JP6198298B2 - Pertussis serodiagnosis method - Google Patents

Description

  The present invention relates to a serodiagnosis method for pertussis, and more particularly to a method for measuring antibody titer for diagnosis of whooping cough patients.

  Pertussis is an acute respiratory infection caused by infection with Bordetella pertussis. The number of whooping cough cases that occurred worldwide in 2008 is estimated at 16 million and the number of deaths is estimated to be about 195,000, with many affected by infants in developing countries. The number of reported cases in Japan in 2010 is 5,388, and the annual number of cases is estimated to be about 50,000. Pertussis is a vaccine-preventable disease that affects unvaccinated infants and adolescents and adults with reduced vaccine efficacy.

There are various reports on how to diagnose pertussis (see Non-Patent Documents 1 and 2).
Bacterial culture tests or serological tests are required for a definitive diagnosis of whooping cough, but since the amount of carriers is small in adolescents and adult patients, it is rare that the culture test is positive. On the other hand, in Japan, pertussis aggregation antigen “Seiken” N and pertussis antibody EIA “Seiken” are available as in vitro diagnostic agents in Japan. In general, the agglutination titer method using an agglutination antigen has the disadvantage of low specificity and sensitivity. Pertussis antibody EIA measures IgG antibodies against pertussis toxin and fibrillary hemagglutinin. However, since pertussis vaccine contains these antigens, WHO requires that 3 years have passed since vaccination. It is said. Furthermore, since induction of IgG antibody takes several weeks after infection, there is a problem that the pertussis EIA method cannot diagnose patients in the early stage of infection.

Pertussis serodiagnosis using the agglutinin titer method. Pathogenic microorganism detection information IASR Vol.32.p.236-237: August 2011 Guiso N, Berbers G, Fry NK, He Q, Riffelmann M, Wirsing von Konig CH, EU Perstrain group.What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories.Eur J Clin Microbiol Infect Dis , 2011, 30: 307-312.

  Current serological tests make it difficult to diagnose whooping cough early and accurately. In order to solve this problem, a diagnostic antigen having high immunogenicity and not included in the vaccine antigen is required. In many infectious diseases, IgM antibody is induced prior to IgG antibody. Therefore, measurement of IgM antibody is considered useful for early diagnosis of pertussis. Accordingly, an object of the present invention is to identify a diagnostic antigen having high immunogenicity and to provide a method for measuring an IgM antibody against the identified diagnostic antigen. Furthermore, an object of the present invention is to provide a serum diagnostic kit using the above IgM measurement method.

  The present invention provides a method for diagnosing serum having superior sensitivity and specificity compared to conventional methods by using virulence-associated gene 8 (Vag8) produced by Bordetella pertussis as a diagnostic antigen, and in particular by measuring anti-Vag8 IgM antibody. It came to be completed.

That is, the present invention is as follows.
[1] A method for detecting an infection of Bordetella pertussis by measuring an IgM antibody against Vag8 of Bordetella pertussis in a blood, serum or plasma sample collected from a subject.
[2] The method for detecting infection of Bordetella pertussis according to [1], wherein an IgM antibody is measured against a fragment consisting of amino acids 36 to 636 of Vag8 consisting of the amino acid sequence represented by SEQ ID NO: 2.
[3] A step in which a carrier on which an anti-human IgM antibody is immobilized is brought into contact with a blood, serum or plasma sample collected from a subject, and the IgM antibody in the sample is captured on the carrier, the tag antibody and Vag8 or A method for detecting an infection of Bordetella pertussis according to [1] or [2], comprising a step of binding a fusion protein of the fragment and a step of binding a labeled anti-tag antibody to the tag protein.
[4] A step in which a carrier on which an anti-human IgM antibody is immobilized is brought into contact with a blood, serum or plasma sample collected from a subject, and the IgM antibody in the sample is captured on the carrier, the tag antibody and Vag8 or The method according to [1] or [2], comprising the step of binding a fusion protein of the fragment, the step of binding a biotin-labeled anti-tag protein antibody to the tag protein, and the step of binding labeled streptavidin or avidin to biotin. To detect infections.
[5] The method for detecting pertussis infection according to any one of [1] to [4], wherein the tag protein is a maltose-binding protein.
[6] A method for detecting an infection of Bordetella pertussis according to any one of [1] to [5], which is detected with a sensitivity of 85% or more and a specificity of 75% or more.
[7] A method for detecting the infection of Bordetella pertussis according to any one of [1] to [6], wherein the presence or absence of Bordetella pertussis is determined based on a cutoff value determined by ROC analysis.
[8] In a sample containing a carrier on which an anti-human IgM antibody is immobilized, a fusion protein of a tag protein and Vag8 or a fragment thereof, a labeled anti-tag protein antibody or a biotinylated anti-tag antibody and labeled streptavidin (avidin) A kit for detecting an IgM antibody against Vag8 of Bordetella pertussis.
[9] The kit according to [8], wherein Vag8 or a fragment thereof is a fragment consisting of amino acids 36 to 636 of Vag8 consisting of the amino acid sequence represented by SEQ ID NO: 2.
[10] The kit according to [8] or [9], wherein the tag protein is a maltose binding protein.

