CN101600965A - Diagnostic kit for leptospirosis - Google Patents
Diagnostic kit for leptospirosis Download PDFInfo
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- CN101600965A CN101600965A CNA2007800509274A CN200780050927A CN101600965A CN 101600965 A CN101600965 A CN 101600965A CN A2007800509274 A CNA2007800509274 A CN A2007800509274A CN 200780050927 A CN200780050927 A CN 200780050927A CN 101600965 A CN101600965 A CN 101600965A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to a kind of composition of diagnosing leptospirosis, described composition comprises from a kind of polysaccharide in the Leptospira lipopolysaccharides (LPS) as antigen; And diagnostic kit that comprises the leptospirosis of described polysaccharide.It is a kind of early stage that diagnostic kit of the present invention provides, accurate and easy leptospirosis diagnostic method.
Description
Invention field
The present invention relates to the diagnosis composition of a kind of leptospirosis (Leptospirosis), comprise a kind of polysaccharide from Leptospira lipopolysaccharides (LPS); And diagnostic kit that comprises the leptospirosis of above-mentioned polysaccharide; Also have a method that detects spirochetal specific antibodies, described method comprises described polysaccharide and biological sample is in contact with one another to confirm the step of antigen-antibody complex formation.
Prior art
Leptospirosis is to be the infectious disease that leptospira interrogans (Leptospirainterrogans) causes by a kind of conveyor screw (spirochetes).Leptospira interrogans can infect humans and animals.Leptospirosis at first was described as a kind of serious febrile disease of following jaundice and renal abnormality in 1887, was named as Wei Ershi disease (Weil ' s disease).
1918, from the Wei Ershi patient, be separated to this bacterium first, and called after jaundice hemorrhage conveyor screw (Spirochaeta icterohaemorrhagiae), be named as jaundice hemorrhage Leptospira (Leptospiraicterohaemorrhagiae) then again.Leptospira belongs to Leptospiraceae on the taxonomy.According to pathogenic, Leptospira is divided into question mark (Leptospira interrogans) and leptospira biflexa (Leptospira biflexa).So far, known pathogenic leptospira interrogans is divided into 19 sero-groups, comprises about 180 kinds of serotypes.The distribution of known serotype changes according to the geographic area.
Wei Ershi disease (or leptospirosis) is a kind of febris acuta disease, and its classical symptom that causes of phase one is as fever, headache, and myalgia and tired unable can also cause more serious symptom such as organ hemorrhage, jaundice and kidney failure.
On the epidemiology, leptospirosis is propagated by the urine of infected animal (as mouse, ox, horse, pig etc.).When soil that touches when the injured skin of people or mucomembranous surface such as nose and mouth and water contain these infected animals urines, will be infected.Therefore, main hazardous occupation crowd comprises the peasant, the soldier of meatworker and outdoor training.Particularly behind the floods, flood has caused large-scale pollution, and the quantity of infection population is increased.
Known leptospirosis can be in Europe, America, Australia, Vietnam, Thailand, Malaysia, Taiwan, China, Japan and other countries and area outburst.In nineteen forty-two, Korea S has at first reported the Leptospira patient with serological evidence, but the report that does not just infect about people's leptospirosis more afterwards.In 1984, at first from the hemoptysis patient, be separated to Leptospira, thereby confirmed the existence of leptospirosis in Korea S.Afterwards, in Korea S's leptospirosis and yochubio, the hemorrhagic fever renal disease syndrome, the mouse typhus is defined as three class infectious diseases together.
Simultaneously, people have also studied the diagnostic method of leptospirosis.Korean Patent Application No. 1990-0000871 discloses a kind of solid phase aggegation experiment reagent, contains a polyvalent antigen I, the positive control serum of a polyvalent antigen II and a diagnosis leptospirosis.The patent No. is that 561687 Korean Patent discloses a kind of diagnostic kit that comprises a recombinant protein, this albumen relies type HY10 serotype to have specificity to leptospira interrogans, Korean Patent 234876 discloses a kind of diagnostic kit that utilizes immuning hybridization, and it comprises the antigen of a leptospira interrogans.
