WO2008108510A1

WO2008108510A1 - Diagnostic kit for leptospirosis

Info

Publication number: WO2008108510A1
Application number: PCT/KR2007/001578
Authority: WO
Inventors: Yoon-Won Kim; Min-Kee Cho; In-Ae Chang; Soo-Dong Woo; Young-Jin Kim; Mi-Jung Lee; Jin-Sook Kim; Yong-Hwan Byun
Original assignee: ImmuneMed Inc
Current assignee: ImmuneMed Inc
Priority date: 2007-03-08
Filing date: 2007-03-30
Publication date: 2008-09-12
Anticipated expiration: 2009-09-08
Also published as: KR100868560B1; CN101600965A; KR20080082337A; CN101600965B

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

DESCRIPTION

DIAGNOSTIC KIT FOR LEPTOSPIROSIS

Technical Field

The present invention relates to a diagnostic composition for leptospirosis comprising a polysaccharide derived from a lipopolysaccharide (LPS) of Leptospira, to a diagnostic kit for leptospirosis comprising the polysaccharide, and to a method for detecting specific antibodies against Leptospira, comprising a step of contacting the polysaccharide with a biological sample to confirm the presence of an antigen-antibody complex.

Background Art

Leptospirosis is an infectious disease caused Leptospira interrogans, a kind of spirochetes, which affects both humans and animals. The disease was first described in 1887 for severe febrile illness with jaundice and renal abnormalities, which are named as Weil's disease.

In 1918, the bacteria was first isolated from a patient with Weil's disease, which was designated as Spirochaeta icterohaemorrhagiae, and then was designated as Leptospira icterohaemorrhagiae again. Leptospira belongs taxonomically to the family Leptospiraceae, and the genus Leptospira is classified into Leptospira interrogans and Leptospira biflexa according to the presence of its pathogenicity. Pathogenic Leptospira interrogans is known to be classified into nineteen serogroups containing about one hundred eighty serovars so far. Serovars distribution is known to vary depending on the geographical regions .

Weil's disease (or leptospirosis) is an acute febrile illness that causes typical symptoms such as fever, headache, muscle aches, and tiredness in the first phase, and also may cause more severe signs such as organ bleeding, jaundice, and renal failure.

Epidemiologically, leptospirosis is transmitted by the urine of an infected animal (rat, cattle, horse, swine, etc.). People become infected when their injured skin or mucosal surface such as nose and mouth comes in contact with soil or water containing the urine from these infected animals. Therefore, the main occupational groups at risk include farmers, slaughter house workers, and soldiers training outdoor. In particular, the number of patients may increase after flooding, which causes wide area contamination.

Leptospirosis is known to occur in Europe, America, Australia, Vietnam, Thailand, Malaysia, Taiwan, China, Japan, or the like. A leptospirosis patient with serologic evidence was first reported in Korea in 1942, but thereafter there had been no report regarding human leptospirosis infection. In 1984, Leptospira was first isolated from a hemoptysis patient, thus confirming domestic presence of leptospirosis. After that, in Korea leptospirosis was designated as a Class III Communicable Disease, along with tsutsugamushi disease, hemorrhagic fever with renal syndrome, murine typhus.

Meanwhile, a diagnostic method for leptospirosis has been studied. Korean Patent Application No. 1990-0000871 discloses a solid agglutination test reagent containing a multivalent antigen I, a multivalent antigen II, and a positive control serum for diagnosis of leptospirosis. Korean Patent No. 561687 also discloses a diagnostic kit containing a recombinant protein, which is specific for Leptospira interrogans serovar lai HYlO, and Korean Patent No. 234876 discloses a diagnostic kit using an immunoblot containing an antigen of Leptospira interrogans.

However, studies for diagnostic method of leptospirosis so far achieved were methods using a protein specific for Leptospira, wherein there is a difference between the protein used in the diagnostic method and the antigen used in MAT

(microscopic agglutination test) , that is a standard method used by WHO (World Health Organization) . MAT is a method using a surface-exposed polysaccharide among the components of LPS (lipopolysaccharide) , which is the serovar-specific surface antigen of Leptospira. The method of the present invention is similar to the principle of the standard method,

MAT, in that a polysaccharide of LPS comprising a specific antigen for the genus Leptospira is used. Therefore, a more accurate diagnosis can be achieved, as well as all types of leptospirosis can be diagnosed by using one antigen. MAT has an advantage which is more accurate than other diagnostic methods, but has a disadvantage that it is difficult to maintain and control the live bacteria used in MAT. Further, expensive equipments are required, and can be handled only by highly skilled experts due to the complicated and difficult process, thereby MAT being hardly used in the hospitals. Thus, the present inventor made an effort to develop a method for diagnosing leptospirosis with ease and simplicity, thereby completing the present invention.

