KR20080082337A - Leptospirosis Diagnostic Kit - Google Patents

Abstract

The present invention relates to a composition for diagnosing leptospirosis comprising a polysaccharide derived from lipopolysaccharide (LPS) of leptospirosis as a antigen, and a kit for diagnosing leptospirosis comprising the polysaccharide. Using the diagnostic kit of the present invention, it is possible to diagnose leptospirosis infection early and precisely and simply.

Description

Diagnostic kit for Leptospirosis

1 is a diagram showing the antigenicity of polysaccharides purified by SDS-PAGE (A) and Western blotting (B) of polysaccharides isolated from patoc and lai.

Figure 2 is a diagram showing the structure of a leptospirosis diagnostic strip using immunochromatography.

3 is a sensitivity and specificity test results of the leptospirosis diagnostic kit.

The present invention provides a composition for diagnosing leptospirosis comprising a polysaccharide derived from lipopolysaccharide (LPS) of leptospira, a leptospirosis diagnostic kit comprising the polysaccharide, and the presence of an antigen-antibody complex formed by contacting the polysaccharide with a biological sample. The present invention relates to a method for detecting an antibody against a bacterium.

Leptospirosis is a common infectious disease of humans and animals caused by the infection of Leptospira interrogans , a type of spiral fungus, and named acute febrile disease with jaundice and kidney disease in 1887 as Weil's disease. It was first introduced as an infectious disease.

Removing the pathogens from Wailea seubyeong patients first in 1918 and was named Spiro Geta wing terrorism MOREY H. Jia (Spirochaeta icterohaemorrhagiae), was later renamed Leptospira wing terrorism MOREY H. Jia (Leptospira icterohaemorrhagiae). Taxonomically, Leptospira belongs to the family Leptospiraceae, and the genus Leptospira is classified as Leptospira interrogans and Leptospira biflexa , depending on the presence or absence of pathogenicity. The pathogenic Leptospira interrogans have been classified into 19 serogroups to date, and have been known to contain about 180 serovars, which are known to have different serotype distributions in different regions.

Wiles disease (or leptospirosis) is an acute febrile disease with typical clinical symptoms such as fever, headache, muscle pain, and deprivation at the onset of the onset, and in severe cases, internal organ bleeding, jaundice, and kidney failure. The epidemiologic characteristics of leptospirosis are mainly because bacteria excreted in the urine of infected animals (eg mice, cattle, horses, pigs, etc.) survive in the soil or water and then invade through the mucous membranes of the wounded skin, nose and mouth of a person. Farmers, slaughterhouse workers, and field-trained soldiers are found as patients, particularly after flooding, which can increase patient incidence because feces contaminate large areas.

The disease is known to occur in Europe, the Americas, Australia, Vietnam, Thailand, Malaysia, Taiwan, China, Japan, etc. In Korea, the occurrence of leptospirosis was reported in 1942. In 1984, leptospirosis was isolated from pulmonary bleeding patients and it was confirmed that the disease exists in Korea. Since then, it has been designated as the third group legal epidemic along with Tsutsugamus disease, nephrotic bleeding, and rash.

Meanwhile, a method for diagnosing leptospirosis has been studied. For example, Korean Patent Application No. 1990-0000871 discloses a slide agglutination reagent for diagnosing leptospirosis composed of polyvalent antigen-I, polyvalent antigen-II, and positive control serum, and Korean Patent No. 561687 discloses leptospira intero. Gans serotype lye ( Leptospira) interrogans Serovar lai HY10) discloses a diagnostic kit comprising a recombinant protein specific, Korean Patent No. 234876 discloses a diagnostic kit using an immunoblot comprising a leptospira interogans antigen.