But up to the present, the leptospirosis diagnostic method study successfully all be the method for utilizing the special albumen of Leptospira, wherein, in this diagnostic method in used albumen and the microscopic agglutination test (MAT) used antigen some difference are arranged.The microscopic agglutination test is by the diagnostic method of WHO (world health organization) accepted standard.The MAT method is to utilize the method for a kind of polysaccharide that is exposed to the surface in lipopolysaccharides (LPS) component, and this polysaccharide is the special surface antigen of Leptospira serotype.Method of the present invention is that the MAT ratio juris is similar to standard method, has promptly adopted a kind of polysaccharide in the lipopolysaccharides that comprises specific antigen of Leptospira.Therefore, realized diagnosis accurately, utilized a kind of antigen just can diagnose all types of leptospirosis simultaneously leptospirosis.It is more accurate that the advantage that the MAT method is compared with other diagnostic method is diagnosed exactly, but shortcoming is also arranged, and promptly is difficult to keep and control used viable bacteria in the MAT method.And the MAT method needs expensive instrument, and owing to its complexity and difficult process, the MAT method can only be operated by the professional of high degree of skill simultaneously.Therefore, in hospital, the MAT method is not almost used.So the inventor has been devoted to develop a leptospirosis diagnostic method that is simple and easy to usefulness, thereby has finished the present invention.
Summary of the invention
Technical scheme
Therefore, an object of the present invention is to provide a kind of neoantigen of diagnosing as leptospirosis from the polysaccharide of Leptospira lipopolysaccharides, and a kind of composition that comprises the diagnosis leptospirosis of described polysaccharide.
Another object of the present invention provides a kind of diagnostic kit that comprises the leptospirosis of described polysaccharide, and utilizes this kit to detect the method for Leptospira specific antibody.
Description of drawings
Fig. 1 represents to separate from leptospira biflexa patoc serotype Patoc 1 bacterial strain and the SDS-PAGE (A) of the bad type serotype polysaccharide of leptospira interrogans and the result of immunoblotting, and this can explain the antigenicity of the polysaccharide that is purified.
Fig. 2 represents to use the structure of the leptospirosis diagnosis test paper of immunochromatographic method.
Fig. 3 represents susceptibility and the specific result of diagnostic kit to leptospirosis detection.
Preferred forms
In one embodiment, the present invention relates to a kind of diagnosis composition of leptospirosis, comprise a kind of polysaccharide from Leptospira lipopolysaccharides (LPS).
The implication of the term that uses among the present invention " diagnosis " is: to the evaluation of pathological state and feature.For the object of the invention, diagnosis is exactly the evaluation to leptospirosis.The implication of term " leptospirosis " is: a kind of disease that is caused by the pathogenic conveyor screw that belongs to Leptospira.
From patient's blood, the antibody in cerebrospinal fluid and the urine in separation of bacterial or the detection serum carries out by directly in the diagnosis of leptospirosis.In a specific embodiment of the present invention, the diagnosis of leptospirosis is to realize by the antibody that detects in the serum.A kind of polysaccharide from the Leptospira lipopolysaccharides is used as the antigen in the method that detects antibody in the serum.The inventor confirms that by the method for western trace and dot blot this polysaccharide of separating can be as the antigen in the leptospirosis diagnosis from the Leptospira lipopolysaccharides.The inventor utilizes Leptospira patient's blood to carry out the ELISA detection, and confirms that the antibody that exists in Leptospira patient's the serum utilizes polysaccharide of the present invention significantly to be identified.Term " conspicuousness " refers to: correctness and reliability with height." height correctness " refers to: diagnosis the result be accurately, and " height reliability " refers to: the result is consistent in the detection that repeats.When the patients serum who diagnosed with the MAT method used the diagnostic method of proteantigen to detect again, susceptibility and specificity can descend a little.But, when as antigen the time, obtaining diagnostic result more accurately with the polysaccharide among the present invention in the MAT method than method with proteantigen.