  According to the present invention, a pertussis patient can be diagnosed with higher accuracy than the conventional method. Moreover, since Vag8 used for the diagnostic antigen is not included in the current pertussis vaccine, it has an advantage that it can be applied to a subject after vaccination. Furthermore, measurement of anti-Vag8 IgM antibody enables early diagnosis of infection and is useful for rapid treatment and prevention of spread of infection.

It is a figure which shows the expression method of diagnostic antigen MBP-Vag8. It is a figure which shows the measurement principle of IgM capture ELISA method using the antigen for MBP-Vag8 diagnosis. It is a figure which shows the result of the induction | guidance | derivation of the anti- Vag8 IgG antibody by the diphtheria, tetanus, and pertussis mixed triple (DTaP) vaccine. It is a figure which shows distribution of the anti- Vag8 antibody in a healthy adult. It is a figure which shows the result of the induction | guidance | derivation of the anti- Vag8 antibody in a pertussis child. It is a figure which shows distribution of the anti- Vag8 IgM antibody titer in a pertussis patient. It is a figure which shows the diagnostic accuracy evaluation result of an anti- Vag8 IgM capture ELISA method.

  Hereinafter, the present invention will be described in detail.

  The present invention is a method for detecting infection of Bordetella pertussis, and the infection is detected by measuring an IgM antibody against virulence-associated gene 8 (Vag8) produced by Bordetella pertussis. In the present invention, Vag8 means Vag8 protein. In the present invention, anti-Vag8 IgM antibody in a sample collected from a subject (patient) is measured. As a sample, blood, serum or plasma is used. In the method of the present invention, a sample collected from a subject is added to a carrier to which an anti-human IgM antibody is bound, and the carrier and the sample are brought into contact with each other. At this time, if an IgM antibody is present in the human sample, it binds to and is captured by the anti-IgM antibody bound to the carrier. Next, a tag-Vag8 fusion protein in which a tag is attached to V. pertussis Vag8 or a fragment thereof is added. When the IgM bound to the anti-IgM antibody is an anti-Vag8 IgM antibody, the tag-Vag8 fusion protein Vag8 and the anti-Vag8 IgM antibody bind to each other, and the anti-human IgM antibody, the anti-Vag8 IgM antibody and the tag-Vag8 fusion protein are supported on the carrier. To form a complex. Next, an anti-tag antibody that is a secondary antibody is added. The anti-tag antibody binds to the tag of the tag-Vag8 fusion protein, and forms a complex of the anti-human IgM antibody, the anti-Vag8 IgM antibody, the tag-Vag8 fusion protein, and the anti-tag antibody on the carrier. The anti-tag antibody may be directly labeled with a labeling substance such as a fluorescent dye, a chemiluminescent substance, or an enzyme, or may be labeled with biotin and used as a biotinylated anti-tag antibody. When the anti-tag antibody is directly labeled, the anti-Vag8 IgM antibody in the test sample can be detected by measuring the signal from the labeling substance bound to the anti-tag antibody. When the anti-tag antibody is labeled with biotin, avidin or streptavidin labeled with a labeling substance such as a fluorescent dye, a chemiluminescent substance, or an enzyme is further added. The labeling substance binds to the anti-tag antibody by binding of avidin or streptavidin and biotin, and the anti-Vag8 IgM antibody in the test sample can be detected by measuring the signal from the labeling substance. In the present invention, an antibody labeled with a labeling substance such as a fluorescent dye, a chemiluminescent substance, an enzyme, or an antibody labeled with biotin is also called a labeled antibody. If anti-Vag8 IgM antibody is detected in a subject sample, the subject can be determined to be infected with Bordetella pertussis.