However, the method for diagnosing leptospirosis studied so far is different from the antigen used in the microscopic agglutination test (MAT), which is a standard method of the World Health Organization (WHO). MAT is a method using an externally exposed polysaccharide among components of the lipopolysaccharide (LPS), which is a surface antigen of a serotype-specific Leptospira bacterium. In the present invention, since the LPS polysaccharide containing the antigen specific to the genus Leptospira is used as an antigen, the diagnostic principle is similar to that of the standard method MAT, which enables not only a more accurate diagnosis but also all leptospirosis as one antigen. Can be diagnosed. In addition, the MAT method has the advantage of being able to make an accurate diagnosis compared to other diagnostic methods, but it is troublesome to maintain and manage it because it uses live bacteria, requires expensive equipment, and can be tested only by skilled professionals. It is difficult to use in hospital because of the disadvantage of difficult method. Therefore, the present inventors have tried to develop a method for diagnosing leptospirosis simply and easily, and thus completed the present invention.

Accordingly, one object of the present invention is to provide a lipopolysaccharide-derived polysaccharide of leptospirosis as a new antigen for diagnosing leptospirosis, and to provide a composition for diagnosing leptospirosis containing the polysaccharide.

It is another object of the present invention to provide a leptospirosis diagnostic kit comprising the polysaccharide and an antibody detection method for leptospira using the same.

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

As used herein, the term “diagnosis” refers to identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to identify leptospirosis. The term "leptospirosis" refers to a disease caused by pathogenic helix bacteria in the genus Leptospira.

The diagnosis of leptospirosis is made by directly identifying and identifying bacteria in the blood, cerebrospinal fluid and urine of the patient, or by detecting antibodies in serum. In a specific embodiment of the present invention, leptospirosis is diagnosed by detecting antibodies in serum. As a method for detecting the antibody in the serum, in the present invention, a polysaccharide in which lipid is separated from lipopolysaccharide (LPS) in leptospira is used as an antigen. The inventors have confirmed that the polysaccharide isolated from lipopolysaccharide of the genus Leptospira can act as an antigen for diagnosing leptospirosis by Western blot and dot blotting. In addition, as a result of performing the experiment on the blood of the actual patient by the ELISA, it was confirmed that the antibody in the serum of patients infected with leptospirosis using the polysaccharide can be significantly diagnosed. The term “significance” means that the result obtained by diagnosis is accurate, so that the validity is high and the reliability is high so that the result is consistent even when repeated measurement is performed. When tested by a diagnostic method using a protein antigen with a patient serum diagnosed with MAT, the sensitivity and specificity are somewhat inferior, but in the present invention, since the polysaccharide used as an antigen in the MAT method is used as an antigen, A more accurate diagnosis can be made.

In order to detect antibodies against leptospira present in biological samples including plasma, serum, blood and the like, polysaccharides derived from lipopolysaccharide of leptospira are used as antigens. Leptospira lipopolysaccharide (LPS) is composed of lipid A (lipid A), core polysaccharide (O-specific side chain). Lipid A and core polysaccharides are linked by 2-keto-3-deoxyoctanic acid (KDO), which consists of glucose, galactose and N-acetylglucosamine. In a specific embodiment of the present invention, a polysaccharide composed of a core-specific O-specific side chain and a core polysaccharide, which is specific to a serotype and genus-specific, in which lipid A is removed to increase antigen antibody reactivity, is used as an antigen.

In another aspect, the present invention relates to a leptospirosis diagnostic kit comprising a polysaccharide derived from lipopolysaccharide (LPS) of leptospira bacteria.

The kit is a kit for diagnosing leptospirosis by measuring the antibody level against leptospira in a biological sample, from the lipopolysaccharide of leptospira acting as an antigen in response to the antibody against leptospira to examine antigen-antibody complex formation Polysaccharides derived.

Immunoassay methods to look at antigen-antibody complex formation include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, FACS, etc. It is not.

Leptospirosis diagnostic kit of the present invention includes not only the polysaccharide that specifically binds to the antibody but also tools, reagents and the like generally used in the art used for immunological analysis. Such tools or reagents include, but are not limited to, suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. In addition, the diagnostic kit of the present invention may be of a type such as, but not limited to, a microplate, a dip-stick device, an immunochromatography test strip, a radiation split immunoassay device, a flow-through device, or the like. In addition, the diagnostic kit of the present invention may include positive and negative standard controls.