Comprise blood plasma in order to detect, the leptospiral antibody in the biological samples such as serum and whole blood adopts a kind of polysaccharide from the Leptospira lipopolysaccharides as antigen.Leptospiral lipopolysaccharides (LPS) comprises lipoid A, the special side chain of core polysaccharide and O-.(2-keto-3-deoxyoctanicacid KDO) connects, and core polysaccharide contains glucose, galactose and N-acetylglucosamine by 2-keto-3-deoxyoctanoic acid for lipoid A and core polysaccharide.In a specific embodiment of the present invention, described polysaccharide has specificity to Leptospira, and contains special side chain of O-and core polysaccharide, does not wherein contain lipoid A.Adopt the above-mentioned polysaccharide can enhancement antigen-antibody response as antigen.
In another embodiment of the present invention, the present invention relates to a kind of diagnostic kit of leptospirosis, comprise from the described polysaccharide in the Leptospira lipopolysaccharides (LPS).
This kit is diagnosed leptospirosis by the Leptospira antibody horizontal in the detection of biological sample.This kit comprises the described polysaccharide from the Leptospira lipopolysaccharides, and it reacts as antigen and Leptospira antibody, confirms the formation of antigen-antibody complex.
The formation of antigen-antibody complex can detect by immunological technique, with the western trace, and enzyme linked immunosorbent detection (ELISA), radio-immunity detects (RIA), radioimmunodiffusion, Ouchterlony immunity double diffusion method, rocket immunoelectrophoresis, immuning tissue's dyeing detects, immuno-precipitation, complement are in conjunction with detection, and immunofluorescence detects, immunochromatographic method and fluorescence activated cell go-on-go instrument (FACS) are example, but are not limited thereto.
Diagnostic kit for leptospirosis of the present invention comprises instrument, reagent or its analog commonly used in the above-mentioned polysaccharide that combines with described antibody specificity and other immunoassay.These instruments or reagent comprise suitable carriers, can produce the labeled substrate of detection signal, solubilizer, and detergent, buffer reagent and stabilizing agent, but be not limited thereto.Diagnostic kit of the present invention can be a microplate, scale (dip-stick device), immuno-chromatographic test paper strip, radial separation immunoassay apparatus, circulation device or its analog.Diagnostic kit of the present invention also comprises contrasting over against shining and bearing of standard.
Diagnostic kit of the present invention preferably utilizes the test paper stripe shape or the type of device of immunochromatography.Adopt in the immunochromatography diagnosis, antibody in the biological sample serum and the reaction of the tracer antibody on the colloid gold particle are then changeed film by the capillary action of micropore on the nitrocellulose filter.Change in the membrane process, antibody with combine with capture antigen on the micropore inside surface, the formation colour band, thus determine that with eyes testing result is the positive or feminine gender." tracer antibody " refers to: combine with coloured particle, and can with the antibody of Leptospira antibody response.
In the diagnostic kit of the present invention, antigen-antibody complex can detect by the coloured particle immunization experiment, and wherein coloured particle can be a colloid gold particle, coloured glass pearl or plastics (as, polystyrene, polypropylene, rubber etc.) pearl, preferred colloid gold particle.
In a specific embodiment, as shown in Figure 2, the diagnostic kit for leptospirosis that is used to detect Leptospira antibody comprises a sample pad that absorbs sample; A collaurum pad, wherein contain can with the tracer antibody of antibodies in the sample; An experimental film comprises the detection line that contains polysaccharide of the present invention, contains control line and preliminary examination survey line of removing non-specific antibody of reference protein; And a test strips, contain the absorption pad that adsorbs remaining sample in this test strips.
