JP4791866B2 - Solid phase used for discrimination of diarrheagenic Escherichia coli infection - Google Patents

Description

  The present invention relates to a solid phase, a method, a kit, and the like that can be used for discrimination of serotypes of E. coli in diarrheagenic E. coli infections.

The abbreviations used in this specification are shown below.
LPS: Lipopolysaccharide Diarrheagenic Escherichia coli infection is known as an infectious disease caused by infection of Escherichia coli such as enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, intestinal aggregating Escherichia coli, intestinal invasive Escherichia coli, etc. It is problematic because symptoms such as diarrhea, water-soluble stool, bloody stool, hemolytic uremic syndrome (hereinafter abbreviated as “HUS”), acute enteritis, abdominal pain, fever and vomiting may occur. .

  On the other hand, the bacterial outer membrane of E. coli as described above is composed of LPS having a structure different depending on its serotype. Generally, when E. coli as described above infects animals such as humans, It is generally known that antibodies specific to the LPS are produced.

  Conventionally, there has been known a method for determining the serotype of E. coli in diarrheagenic E. coli infection by detecting antibodies produced in serum by the mechanism as described above by an immunological measurement method. .

  Patent Document 1 discloses an array in which different polypeptides derived from a target organism are arranged at respective positions. An example of a target organism is diarrheagenic Escherichia coli. However, there is no disclosure or suggestion about an array in which LPS is arranged.

  In diarrheagenic Escherichia coli infection, it is desirable to determine the serotype of E. coli that is infected very quickly and accurately. Therefore, the serotype of E. coli in diarrheagenic Escherichia coli infection is more efficient and reliable. There was a need for a method that could do this.

JP 2005-515754 A

  An object of the present invention is to provide a purified LPS, a solid phase, a method, a kit, and the like that can be used to more quickly and accurately discriminate E. coli in diarrheagenic E. coli infections.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor efficiently and quickly discriminates E. coli in diarrheagenic E. coli infection by using a solid phase to which LPS is fixed. The present invention was completed by finding out that it can be carried out.

That is, the present invention provides the following.
(1) A solid phase to which LPS is fixed (hereinafter referred to as “the solid phase of the present invention”).
(2) The solid phase according to (1) above, wherein a plurality of types of LPS are individually fixed on the same solid phase.
(3) The solid phase according to (1) or (2) above, wherein the LPS is derived from one or more E. coli classified according to an O serotype selected from the following group;
O1, O25, O26, O63, O113, O125, O125ac, O144, O153, O168, O3, O77, O114, O126, O145, O157, O169, O6, O27, O78, O115, O127, O146, O158, O8, O28, O86, O117, O128, O148, O159, O11, O29, O91, O119, O131, O149, O164, O15, O32, O103, O121, O136, O150, O18, O44, O111, O111ab, O123, O142, O151, O166, O20, O55, O112, O112ac, O124, O143, O152, O167, O165.
(4) A method for discriminating serotypes of Escherichia coli in diarrheagenic Escherichia coli infection (hereinafter referred to as “the present invention discriminating method”), which comprises at least the following steps.
Step 1: LPS affixed to the solid phase of the present invention is contacted with serum from an animal suspected of having diarrheagenic E. coli infection to form a conjugate comprising the LPS and an anti-LPS antibody contained in the serum. Process.
Step 2: contacting the conjugate with an antibody capable of binding to an anti-LPS antibody, and detecting the conjugate.
(5) The discrimination method according to (4) above, wherein the serotype of E. coli to be discriminated is the O serotype.
(6) The discrimination method according to (4) or (5) above, wherein the antibody in step 2 is an anti-IgM antibody.
(7) A serotype discriminating kit for Escherichia coli in diarrheagenic Escherichia coli infection (hereinafter referred to as “invention kit”) comprising at least an antibody capable of binding to the solid phase of the present invention and an anti-LPS antibody as a constituent component.

According to the discrimination method of the present invention, the serotype of E. coli in diarrheagenic E. coli infection can be determined with very high accuracy. In addition, by using the solid phase of the present invention, the present invention discrimination method can be performed. Moreover, by using this invention discrimination kit, this invention discrimination method can be performed more efficiently and simply.

Hereinafter, the present invention will be described in detail by the best mode for carrying out the invention.
<1> Solid phase of the present invention The solid phase of the present invention is a solid phase to which LPS is fixed.

