kind of albumen of 1ml.After being concentrated using Amicon Ultra 30K, BCA albumen is used
Measurement determines the concentration of albumen in each fraction.
For include embodiment 1 tsutsugamushi antigen albumen in Orientia Tsutsugamushi Kangwon 56kDa albumen tool
Body amino acid sequence, reference can be made to amino acid sequence disclosed in Korean Patent No. 10-0796772.
Embodiment 2. relies on the reaction of the kit of antigen concentration (determine false positive)
With the tsutsugamushi antigen egg of the concentration preparation embodiment 1 of 0.6mg/ml, 0.9mg/ml, 1.2mg/ml and 1.5mg/ml
It is white.The kit of the four tsutsugamushi antigen albumen comprising respective concentration of preparation.Using immunofluorescence antibody test, (it is examined for one kind
The standard test method of disconnected yochubio) evaluation patient sample, prepare a negative serum (DK13) and three positive bloods later
They are added to each diagnostic kit with different antigen concentrations with observing response by (90-202,00-350,89-129) clearly.
Fig. 3 shows the kit reaction of the dependence tsutsugamushi antigen protein concentration of embodiment of the present invention.
The kit reaction for relying on tsutsugamushi antigen protein concentration is given in Table 2 below.
[table 2]
From table 2 and Fig. 3 it will be evident that wherein the concentration of tsutsugamushi antigen albumen be 0.9mg/ml, 1.2mg/ml and
The diagnostic kit of 1.5mg/ml is false positive, and wherein the concentration of tsutsugamushi antigen albumen be 0.6mg/ml diagnostic kit not
It is false positive.
Embodiment 3. relies on the kit reaction of hybrid antigen composition
With the tsutsugamushi antigen albumen of 0.6mg/ml preparation embodiment 1, and conventional tsutsugamushi antigen egg is prepared with identical concentration
It is white.Conventional tsutsugamushi antigen albumen is made of cr56, kr56 and r21 albumen, and cr56, kr56 and r21 albumen is with 5: 2: 1 ratio
Example mixing.The diagnostic kit of tsutsugamushi antigen albumen of the manufacture comprising embodiment 1 and examining comprising conventional tsutsugamushi antigen albumen
Disconnected kit, and it is measured for negative and positive serum reactivity.As a control group, negative and sun is measured by IFA
The Ab potency of property serum.
Fig. 4 shows the kit reaction for relying on tsutsugamushi antigen albumen composition of embodiment of the present invention.
The sensitivity results of the antibody response of conventional tsutsugamushi antigen albumen and the tsutsugamushi antigen albumen of embodiment 1 by -=0 ,+
=1, ++=2, +++=3, +++ +=4 expressions, as shown in table 3 below.
[table 3]
From table 3 and Fig. 4 it will be evident that when tsutsugamushi antigen albumen does not contain the amount increase of r21 and kr56, antibody
The sensitivity of response increases.
For the embodiment of Leptospira antigen
4. Leptospira antigen of embodiment
The culture of 4-1. Leptospira strain
By EMJH (leptospira medium substrate Ellinghausen McCullough Johnson Harris) with
0.23g/90ml is dissolved and is sterilized, and EMJH of the 10ml rich in Leptospira is added then to prepare culture medium.By the culture medium
With Leptospira inoculation and shake culture 5 days at 30 DEG C.
The preparation of 4-2. Leptospira antigen
The Leptospira cultivated in EMJH culture medium 5 days is centrifuged 20 minutes at 4000 × g, using 1 × PBS
Buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4With 2mM KH2PO4) wash three times, then add at 100 DEG C
Heat 1 hour.It reacts it at 37 DEG C 16 hours with 150 μ g/ml Proteinase Ks, 2mM EGTA is added, reacts it at 70 DEG C
It 15 minutes, is then centrifuged 2 hours at 180000 × g at 4 DEG C.Gained pellets are suspended in H2It is more to obtain rouge in O
Sugared (Westpha and Jann, 1965).By 1% acetic acid treatment of the LPS antigen so purified, heated 1 hour at 100 DEG C, and
It is centrifuged 20 minutes at 3000 × g, the lipid A of Precipitation is removed, polysaccharide antigen is obtained.It is measured using standard polysaccharide such
The amount of the antigen of acquisition, the standard polysaccharide are by being handled in the same manner as described above from Escherichia coli (Escherichia
Coli obtained from the LPS (Sigma) purified in).
