JP2009288065A - Detection method of shigatoxin2e and antibody to shigatoxin2e - Google Patents

Abstract

An object of the present invention is to provide a method for detecting an antibody against Shiga toxin 2e (Stx2e) or Stx2e in an animal specimen by utilizing the selective binding property of Globotetraosylceramido (Gb4) to Shiga toxin 2e (Stx2e).
A method for detecting Shiga toxin 2e (Stx2e) in a specimen, wherein the specimen is brought into contact with a carrier on which globotetraosylceramide (Gb4) is solid-phased, and Gb4 formed on the carrier by the contact. Detection of Shiga toxin2e (Stx2e) in a sample, including the use of a labeled anti-Stx2e antibody or an anti-Stx2e antibody and a labeled anti-immunoglobulin antibody that binds to it to detect the presence of a complex between the sample and Stx2e in the sample how to.
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Description

  The present invention utilizes the selective binding property of Globotetraosylceramido (Gb4) to Shiga toxin2e (Stx2e) to detect an antibody against Stx2e or Stx2e in a pig sample, thereby producing an toxin-producing Escherichia coli animal. The present invention relates to a technique for detecting the presence or absence of infection and edema disease in pigs.

  Shiga toxin 2e (Stx2e) is a toxin produced by toxin-producing Escherichia coli and is an important toxin that causes swine edema disease. Stx2e is a variant of Shiga toxin 2 (Stx2), and it is extremely important to distinguish it from Stx2 in the diagnosis of this disease.

  The method of gene search by PCR is the mainstream, but in the diagnosis of this disease, it is better to detect Stx2e directly and prove it directly than to prove the presence of Stx2e indirectly by gene search It is reliable and has a wide range of applications.

  Conventionally, the presence of an antibody that neutralizes the toxicity of Shiga toxin 2e to Vero cells has been determined by the presence or absence of cytopathy (see Non-Patent Documents 1 to 3). However, since the conventional method uses cells, it took one week or more to prepare and determine the result. In addition, since the presence / absence of cell degeneration is visually confirmed and the result is determined, experience is necessary to make an objectively reliable determination. In addition, since the work is complicated, it is difficult to test multiple samples at a time. In addition, substances other than antibodies may act on neutralization of toxins, and the obtained results are not necessarily valid.

  Since Stx2e has a high amino acid homology with Stx2, it was extremely difficult to distinguish and detect it by a conventional immunoassay system depending on the specificity of the antibody.

  On the other hand, detection of Stx2e-infected antibodies is also essential for the diagnosis and epidemiological investigation of porcine edema disease. The only detection method currently used is a bioassay method using Vero cells. However, the bioassay method has a problem that the work is complicated and it takes several days to make a determination, and considerable skill is required to make a reliable determination with reproducibility.

J. KONOWALCHUK et al., INFECTION AND IMMUNITY, Dec. 1977, p.775-779 Mohamad A, et al, The journal of Infectious Diseases, Vol.151, No.5, May 1985, p.775-782 Mohamad A. Karmali et al., THE LANCET, MARCH 19, 1983, p.619-620

  The present invention provides a method for detecting an antibody against Shiga toxin2e (Stx2e) or Stx2e in an animal sample by utilizing the selective binding property of Globotetraosylceramido (Gb4) to Shiga toxin2e (Stx2e). Objective.

  The present inventors applied the fact that Stx2e selectively binds to Globotetraosylceramide (Globotetraosylceramide: Gb4) in vivo, and conducted intensive studies on a method for detecting Stx2e simply and specifically. The present inventors have found that Stx2e can be detected with extremely high specificity by a sandwich ELISA method (Gb4ELISA) in which Gb4 is coated on a microplate and Stx2e bound to Gb4 is detected with an anti-Stx2e polyclonal antibody.

  Furthermore, the present inventors also examined a method for detecting infectious antibodies by applying the aforementioned Gb4 ELISA. As a result of trying a method to detect the infectious antibody by reacting a certain amount of Stx2e on the plate coated with Gb4, and then sensitizing the porcine serum of the test material, this method detects the infectious antibody quickly and accurately I found out.