In the above, LPS can be obtained by extraction from a gram-negative bacterium exemplified by E. coli by a known method such as a hot phenol method. In the present invention, the origin of LPS is not particularly limited, but Escherichia coli is preferable. Among the Escherichia coli, it is more preferable that the Escherichia coli is selected from enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, intestinal agglutinating Escherichia coli, intestinal invasive Escherichia coli, and the serotype is O serotype. More preferably, it is a certain Escherichia coli, in particular, one or more E. coli selected from the following group;
O1, O25, O26, O63, O113, O125, O125ac, O144, O153, O168, O3, O77, O114, O126, O145, O157, O169, O6, O27, O78, O115, O127, O146, O158, O8, O28, O86, O117, O128, O148, O159, O11, O29, O91, O119, O131, O149, O164, O15, O32, O103, O121, O136, O150, O18, O44, O111, O111ab, O123, O142, O151, O166, O20, O55, O112, O112ac, O124, O143, O152, O167, O165.

  The LPS in the solid phase of the present invention is not limited by the degree of purification and the like, but is preferably the purified LPS of the present invention described later.

  In the present specification, LPS derived from E. coli is abbreviated as Ec-LPS.

  In the solid phase of the present invention, as the solid phase to which LPS is fixed, for example, a carrier such as a plate, a membrane, or a bead can be used.

  In the solid phase of the present invention, the method for fixing LPS to the solid phase is not particularly limited. However, when a membrane is used as the solid phase to fix LPS, for example, LPS is dotted on the membrane (Example 1 and FIG. 1 described later). And a method of fixing LPS on a membrane using a buffer or the like and drying it, as appropriate.

  In the solid phase of the present invention, the type of LPS fixed to the solid phase may be singular or plural. However, for example, when the solid phase of the present invention is used in the determination method of the present invention described later, different types of anti-LPS antibodies present in or possibly in the serum of patients suspected of having diarrheagenic E. coli infection It is desirable to quickly and efficiently confirm the specificity of the reaction to a plurality of E. coli-derived LPS having a serotype of serotype. In such a case, a plurality of types of LPS are individually fixed on the same solid phase. It is preferable to make it. Here, the plural types are not particularly limited as long as they are two or more types, but are preferably three or more types, more preferably five types or more, and most preferably ten types or more.

  In the above, the state in which a plurality of types of LPS are fixed “individually” on the same solid phase is not particularly limited as long as the plurality of types of LPS are not in contact with or mixed with each other. Specifically, refer to Example 1 described later.

The solid phase of the present invention is not limited by the method using the same, but can be used, for example, as a solid phase in step 1 of the method for discriminating the present invention described later or as a component of the kit for discriminating the present invention described later.
<2> Discrimination method of the present invention The discrimination method of the present invention is a method for discriminating serotypes of Escherichia coli in diarrheagenic Escherichia coli infection characterized by including at least the following steps.
Step 1: LPS affixed to the solid phase of the present invention is contacted with serum from an animal suspected of having diarrheagenic E. coli infection to form a conjugate comprising the LPS and an anti-LPS antibody contained in the serum. Process.
Step 2: contacting the conjugate with an antibody capable of binding to an anti-LPS antibody, and detecting the conjugate.

  In the above, regarding the terms “the solid phase of the present invention” and “LPS” and the method for fixing LPS to the solid phase of the present invention, refer to the above description of <1> the solid phase of the present invention.

  In step 1, the LPS adhered to the solid phase of the present invention is contacted with serum derived from an animal suspected of having diarrheagenic E. coli infection, and a conjugate comprising the LPS and an anti-LPS antibody contained in the serum To form.

  In general, when animals such as humans are infected with diarrheagenic Escherichia coli, it is known that symptoms such as diarrhea, water-soluble stool, bloody stool, HUS, acute enteritis, abdominal pain, fever, and vomiting appear. In the above, examples of the state of “suspected diarrheagenic Escherichia coli infection” include, for example, a state in which one or more of the symptoms exemplified above have appeared, Even if there is no symptom as described above, if for some reason it is necessary to determine the serotype of E. coli in diarrheagenic E. coli infection, "Diarrheagenic E. coli infection is suspected. ”State. Examples of the “animal” include mammals such as humans, dogs and cats. The above-mentioned “serum” can be obtained by using a blood collected from such an animal as a material and preparing it by a known method.