Disclosure Technical Solution Accordingly, it is an object of the present invention to provide a polysaccharide derived from a lipopolysaccharide of Leptospira as a novel antigen for leptospirosis diagnosis, and a diagnostic composition for leptospirosis comprising the polysaccharide . It is another object of the present invention to provide a diagnostic kit for leptospirosis comprising the polysaccharide, and a method for detecting specific antibodies against Leptospira using the same. Description of Drawings

Fig. 1 shows the results of SDS-PAGE (A) and western blotting (B) of a polysaccharide isolated from Leptospira biflexa serovar patoc strain Patoc 1 and Leptospira interrogans serovar lai, which can explain the antigenicity of the purified polysaccharide.

Fig. 2 shows the structure of a diagnostic strip for leptospirosis using immunochromatography.

Fig. 3 shows the results of measurement of the sensitivity and specificity of a diagnostic kit for leptospirosis .

Best Mode

In one embodiment, the present invention relates to a diagnostic composition for leptospirosis comprising a polysaccharide derived from a lipopolysaccharide (LPS) of Leptospira .

The term "diagnosis" as used herein means identification of pathological condition and features. For the purpose of the invention, a diagnosis is to identify leptospirosis. The term "leptospirosis" means a disease caused by pathogenic spirochete, which belongs to the genus Leptospira.

Leptospirosis diagnosis is performed by directly isolating bacteria from blood, cerebrospinal fluid, and urine of a patient, or by detecting antibodies in a serum. In specific embodiments of the invention, leptospirosis diagnosis is performed by detecting antibodies in the serum.

A polysaccharide, which is derived from a lipopolysaccharide

(LPS) of the genus Leptospira, is used as an antigen in a method for detecting antibodies in the serum. The present inventors performed western blotting and dot-blotting to confirm that the polysaccharide isolated from the lipopolysaccharide of the genus Leptospira acts as an antigen for leptospirosis diagnosis. The present inventors performed an ELISA assay using the blood from patients with leptospirosis, and confirmed that antibodies, which is present in the serum from a patient with leptospirosis, can be significantly identified using the polysaccharide. The term "significance" means that validity and reliability are high. High validity means that the result of diagnosis is accurate and high reliability means that the results are consistent in repeated measurements. When sera from patients diagnosed by MAT were examined by a diagnostic method using a protein antigen, sensitivity and specificity slightly decreased. However, the polysaccharide, which is used as the antigen in MAT method, is used in the present invention, whereby a more accurate diagnosis can be achieved than the method using a protein antigen. In order to detect antibodies against Leptospira in biological samples including blood plasma, serum, and whole blood, a polysaccharide derived from a lipopolysaccharide of Leptospira is used as an antigen. The lipopolysaccharide (LPS) of Leptospira consists of a lipid A, a core polysaccharide, and an O-specific side chain. The lipid A and core polysaccharide is connected with 2-keto-3- deoxyoctanic acid (KDO) , and the core polysaccharide consists of glucose, galactose, and N-acetylglucosamine . In specific embodiments of the invention, the polysaccharide being specific for the genus and consisting of an O-specific side chain and a core polysaccharide, in which a lipid A is removed, is used as an antigen in order to improve antigen- antibody reactivity.

In another embodiment of the invention, the present invention relates to a diagnostic kit for leptospirosis comprising the polysaccharide derived from the lipopolysaccharide (LPS) of Leptospira.

This kit is for diagnosing leptospirosis by measuring an antibody level against leptospira in a biological sample. The kit comprises the polysaccharide derived from the lipopolysaccharide of Leptospira, which acts as an antigen reactive to an antibody against Leptospira, in order to confirm an antigen-antibody complex formation. Antigen-antibody complex formation may be detected by immunological techniques, which are exemplified by western blotting, ELISA (enzyme linked immunosorbent assay) , radioimmunoassay (RIA) , radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining assay, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, and FACS, but are not limited thereto.