Preferred diagnostic kits are strip type diagnostic kits using immunochromatography or diagnostic kits in the form of devices. Diagnosis using immunochromatography involves the reaction of an antibody in serum of a biological sample with a tracer antibody bound to colloidal gold particles, followed by micropores of the nitrocellulose membrane by capillary action. While moving through the micropore, it combines with a capture antigen fixed to the inner surface of the micropores to form a color band, thereby making it possible to visually distinguish between positive and negative. The "marker antibody" refers to an antibody which binds to an antibody against leptospira and is associated with a color particle.

In the diagnostic kits of the present invention, the antigen-antibody complex can be detected by color particle binding methods, which may include, for example, colloidal gold particles or colored glass or plastic (eg polystyrene, polypropylene, latex, etc.) beads. (bead) can be used. More preferably, gold colloids are used.

In one specific embodiment, as can be seen in Figure 2, the sample pad (sample pad) is absorbed; Gold conjugation pads comprising marker antibodies that bind to antibodies in the sample; Test membrane is treated with a test line containing a polysaccharide of the present invention, a control line containing a control protein and a pre-test line for removing nonspecific antibodies. ; And a leptospirosis diagnostic kit for detecting an antibody against leptospira in a biological sample, which includes a test strip including an absorption pad on which a residual amount of the sample is absorbed. The reaction line contains the polysaccharide of the present invention at a concentration of 0.5 to 2.5 mg / ml, preferably 1 to 2 mg / ml, and the control line comprises 0.1 control protein such as rabbit anti-goat IgG and rabbit anti-protein A. To 1 mg / mL, preferably 0.1 to 0.2 mg / mL. In addition, the whole reaction line contains a gram-negative bacterium similar in structure to the lipopolysaccharide (LPS) of leptospira of the present invention, for example, lipopolysaccharide (LPS) such as E. coli and Pseudomonas at a concentration of 0.5 to 1 mg / ml. have. The gold binding pad comprises gold-labeled goat anti-human IgM, goat anti-human IgG or gold-labeled protein A and the like.

The method for diagnosing leptospirosis using the strip type diagnostic kit using the immunochromatography method is as follows. First, when a biological sample to be sampled is dropped onto a sample pad, which is a site where a sample of the diagnostic kit is absorbed, the biological sample reaches a gold conjugation pad by capillary action to prevent the antibody in the sample. Marker antibodies in gold binding pads such as gold-labeled anti-human IgM goat antibody and gold-labeled anti-human IgG goat antibody ) Or gold-labeled protein A and the like to form a colloid. At this time, the biological sample to be sampled continues to move without being immobilized on the gold binding pad to reach a test line in which the polysaccharide antigen of the present invention is immobilized. In biological samples of patients infected with leptospira, an antigen-antibody reaction with the polysaccharide antigen occurs, i.e., an antibody against leptospira bound to a specific marker antibody in the gold binding pad of the patient is directed to a polysaccharide antigen immobilized on the reaction line. By combining, they form a reddish purple band that can be visually observed. In addition, the marker antibody in the remaining gold binding pads that did not react with the antibody in the biological sample reached the control line and reacted with the control protein to form a red purple band, indicating the suitability of the test. As described above, the diagnostic kit of the present invention can quickly check the test result with the naked eye without special equipment by using the immunochromatography method by the antigen-antibody reaction. In addition, the diagnostic kit improves the accuracy of the diagnosis of leptospirosis by eliminating the possibility that the diagnostic kit may be mistaken as having the antibody against the leptospira by the pretest line.

In another aspect, the present invention relates to a method for detecting an antibody against leptospira, comprising detecting an antigen-antibody complex formed by contacting the polysaccharide with a biological sample.

Biological samples from which antibodies against leptospira are detected include, but are not limited to, blood, serum, plasma, and the like.

 Hereinafter, the present invention will be described in more detail with reference to the following examples. However, this is only added to help the understanding of the present invention, the scope of the present invention is not limited by these examples.