  In addition, the method of bringing the serum derived from an animal suspected of having diarrheagenic E. coli infection into contact with LPS adhered to the solid phase of the present invention is the method of contacting LPS adhered to the solid phase of the present invention with diarrheagenic E. coli infection. The method is not particularly limited as long as it can cause a binding reaction with an anti-LPS antibody contained in serum derived from a suspected animal. For example, diarrheagenic Escherichia coli infection on the solid phase surface of the present invention to which LPS is fixed It is possible to make contact by adding serum derived from an animal suspected of being in contact and preferably incubating for about 5 minutes to 1 hour, more preferably about 30 minutes. Specifically, refer to Example 1 described later.

  In step 2, the conjugate formed in step 1 is contacted with an antibody that can bind to the anti-LPS antibody, and the conjugate is detected.

  In the above, examples of the antibody that can bind to the anti-LPS antibody include antibodies such as anti-IgG antibody, anti-IgM antibody, anti-IgA antibody, anti-IgD antibody, and anti-IgE antibody. Anti-IgM antibody is preferable from the viewpoint of being capable of binding to anti-LPS antibody, that is, leading to accurate determination of the type of anti-LPS antibody contained in serum. Furthermore, it is preferable to use a combination of an anti-IgM antibody and an antibody other than the anti-IgM antibody (for example, an anti-IgA antibody and / or an anti-IgG antibody) because more reliable discrimination can be expected.

  The antibody capable of binding to the anti-LPS antibody is preferably labeled with, for example, an enzyme such as peroxidase. Such an antibody can be prepared or obtained by a known method.

  In the above method, the method of bringing the conjugate formed in Step 1 into contact with an antibody capable of binding to the anti-LPS antibody includes the conjugate formed in Step 1 and the antibody capable of binding to the anti-LPS antibody. Is not particularly limited as long as it can elicit an immune reaction between, for example, a solution containing an antibody capable of binding to an anti-LPS antibody is added to the surface of the solid phase after step 1, for example, preferably 5 It can be contacted by incubating for 1 minute to 1 hour, more preferably about 30 minutes. Specifically, refer to Example 1 described later.

  In step 2, the conjugate is detected based on the above-described immune reaction elicited between the conjugate formed in step 1 and an antibody that can bind to the anti-LPS antibody. Here, in such an immune reaction, for example, when the detected signal is enhanced in accordance with the increase in the concentration of the conjugate formed in Step 1 described above, it can be determined that the reaction has been triggered. .

  The detection method in step 2 is not particularly limited. For example, enzyme immunoassay methods such as EIA method and ELISA method, labeled immunoassay methods such as fluorescent immunoassay method (FIA method) and radioimmunoassay method (RIA method), and chemiluminescent enzyme immunoassay. Known methods exemplified by the labeling method (CLIA method), latex agglutination reaction method, unlabeled immunoassay method such as laser immunoassay and the like can be appropriately employed.

In the discrimination method of the present invention, in the detection in Step 2, the signal detected in the part to which Ec-LPS derived from Escherichia coli having a specific O serotype is fixed is detected in the part to which other Ec-LPS is fixed. If it is stronger than the detected signal, it can be determined that the infected E. coli is likely to be an E. coli having the specific O serotype from which a stronger signal was detected. Furthermore, in such a determination, as described above, each detection result is obtained using a plurality of types of antibodies that can bind to the anti-LPS antibody, and for example, the strongest signal among the plurality of detection results is detected. It is desirable to determine the most probable serotype of the infected E. coli by comprehensively judging each detection result by confirming the detected part.
<3> Discrimination Kit of the Present Invention The discriminating kit of the present invention is a kit for discriminating the serotype of E. coli in diarrheagenic E. coli infection, which contains at least an antibody capable of binding to the solid phase of the present invention and an anti-LPS antibody.

  In the above description, regarding the “solid phase of the present invention”, “antibody capable of binding to anti-LPS antibody”, “diarrheagenic Escherichia coli infection”, and “discrimination of serotype of E. coli”, <2> Discrimination of the present invention See method.

  In addition to the antibody capable of binding to the solid phase of the present invention and the anti-LPS antibody, the discrimination kit of the present invention may contain, for example, a reaction buffer, a washing solution, a coloring reagent and the like used in immunological measurement as other components. .

By using the discrimination kit of the present invention, the discrimination method of the present invention can be performed more efficiently and simply. For a specific method using the discrimination kit of the present invention, see <2> Discrimination method of the present invention and Example 1 described later.
<4> Purified LPS of the present invention
The purified LPS of the present invention is a purified LPS characterized by SDS gel electrophoresis characteristics shown in any lane of FIG. 2 of the accompanying drawings.

  One feature of the purified LPS of the present invention is that it has a very high purity. Such purified LPS can be obtained, for example, by extracting LPS from E. coli by the method described in Example 1 described later.

