CN101600965B - Diagnostic kit for leptospirosis - Google Patents
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- CN101600965B CN101600965B CN2007800509274A CN200780050927A CN101600965B CN 101600965 B CN101600965 B CN 101600965B CN 2007800509274 A CN2007800509274 A CN 2007800509274A CN 200780050927 A CN200780050927 A CN 200780050927A CN 101600965 B CN101600965 B CN 101600965B
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- 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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Abstract
The present invention relates to a diagnostic composition for leptospirosis comprising a polysaccharide derived from a lipopolysaccharide (LPS) of Leptospira as an antigen, and to a diagnostic kit for leptospirosis comprising the polysaccharide. The diagnostic kit of the invention provides an early, accurate, and simple diagnosis of leptospirosis.
Description
Invention field
The present invention relates to the diagnosis composition of a kind of leptospirosis (Leptospirosis), comprise a kind of polysaccharide from Leptospiral lipopolysaccharide (LPS); And diagnostic kit that comprises the leptospirosis of above-mentioned polysaccharide; Also have a method that detects spirochetal specific antibodies, described method comprises the step that described polysaccharide and biological sample is in contact with one another to confirm 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 first this bacterium, and called after jaundice hemorrhage conveyor screw (Spirochaeta icterohaemorrhagiae), then be named as again jaundice hemorrhage Leptospira (Leptospiraicterohaemorrhagiae).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 sick (or leptospirosis) is that a kind of febris acuta is sick, and its classical symptom that causes of phase one is such 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 (such as mouse, ox, horse, pig etc.).When the 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, leptospirosis diagnosis method research success 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 namely adopted a kind of polysaccharide in the lipopolysaccharides that comprises specific antigen of Leptospira.Therefore, realize the accurately diagnosis to leptospirosis, utilized simultaneously a kind of antigen just can diagnose all types of 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 namely 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 diagnosis 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 polysaccharide from the Leptospira lipopolysaccharides as the neoantigen of leptospirosis diagnosis, 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 strips of immunochromatographic method.
Fig. 3 represents that diagnostic kit is to susceptibility and the specific result of 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 Leptospiral lipopolysaccharide (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: the disease that a kind of pathogenic conveyor screw by belonging to Leptospira causes.
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 detecting the antigen in the method for Serum Antibody.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 than the method with proteantigen more accurately diagnostic result with the polysaccharide among the present invention in the MAT method.
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 Leptospiral lipopolysaccharide as antigen.Leptospiral lipopolysaccharides (LPS) comprises lipoid A, the special side chain of core polysaccharide and O-.Lipoid A and core polysaccharide connect by 2-keto-3-deoxyoctanoic acid (2-keto-3-deoxyoctanicacid, KDO), and core polysaccharide contains glucose, galactose and N-Acetyl-D-glucosamine.In a specific embodiment of the present invention, described polysaccharide has specificity to Leptospira, and contains the 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 Leptospiral lipopolysaccharide (LPS).
This kit is diagnosed leptospirosis by the Leptospira antibody horizontal in the detection of biological sample.This kit comprises the described polysaccharide from Leptospiral lipopolysaccharide, 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 staining examine, 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 to this.
Diagnostic kit for leptospirosis of the present invention comprises the above-mentioned polysaccharide with described antibody specific binding, and instrument, reagent or its analog commonly used in other immunoassay.These instruments or reagent comprise suitable carrier, can produce the labeled substrate of detection signal, solubilizer, and detergent, buffer reagent and stabilizing agent, but be not limited to this.Diagnostic kit of the present invention can be 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 positive control and the negative contrast of standard.
Diagnostic kit of the present invention preferably utilizes test paper stripe shape or the type of device of immunochromatography.Adopt in the immunochromatography diagnosis, the antibody in the biological sample serum and the reaction of the tracer antibody on the colloid gold particle are then carried out transferring film by the capillary action of micropore on the nitrocellulose filter.In the transferring film process, antibody is combined with the capture antigen on the micropore inside surface, forms colour band, thereby determines that with eyes testing result is positive or negative." tracer antibody " refers to: be combined 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 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, comprise the sample pad of an absorption sample for detection of the diagnostic kit for leptospirosis of Leptospira antibody; A collaurum pad, wherein contain can antibody be combined in sample tracer antibody; 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 the anti-sheep IgG of rabbit, and the anti-albumin A of rabbit.The preliminary examination survey line contains such 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 the 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 wherein polysaccharide antigen of the present invention is immobilized.If come self-infection leptospirosis patient's sample, this sample and polysaccharide antigen react inducing antigen-antibody response.Namely, the specific tracer antibody of Leptospira antibody in the sample pad that contains collaurum is combined, and the polysaccharide antigen in being fixed on detection line is combined again, thereby forms mauve band.Therefore, formed band can with the naked eye directly be observed.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 with the reference protein reaction, thereby guarantee the consistance of testing.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 an antigen-antibody complex step that forms after described polysaccharide contacts with biological sample.
The example that can detect the biological sample of Leptospira antibody comprises blood, serum and plasma, but be not limited to this.