The diagnostic kit for leptospirosis of the present invention includes the polysaccharide, which specifically binds to the antibody, as well as tools, reagents, or the like, which are generally used in the art for immunological analysis. These tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, solubilizing agents, detergents, buffering agents and stabilizing agents. The diagnostic kit of the present invention may be of a type of a microplate, a dip-stick device, an immunochromatography test strip, a radial partition immunoassay device, a flow-through device, or the like. Also, the diagnostic kit of the present invention may include positive and negative standard controls .

The diagnostic kit of the present invention is preferably of a strip type or a device type using immunochromatography. In the diagnosis using immunochromatography, antibodies in the serum of a biological sample react with tracer antibodies on colloidal gold particles, and then migrate through micropores of a nitrocellulose membrane by capillary action. During migration, the antibodies bind to capture antigens on the internal surface of micropores to form a color band, thereby determining with the naked eye whether the test result is positive or negative. The "tracer antibody" means the antibody that is conjugated with colored particles and reacts with antibodies against Leptospira.

In the diagnostic kit of the invention, the antigen- antibody complex can be detected by a colored particle immunoassay, in which a colored particle may be a colloidal gold particle, a color glass or a plastic (for example, polystyrene, polypropylene, latex etc.) bead, more preferably a colloidal gold particle.

In a specific embodiment, as shown in Fig. 2, the diagnostic kit for leptospirosis to detect antibodies against Leptospira consists of a sample pad absorbing the sample; a gold conjugation pad containing tracer antibodies to bind with antibodies in the sample; a test membrane comprising a test line containing the polysaccharide of the invention, a control line containing a control protein, and a pre-test line for removing nonspecific antibody; and a test strip containing an absorption pad absorbing the residual sample.

The test line contains the polysaccharide of the invention at a concentration of 0.5 to 2.5 mg/ml, preferably 1 to 2 mg/ml. The control line contains a control protein such as rabbit anti-goat IgG, and rabbit anti-protein A, at a concentration of 0.1 to 1 mg/ml, preferably 0.1 to 0.2 mg/ml. The pre-test line contains a lipopolysaccharide (LPS) of gram negative bacteria such as E. coli and Pseudomonas sp., of which structure is similar to that of Leptospira in the invention, at a concentration of 0.5 to 1 mg/ml. The gold conjugation pad contains a gold-labeled goat anti-human IgM, a goat anti-human IgG, a gold-labeled protein A or the like.

The method for diagnosing leptospirosis by the strip type diagnostic kit using immunochromatography is as follows. First, when a biological sample for analysis is added dropwise to the sample pad of the diagnostic kit, which is a portion absorbing the sample, the biological sample migrates to the gold conjugation pad by capillary action. At this time, the antibodies in the sample bind to tracer antibodies, for example a gold-labeled anti-human IgM goat antibody, gold-labeled anti-human IgG goat antibody and a gold-labeled protein A, in the gold conjugation pad, so as to form a colloid. The biological sample for analysis is not immobilized in the gold conjugation pad, but continues to migrate to the test line, in which the polysaccharide antigen of the invention is immobilized. In the case of the sample from a patient infected with Leptospira, the sample reacts with the polysaccharide antigen to induce an antigen-antibody reaction. That is, antibodies against Leptospira, which are bound to specific tracer antibodies in the gold conjugation pad, are bound to the polysaccharide antigen immobilized in the test line to form a red-purple band. Therefore, the band can be observed with the naked eye. The residual tracer antibodies in the gold conjugation pad, which are not reacted with antibodies in the biological sample, migrate to the control line to react with a control protein. Subsequently, a red-purple band is formed, thereby ensuring conformance to the test. As described above, the diagnostic kit of the invention is performed using immunochromatography by an antigen-antibody reaction. Accordingly, any equipment is not required and the results can be quickly observed with the naked eye. Further, the pre-test line of the diagnostic kit eliminates any possibility of misdiagnosing the presence of antibodies against Leptospira, thereby improving the accuracy of the diagnosis for leptospirosis.

In another embodiment, the present invention relates to a method for detecting antibodies against Leptospira comprising a step of detecting antigen-antibody complex, which is formed by contacting the polysaccharide with a biological sample.