The antigenicity evaluation of 5. Leptospira polysaccharide of embodiment
5-1. immunoblotting
SDS- is carried out on 12% gel with a thickness of 1mm or 1.5mm using Bio-Rad Protean II electrophoretic apparatus
PAGE.Using Bio-Rad transfer groove (Transblot cell) at 80V in 30 minutes by albumen from the gel after electrophoresis
It is transferred on film.By 1 × TBS buffer blind of the film after transfer containing 5% skimmed milk and 0.1% polysorbas20, primary antibody is
Rabbit immune serum (leptospira interrogans (L.interrogans) strain WH-19), secondary antibody is the horseradish mistake for being diluted to 1: 10000
The anti-rabbit IgG (Bio-Rad) of oxide enzyme conjugation.After the completion of immune response, ECL kit (Amersham is used
Pharmacia Biotech) it develops the color.
Fig. 5 shows the antigenicity of the polysaccharide of purifying, is embodiment according to the present invention from patoc type and lai type
The SDS-PAGE (A) of middle isolated polysaccharide and the result of immunoblotting (B).
As shown in figure 5, the polysaccharide that leptospira interrogans (Leptospira interrogans) serum becomes lai type exists
It is similar to the polysaccharide of leptospira interrogans serovar patoc type strain Patoc in terms of PAGE gel electrophoretogram, and
14kDa or bigger polysaccharide are present in lai type and patoc type the two and are considered serving as antigen.
5-2. dot blotting
1 μ g (1 μ g/1 μ l) antigen is added drop-wise on nitrocellulose filter, it is then 1 hour dry at 37 DEG C.After dry
Film closed 1 hour with 5% skimmed milk-TBST, so that it with patients serum's (being diluted to 1: 3000) as primary antibody is reacted 1 small
When, and reacted with the anti-human igg (being diluted to 1: 10000) of the horseradish peroxidase conjugation as secondary antibody.After the reaction was completed, make
It is developed the color with ECL kit, and its result is compared with MAT result.In Most patients serum, the positive is observed
Signal.
The following table 4 shows the comparison result of MAT and dot blotting.
[table 4]
5-3. enzyme linked immunosorbent assay (ELISA) (ELISA)
Polysaccharide ELISA is carried out on the basis of the method for Engvall and Perlmann (1972).By poly-L-Lysine (10
μ g/ml) it is diluted with 0.01M PBS with pre-processed board, and react it at room temperature 24 hours.By the plate with containing 0.05%
The PBS buffer solution of polysorbas20 washes twice, and polysaccharide is suspended in PBS handles the plate with 100 holes μ l/ later.Make through handling
Plate reacted 1 hour at 37 DEG C, using containing 0.05% polysorbas20 PBS buffer solution washing three times, at lowlenthal serum
Reason, and closed at 37 DEG C.The serum of the serum and 20 normal persons that use 20 Leptospira patients is as one
It is anti-, and the anti-human igg (Bio-Rad) (being diluted to 1: 10000) for using horseradish peroxidase to be conjugated is as secondary antibody.It uses in each hole
Substrate processing measures absorbance using microplate reader thus to observe its colour developing at 490nm.As a result, discovery Leptospira whip
Hair (Leptospira flagella) recombinant protein shows 72% to 88% sensitivity and 88% to 90% specificity,
And polysaccharide of the invention shows 95% sensitivity and 95% specificity.Therefore, as leptospirosis early stage
The antigen candidate object of diagnosis finds the polysaccharide better than the albumen.