  The method for detecting Stx2e and the method for detecting an antibody against Stx2e of the present invention is considered to be an excellent test method for the presence or absence of infection with toxin-producing Escherichia coli in animals and swine edema disease because of its simplicity and high specificity. .

That is, the present invention is as follows.
[1] A method for detecting Shiga toxin 2e (Stx2e) in a specimen, wherein the specimen is brought into contact with a carrier on which globotetraosylceramide (Gb4) is solid-phased, and Gb4 formed on the carrier by the contact. Detect Shiga toxin2e (Stx2e) in a sample, including detecting the presence of a complex with Stx2e in the sample using a labeled anti-Stx2e antibody or a labeled anti-immunoglobulin antibody that binds to the anti-Stx2e antibody Method.
[2] A method for detecting Shiga toxin2e (Stx2e) in a specimen of [1], which is an enzyme immunoassay.

[3] A method for detecting an antibody against Shiga toxin2e (Stx2e) in a specimen, wherein the specimen is brought into contact with a carrier on which a complex of globotetraosylceramide (Gb4) and Stx2e is immobilized, and the carrier is thereby contacted. A method for detecting an antibody against Shiga toxin2e (Stx2e) in a specimen, comprising detecting the presence of a complex of Gb4, Stx2e and an anti-Stx2e antibody formed above using a labeled anti-immunoglobulin antibody.
[4] A method for detecting an antibody against Shiga toxin2e (Stx2e) in a specimen according to [3], which is an enzyme immunoassay.

[5] A method for detecting the presence or absence of infection of an animal-producing toxin-producing Escherichia coli by detecting Shiga toxin2e (Stx2e) in an animal specimen by the method of [1] or [2].
[6] A method for detecting the presence or absence of infection with an toxin-producing Escherichia coli in an animal by detecting an antibody against Shiga toxin2e (Stx2e) in the animal specimen by the method of [3] or [4].
[7] A method for diagnosing porcine edema disease by detecting Shiga toxin2e (Stx2e) in a pig specimen by the method of [1] or [2].

[8] A method for diagnosing porcine edema disease by detecting an antibody against Shiga toxin2e (Stx2e) in a pig specimen by the method of [3] or [4].
[9] A kit for detecting Shiga toxin 2e (Stx2e) in a specimen, comprising at least a carrier on which globotetraosylceramide (Gb4) is immobilized and a labeled antibody.
[10] A kit for detecting an antibody against Shiga toxin2e (Stx2e) in a sample containing a carrier on which at least globotetraosylceramide (Gb4) is immobilized, Shiga toxin2e (Stx2e) and a labeled antibody.
[11] A kit for detecting the toxin-producing Escherichia coli infection of animals, which is a kit according to [9] or [10].
[12] A diagnostic kit for porcine edema disease, which is the kit according to [9] or [10].

  As described in the Examples, the method of the present invention makes use of the selective binding property of Globotetraosylceramido (Gb4) to Shiga toxin2e (Stx2e), and the presence of Stx2e in the specimen or an antibody against Stx2e. Can be detected easily and rapidly specifically to detect whether the test animal is infected with toxin-producing Escherichia coli. In the case of pigs, it can be diagnosed whether the pig suffers from edema disease.

Hereinafter, the present invention will be described in detail.
The present invention is a method for detecting Shiga toxin 2e (Stx2e) in an animal-derived specimen, wherein globotetraosylceramide (Gb4) is immobilized on a carrier, and Shiga toxin 2e (Stx2e) is immobilized on the immobilized carrier. A specimen that may be contained is brought into contact, and a complex of Gb4 and Stx2e is formed on the solid-phase support. Furthermore, by contacting an antibody against Stx2e (anti-Stx2e antibody), binding the anti-Stx2e antibody to the complex of Gb4 and Stx2e on the solid-phased carrier, and detecting the bound anti-Stx2e antibody, Stx2e in the specimen is detected. It is a method of detection. Furthermore, the present invention is a method for detecting an anti-Stx2e antibody in a specimen, wherein globotetraosylceramide (Gb4) is immobilized on a carrier, Shiga toxin 2e (Stx2e) is contacted with the immobilized carrier, A complex of Gb4 and Stx2e is formed on the immobilized carrier, and a specimen that may contain an anti-Stx2e antibody is contacted, and a complex of Gb4, Stx2e, and anti-Stx2e antibody (Gb4 -Stx2e-anti-Stx2e antibody), contact with an antibody that binds to the labeled anti-Stx2e antibody, bind the labeled antibody to the complex, and measure the bound labeled antibody. Detect antibodies.