Examples of the biological sample, in which antibodies against Leptospira are detected, include blood, serum, and blood plasma, but are not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these

Examples are for the illustrative purpose only, and the invention is not intended to be limited by Examples.

Mode for Invention

Example 1: Preparation of leptospira antigen 1-1. Culture of leptospira strain

EMJH (leptospira medium base Ellinghausen McCullough Hohnson Harris) was dissolved in an amount of 0.23 g/90 ml and sterilized. 10 ml of leptospira enrichment EMJH was added thereto to prepare a medium. In this medium, leptospira bacteria were inoculated, and shaking-cultured at 3O⁰C for 5 days.

1-2. Preparation of leptospira antigen

Leptospira bacteria cultured in the EMJH medium for 5 days were centrifuged at 4,000 xg for 20 minutes. The pellet was washed with 1 PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4) three times, and heated at 100 C for 1 hour. 150 g/ml of proteinase K was added thereto, and subjected to reaction at 37 C for 16 hours. EGTA was added thereto to a concentration of 2 mM, and subjected to reaction at 70 C for 15 minutes, and then centrifuged at 180,000 xg, 4 C for 2 hours. H20 was added to the obtained pellet, and suspended therein to prepare a lipopolysaccharide (Westpha & Jann, 1965) . The purified LPS antigen was treated with 1% acetic acid and heated at 100 C for 1 hour to centrifuge at 3,000 xg for 20 minutes. The precipitated lipid A was removed to prepare a polysaccharide antigen. The prepared antigen was quantitated using a standard polysaccharide, which had been prepared using LPS purified from Escherichia coli (Sigma) in the same manner as described above.

Example 2 : Antigenicity analysis of leptospira polysaccharide 2-1. Western blotting analysis

For SDS-PAGE, a 12% gel with a thickness of 1 mm or 1.5 mm was prepared, and a Bio-Rad Protean II electrophoresis system was used. The proteins were transferred from the electrophoresis gel to a membrane at 80 V for 30 minutes using a Bio-Rad Transblot cell. The membrane was blocked using 1 x TBS buffer, to which 5% skim milk and 0.1% Tween 20 had been added. A rabbit immunoserum (L. interrogans strain WH-19) was used as a primary antibody, and a horse-radish peroxidase conjugated anti-rabbit IgG (Bio-Rad) as diluted to 1:10,000 was used as a secondary antibody. After completing the immunoreaction, the immunoblots were developed with an ECL Kit (Amersham Pharmacia Biotech) . The results are shown in Fig. 1.

As shown in Fig. 1, the polysaccharides of leptospira interrogans serovar lai and leptospira biflexa serovar patoc strain Patoc 1 were similar to each other in SDS-PAGE gel profile. The polysaccharide with position of 14 kDa or more was present in both lai and patoc. It was assumed that the polysaccharide acted as an antigen.

2-2. Dot-blotting analysis

1 ug (1 ug/1 ul) of the antigen was added dropwise to a nitrocellulose membrane, and dried at 37°C for 1 hour. The dried membrane was blocked using 5% skim milk-TBST for 1 hour, and reacted with the patient serum (diluted to 1:3,000) as a primary antibody for 1 hour. A horse-radish peroxidase conjugate anti-human IgG (diluted to 1:10,000) was used as a secondary antibody. After completing the reaction, the membrane was developed with the ECL Kit, was then compared with the MAT results (Table 1) . It was found that positive signals were observed in most of the sera of the patients.

Table 1

2-3. Enzyme Linked Immunosorbent Assay (ELISA) ELISA for polysaccharide was performed on the basis of the method introduced by Engvall and Perlmann (1972) . PoIy-

L-lysine (10 μg/ml) was diluted in 0.01 M PBS to pretreat a plate, and then subjected to reaction at room temperature for 24 hours. After the plate was washed with a PBS buffer containing 0.05% Tween 20 twice, the polysaccharide was suspended in a PBS buffer to treat the plate with 100 μl per well. The treated plate was left to react at 37°C for 1 hour, and then washed with a PBS buffer containing 0.05% Tween 20 three times. The plate was blocked with goat serum at 37°C. Sera from twenty patients with leptospirosis and twenty healthy persons were used as a primary antibody, and a horse-radish peroxidase conjugated anti-human IgG (Bio-Rad) as diluted to 1:10,000 was used as a secondary antibody. Each well was treated with a substrate to develop, and the absorbance was measured using an ELISA reader at 490 nm. It was found that while a recombinant protein of leptospira flagella had a sensitivity of 72 to 88%, and a specificity of 88 to 90%, the polysaccharide of the invention had a sensitivity of 95%, and a specificity of 95%. Accordingly, as a candidate antigen for early diagnosis of leptospirosis, the polysaccharide was found to be better than the protein (Table 2) .