The following table 5 is shown in using the result that sensitivity and specificity of the ELISA to the polysaccharide measure.
[table 5]
A: the patients serum's number diagnosed by MAT: 20
B: normal serum number: 20
For the embodiment of HFRS Antigen albumen
Embodiment 6.Soochong virus and culture cell
The Soochong Strain SooV-2 separated from the apodemus speciosus of South Korea (Apodemus peninsulae) is existed
Secondary culture in vero cell.For the proliferation and maintenance of vero cell, is used in 37 DEG C of incubator and contain 10% tire ox
The EMEM (Eagle minimum essential medium) of serum (PBS) is in 5%CO2In the presence of cultivated, when culture is to about 90%
Cell is used when single layer.
Embodiment 7: the separation of viral RNA
Soochong virus is centrifuged 3 minutes at 1075g (3000rpm) in the cell being wherein proliferated, 750 μ l are added
Trizol LS (Gibco) solution, makes it stand 5 minutes at room temperature, and 200 μ l chloroforms are added, then stand 10 at room temperature again
Minute.It is centrifuged at 17000g (12000rpm) at 4 DEG C 15 minutes, removes the pellets of Precipitation, Xiang Shangqing later
In liquid be added same volume isopropanol, so that it is stood 10 minutes at room temperature, at 4 DEG C at 17000g (12000rpm) from
The heart 15 minutes, so that RNA precipitate is precipitated, it is later that obtained RNA pellets are primary with 75% ethanol washing, in air
Middle drying 10 minutes, is then dissolved in DEPC aqueous solution.
Implement synthesis and the PCR of 8:cDNA
Fig. 6 shows the nuclear shell protein (NP), complete NP (CNP) and truncated NP of embodiment of the present invention
(CNP) in the gene structure of the S section of Soochong virus, Fig. 7 shows TNP of the embodiment of the present invention after PCR
DNA band (about 370bp) and CNP DNA band (about 1300bp).
The PCR primer for being used to prepare Soochong virus recombinant antigen is shown in the following table 6.
[table 6]
From Fig. 6 and table 6 it will be evident that in order to from coding NP nucleic acid sequence (GenBank registration number AY675350)
SEQ ID NO:1 synthesizes cDNA, and each primer (10pmol) being added in 1 μ l table 6 into the RNA solution of 4 μ l embodiments 7 is simultaneously added
Include 5 × reverse transcriptase buffer, 10mM dNTPs, 100pmol/ μ l Oligo (dT), 40 units/μ l RNase inhibitor
(RNasin) and 200 units/μ l MMLV reverse transcriptase mixture.Reaction carries out 10 minutes at 30 DEG C, carries out at 45 DEG C
It 30 minutes, carries out 5 minutes at 99 DEG C and carries out 5 minutes at 5 DEG C, obtain cDNA.
Include 5 μ l cDNA products, 10pmol primer pair, 10mM dNTPs, 10 × taq buffer and 5 units/μ l Taq
The PCR of the mixture of polymerase is carried out primary with following circulation: 95 DEG C 5 minutes, 52 DEG C 2 minutes and 72 DEG C 2 minutes；With as follows
Circulation carry out five times: 95 DEG C 1 minute, 52 DEG C 1 minute and 72 DEG C 1 minute；With following circulation carry out 20 times: 95 DEG C 1 minute,
60 DEG C 1 minute and 72 DEG C 1 minute；And carried out with following circulation primary: 95 DEG C 1 minute, 60 DEG C 1 minute and 72 DEG C 7 minutes, from
And DNA amplification.The DNa of amplification is saved at -20 DEG C.
Fig. 7 shows the DNA band (about 370bp) of the TNP after PCR and the DNA band (about 1300bp) of CNP.