  The method of the present invention is both a test method for diagnosing pertussis infection and a method for obtaining auxiliary data for diagnosing pertussis infection.

  The method of the present invention is a method including capturing an IgM antibody in a sample, and is also referred to as an IgM capturing method. The method of the present invention is preferably an ELISA (Enzume-Linked ImmunoSorbent Assay) method, in which case it is referred to as an IgM capture ELISA method.

1. Preparation of Reagent for Use in IgM Capture Method In the present invention, a “tag” is a substance bound to Vag8 or a fragment thereof that specifically binds to an anti-Vag8 IgM antibody of a subject sample to be measured, and is labeled A protein that specifically binds to a secondary antibody, also called a tag protein.

  Vag8 is a highly immunogenic antigen that is recognized by patients infected with whooping cough and induces antibodies, particularly IgM antibodies, early in the patient. Vag8 may use the full-length Vag8 protein or a partial fragment thereof. The sequence of DNA encoding Vag8 is shown in SEQ ID NO: 1, and the amino acid sequence of Vag8 is shown in SEQ ID NO: 2. The fragment of Vag8 is particularly preferably a fragment with high immunogenicity. Examples of the fragment of Vag8 include a fragment protein consisting of amino acids 36 to 636 in the amino acid sequence shown in SEQ ID NO: 2. Vag8 is a membrane protein and is hardly soluble as it is. By fusing Vag8 with other tag proteins that are soluble, it is solubilized and can be used in immunological assays.

  Since Vag8 is not included in the commonly used pertussis vaccine, anti-Vag8 antibody is produced in a subject who has been vaccinated and is not infected with pertussis. Rather, subjects with Vag8 antibody can be determined to be infected with Bordetella pertussis.

Any tag can be used as long as it has antigenicity and can bind to a labeled secondary antibody by antigen-antibody reaction. In the present invention, MBP (maltose binding protein), His ₆ (histidine 6) can be used. A protein or peptide capable of producing a fusion protein with an antigen protein by gene recombination, such as a mer), GST (Glutathione S-Transferase), galactose binding protein, and thioredoxin can be preferably used.

  The tag is bound to Vag8 or a fragment thereof. The tag and Vag8 or a fragment thereof may be bound chemically or enzymatically, or a fusion protein of a DNA encoding the tag protein and a DNA encoding Vag8 or a fragment thereof is expressed. Can also be produced. In the present invention, when a tag and Vag8 or a fragment thereof are bound chemically or enzymatically, it is also called a fusion protein.

  Fusion of the DNA encoding the tag protein and the DNA encoding Vag8 or a fragment thereof can be carried out by an ordinary gene recombination technique. In this case, it can be carried out by introducing an appropriate restriction enzyme recognition cleavage sequence. The tag protein may be added to the C-terminal side or V-terminal side of Vag8 or a fragment thereof. At this time, a stop codon is prevented from appearing between the gene encoding the tag protein and the DNA encoding Vag8 or a fragment thereof. The distance between the tag protein and Vag8 or a fragment thereof is not limited, and a linker may be included between the two. The constituent amino acids and the number of amino acids of the linker are not limited, but for example, those composed of one or more amino acids of glycine, serine, alanine, threonine or methionine can be used. Moreover, generally the number of amino acids is 2-20 residues, Preferably it is 2-15 residues, More preferably, it is 2-10 residues, More preferably, it is 2-5 residues. In order for the tag protein and Vag8 or a fragment thereof to be translated, the open reading frames of both DNAs are matched. Moreover, you may have a base sequence which a specific amino acid is contained in a linker part between both DNA.

  The antibody against the tag used in the present invention can be obtained by immunizing an animal using the tag as an immunogen, collecting antiserum and purifying it, and can also be obtained as a monoclonal antibody using a cell fusion method. Commercially available products can also be used.

  Fluorescent dyes such as fluorescein isothiocyanate (FITC), horseradish peroxidase (HRP), alkaline phosphatase (ALP) as labeling substances that bind to anti-tag antibodies through anti-tag antibodies, biotin-streptavidin bonds or biotin-avidin bonds Enzymes such as luminescent materials, radioisotopes, etc. are used. When using an enzyme, TMB (tetramethylbenzene) for HRP and p-nitrophenyl phosphate (pNPP) for ALP may be used as the chromogenic substrate. The method of binding the labeling substance to the antibody to the tag can be performed by a method known to those skilled in the art. For example, in the case of enzyme labeling, maleimide method or the like (Eiji Ishikawa “Ultrasensitive Enzyme Immunoassay”) (Described in the Society Publishing Center, etc.)) can be used.