Table 2 Sensitivity and specificity of ELISA

a: Number of sera of patients diagnosed by MAT: 20 b: Number of normal sera: 20

Example 3: Diagnostic kit production 3-1. Immobilization of leptospira antigen (polysaccharide) onto nitrocellulose membrane

Immobilization of a specific antigen to capture a specific antibody is performed on a nitrocellulose (NC) membrane (pore size: 5 urn) by means of electrostatic interaction. The purified antigen (polysaccharide) was added to line a NC membrane strip (5 x 25 mm) at a point of 1 cm from the bottom of it with a backing plastic, and a control antibody (rabbit anti-goat IgG or rabbit anti-protein A) was added to line it at a point of 1.4 cm from the bottom using a dispenser at a speed of 1 ul/cm. The membrane was dried at 37°C for 1 hour.

The experiment was carried out at varying concentrations of the purified polysaccharide. As a result, the maximum signal was observed at an antigen concentration of 1 mg/ml. The signals were observed to decrease at higher or lower concentrations than 1 mg/ml. Accordingly, in order to maximize the signal, a sufficient amount of the antigen was required to bind to a target antibody. However, an excessive amount of the antigen in the reaction area made that it was difficult to give a necessary distance of space required for an antigen-antibody reaction. As a result, the chance for immune adherence reaction was reduced.

3-2. Antigen buffer for lining

A Tween 20 affects nonspecific reactions of the antigen. As its concentration increases, the signal strength decreases. However, in order to remove a cross-reactivity, addition of a Tween 20 solution is required. The effect of

Tween 20 was tested in the range of 0.01, 0.05 and 0.1%. The most effective concentration is 0.05% for removing a cross- reactivity. Accordingly, it was determined that the optimum concentration of Tween 20 be 0.05%.

3-3. Determination of antigen used in pre-test line

In the case where nonspecific signals were generated by the antigen (polysaccharide) , the pre-test line was placed before the point of the antigen line to remove the nonspecific antibodies. Subsequently, in the present invention, LPSs of Leptospira and other similar bacteria (E. coli, Psudomonas aeruginosa) were used individually or in combination with each other. From the result, it was found that 0.6 mg/ml of E. coli LPS be most effective.

3-4. Deposition of tracer antibody-gold conjugate on glass fiber

(1) Selection of glass fiber, and determination of pretreatment condition

A goat anti-human IgM (μ-specific) colloidal gold conjugate solution, a goat anti-human IgG (H+L) colloidal gold conjugate solution, or a solution of colloidal gold conjugate labeled with protein A was diluted with a 50% trehalose solution (final cone. 5% trehalose) and distilled water to be its optical density of 3. A glass fiber membrane

(Millipore) was immersed in the prepared solution, and then dried at 37°C for 2 hours. The membrane was cut with a width of 0.5 cm.

(2) Determination of amount of gold-conjugate for lining, and of drying condition The strength of specific signal generated from analysis system increases in proportion to the amount of gold-antibody conjugate used. However, if an excessive amount of gold- antibody conjugate is used, nonspecific signals, which are generated from the relatively low concentration antibody or the nonspecific antibody in test sample, increase to cause a false-positive result. In this invention, a goat anti-human IgM (μ-specific) labeled gold (40 nm) , a goat anti-human IgG (γ-specific) labeled gold(40 nm) , a goat anti-human IgG (H+L) labeled (40 nm) or a protein A labeled gold (40 nm) were used as a gold-conjugate. Comparison tests were carried out according to an optical density of each conjugate. IgG was not detected well in all gold conjugates (Table 3) . It can be understood that the LPS is generally a T-cell non- dependent antigen. It was assumed that T-cell non-dependent antigen generates IgM but does not IgG well in immune response. Accordingly, it was concluded that IgM measurement is more important than IgG measurement in leptospirosis diagnosis (Table 3) . It was found that the optimum OD for gold labeled IgM was 3.