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 for subsequent use to sterilize.Add the EMJH of 10ml Leptospira enrichment in above-mentioned nutrient culture media, the used nutrient culture media of preparation the present invention.The above-mentioned inoculation of medium Leptospira that makes, 30 ℃ of shaking flasks were cultivated 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
4, and 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, namely obtains lipopolysaccharides (Westpha and Jann, 1965).After the LPS antigen of purifying was processed with 1% acetic acid, 100 ℃ were heated 1 hour, and then 3000g is centrifugal 20 minutes.The lipoid A that removal precipitates makes polysaccharide antigen.The antigen that makes quantitatively detects 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.With Bio-Rad transferring film box 80V, 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 as primary antibodie, 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 larger 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 patients serum (1: 3, the 000 dilution) reaction of primary antibodie 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, then the result with the MAT method compares (table 1).The result shows: Most patients serum has positive signal.
Table 1. dot blot and MAT method result are relatively
2-3 enzyme linked immunosorbent detection (ELISA) detects
The ELISA that polysaccharide is carried out on the basis of the method for introducing at Engvall and Perlmann (1972) detects.Poly-L-Lysine (10 μ g/ml) is with 0.01MPBS dilution pre-service microwell plate, then room temperature reaction 24 hours.Then microwell plate washs secondary with the PBS damping fluid that contains 0.05% Tween-20.Described polysaccharide is suspended in the PBS damping fluid, and every hole adds 100 μ l and processes 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.As primary antibodie, anti-as two after 10000 times of the anti-human IgG (Bio-Rad) of the horseradish peroxidase combination dilutions from the serum of 20 Leptospira patients and 20 Healthy Peoples.Each hole 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, cellulose nitrate (NC) film (aperture: 5 μ m) fixedly specific antigen to catch specific antibodies.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 (the 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 is tested.As a result, the signal that detects when antigen concentration is 1mg/ml is the strongest.Concentration is higher or lower than the signal that 1mg/ml detects and all weakens.Therefore, in order to obtain the strongest signal, need the antigen of q.s to remove combining target antibody.But the antigen excess of conversion zone so that the requisite space distance that antigen-antibody reaction needs seldom arrive.Thereby so that the chance of immune adherence reaction reduces.
3-2. become the antigen damping fluid of linear usefulness
Tween-20 affects 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) in 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 mentioned solution of preparation, and then 37 ℃ of dryings are 2 hours.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 that employed collaurum is combined with antibody.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: have the nitrocellulose filter of plastic back plate 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, absorption has 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 by prolonging it to retention time of top migration, induce the effectively reaction of detected materials and dissolved gum body gold.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 adsorption (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 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 entirely positive, so the diagnosis sensitivity of IgM (μ-special) is 100% (Fig. 3).
With the serum of 6 Healthy Peoples, 3 tarbadillo patients' serum, the serum of 4 yochubio patients' serum and 6 Hemorrhagic fever Levels in Nephrotic Syndrome Patients carries out the cross reaction experiment, and its specificity is 95% (Fig. 3).
3-8. the result in the efficient of other research institution's test experience product
In order to determine sensitivity and the specificity of diagnostic kit for leptospirosis of the present invention, leptospirosis patients serum and the sick (tarbadillo of other febris acuta from one member of Imperial Academy university of joint study mechanism (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 entirely positive.Therefore the diagnosis sensitivity of IgM (μ-special) is 100% (table 4).
With the serum of 10 Healthy Peoples, 10 tarbadillo patients' serum, the serum of yochubio patient's serum and Hemorrhagic fever Levels in Nephrotic Syndrome Patients 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 contrast (n=10) | 0 |
A: serum diluting multiple
Sensitivity: 5/5=100%
Specificity and normal healthy controls in other febris acuta sick (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 antigen from Leptospiral lipopolysaccharide, described polysaccharide antigen is comprised of core polysaccharide and the special side chain of O-, and the lipoid A in the wherein said Leptospiral lipopolysaccharide is removed.
2. be used for the diagnostic kit of leptospirosis, described kit comprises composition claimed in claim 1.
3. diagnostic kit according to claim 2, wherein said diagnostic kit are the test paper types that adopts immunochromatography, adopt device type or the microplate type of immunochromatography.
4. diagnostic kit according to claim 2 wherein passes through coloured particle immunoassay detectable antigens-antibody complex.
5. method that detects Leptospira antibody, described method comprise detecting by the composition described in the claim 1 and contact afterwards formed antigen-antibody complex 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, radio-immunity detects 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 |
|---|---|---|---|
| KR1020070023093 | 2007-03-08 | ||
| KR10-2007-0023093 | 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 |
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| 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 |
| WO2016195138A1 (en) * | 2015-06-05 | 2016-12-08 | 주식회사 이뮨메드 | Igm semi-quantitative leptospirosis diagnosis kit |
| KR101920961B1 (en) * | 2016-03-07 | 2018-11-21 | 주식회사 이뮨메드 | Multiple Diagnostic kit |
| 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 |
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| 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 |
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| JPH11503764A (en) | 1996-01-22 | 1999-03-30 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Leptospira outer membrane protein |
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| CN101600965A (en) | 2009-12-09 |
| KR20080082337A (en) | 2008-09-11 |
| KR100868560B1 (en) | 2008-11-13 |
| WO2008108510A1 (en) | 2008-09-12 |
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