As shown in fig. 7, carrying out PCR using each primer, the DNA band of TNP is thus identified at about 370bp, and about
The DNA band of CNP is identified at 1300bp.
Implement the clone of 9. amplified productions
Fig. 8 shows the DNA clone by the DNA of TNP and CNP of embodiment of the present invention to pGem-T-Easy PCR
In cloning vector.
As shown in figure 8, by being carried out on Ago-Gel by the DNA at about 370bp and about 1300bp of PCR amplification
Electroelution is simultaneously cloned into pGem-TEasy PCR cloning vector (Invitrogen).By using restriction enzyme Bgl II and Hind
The DNA of III digested to identify TNP and CNP.This is intended to prepare CNP (1287bp:37-1323) and TNP (357bp:37-393)
Polypeptide.Its amino acid sequence is shown in SEQ ID NO:2 and 3.The plasmid for wherein inserting the 369bp containing TNP is known as pGT-
The plasmid for wherein inserting the 1299bp containing CNP is known as pGT-SooV2-CNP by SooV2-TNP.
Embodiment 10. is cloned into expression vector
Fig. 9 shows the PCR product gram by pGT-SooV2-TNP and pGT-SooV2-CNP of embodiment of the present invention
It is grand to the end N- with His-Taq baculovirus plasmid carrier in.
As shown in figure 9, the expression fusion vector that will there is His-Taq in the end N-, i.e. pBlue-Bac-His2B
(Invitrogen), it is handled using restriction enzyme Bgl II and Hind III, and by pGT-SooV2-TNP and pGT-SooV2-CNP
PCR product cloned using restriction enzyme Bgl II and Hind III, to obtain pBac-SooV2-TNP and pBac-
SooV2-CNP。
The formation of 11. recombinant baculovirus of embodiment
Figure 10 show embodiment of the present invention using rod string design from pBac-SooV2-TNP
Bac-SooV2-TNP and Bac-SooV2-CNP is formed with pBac-SooV2-CNP.
As shown in Figure 10, using Bac-N-Blue rod string design by pBac-SooV2-TNP and pBac-
Bac-SooV2-TNP and Bac-SooV2-CNP is made in SooV2-CNP.
By the Sf9 cell (2 × 10 of logarithmic phase6) be placed in the plate of 60mm to form single layer.In order to prepare transfection mixing
Object, by 10 μ l (0.5 μ g) Bac-N-BlueTMDNA is placed in microcentrifuge, and following solution is added.
PBac-SooV2-TNP or pBac-SooV2-CNP (1 μ g/ μ l): 4 μ l
Grace Insect culture medium (without replenishers or FBS): 1ml
Cellfectin reagent (using preceding uniformly mixed and constantly mixing in use): 20 μ l
So that plate is stood 15 minutes at room temperature, culture medium is removed from plate, 2ml is added later and does not both contain replenishers
Also the Grace Insect culture medium for not containing FBS removes culture medium again from single layer, and prepared transfection mixture is dripped
It is added in the plate of 60mm.So that the plate is stood about 4 hours at room temperature, 1ml TNM-FH culture medium is added, seals, and 27
It is incubated for 72 hours at DEG C.When carrying out cotransfection in this way, the recombinant baculovirus of duplication is used as virus stocks
(stock)。
The purifying of 12. recombinant baculovirus of embodiment
The concentration of the virus stocks is measured using Endpoint Dilution Method.
For determining the end dilution of virus titer, 10 are carried out to virus inoculation solution-5To 10-8Serial dilution again, will
Each dilute solution is inoculated into 1 × 104The cell concentration in/hole is distributed in the culture cell on 96 orifice plates, and at 27 DEG C into
The continuous culture of row.Virus titer is calculated from the ratio in the hole in the hole and uninfecting virus of having infected virus, to determine PFU (plaque
Form unit).