It is 0.5-2.5mg/ml that detection line contains polysaccharide concentration of the present invention, preferred 1-2mg/ml.It is 0.1-1mg/ml that control line contains reference protein concentration, preferred 0.1-0.2mg/ml, and reference protein can be anti-sheep IgG of rabbit and the anti-albumin A of rabbit.The preliminary examination survey line contains as Escherichia coli and these Gram-negative bacteria lipopolysaccharides of pseudomonad, and its concentration is 0.5-1mg/ml, wherein the leptospiral lipopolysaccharides structural similarity among lipopolysaccharides and the present invention.The collaurum pad contains the goat-anti people IgM of golden mark, the goat anti-human igg of golden mark, the albumin A of golden mark or its analog.
Adopt immunochromatographic method as follows with the method for strip-type diagnostic kit diagnosis leptospirosis.At first, biological sample to be analyzed is added drop-wise on the absorption of sample pad of diagnostic kit, the absorption of sample pad is the part that absorbs sample.Biological sample is moved on the pad that contains collaurum by capillary action.At this moment, in containing the sample pad of collaurum, the goat-anti people IgM of antibody in the sample and tracer antibody such as golden mark, the albumin A combination of the goat anti-human igg of golden mark or golden mark, thus form colloid.Biological sample to be analyzed is not to be fixed in the sample pad that contains collaurum, but continues to move to detection line, and polysaccharide antigen wherein of the present invention is immobilized.If come self-infection leptospirosis patient's sample, this sample and polysaccharide antigen react inducing antigen-antibody response.Just, Leptospira antibody combines with specific tracer antibody in the sample pad that contains collaurum, again be fixed on detection line in polysaccharide antigen combine, thereby form mauve band.Therefore, formed band Direct observation with the naked eye.Contain the remaining tracer antibody of the sample pad of collaurum not with biological sample in antibody response, move to control line forming mauve band, thereby guarantee the consistance of testing with the reference protein reaction.As mentioned above, diagnostic kit of the present invention has utilized the immunochromatographic method of antigen-antibody reaction to carry out.Therefore, any equipment does not need, and the result can directly with the naked eye observe rapidly.And then the preliminary examination survey line of diagnostic kit is removed the mistaken diagnosis that any possible Leptospira antibody causes, thereby improves the accuracy of leptospirosis diagnosis.
In another embodiment, the present invention relates to a method that detects Leptospira antibody, comprise that is detected described polysaccharide contacts back formation with biological sample an antigen-antibody complex step.
The example that can detect the biological sample of Leptospira antibody comprises blood, serum and blood plasma, but be not limited thereto.
Hereinafter, reference example is described the present invention in detail.But these examples just for illustrative purposes, and the invention is not restricted to these examples.
Embodiment
The preparation of embodiment 1. Leptospira antigens
1-1. the cultivation of Leptospira bacterial strain
EMJH (the Leptospira nutrient culture media is based on Ellinghausen McCullough Hohnson Harris) 0.23g is dissolved in the 90ml water, and it is standby to sterilize.The EMJH that adds the enrichment of 10ml Leptospira prepares the used nutrient culture media of the present invention in above-mentioned nutrient culture media.The above-mentioned inoculation of medium Leptospira that makes, 30 ℃ of shake-flask culture 5 days.
1-2. the preparation of Leptospira antigen
The Leptospira bacterium was cultivated in the EMJH nutrient culture media after 5 days, centrifugal 20 minutes of 4000g.Precipitation 1PBS damping fluid (137mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4And 2mM KH
2PO
4) washing three times after, 100 ℃ the heating 1 hour.Add the 150g/ml Proteinase K, 37 ℃ were reacted 16 hours.Add EGTA to final concentration 2mM, 70 ℃ were reacted after 15 minutes, 180000g, and 4 ℃ are centrifugal 2 hours.The gained precipitation is used aqueous suspension, promptly obtains lipopolysaccharides (Westpha and Jann, 1965).After the LPS antigen of purifying was handled with 1% acetic acid, 100 ℃ were heated 1 hour, and 3000g is centrifugal 20 minutes then.The lipoid A that removal precipitates makes polysaccharide antigen.The antigen that makes carries out detection by quantitative with the standard polysaccharide, and wherein said standard polysaccharide is to prepare from the lipopolysaccharides of the purifying of Escherichia coli (Sigma) with method same as described above.