Example  1: Leptospira antigen acquisition

1-1. Leptospira Strain Culture

After sterilization by melting 0.23 g / 90 ml of EMJH (Leptospira medium base Ellinghausen McCullough Hohnson Harris), a medium was prepared by adding 10 ml of leptospira enrichment EMJH. The medium was inoculated with leptospira and shaken for 5 days at 30 ° C.

1-2. Leptospira Antigen Preparation

Centrifuge the leptospira bacteria cultured in EMJH medium for 5 days at 4,000xg for 20 minutes and wash the cells three times with 1X PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO _{4 ,} 2mM KH ₂ PO ₄ ) and then 100 Heated at < RTI ID = 0.0 > Proteasease (Proteinase) K was treated here to 150㎍ / ㎖ and reacted for 16 hours at 37 ℃. Thereafter, EGTA was added to 2 mM and reacted at 70 ° C. for 15 minutes, followed by centrifugation at 4 ° C. and 180,000 × g for 2 hours. H ₂ O was added to the precipitate and suspended to prepare a lipopolysaccharide (Westpha & Jann, 1965). The purified LPS antigen was treated with acetic acid at 1%, warmed at 100 ° C. for 1 hour, and then centrifuged at 3,000 × g for 20 minutes to remove lipid A (lipid A), thereby preparing a polysaccharide antigen. The prepared antigen is Escherichia coli ), the amount of polysaccharide obtained by treating LPS (Sigma) purified in the same manner as above was used as a control (standard).

Example  2: antigenicity investigation of leptospira polysaccharide

2-1. Weston Blot  analysis

SDS-PAGE used a Bio-Rad Protean II electrophoresis device to make a 12% gel of 1 mm or 1.5 mm thickness. Transcription of the protein from the electrophoretic gel to the membrane (membrane) was performed for 30 minutes at 80V using a Bio-Rad Transblot cell. The transcribed membrane was blocked using 1 × TBS buffer containing 5% skim milk and 0.1% Tween 20, and rabbit immune serum (WH-19) was used as the primary antibody. Was used to dilute horse-radish peroxidase conjugated anti-rabbit IgG (Bio-Rad) to 1: 10,000. After completion of the immune response, ECL kit (Amersham Pharmacia Biotech) was developed. The results are shown in FIG. 1.

As seen in Figure 1,Leptospira interrogans The polysaccharide of serovar laiLeptospira biflexa Similar results were observed in serovar patoc and SDS-PAGE gel profiles, and polysaccharides above 14kDa were commonly present in lai and patoc.

2-2. Dot blotting

  1 ug (1 ug / 1 ul) of the antigen is added to the nitrocellulose membrane and then dried at 37 ° C. for 1 hour. The dried membrane was blocked with 5% skim milk-TBST for 1 hour, and the patient serum, the primary antibody, was diluted 1: 3,000 and reacted for 1 hour. The secondary antibody was reacted by diluting the horse-radish peroxidase conjugate anti-human IgG to 1: 10,000. After the reaction, the color was developed using the ECL kit and compared with the MAT results (Table 1). It was confirmed that most patients sera showed a positive signal.

2-3. Enzyme Immunoassay (ELISA)

  Polysaccharide ELISA was performed based on the method used by Engvall and Perlmann (1972). After diluting poly-L-lysine (10µg / ml) in 0.01M PBS, the plate was pretreated and reacted at room temperature for 24 hours. After washing twice with PBS buffer containing 0.05% Tween 20, polysaccharides were suspended in PBS and treated with 100 μl per plate. After reacting at 37 ° C. for 1 hour, the plate was washed three times with PBS buffer containing 0.05% Tween 20, and treated with goat serum to block at 37 ° C. The primary antibody consisted of 20 patient sera confirmed with leptospirosis and the serum of 20 normal subjects. The secondary antibody was diluted 1: 10,000 with horse-radish peroxidase conjugated anti-human IgG (Bio-Rad). . Each well was treated with a substrate to observe color development, and the value was measured at 490 nm using an ELISA reader. As a result, the leptospira flagella recombinant protein showed 72-88% sensitivity and 88-90% specificity, whereas the polysaccharide showed 95% sensitivity and 95% specificity, so that the early diagnosis of leptospirosis was possible. Antigen candidates showed better polysaccharides than proteins (Table 2).