In order to select pure virus, virus inoculation solution is diluted 10-2To 10-8Times, each dilute solution is inoculated into 1
×104The cell concentration in/hole is distributed in the culture cell on 96 orifice plates, and is continuously cultivated at 27 DEG C.After inoculation
From 4th day, the formation and virus infection of intracellular polygonal crystal are observed using microscope, select the maximum dilution factor
Hole in virus, and purify finally obtain pure virus at least three times in an identical manner.
The expression and separation of 13. recombinant protein of embodiment
By Sf9 insect cell with about 5 × 106Density sow in 75cm2In flask, and it is inoculated with 5MOI recombinant virus.?
The 5th day after infection, observes the formation of occlusion body and recycle all cells.By the cell of recycling at 6000g (7000rpm)
Centrifugation, and pellets are collected, it is suspended in 1 × combination buffer (5mM imidazoles, 20mM Tris-HCl pH 7.9,0.5M
NaCl it in), and is stirred at 18Hz six times, every time 15 seconds using ultrasonoscope.15 points are centrifuged at 23400g (14000rpm)
Clock, and supernatant is saved with soluble form.Equally, the pellets of insoluble form are suspended in 1 × combination containing 6M urea
In buffer, it is made to stand 1 hour at 4 DEG C, and is centrifuged 30 minutes at 23400g (14000rpm) and (is split with recycling supernatant
The pellets of solution).The pellets of recovered cracking are granular with what is purified by the purifying of His- combination chromatography
Precipitating.Make the soluble form, the pellets of cracking and the pellets of purifying carry out SDS-PAGE, and print by protein
Mark method identifies any fraction that wherein there is the recombinant protein.As control, using wild type infection cell can
Molten form.For immunoblotting, made using the serum and normal serum of the patient with hemorrhagic fever with renal syndrome (HFRS)
For primary antibody.
Figure 11 shows the protein print that TNP is detected using patients serum and normal serum of embodiment of the present invention
Mark method as a result, wherein the band at lower arrow is pointed out Partial digestion for TNP.The image on the left side shows use and suffers from
The serum of the patient of hemorrhagic fever with renal syndrome is as primary antibody as a result, the image on the right shows and uses normal serum as primary antibody
Result (the 1st: infected the solution form of the cell of wild-type baculovirus, the 2nd: having infected recombinant baculovirus
The soluble form of cell, the 3rd: the cracking after being then centrifuged for using the insoluble form of the buffer processing cell containing urea
Pellets, the pellets of the 4th: 3 purifying).
As shown in figure 11, for TNP, the albumen is bigger than existing amount in an insoluble form with amount existing for soluble form.
In addition to the specific band at 22kDa other than --- it corresponds to TNP fraction ---, the immunoblotting based on patients serum
As a result, it can be seen that though in the pellets and soluble form of purifying, the lower strip that should be shown as weak be it is strong,
Therefore, it is considered that this is as caused by specific reaction.For normal serum, two bands do not occur.Lower strip is considered as
As caused by the Partial digestion of TNP.
Figure 12 shows the immunoblotting of the pellets of the purifying of the TNP and CNP of embodiment of the present invention
As a result (the 1st: the cell of uninfecting virus, the 2nd: infect the cell of baculoviral, the 3rd: having infected CNP clone's
The cell of recombinant baculovirus, the 4th: infected TNP clone recombinant baculovirus cell, the 5th: having infected CNP grams
The cell of grand recombinant baculovirus, the 6th: having infected the cell of the recombinant baculovirus of TNP clone).
As shown in figure 12, CNP is only observed in the patient with hemorrhagic fever with renal syndrome (HFRS), and confirms to produce
The recombinant protein of about 50kDa.