Embodiment 2: the antigenicity analysis of Leptospira polysaccharide
2-1.Western engram analysis
12% the SDS-PAGE gel that preparation 1mm or 1.5mm is thick, and utilize Bio-Rad II electrophoresis apparatus to carry out SDS-PAGE.Change bellows 80V with Bio-Rad, 30min makes protein transfer on the film from running gel.Film is with containing 5% skimmed milk, 1 * TBS damping fluid sealing of 0.1% Tween-20.Rabbit immune serum (L.interrogans strainWH-19) is anti-as one, and is anti-as two after the anti-rabbit igg (Bio-Rad) of horseradish peroxidase combination dilutes 10000 times.After immune response was finished, (Amersham Pharmacia biotech company) carried out Western blotting with the ECL kit.The result as shown in Figure 1.
As shown in Figure 1, it is closely similar that leptospira interrogans relies the SDS-PAGE gel electrophoresis figure of serotype and leptospira biflexa patoc serotype Patoc 1 bacterial strain polysaccharide.The polysaccharide of 14KDa or bigger position appears at and relies in serotype and the patoc serotype.Suppose with above-mentioned polysaccharide as antigen.
2-2. dot blot analysis
1 μ g (1 μ g/ μ l) antigen is added drop-wise on the nitrocellulose membrane, 37 ℃ of dryings 1 hour.Dried film is with the TBST sealing that contains 5% skimmed milk 1 hour, and as anti-patients serum (1: 3,000 a dilution) reaction 1 hour, the anti-human IgG of horseradish peroxidase combination (dilute 10000 times) resists as two.After immune response is finished, carry out Western blotting with the ECL kit, the result with the MAT method compares (table 1) then.The result shows: Most patients serum all has positive signal.
Table 1. dot blot and MAT method result are relatively
2-3 enzyme linked immunosorbent detection (ELISA) detects
The ELISA that carries out polysaccharide on the basis of the method that Engvall and Perlmann (1972) introduce detects.Poly-L-Lysine (10 μ g/ml) is with 0.01MPBS dilution pre-service microwell plate, then room temperature reaction 24 hours.Microwell plate washs secondary with the PBS damping fluid that contains 0.05% Tween-20 then.Described polysaccharide is suspended in the PBS damping fluid, and every hole adds 100 μ l and handles microwell plate.37 ℃ of reactions of microwell plate 1 hour are then with the PBS damping fluid washing that contains 0.05% Tween-20 three times.Microwell plate seals at 37 ℃ with sheep blood serum.Serum from 20 Leptospira patients and 20 healthy people is anti-as one, and is anti-as two after the anti-human IgG (Bio-Rad) of horseradish peroxidase combination dilutes 10000 times.Each hole all adds substrate hatches, and detects the absorbance in each hole at 490nm with microplate reader.The result shows: the recombinant protein of Leptospira flagellum has the sensitivity of 72-88%, the specificity of 88-95%; And polysaccharide of the present invention has 95% sensitivity and 95% specificity.Correspondingly, as the candidate antigens of Leptospira disease early diagnosis, described polysaccharide is better than above-mentioned albumen (table 2).
Sensitivity and the specificity of table 2.ELISA
Patients serum's number that the a:MAT method detects: 20
B: normal serum number: 20
The production of embodiment 3. diagnostic kits
3-1. the immobilization of Leptospira antigen (polysaccharide) on nitrocellulose filter
By electrostatic interaction, (aperture: 5 μ m) last fixedly specific antigen is to catch specific antibodies at cellulose nitrate (NC) film.With the speed of distributor with 1 μ l/cm, with the antigen (polysaccharide) of purifying linear be added to apart from the nitrocellulose filter bar (5 * 25mm) bottom 1cm the place, there is plastic back plate at the nitrocellulose filter bar back side, and control antibodies (anti-sheep IgG of rabbit or the anti-albumin A of rabbit) linearity is added to apart from the place of nitrocellulose filter bar bottom 1.4cm.Nitrocellulose filter was 37 ℃ of dryings 1 hour.