  An anti-human IgM antibody can be bound to an appropriate carrier to produce a solid phase carrier capable of capturing the IgM of the present invention. The antibody solid phase carrier used in the present invention can be obtained by immobilizing an anti-human IgM antibody on a carrier such as an ELISA titer plate or beads. As the anti-human IgM antibody, a commercially available one can be used, or it can be prepared by immunizing an animal with human IgM (μ chain). The anti-human IgM antibody is immobilized on a carrier such as a titer plate by physical adsorption or chemical bonding. The amount of the solid phase is not particularly limited, but when the carrier is a microtiter plate, it is preferably several ng to several tens of μg per well. Immobilization can be carried out by dissolving the antibody to be immobilized in an appropriate buffer and bringing it into contact with a carrier. For example, when microtiter wells are used, the antibody solution can be solidified by dispensing into a well of a microtiter plate and placing it for a certain period of time.

2. Immunological measurement of IgM antibody by IgM capture method The carrier on which the above-mentioned anti-human IgM antibody is immobilized, Vag8 or a fragment thereof bound to the tag, and a labeled secondary antibody or biotinylated anti-tag antibody and labeled streptavidin against the tag The target anti-Vag8 IgM antibody can be measured using (avidin).

  For example, antiplate ELISA by IgM capture method using microplate with antibody against human IgM antibody, MBP (maltose binding protein) as tag, biotinylated anti-MBP antibody, streptavidin conjugated with enzyme as labeling substance Vag8 IgM antibody measurement can be performed by the method shown below.

  Serum collected from a subject is diluted with a buffer solution, added to an anti-human IgM antibody solid phase plate, and reacted at 15 to 30 ° C. for 0.5 to 8 hours. After the reaction, it is washed with a buffer solution or physiological saline, and a fusion of MBP and Vag8 or a fragment thereof (MBP-Vag8) is added and reacted at 15-30 ° C. for 0.5-8 hours. After the reaction, it is washed with a buffer or physiological saline, a biotinylated anti-MBP antibody is added, and the reaction is carried out at 15-30 ° C. for 0.5-8 hours. After the reaction, it is washed with a buffer or physiological saline, and peroxidase-labeled streptavidin is added. The mixture is reacted at 15 to 30 ° C. for 0.1 to 4 hours, added with a chromogenic substrate solution (TMB), developed for 0.1 to 2 hours, added with a chromogenic stop solution, and the absorbance at 450 nm is measured. A calibration curve may be prepared in advance, and the antibody concentration in the sample may be calculated from the obtained absorbance.

  If an anti-Vag8 antibody is present in a sample collected from a subject, ie, is positive, it can be determined that the subject is infected with pertussis. Whether the anti-Vag8 antibody in the sample is positive or not is determined in advance by setting a cut-off value for the absorbance or antibody measurement value and exceeding the cut-off value with reference to the cut-off value. be able to.

  The cutoff value can be determined by, for example, ROC (receiver operating characteristic curve) analysis. Moreover, the diagnostic accuracy (sensitivity and specificity) by the method of the present invention can be determined by ROC analysis. ROC analysis measures anti-Vag8 IgM antibody for samples collected from whooping cough patients and samples collected from healthy subjects, calculates sensitivity and false positive rate (1-specificity) at each cutoff value, Plotting on the coordinate with the axis and 1-specificity and the vertical axis as sensitivity. An example of the ROC curve of the measured value measured by the method of the present invention is shown in FIG.

  When the diagnostic accuracy is analyzed by ROC analysis for the measurement results of the method of the present invention, the area under the curve (AUC) is as high as 0.9 or more, and the sensitivity is 80% or more, preferably 85% or more, more preferably Is 90% or more, and the specificity is 75% or more, preferably 80% or more. By the method of the present invention, it is possible to detect Bordetella pertussis infection with very high accuracy.