  Globotetraosylceramide is a glycosphingolipid contained in the living body and contains a β1-3 linked galNac residue. Globotetraosylceramide can be purified from mammalian erythrocytes such as porcine erythrocytes. Commercially available refined products can also be used.

  As the carrier, various materials such as ELISA microtiter plate, slide glass, nitrocellulose membrane, resin beads, magnetized beads and the like can be used. The bonding can be performed by a known method such as physical adsorption or covalent bonding using a functional group. The amount of the solid phase is not particularly limited, but when the carrier is a 96-well microtiter plate, it is preferably several ng to several tens of μg per well. The solid phase can be obtained by dissolving Gb4 to be solid phase in a suitable organic solvent such as methanol and bringing it into contact with a carrier. For example, when a microtiter plate is used, the Gb4 solution can be solidified by dispensing into a well of the microtiter plate and placing it for a certain period of time. After immobilizing Gb4, it is preferable to perform blocking using a blocking solution containing bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin and the like in order to prevent non-specific binding during the assay. Stx2e or anti-Stx2e antibody in a specimen can be detected using the Gb4 solid-phase support thus prepared.

  When detecting Stx2e in a sample, the carrier on which Gb4 is immobilized is reacted with the sample, Gb4 and Stx2e in the sample are bound, and a complex of Gb4 and Stx2e is formed on the carrier. After washing, an anti-Stx2e antibody labeled with an enzyme or the like or an unlabeled anti-Stx2e antibody is brought into contact with and bound to Stx2e in a complex of Gb4 and Stx2e. When a labeled anti-Stx2e antibody is used, the labeled anti-Stx2e antibody bound to the complex of Gb4 and Stx2e is measured. When using an anti-Stx2e antibody that is not labeled, an antibody that binds to the anti-Stx2e antibody labeled with an enzyme or the like after washing (a labeled antibody against immunoglobulin) is further brought into contact with the Gb4 / Stx2e / anti-Stx2e antibody complex. The labeled antibody is measured by binding to the anti-Stx2e antibody.

  When detecting an anti-Stx2e antibody in a specimen, a known amount of Stx2e is brought into contact with a carrier on which Gb4 is immobilized, thereby forming a complex of Gb4 and Stx2e. At this time, for example, an excess amount of Stx2e in a molar ratio may be brought into contact with the amount of Gb4 immobilized on the carrier, and Stx2e may be bound to almost all of the immobilized Gb4. After washing, the specimen is brought into contact with the carrier on which the complex of Gb4 and Stx2e has been formed. When the sample contains an anti-Stx2e antibody, a complex of Gb4, Stx2e, and anti-Stx2e antibody (Gb4-Stx2e-anti-Stx2e antibody) is formed on the carrier. Next, after washing, an antibody that binds to the anti-Stx2e antibody labeled with an enzyme or the like (labeled antibody against immunoglobulin) is contacted, and bound to the anti-Stx2e antibody in a complex of Gb4, Stx2e, and anti-Stx2e antibody, The labeled antibody is measured.

  The labeling of the antibody can be performed using an enzyme such as β-D-galactosidase, peroxidase, alkaline phosphatase or glucose oxidase, a fluorescent substance, a chemiluminescent substance, a radioisotope, or other coloring substances. In the measurement, when the label is an enzyme, a substrate for each enzyme is added to cause an enzyme reaction, and the color development may be measured by measuring the absorbance. When the label is a fluorescent substance, the fluorescence may be measured using a fluorescence detector. The anti-Stx2e antibody may be a monoclonal antibody or a polyclonal antibody. The antibody can be produced using, for example, a mouse, rabbit, goat, horse, sheep or the like. At this time, for example, Stx2e B subunit or a partial peptide thereof can be used as the Stx2e antigen. Stx2e can be obtained by culturing enterohemorrhagic Escherichia coli that produces Stx2e, isolated and purified from the culture, chemically synthesized based on the amino acid sequence information of Stx2e, or the gene encoding Stx2e. It can also be produced as a recombinant protein by genetic engineering techniques based on the base sequence information. As an antibody that binds to the anti-Stx2e antibody, an antibody against the immunoglobulin of the animal used to produce the anti-Stx2e antibody may be used.