Table 3

Comparison of MAT and diagnostic kit results

3-5. Preparation of strip

The designed system consists of a NC membrane with a backing plastic being immobilized antigens as a main structure, a glass fiber membrane (conjugate pad) being deposited with conjugates at lower part, and a cellulose membrane absorbing an excessive amount of solution at the both end points (sample pad, absorption pad) that are connected with each other. The glass fiber membrane placed on the lower part of the system can not only quickly absorb a sample because of high absorbing power, but also induces the effective reaction between test material and the dissolved conjugates by extending the retention time before migrating to the upper part. On the middle part of the immunostrip, antigens to measure a test material (test line or capture line) and specific antibodies capable of detecting conjugates

(control line) were immobilized separately. And a cellulose membrane was placed on the upper part of the immunostrip

(absorbent pad) to quickly absorb any excessive amounts of the sample (Fig. 2) .

3-6. Determination of human serum concentration As a result of testing the human serum concentration, an excessive amount of serum may induce a nonspecific reaction. The test was performed with 100 μl of sera, which had been diluted to 1/1000, 1/100, 1/50, and 1/10, or with 100 μl of undiluted serum. Consequently, it was found that the optimum result was obtained with the serum diluted to 1/100.

3-7. Sensitivity and specificity of rapid diagnostic kit

To confirm the sensitivity and the specificity of a diagnostic kit for leptospirosis, the sera from patients with leptospirosis, and the sera from patients with other acute febrile illnesses (murine typhus, hemorrhagic fever with renal syndrome, tsutsugamushi disease) were used as samples. The sera from six patients with leptospirosis diagnosed by MAT were used. After 5 minutes, all of the sera were found to be positive, thereby the diagnostic sensitivity of IgM (μ- specific) being 100% (Fig. 3) . Tests for cross-reactivity were carried out with the sera from healthy persons (n=6) , the sera from patients with murine typhus (n=3) , the sera from patients with tsutsugamushi disease (n=4) , and the sera from patients with hemorrhagic fever with renal syndrome (n=6) , whereby the specificity was found to be 95% (Fig. 3) .

3-8. Result of efficiency test for trial product studied in other organization To confirm sensitivity and specificity of a diagnostic kit for leptospirosis of the present invention, sera from patients with leptospirosis and sera from patients with other acute febrile illness (murine typhus, hemorrhagic fever with renal syndrome, tsutsugamushi disease) obtained from a collaborative institute, Hallym University, were used as a test sample. Sera from five patients with leptospirosis diagnosed by MAT were used. After 5 minutes, all sera were found to be positive, whereby diagnostic sensitivity of IgM (μ-specific) was found to be 100% (Table. 4).

Tests for cross-reactivity were carried out with the sera from healthy persons (n=10) , the sera from patients with murine typhus, the sera from patients with tsutsugamushi disease, and the sera from patients with hemorrhagic fever with renal syndrome (n=10) , whereby the specificity was found to be 95% (Table. 4) .

Table 4

a: dilution ratio of sera Sensitivity: 5/5 = 100%

Specificity among other acute febrile illness (hemorrhagic fever with renal syndrome, murine typhus, tsutsugamushi disease) and healthy control: 19/20 = 95%

Industrial Applicability

As described above, use of a novel antigen of the present invention allows an accurate, quick, and simple diagnosis of leptospirosis.

Claims

1. A diagnostic composition for leptospirosis comprising a polysaccharide derived from a lipopolysaccharide (LPS) of leptospira.

2. A diagnostic kit for leptospirosis comprising the composition of claim 1.

3. The diagnostic kit according to claim 2, which is of a strip type using immunochromatography, a device type using immunochromatography, or a microplate type.

4. The diagnostic kit according to claim 2, wherein an antigen-antibody complex is detected by a colored particle immunoassay.

5. A method for detecting antibodies against Leptospira, comprising detecting an antigen-antibody complex formed by contacting the composition of claim 1 with a biological sample .

6. The method according to claim 5, which is selected from the group consisting of western blotting, ELISA (enzyme linked immunosorbent assay) , radioimmunoassay (RIA) , radioimmunodiffusion, Ouchterlony immunodiffusion, rocket

Immunoelectrophoresis, immunohistostaining assay, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, and FACS.