Sensitivity and specificity of ELISA Antigen Polysaccharide patoc lai Sensitivity (%) ^a 95 95 Specificity (%) ^b 95 95

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

  b: Number of normal sera: 20

Example 3 Preparation of Diagnostic Kit

3-1. Immobilization of Leptospira Antigen (polysaccharide) on Nitrocellulose Membrane

Immobilization of specific antigens, which serve to capture assay specific antibodies, was performed by electrostatic interaction on nitrocellulose (NC) membranes (5um pore size). The purified polysaccharide was placed 1 cm from the bottom of the plastic backed NC membrane strip (5 × 25 mm) and the control antibody (rabbit anti-goat IgG or rabbit anti-protein A) was placed 1.4 cm away from the dispenser. Was used at a rate of 1 ul / cm. The deposited membrane was dried at 37 ° C. for 1 hour.

As a result of experiments with different concentrations of purified polysaccharides, the signal developed showed maximum at 1 mg / ml antigen concentration, and decreased at concentrations below or higher. This is because the signal development requires a sufficient amount of antigen to bind to the analytical antibody, but when the excess antigen is concentrated in a certain area, the spatial approach for antigen-antibody reaction is difficult, which lowers the probability of the immunoadhesion reaction.

3-2. Antigen Application Buffer

  In the case of Tween 20, it affects the nonspecific reaction of antigenic proteins. The higher the concentration, the lower the signal strength, but the addition of Tween 20 is necessary to eliminate the cross reaction. Tween 20 concentrations range from 0.01, 0.05 and 0.1%. The result was the most effective at eliminating the cross reaction at 0.05%. Therefore, 0.05% tween 20 was determined as the optimal concentration.

3-3. Determination of antigen to use for pre-test line

When a nonspecific signal generated by an antigen (polysaccharide) occurred, it could be removed by dropping a pretest line in front of the antigen drop site to remove the nonspecific antibody. Therefore, in this study, LPS of leptospira LPS and some similar bacteria ( E. coli , Psudomonas aeruginosa ) were individually or mixed and then instilled. As a result, E. coli LPS was the most effective at 0.6mg / ml.

3-4. Accumulation of Detection Antibody-gold Polymers on Glass Fibers

(1) Selection of glass fiber and determination of pretreatment conditions

The goat anti-human IgM (colloidal gold polymer) and the goat anti-human IgG (H + L) colloidal gold polymer or protein A labeled colloidal gold polymer solution were 50% trehalose (OD 3). trehalose) (final concentration 5% trehalose) was prepared by diluting with distilled water. The solution prepared in the glass fiber membrane (Millipore Co., Ltd.) was sufficiently wetted, dried at 37 ° C. for 2 hours, and cut into 0.5 cm in width.

(2) Determination of Dose and Drying Conditions of Gold-Polymers

The specific signal intensity generated from the assay system is increased in proportion to the amount of gold-antibody polymer used. Excessive use, however, increases the non-specific signal in areas with relatively low analyte concentrations, resulting in false positive results. Thus, in this experiment, gold anti-human goat anti-human IgM (μ-specific) labeled gold (40 nm) and goat anti-human IgG (γ-specific) or (H + L) labeled gold (40 nm) or protein A labeled gold (40 nm) was used. As a result of comparative tests according to the concentration (OD) of each polymer, IgG showed poor detection in all the gold polymers used (Table 3). This result was judged because LPS is usually T-cell independent antigen. Since the antibody produced by the T-cell independent antigen immune response is IgM, it has been estimated that only a few IgGs are detected. Therefore, it was concluded that the measurement of IgM is important in the diagnosis of leptospirosis and that the detection of IgG is relatively insignificant (Table 3). Gold-labeled IgM was determined to be the optimal concentration by showing effective results at OD 3.