  The method of the present invention includes any method based on an antigen-antibody reaction using a carrier to which Gb4 is bound, such as enzyme immunoassay such as ELISA, fluorescence immunoassay, immunochromatography and the like. In immunochromatography, Gb4 or a complex of Gb4 and Stx2e is solid-phased on a suitable solid phase carrier such as a nitrocellulose membrane, and the substance to be detected (Stx2e or anti-antigen is immobilized on the solid-phased carrier using capillary action. A complex of an antibody labeled with an appropriate labeling substance such as colloidal gold that can be bound to the Stx2e antibody) and a substance to be detected is transferred. As a result, a solid-phased Gb4-Stx2e-labeled antibody or a solid-phased Gb4 and Stx2e complex-anti-Stx2e antibody-labeled antibody complex is formed on the solid phase carrier, and the labeling reagent emitted from the complex In the case of colloidal gold (in the case of gold colloid), the substance to be detected can be detected by detecting the solid phase carrier on which Gb4 is immobilized. Production of the immunochromatography apparatus and detection by immunochromatography can be performed by a known method.

  The present invention is intended for animals, and examples include pigs, cows, horses, sheep, goats, humans, dogs, cats and the like. As the specimen, whole blood, serum, plasma, and feces collected from these animals can be used. It is also possible to use organs of these dead animals. In the case of pigs, detection of Stx2e or anti-Stx2e antibody enables diagnosis of porcine edema disease.

  When Stx2e or anti-Stx2e antibody is detected in the specimen, the test animal can be detected or diagnosed as infected with toxin-producing Escherichia coli. Alternatively, it can be determined that the toxin-producing Escherichia coli has been infected in the past. Further, when the test animal is a pig, it can be detected or diagnosed that the test pig is suffering from edema disease, or it can be determined that the subject pig has suffered from edema disease in the past. In the present invention, detecting, diagnosing, or judging that an animal has been infected with toxin-producing Escherichia coli and that the animal has been infected with toxin-producing Escherichia coli is referred to as detection or diagnosis of the presence or absence of infection of the animal with toxin-producing Escherichia coli. Further, detecting, diagnosing, or judging that a pig suffers from edema disease and that a pig has a history of suffering from edema disease is referred to as detection or diagnosis of pig edema disease.

  The present invention further includes a kit for detecting Stx2e or anti-Stx2e antibody in an animal sample, comprising a carrier on which at least Gb4 is immobilized. The kit can detect or diagnose that the test animal is infected with toxin-producing Escherichia coli. Alternatively, it can be determined that the toxin-producing Escherichia coli has been infected in the past. The present invention also includes a kit for diagnosing porcine edema disease in a specimen, comprising a carrier having at least Gb4 immobilized thereon. With this kit, edema disease can be diagnosed by detecting Stx2e in a pig specimen.

  The present invention further includes a kit for detecting Stx2e or anti-Stx2e antibody in an animal sample, comprising a carrier on which a complex of at least Gb4 and Stx2e is immobilized. The kit can detect or diagnose that the test animal is infected with toxin-producing Escherichia coli. The present invention also includes a kit for diagnosing porcine edema disease, comprising a carrier on which a complex of at least Gb4 and Stx2e is immobilized. With this kit, edema disease can be diagnosed by detecting an anti-Stx2e antibody in a pig specimen. Kits for diagnosing edema disease by detecting anti-Stx2e antibody in animal specimens and kits for diagnosing edema disease by detecting anti-Stx2e antibody in pig specimens have at least Gb4 immobilized The carrier and Stx2e may be included independently. In this case, Stx2e may be bound to Gb4 immobilized on a carrier at the time of use. Alternatively, a carrier on which a complex of Gb4 and Stx2e is immobilized may be included.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