Purified polysaccharide with variable concentrations experimentizes.As a result, detected signal is the strongest when antigen concentration is 1mg/ml.Concentration is higher or lower than the detected signal of 1mg/ml and all weakens.Therefore, in order to obtain the strongest signal, need the antigen of q.s to remove combining target antibody.But the requisite space distance that the antigen excess of conversion zone makes antigen-antibody reaction need seldom arrives.Thereby make the chance of immune adherence reaction reduce.
3-2. become the antigen damping fluid of linear usefulness
Tween-20 influences the nonspecific reaction of antigen.Along with the increase of its concentration, signal strength weakening.But in order to remove cross reaction, the essential Tween-20 solution that adds.Detected the effect of 0.01,0.05 and 0.1% Tween-20.Removing the most effective concentration of cross reaction is 0.05%.Therefore, the optimum concentration of Tween-20 is 0.05%.
3-3. determining of the antigen that uses in the preliminary examination survey line
Produce at antigen (polysaccharide) under the situation of non-specific signal, before the antigen line, be provided with the preliminary examination survey line to remove non-specific antibody.Then, among the present invention, the lipopolysaccharides of Leptospira and its class bacteroid (Escherichia coli, pseudomonas aeruginosa) uses separately or together respectively.From the result, the Escherichia coli LPS of 0.6mg/ml is very effective.
3-4. the deposition of tracer antibody-gold complex on glass fibre
(1) selection of glass fibre and pretreatment condition determines
Goat-anti people IgM (μ-special) collaurum coupling solution, goat anti-human igg (H+L) collaurum coupling solution, or with the colloidal gold solution of albumin A mark, being diluted to optical density with 50% aqueous trehalose (trehalose final concentration 5%) and aquae destillata is 3.Glass fibre membrane (Millipore) immerses in the above-mentioned solution of preparation, and 37 ℃ of dryings are 2 hours then.Then film is cut into wide rectangular of 0.5cm.
(2) become the amount of line style collaurum couplet and determining of drying condition
The increase of analyzing the signal specific intensity of decorum generation is proportional to the amount of employed collaurum and antibodies.But if collaurum-antibody conjugates is excessive, the non-specific signal that relative low concentration antibody or non-specific antibody produce in the specimen can increase, thereby produces false positive results.In the present invention, adopt the goat-anti people IgM (μ-special) of collaurum (40nm) mark, the goat anti-human igg (γ-special) of collaurum (40nm) mark, the albumin A of the goat anti-human igg (H+L) of collaurum (40nm) mark or collaurum (40nm) mark is as the collaurum couplet.Optical density according to every kind of couplet compares experiment.Bad (table 3) that IgG detects in all golden couplets.This is because normally a kind of T cell of LPS independent form antigen.Think that in immune response T cell independent form antigen produces IgM, does not but produce IgG.Therefore, in leptospirosis detection, can conclude that IgM detects than IgG detection more important (table 3).Can see, be 3 to the suitableeest optical density of the IgM of colloid gold label.
Table 3.MAT method and diagnostic kit result's comparison
3-5. the preparation of test strips
Designed system comprises: the nitrocellulose filter with plastic back plate is as the primary structure immobilized antigen, deposit the glass fibre membrane (couplet pad) of golden couplet in the lower position of nitrocellulose filter, with be positioned at the nitrocellulose filter two ends, be connected with each other, be adsorbed with the cellulose membrane (sample pad and absorption layer) of excess solution.Glass fibre membrane is in the lower position of system and since its higher adsorptive power it not only can very fast absorption sample, and can induce the effectively reaction of detected materials and dissolved gum body gold by prolonging its retention time to the top migration.At the middle part of immunity test strip, the specific antibodies (control line) that detects the antigen (detection line or capture line) of detected materials and can detect collaurum is fixed respectively.And cellulose membrane is positioned at the top (absorption layer) of immunity test strip with any too much sample of quick absorption (Fig. 2).