Embodiment 14: pass through the purifying of the recombinant protein of His- combination affinity chromatography
Using 2ml His- binding resin (Novagen) packed column, and use distilled water, 1 × loading buffer and 1 × knot
Buffer is closed to be prepared.Antigen protein is set to pass through the column, using 1 × combination buffer (5mM imidazoles, 20mM Tris-HCl
PH 7.9,0.5M NaCl) and 1 × washing buffer (40mM imidazoles, 20mM Tris-HCl pH 7.9,0.5M NaCl) washing
The column.Elution buffer (300mM imidazoles, 0.5M NaCl, 20mM Tris-Cl pH 7.9) is set to pass through the column to recycle
Every kind of albumen of 1ml.The concentration of albumen in each fraction is determined by Lowry measuring method.Carry out SDS-PAGE and Western blotting
Method is to identify albumen.
15. recombinant protein measuring method of embodiment
15-1. immunoblotting
To recombinant protein/Kong Jinhang electrophoresis of 10 μ g purifying on 10% gel with a thickness of 1mm or 1.5mm.It uses
Gel of the Bio-Rad conveyer at 80V in 40 minutes after electrophoretic blotting.By the film after transfer with 5% skimmed milk-TBST
(saline-Tween 20 of Tris buffering) closing 1 hour, makes it react 1 with patients serum's (being diluted to 1: 3000) as primary antibody
Hour, and reacted with the anti-human igg (being diluted to 1: 10000) of the horseradish peroxidase conjugation as secondary antibody.After the reaction was completed,
It is developed the color using ECL kit.
It is based on immunoblotting as a result, recombinant C NP and TNP is only in the serum of the patient with hemorrhagic fever with renal syndrome
It is middle to be reacted at about 50kDa and 22kDa respectively, but do not reacted with normal serum.
15-2. dot blotting
1 μ g recombinant protein is added drop-wise on nitrocellulose filter and 1 hour dry at 37 DEG C.Film after drying is used
5% skimmed milk-TBST close 1 hour, make its with as primary antibody patients serum and normal serum (being diluted to 1: 3000) react 1
Hour, and reacted with the anti-human igg (being diluted to 1: 10000) of the horseradish peroxidase conjugation as secondary antibody.After the reaction was completed,
It is developed the color using ECL kit.
Using the patient with hemorrhagic fever with renal syndrome (HFRS) for having infected hantavirus or Seoul Virus serum into
Row dot blotting.As shown in table 7 below, the positive signal shown in CNP is more stronger than what is shown in TNP, and with it is normal
There is no reactions for serum.
[table 7]
A: data are indicated in the form of strong positive (+++), middle positive (++) or weakly positive (+) dot blotting result.
* ten parts of normal serums not with recombination NP antigen-reactive.
15-3.ELISA
Recombinant protein antigen is diluted at 1 μ g/ml with 0.05M carbonate buffer solution (pH 9.6), 96 holes are added
In micro plate, react it 18 hours at 4 DEG C.It is washed twice using the PBST containing 0.05% polysorbas20, and uses 3%
BSA is closed 2 hours at 37 DEG C.Using the infection hantavirus of dilute form, Seoul Virus, Pummala virus or
For the serum of the rat of Prospect Hill virus as primary antibody, secondary antibody is the anti-rat IgG of horseradish conjugate for being diluted to 1: 6000
(IgM).It is developed the color using substrate solution, measures OD under the wavelength of 490nm using microplate reader.
From the following table 8 can, it is evident that recombinant antigen CNP reacted with hantavirus and Seoul Virus but not with Pummala
Virus or the reaction of Prospect Hill virus.
[table 8]
| For the rat antisera of following virus | The Chinese he | Seoul | Pummala | Prospect Hill |
| Pass through the reactivity of the CNP of ELISA measurement | +++ | +++ | - | - |
Although disclosing preferred embodiment of the invention for illustrative purposes, those skilled in the art will be managed
Solution, can be from the embodiment party without departing substantially from scope and spirit of the present invention as disclosed in the accompanying claims
Various modifications and other equivalent embodiments are made in case.Disclosed embodiment should be considered as illustrative rather than limit
Property processed.The scope of the present invention is not shown in description above and is illustrated in claims, in the range being equal with it
Interior all variations are interpreted as incorporated herein.