  In this example, Globotetraosylceramide (Globoside from human erythrocytes, sigma) was used as purified Gb4. A 96-well microplate (Nunc Polysorp) was used as the ELISA plate. As the secondary serum, anti-rabbit IgG HRP-labeled antibody (Upstate) and anti-pig IgG rabbit HRP-labeled antibody (DaKoCytomation) were used. Furthermore, as Stx2eB, a recombinant protein produced by Escherichia coli recombined with the gene for the B subunit of Shiga toxin type 2e was used. As the anti-Stx2eB rabbit polyclonal antibody, a rabbit polyclonal antibody prepared using Stx2eB as an antigen was used. Furthermore, tetramethylbenzidine (TMB, DaKoCytomation) was used as the chromogenic substrate, and 2N sulfuric acid was used as the reaction stop solution.

  As test materials for the Stx2e detection system, 26 strains of E. coli derived from swine, 1 strain of E. coli derived from deer, and 3 strains of ATCC standard E. coli were used. Test materials for infectious antibody detection systems include 25 samples of pig serum from affected farms (33, 63, 90, 122, 180 days of age: 5 samples each), 20 samples of pig serum from unaffected farms (40, 90, 140, 180 days of age: 5 samples each) were used. The positive control serum and the negative control serum were one sample each of neutralizing antibody positive pig serum and neutralizing antibody negative pig serum.

Preparation of Gb4ELISA plate
The solid phase of Gb4 on the plate was performed by adding 10 μl of Gb4 adjusted to 10 μg / ml with 100% methanol to each 96-well microplate and allowing to stand at room temperature for 1 hour. Blocking was performed by adding 200 μl of bovine serum albumin adjusted to 1% with PBS (phosphate buffer, pH 7.4) to each well and allowing to stand at room temperature for 1 hour.

Detection of Stx2e First, a test material was sensitized with a Gb4 ELISA plate, followed by sensitization with an anti-Stx2eB rabbit polyclonal antibody, followed by detection with an anti-rabbit IgG HRP-labeled antibody. The determination was made by measuring the absorbance value (OD value) with a microplate reader (450 nm) after stopping the reaction. As the test material, E. coli to be tested was treated with polymyxin B solution, and the supernatant was used. (Table 1)

Stx detection
Stx was detected by a commercially available reverse passive latex agglutination method (VTEC-RPLA “SEIKEN”) according to the operation method in the package insert.

Stx gene search The E. coli gene search was carried out by the PCR method according to the reference to search for the presence and mutation of the Stx gene.

Infectious antibody detection
The Stx2e crude extract was sensitized on a Gb4 ELISA plate, then the test material was sensitized and detected with an anti-pig IgG rabbit HRP-labeled antibody. The determination was made by measuring the OD value with a microplate reader (450 nm) after stopping the reaction. The Stx2e crude extract was obtained by extracting Stx2e from Escherichia coli having Stx2e-producing ability using polymyxin B solution. As the test material, porcine serum diluted 1: 800 with 0.1% BSA-TPBS (0.1% bovine albumin, phosphate buffer solution with 0.05% Tween20, pH 7.4) was used. (Table 2)

In this example, the following results were obtained.
Stx2e detection system The detection sensitivity was examined using Stx2eB diluted 2-fold in steps with PBS containing 0.1% BSA (phosphate buffer solution with 0.1% bovine albumin, pH 7.4) as a test material. As a result, the minimum detection sensitivity was 1 ng (20 ng / ml) (FIG. 1).

  As a result of performing Gb4ELISA and VTEC-RPLA on five specimens derived from the Stx2e gene-bearing strain, the average OD value of Gb4ELISA was 0.46 (minimum value 0.24 to maximum value 1.15). On the other hand, VTEC-RPLA determined that 2 specimens with high Gb4 ELISA OD values were Stx2 positive and 3 specimens with low values were Stx2 negative. As a result of carrying out three specimens derived from the strains whose presence of Stx1 or 2 was confirmed by VTEC-RPLA, the average OD value was 0.00. As a result of performing 22 samples derived from strains in which Stx-producing genes were not detected by PCR, the OD value of all samples was 0.00 (Table 3).