3-6. determining of human serum concentration
Human serum concentration experimental result shows that excessive serum can cause non-specific responding.Test with 100 μ l serum, and be diluted to 1/1000,1/100,1/50, the 1/10 or 100 undiluted serum of μ l and test.The result: serum is diluted to 1/100 can obtain best result.
3-7. the sensitivity of quick diagnosis reagent kit and specificity
In order to determine the sensitivity and the specificity of diagnostic kit for leptospirosis, leptospirosis patients serum and other febrile disease (tarbadillo, hemorrhagic fever renal disease syndrome, yochubio) patient's serum is as sample.6 leptospirosis patients serums diagnose with the MAT method.After 5 minutes, 6 serum are positive entirely, so the diagnosis sensitivity of IgM (μ-special) is 100% (Fig. 3).
With 6 healthy people's serum, 3 tarbadillo patients' serum, 4 yochubio patients' serum and 6 hemorrhagic fever renal disease syndrome patients' serum carries out the cross reaction experiment, and its specificity is 95% (Fig. 3).
3-8. result in the efficient of other research institution's test experience product
In order to determine the sensitivity and the specificity of diagnostic kit for leptospirosis of the present invention, leptospirosis patients serum and other febris acuta disease (tarbadillo from joint study mechanism-member of Imperial Academy university (Hallym University) acquisition, hemorrhagic fever renal disease syndrome, yochubio) patient's serum is as specimen.Detect 5 leptospirosis patients serums with the MAT method, after 5 minutes, 5 serum are positive entirely.Therefore the diagnosis sensitivity of IgM (μ-special) is 100% (table 4).
With 10 healthy people's serum, 10 tarbadillo patients' serum, yochubio patient's serum and hemorrhagic fever renal disease syndrome patient's serum carries out the cross reaction experiment, and its specificity is 95% (table 4).
Table 4
| RDK(1∶100)a | |
| Leptospirosis (n=5) | 5 |
| Other AFI (n=10) | 1 |
| Healthy people's contrast (n=10) | 0 |
A: serum diluting multiple
Sensitivity: 5/5=100%
Specificity and normal healthy controls in other febris acuta disease (tarbadillo, hemorrhagic fever renal disease syndrome, yochubio): 19/20=95%
Industrial applicibility
As mentioned above, the application of neoantigen of the present invention can realize to leptospirosis accurately, fast, easy diagnosis.
Claims (6)
1. the diagnosis composition that is used for leptospirosis, described composition comprises the polysaccharide from the Leptospira lipopolysaccharides.
2. the diagnostic kit that is used for leptospirosis, described kit comprise the described composition of claim 1.
3. diagnostic kit according to claim 2, wherein said diagnostic kit are the test paper types that adopts immunochromatography, adopt the device type or the microplate type of immunochromatography.
4. diagnostic kit according to claim 2 wherein detects antigen-antibody complex by the coloured particle immunoassay.
5. method that detects Leptospira antibody, described method comprise that detection contacts the formed antigen-antibody complex in back by the composition described in the claim 1 with biological sample.