  The purified LPS of the present invention is not limited by its method of use, but is suitable as, for example, the above <1> LPS on the solid phase of the present invention or the LPS in step <1> of the above <2> discrimination method of the present invention Can be used. More specifically, refer to Example 1 described later.

  Hereinafter, the present invention will be described in detail by way of examples.

(1) Production of Ec-LPS-adhered Membrane 58 types of E. coli having the serotypes listed in Table 4 described below were cultured in Gmeiner medium at 37 ° C. for about 15 to 20 hours, respectively. The cultured Escherichia coli was collected, washed twice with distilled water, and lyophilized. The dried cells were dissolved in a 45% aqueous phenol solution, heated and stirred at 68 ° C. for 20 minutes, and then cooled to room temperature. The solution was centrifuged at 2500 rpm for 20 minutes to remove the water fraction, and an equal amount of distilled water was added to the phenol fraction to perform extraction again. The obtained water fraction was collected, put into a dialysis tube, and dialyzed for 3 days while changing water every day. After dialysis, the contents were lyophilized to obtain crude LPS. This crude LPS was dissolved in distilled water at a concentration of 10 mg / ml, and ultracentrifuged at 100,000 × g for 3 hours. An appropriate amount of distilled water was added to the precipitate for dissolution, ultracentrifugation was performed again, and the precipitate was lyophilized to obtain purified LPS.

  The purified LPS was dissolved in 0.1 M sodium carbonate buffer so as to have a concentration of 10 μg / ml, and 50 μl was spotted on a PVDF membrane (Nippon Millipore). This membrane was treated with 5% TweenX20-phosphate buffered saline (hereinafter referred to as “phosphate buffered saline” as “PBS”, and “5% TweenX20-phosphate buffered saline” as “washing solution”). After washing twice with (abbreviated), 3% skim milk (Difco) -PBS was mounted on the membrane. After standing at room temperature for 30 minutes, this was washed again with a washing solution, dried and stored.

  In addition, the step of spotting Ec-LPS on the solid phase is shown in FIG. 1. The result of SDS gel electrophoresis of Ec-LPS extracted as described above and specific Hitchcock silver staining for carbohydrates Is shown in FIG. Moreover, the result of having confirmed the reactivity of said Ec-LPS and commercially available antiserum by Western blotting is shown in FIG. 3, and the result of having confirmed by ELISA method is shown in Table 1. Moreover, the result of having confirmed the reactivity of said Ec-LPS and patient serum by Western blotting is shown in FIG. 4, and the result of having confirmed by ELISA method is shown in Table 2.

  In the table, + indicates that the reaction was confirmed, and-indicates that the reaction was not confirmed. Moreover, it shows that the strong reaction was confirmed, so that there are many numbers of + (same also in Table 2 below).

  In addition, the serum numbers 1, 2-1 and 2-2, 3-1 to 3-3, 4-1 and 4-2, 5-1 and 5-2, 6-1 and 6-2 in the table are: The sera collected from patients No. 1, 2, 3, 4, 5, and 6 in Table 3 are shown.

^{* 1} Indicates the number of days from blood collection to implementation of this example.
^* Indicates serotype determined by identification of E. coli isolated from stool.

(2) Discrimination of Escherichia coli serotypes in diarrheagenic E. coli infection About 30 μl of human serum is added to 3 ml of 3% skim milk-PBS in the portion where LPS is fixed on the Ec-LPS fixing membrane prepared in (1) above. The diluted product was mounted and allowed to stand at room temperature for 30 minutes. This was washed twice with a washing solution, and then a peroxidase-labeled secondary antibody (anti-IgG antibody, anti-IgM antibody, anti-IgA antibody, each manufactured by Dako) 6 μl diluted in 3% skim milk-PBS was mounted, and For 30 minutes. This was washed four times with a washing solution, developed with diaminobenzidine (DAB), then washed again with the washing solution four times and dried. As described above, anti-LPS antibody in each serum was detected.

  A part of a photograph showing the detection result is shown in FIGS. Please refer to Table 3 for patient data from the above serum. In addition, Table 4 shows the serotypes of E. coli derived from Ec-LPS fixed to each spot in FIGS.

The results of serotype discrimination based on the detection results are shown in Serotype ^{* 3} in Table 3. The details up to the determination will be specifically shown below.