  For the 30 samples, the Stx2e gene detection results by PCR were compared with the Gb4 ELISA test results (samples with an OD value of 0.01 or more were determined to be positive). As a result, the positive coincidence rate, negative coincidence rate, positive predictive value, and negative predictive value were all 100% (Table 4).

  These results suggest that Gb4ELISA can detect Stx2e with high sensitivity and specificity.

Infectious antibody detection system As a result of carrying out the serum obtained by serially diluting the positive control serum and the negative control serum with 0.1% BSA-TPBS, the OD value of the positive control serum was changed reflecting the dilution ratio of the serum. Negative control serum remained constant at low values (FIG. 2).

  As a result of investigating the relationship between the age of pigs and OD values in affected and unaffected farms, OD values tended to increase significantly after 90 days of age (90 days and 122 days). There was a statistically significant difference in age and OD values between 90 days and 180 days (p <0.01, t test). On the other hand, in the unaffected farm, it remained constant at a low value regardless of the age (FIG. 3).

  From the above results, it was suggested that the antibody test results by Gb4ELISA reflect the amount of Stx2e-infected antibody.

  By the method of the present invention, it is possible to easily and accurately detect toxin-producing Escherichia coli infection in animals and edema disease in pigs, and contribute to the development of medicine, veterinary medicine and livestock industry.

It is a figure which shows the relationship between the antigen concentration and OD value in Stx2e measurement ELISA. It is a figure which shows the relationship between the serum dilution rate and OD value in Stx2e measurement ELISA. It is a figure which shows the relationship between the OD value by the age of a pig, and Stx2e measurement ELISA in the onset farm of an edema disease, and an unaffected farm.

Claims (12)

  A method for detecting Shiga toxin2e (Stx2e) in a specimen, wherein the specimen is brought into contact with a carrier on which globotetraosylceramide (Gb4) is immobilized, and the Gb4 formed on the carrier by the contact and A method for detecting Shiga toxin2e (Stx2e) in a specimen, comprising detecting the presence of a complex with Stx2e using a labeled anti-Stx2e antibody or an anti-Stx2e antibody and a labeled anti-immunoglobulin antibody that binds thereto.   The method for detecting Shiga toxin2e (Stx2e) in a specimen according to claim 1, which is an enzyme immunoassay.   A method for detecting an antibody against Shiga toxin2e (Stx2e) in a specimen, wherein the specimen is contacted with a carrier on which a complex of globotetraosylceramide (Gb4) and Stx2e is immobilized, and formed on the carrier by the contact. A method for detecting an antibody against Shiga toxin2e (Stx2e) in a specimen, comprising detecting the presence of a complex of Gb4, Stx2e and an anti-Stx2e antibody using a labeled anti-immunoglobulin antibody.   The method for detecting an antibody against Shiga toxin2e (Stx2e) in a specimen according to claim 3, which is an enzyme immunoassay.   A method for detecting the presence or absence of infection of an animal-producing toxin-producing Escherichia coli by detecting Shiga toxin2e (Stx2e) in an animal specimen by the method according to claim 1 or 2.   A method for detecting the presence or absence of infection of an animal-producing toxin-producing Escherichia coli by detecting an antibody against Shiga toxin2e (Stx2e) in an animal specimen by the method according to claim 3 or 4.   A method for diagnosing porcine edema disease by detecting Shiga toxin2e (Stx2e) in a pig specimen by the method according to claim 1 or 2.   A method for diagnosing porcine edema disease by detecting an antibody against Shiga toxin2e (Stx2e) in a pig specimen by the method according to claim 3 or 4.   A kit for detecting Shiga toxin 2e (Stx2e) in a specimen, comprising at least a carrier on which globotetraosylceramide (Gb4) is immobilized and a labeled antibody.   A kit for detecting an antibody against Shiga toxin 2e (Stx2e) in a specimen containing at least a carrier on which globotetraosylceramide (Gb4) is immobilized, Shiga toxin 2e (Stx2e) and a labeled antibody.   A kit for detecting an toxin-producing Escherichia coli infection in animals, which is the kit according to claim 9 or 10.   A porcine edema disease diagnostic kit, which is the kit according to claim 9 or 10.

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