6. method according to claim 5, wherein said method is selected from the group that comprises following method: immunoblotting, enzyme-linked immunosorbent assay, the radio-immunity detection method, radioimmunodiffusion, Ouchterlony immune double diffusion method, rocket immunoelectrophoresis, immuning tissue's decoration method, immuno-precipitation, complement combined techniques, immunofluorescence technique, immunochromatographic method and fluorescence activated cell go-on-go instrument.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2007-0023093 | 2007-03-08 | ||
| KR1020070023093 | 2007-03-08 | ||
| KR1020070023093A KR100868560B1 (en) | 2007-03-08 | 2007-03-08 | Diagnostic kit for Leptospirosis |
| PCT/KR2007/001578 WO2008108510A1 (en) | 2007-03-08 | 2007-03-30 | Diagnostic kit for leptospirosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101600965A true CN101600965A (en) | 2009-12-09 |
| CN101600965B CN101600965B (en) | 2013-03-06 |
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| CN2007800509274A Active CN101600965B (en) | 2007-03-08 | 2007-03-30 | Diagnostic kit for leptospirosis |
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| KR (1) | KR100868560B1 (en) |
| CN (1) | CN101600965B (en) |
| WO (1) | WO2008108510A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106687813A (en) * | 2015-06-05 | 2017-05-17 | 免疫制药公司 | Igm semi-quantitative leptospirosis diagnosis kit |
| CN107167606A (en) * | 2016-03-07 | 2017-09-15 | 疫免医学有限公司 | Many diagnostic kits |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101054951B1 (en) * | 2009-01-22 | 2011-08-05 | 재단법인 한국원자력의학원 | CAPN12, a molecule for diagnosing and treating liver cancer, and a kit comprising the same |
| CN102662058B (en) * | 2012-04-17 | 2015-01-07 | 中国农业科学院兰州兽医研究所 | Indirect enzyme-linked immuno sorbent assay (ELISA) method for ovinetheileriasis detection |
| EP2717055A1 (en) * | 2012-10-04 | 2014-04-09 | Institut Pasteur | Use of Leptospira fainei serovar Hurstbridge bacteria for diagnosing leptospirosis |
| KR101774749B1 (en) | 2015-03-20 | 2017-09-19 | 베트올 (주) | Recombinant antigenic protein for diagnosis of canine leptospira IgG,IgM antibody and diagnostic kit by using the same simultaneously |
| KR102141591B1 (en) | 2016-05-13 | 2020-09-14 | 광주과학기술원 | Diagnostic kit |
| WO2019060384A1 (en) * | 2017-09-19 | 2019-03-28 | The Regents Of The University Of California | Composition and methods of making and using leptospiral lps |
| KR102417665B1 (en) | 2020-11-16 | 2022-07-05 | 조선대학교산학협력단 | Composition for detecting Leptospirosis and method of diagnosing Leptospirosis using the same |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2215900A1 (en) | 1996-01-22 | 1997-07-24 | David A. Haake | Leptospiral outer membrane proteins |
| KR100421539B1 (en) * | 2001-04-06 | 2004-03-18 | 대한민국 | Recombinant antigenic protein of Fla B gene of Leptospira interrogans Icterohaemorrhagiae serovar lai and diagnostic kit using said protein |
| BR0308180A (en) * | 2002-02-28 | 2007-04-10 | Fundacao De Amparo A Pesquisa | leptospira surface proteins |
| CN1443774A (en) * | 2002-03-11 | 2003-09-24 | 中国科学院上海生物工程研究中心 | Leptospira hemolysin and its code sequence |
| CN1514247A (en) * | 2003-05-06 | 2004-07-21 | 浙江大学 | Monoclone antibody of anti leptospira and detection method of anti leptospira |
| KR200357469Y1 (en) * | 2004-02-03 | 2004-07-30 | 주식회사 녹십자상아 | Diagnosis Kit for Analyzing Immuno-Chromatography |
-
2007
- 2007-03-08 KR KR1020070023093A patent/KR100868560B1/en active IP Right Grant
- 2007-03-30 WO PCT/KR2007/001578 patent/WO2008108510A1/en active Application Filing
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106687813A (en) * | 2015-06-05 | 2017-05-17 | 免疫制药公司 | Igm semi-quantitative leptospirosis diagnosis kit |
| CN106687813B (en) * | 2015-06-05 | 2019-03-08 | 免疫制药公司 | IgM sxemiquantitative leptospirosis diagnostic kit |
| CN107167606A (en) * | 2016-03-07 | 2017-09-15 | 疫免医学有限公司 | Many diagnostic kits |
| CN107167606B (en) * | 2016-03-07 | 2019-03-15 | 疫免医学有限公司 | More diagnostic kits |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008108510A1 (en) | 2008-09-12 |
| KR20080082337A (en) | 2008-09-11 |
| KR100868560B1 (en) | 2008-11-13 |
| CN101600965B (en) | 2013-03-06 |
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