MAT vs. Diagnostic Kit Results Patient NO. MAT Kit lai canicola yeonchon proteinA IgG (H + L) IgG (r) IgM 2000-100 - 640 - - - - ++ 2000-224 40 80 - +++ + + +++ 2000-307 40 320 320 - - - ++ 2000-355 - 80 - +/- - - ++ 2000-407 80 640 160 - - - ++ 2001-21 40 160 160 - - - ++ 2001-116 - 80 - - - - ++ 2001-404 80 1280 160 +/- - - +++ 2001-478 80 640 80 - - - ++ 2001-479 1280 40 1280 ++ - +/- +++

3-5. Strip manufacturer

The designed system fixed the antigen in the middle with a plastic backing NC membrane as a base skeleton, and attached a glass fiber membrane (conjugate pad) where polymer was accumulated, and a cellulose membrane absorbing excess aqueous solution at both ends (samples). Pads, absorbent pads) are formed in contact with each other. The glass fiber membrane located at the bottom can absorb the sample in a short time due to its high absorption ability, and can induce an efficient reaction between the analyte and the polymer by prolonging the retention time before the dissolved polymer is transferred to the upper immune strip. On the intermediate immune strip, the analyte measuring antigen (test line or capture line) and the specific antibody (control line) that can detect the polymer were spatially separated and immobilized. The cellulose membrane, an absorbent pad placed, allowed the excess sample to be rapidly absorbed (FIG. 2).

3-6. Determination of Human Serum Concentration

As a result of testing human serum concentrations, serum concentrations, like polymers, can also induce nonspecific reactions when in excess. The serum was diluted to 1/1000, 1/100, 1/50, and 1/10 or tested by 100 μl in the form of undiluted serum, and found to be optimal when 1/100 serum was used.

3-7. Rapid diagnostic kit Sensitivity and specificity of

In order to evaluate the diagnostic sensitivity and specificity of the leptospirosis diagnostic kit, the sera of leptospirosis patients and the sera of other patients with acute febrile illnesses (rash, renal hemorrhage, Tsutsugamus disease) were tested. Serum from 6 patients with leptospirosis diagnosed with MAT was used. After 5 minutes, all 6 samples were positive, and thus the IgM (μ-speicific) diagnostic sensitivity was 100% (FIG. 3).

Cross-reaction tests were performed with healthy serum (n = 6), rash sera (n = 3), Tsutsugamus serum (n = 4) and nephrotic bleeding serum (n = 6). % Is shown (FIG. 3).

3-8. To other institutions  Prototype Performance test  request

In order to determine the diagnostic sensitivity and specificity of the leptospirosis diagnostic kit of the present invention, the serum of patients with leptospirosis patients and sera of other acute febrile diseases (rash, renal bleeding hemorrhage, Tsutsuga musiosis) were tested at Hallym University. Serum from 5 patients with leptospirosis diagnosed with MAT was used. All 5 samples were positive after 5 minutes, and the IgM (μ-speicific) diagnostic sensitivity was 100% (Table 4).

Cross-reactivity was tested with healthy serum (n = 10), rash sera, Tsutsuga mucosa serum and nephrotic bleeding serum (n = 10). The specificity was 95% (Table 4).

RDK  (1: 100) ^a Leptospirosis  (n = 5) 5 Other AFI  (n = 10) One Healthy control  (n = 10) 0

a: dilution ratio of sera

Sensitivity: 5/5 = 100%

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

As described above, the use of the novel antigen of the present invention enables accurate, rapid and simple diagnosis of leptospira infection.

Claims (6)

Leptospirosis diagnostic composition comprising a polysaccharide derived from lipopolysaccharide (LPS) of leptospira. Leptospirosis diagnostic kit comprising the composition of claim 1. The diagnostic kit according to claim 2, wherein the strip type using immunochromatography, the device type or microplate type using immunochromatography. The diagnostic kit according to claim 2, wherein the antigen-antibody complex is detected by chromosomal particle binding. A method for detecting an antibody against leptospira comprising detecting the antigen-antibody complex formed by contacting the composition of claim 1 with a biological sample. The method of claim 5, wherein the detection method is Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography and FACS.

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