  As is apparent from FIGS. 5 and 6, for serum Nos. 3, 5, 6, and 7, particularly when anti-IgM antibody was used as the secondary antibody, serum No. 7 was further added with anti-IgA. When an antibody was used as a secondary antibody, a very strong signal was detected in the part where Ec-LPS derived from E. coli having a serotype of O157 was fixed. It was determined that there is a high possibility that the serotype of E. coli infected with (patient Nos. 3, 5, 6, 7) is O157.

  For serum No. 9, particularly when an anti-IgM antibody was used as the secondary antibody, a strong signal was detected in the portion where Ec-LPS derived from Escherichia coli having a serotype of O26 was fixed. Only a relatively weak signal is detected in the part to which Ec-LPS other than Ec-LPS is adhered, that is, in this case, the part to which Ec-LPS derived from Escherichia coli having high serotype O26 is adhered. Since the signal was detected in, the possibility that the serotype of Escherichia coli infected with patient No. 9 is O26 is high.

  For serum No. 11, especially when an anti-IgG antibody is used as a secondary antibody and when an anti-IgM antibody is used as a secondary antibody, Ec-LPS derived from Escherichia coli whose serotype is O150 is used. A relatively strong signal is detected in the fixed part, and particularly when an anti-IgM antibody is used as the secondary antibody, only a relatively weak signal is present in the part where Ec-LPS other than the above is fixed. Since no signal was detected, that is, in this case, a signal was detected in the portion where Ec-LPS derived from E. coli having high specificity and serotype O150 was fixed, the serum of E. coli infected with patient No. 11 It was determined that the type is likely to be O150.

  For serum Nos. 12 and 21, particularly when an anti-IgM antibody was used as the secondary antibody, a relatively strong signal was observed in the portion where Ec-LPS derived from E. coli having a serotype of O136 was fixed. From the detection, it was determined that there is a high possibility that the serotype of Escherichia coli infected with patients No. 12 and 21 is O136.

  For serum No. 15, particularly when an anti-IgG antibody was used as the secondary antibody, a relatively strong signal was detected in the portion where Ec-LPS derived from E. coli having a serotype of O121 was fixed. Therefore, it was determined that there is a high possibility that the serotype of E. coli infected with patient No. 15 is O121.

  For serum No. 18, especially when an anti-IgG antibody is used as a secondary antibody and when an anti-IgM antibody is used as a secondary antibody, Ec-LPS derived from Escherichia coli whose serotype is O153 is used. A strong signal is detected in the fixed part, and only a relatively weak signal is detected in the part where Ec-LPS other than the above is fixed, that is, in this case, the serotype is O153 with high specificity. Since a signal was detected in the part where Ec-LPS derived from a certain Escherichia coli was fixed, the anti-IgA antibody was used as a secondary antibody. A signal with high specificity was detected (in other parts where Ec-LPS other than the above was fixed, almost no sigma Le is not detected), serotypes infected E. coli patient No.18 is judged that it is likely to be O153.

  As for serum No. 20, particularly when an anti-IgM antibody was used as a secondary antibody, a strong signal was detected in a portion where Ec-LPS derived from E. coli having a serotype of O150 was fixed. It was determined that there is a high possibility that the serotype of E. coli infected with patient No. 20 is O150.

  For serum No. 13, particularly when an anti-IgM antibody is used as the secondary antibody, the signal is relatively weak in the portion where Ec-LPS derived from E. coli having a serotype of O25 is fixed. However, since a signal having high specificity for the above-mentioned part was detected (almost no signal was detected in the part where Ec-LPS other than the above was fixed), the patient No. 13 was infected. It was determined that the serotype of E. coli is likely to be O25.

  For serum No. 16, Ec-LPS derived from Escherichia coli whose serotype is O166 is particularly obtained when an anti-IgM antibody is used as a secondary antibody and when an anti-IgA antibody is used as a secondary antibody. A relatively strong signal is detected in the fixed part, and particularly when an anti-IgA antibody is used as the secondary antibody, almost no signal is detected in the part to which Ec-LPS other than the above is fixed, That is, in this case, since a signal was detected in the portion to which Ec-LPS derived from E. coli having high specificity and serotype O166 was fixed, the serotype of E. coli infected with patient No. 16 was O166. It was determined that there was a high probability.

  For serum No. 17, especially when an anti-IgG antibody is used as a secondary antibody and when an anti-IgA antibody is used as a secondary antibody, Ec-LPS derived from Escherichia coli whose serotype is O115 is used. A relatively strong signal is detected in the fixed part, and particularly when an anti-IgA antibody is used as the secondary antibody, almost no signal is detected in the part to which Ec-LPS other than the above is fixed, That is, in this case, since a signal was detected in the part where Ec-LPS derived from E. coli having high specificity and serotype O115 was fixed, the serotype of E. coli infected with patient No. 17 was O115. It was determined that there was a high probability.