Claims (9)
- Diagnostic kit more than 1. comprising test strips,Wherein the test strips include:Tsutsugamushi antigen albumen, the 56kDa fusion protein (cr56) containing Orientia Tsutsugamushi Gilliam, Karp and Kato and The 56kDa albumen (kr56) of Orientia Tsutsugamushi Kangwon；Leptospira antigen；WithHFRS Antigen albumen；Wherein the first test strips include the second pre-reaction line containing Sf (Spodopterafrugiperda) extract, the Sf extract and Two non-specific antibodies combine, andSecond non-specific antibody be and be originated from Sf protein bound antibody.
- 2. more diagnostic kits of claim 1, wherein the test strips include the first test strips and the second test strips,The first test strips detection tsutsugamushi mite IgM antibody, Leptospira IgM antibody and hemorrhagic fever with renal syndrome IgM antibody, and AndThe second test strips detection tsutsugamushi mite IgG antibody, Leptospira IgG antibody and hemorrhagic fever with renal syndrome IgG antibody.
- 3. more diagnostic kits of claim 1, included in Orientia Tsutsugamushi Gilliam, Karp and Kato The volume ratio of the 56kDa albumen (kr56) of 56kDa fusion protein (cr56) and Orientia Tsutsugamushi Kangwon is 5:3.
- 4. more diagnostic kits of claim 1, wherein the Leptospira antigen includes the lipopolysaccharides from Leptospira (LPS) core polysaccharide and O- specific side chain prepared in.
- 5. more diagnostic kits of claim 1, wherein the HFRS Antigen albumen includes nuclear shell protein, institute Stating nuclear shell protein is amino prepare from Soochong virus and with the SEQ ID NO:2 or 3 from eukaryocyte Acid sequence.
- 6. more diagnostic kits of claim 1, wherein the test strips include:The first response line comprising Leptospira antigen；The second response line comprising tsutsugamushi antigen albumen；WithThird response line comprising HFRS Antigen albumen.
- 7. more diagnostic kits of claim 1, wherein the test strips include the gold-labelled pad containing tag antibody, the mark Antibody in conjunction with tsutsugamushi mite antibody, antibodies against Leptospira and nephrotic syndrome hemorrhagic fever antibody, andThe tag antibody includes to mark at least one of anti-human IgM, gold mark anti-human igg and gold mark chicken IgY selected from gold.
- 8. more diagnostic kits of claim 1, wherein first test strips include first containing E. coli extract Pre-reaction line, the E. coli extract in conjunction with the first non-specific antibody, andFirst non-specific antibody be and be originated from Escherichia coli protein bound antibody.
- 9. more diagnostic kits of claim 1, wherein the test strips include the control line containing reference protein, the control Albumen in conjunction with tag antibody, andThe reference protein includes more selected from the anti-chicken IgY antibody of goat, rabbit-anti anti-goat IgG polyclonal antibody and rabbit-anti goat IgM At least one of clonal antibody.
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| CN101379398A (en) * | 2006-09-04 | 2009-03-04 | 株式会社疫免美德 | Diagnostic formulation for tsutsugamushi disease |
| CN101600965A (en) * | 2007-03-08 | 2009-12-09 | 疫免美德有限公司 | Diagnostic kit for leptospirosis |
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| KR100561687B1 (en) * | 2004-02-03 | 2006-03-20 | 신진메딕스(주) | A kit capable of detecting tsutsugamushi, Leptospira and Hantaan virus infection simultaneously |
| KR101032956B1 (en) * | 2008-09-12 | 2011-05-09 | 주식회사 이뮨메드 | Rapid diagnostic kit of hemorrhagic fever with renal syndrome detecting specific IgM and IgG using nucleocapsid protein derived from Soochong virus |
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| CN101600965A (en) * | 2007-03-08 | 2009-12-09 | 疫免美德有限公司 | Diagnostic kit for leptospirosis |
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