  For serum No. 19, especially when an anti-IgM antibody is used as a secondary antibody and when an anti-IgA antibody is used as a secondary antibody, Ec-LPS derived from Escherichia coli whose serotype is O166 is used. Since a strong signal was detected in the adhered part, it was determined that there is a high possibility that the serotype of E. coli infected with patient No. 19 is O166.

For serum Nos. 7 and 9, as shown in Table 3, it was confirmed that the serotype determined by identification of Escherichia coli separated from stool and the serotype determined by the method of this example were the same. . This confirms that the accuracy of the discrimination method of the present invention is high.

It is a figure which shows the step which dots Ec-LPS with respect to a solid phase. The extracted Ec-LPS is subjected to SDS gel electrophoresis by a conventional method, and the result of Hitchcock silver staining specific to carbohydrate is shown. The numbers shown at the top of each lane indicate the number of the O serotype of each Ec-LPS. It is a figure which shows the reactivity with the commercially available antiserum by Western blotting of extracted Ec-LPS. In the figure, anti-O26, anti-O55, anti-O86, anti-O103, anti-111, anti-119, anti-128, anti-142, and anti-157 indicate the types of commercially available antisera used. . The numbers shown at the top of each lane indicate the number of the O serotype of each Ec-LPS. It is a figure which shows the reactivity with the patient serum by the western blotting method of extracted Ec-LPS. The numbers shown at the top of each lane indicate the number of the O serotype of each Ec-LPS. The discrimination | determination result of a serotype is shown. IgG, IgM, and IgA described at the top of the figure indicate that the secondary antibodies used are an anti-IgG antibody, an anti-IgM antibody, and an anti-IgA antibody, respectively. The serotypes of E. coli derived from Ec-LPS fixed to each part specified by the row number and column number are shown in Table 4, so please refer to them. The discrimination | determination result of a serotype is shown. IgG, IgM, and IgA described at the top of the figure indicate that the secondary antibodies used are an anti-IgG antibody, an anti-IgM antibody, and an anti-IgA antibody, respectively. The serotypes of E. coli derived from Ec-LPS fixed to each part specified by the row number and column number are shown in Table 4, so please refer to them.

Claims (2)

A method for distinguishing O serotype Escherichia coli in diarrheagenic E. coli infection, comprising at least the following steps.
Step 1: Diarrheagenic Escherichia coli infection to lipopolysaccharide fixed to a solid phase in which each lipopolysaccharide derived from E. coli having the O serotype described in group (A) below is individually fixed on the same solid phase A step of contacting a serum derived from an animal suspected of having a disease to form a conjugate comprising the lipopolysaccharide and an anti-lipopolysaccharide antibody contained in the serum.
Step 2: A step of bringing the conjugate into contact with an anti-IgM antibody, an anti-IgG antibody, and an anti-IgA antibody, which are antibodies that can bind to an anti-lipopolysaccharide antibody, and obtaining each detection result of the conjugate.
(A) O1, O25, O26, O63, O113, O125, O144, O153, O168, O3, O77, O114, O126, O145, O157, O169, O6, O27, O78, O115, O127, O146, O158, O8 , O28, O86, O117, O128, O148, O159, O11, O29, O91, O119, O131, O149, O164, O15, O32, O103, O121, O136, O150, O18, O44, O111, O123, O142, O151 , O166, O20, O55, O112, O124, O143, O152, O167, O165. A serotype discrimination kit of O serotype Escherichia coli in diarrheagenic Escherichia coli infection, comprising at least the following (1) and (2) as components.
(1) A solid phase in which each lipopolysaccharide derived from E. coli having the O serotype described in the group (A) below is individually fixed on the same solid phase.
(2) An anti-IgM antibody, an anti-IgG antibody and an anti-IgA antibody, which are antibodies that can bind to an anti-lipopolysaccharide antibody.
(A) O1, O25, O26, O63, O113, O125, O144, O153, O168, O3, O77, O114, O126, O145, O157, O169, O6, O27, O78, O115, O127, O146, O158, O8 , O28, O86, O117, O128, O148, O159, O11, O29, O91, O119, O131, O149, O164, O15, O32, O103, O121, O136, O150, O18, O44, O111, O123, O142, O151 , O166, O20, O55, O112, O124, O143, O152, O167, O165.

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