3. Anti-Vag8 IgM Antibody Measuring Reagent The present invention also includes an anti-Vag8 IgM antibody measuring reagent. The reagent includes a carrier on which an anti-human IgM antibody is immobilized, a fusion protein of a tag such as MBP and Vag8 or a fragment thereof, a labeled anti-tag antibody or a biotinylated anti-tag antibody and labeled streptavidin (avidin). The reagent may further contain a blocking solution, a reaction solution, a reaction stop solution and the like.

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

Example 1 Preparation of a Maltose Binding Protein and Pertussis Vag8 Partial Fusion Protein A part of the vag8 gene (position 112-1908, 1797 bp) was amplified by PCR using pertussis Higashihama strain DNA as a template. This gene was inserted into an expression vector pMAL-c2X and expressed as a fusion protein (MBP-Vag8) with a maltose binding protein.

  Specifically, a part of Vag8 gene (position 112-1908) was amplified by PCR using pertussis Higashihama strain DNA as a template, and inserted into BamHI-HindIII site of expression vector pMAL-c2X by infusion method (references) : Finn & Amsbaugh, Infect Immun, 1998). The constructed pMAL-c2X Vag8 was transformed into E. coli BL21 and MBP-Vag8 was expressed according to a conventional method. The cells were suspended in a column buffer (20 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, pH 7.5) containing 6 M urea, and the cells were disrupted by ultrasonication. Next, the supernatant was collected by centrifugation and dialyzed sequentially against a column buffer containing 4 M, 2 M, and 0 M urea. After dialysis, insoluble substances were removed by centrifugation, the supernatant was added to an amylose resin column, washed with column buffer, and MBP-Vag8 was eluted with column buffer containing 10 mM maltose. The MBP-Vag8 fraction was dialyzed against 25 mM Tris-HCl (pH 7.5), added to an ion exchange column (Resource Q), and MBP-Vag8 was eluted with a concentration gradient from 0 to 0.5 M NaCl. This MBP-Vag8 was used as the final purified product.

  FIG. 1 shows the structure of the expression vector pMAL-c2X into which a part of the vag8 gene has been inserted and the structure of the resulting fusion protein MBP-Vag8.

Example 2 Development of IgM capture ELISA method for measurement of anti-Vag8 IgM antibody
Anti-human IgM antibody is bound to the ELISA plate and the IgM antibody in the patient serum is captured. After binding MBP-Vag8 to the captured anti-Vag8 IgM antibody, a biotin-labeled anti-MBP IgG antibody is bound to MBP. Subsequently, peroxidase-labeled streptavidin is reacted, and color development is performed using a TMB substrate.

Specifically, the anti-human IgM antibody was immobilized on a commercially available 96-well ELISA plate, washed, and 100 μl of patient serum diluted 100-fold with an appropriate buffer was added to the well. After standing at 25 ° C. for 1 hour, the wells were washed 3 times with 200 μl of washing solution (physiological saline containing 0.05% Tween-20). MBP-Vag8 was diluted with buffer to 0.5 μg / μl and 100 μl was added to the wells. After standing at 25 ° C. for 1 hour, the wells were washed three times, and 100 μl of biotin-labeled anti-MBP IgG antibody diluted with a buffer solution was added to the wells. After allowing to stand at 25 ° C. for 1 hour, the wells were washed three times, and 100 μl of a peroxidase-labeled streptavidin solution diluted with a buffer solution was added. After standing at 25 ° C. for 30 minutes, the wells were washed 5 times with a washing solution, 100 μl of TMB coloring substrate solution (3, 3 ′, 5, 5′-tetramethylbenzidine) was added, and color was developed in the dark at 25 ° C. After 30 minutes, 100 μl of a color stop solution (0.3 M sulfuric acid solution) was added, and the absorbance at 450 nm was measured (reference wavelength: 650 nm).
FIG. 2 shows the principle of the IgM capture ELISA method of this example.

Example 3 Induction of anti-Vag8 IgG antibody by diphtheria / tetanus / pertussis mixed vaccine (DTaP) vaccine DTAP vaccine from 4 domestic factories was inoculated into mice (n = 3), and serum was obtained from mice 14 and 28 days later. Were collected, and anti-Vag8 IgG antibody in serum and IgG antibodies against vaccine antigens PT (pertussis toxin), Prn (pertactin), and FHA (filamentous hemagglutinin) were measured. IgG measurement was performed by the conventional indirect ELISA method. First, the antigen protein was immobilized on a 96-well ELISA plate, and mouse serum was added. IgG antibody in mouse serum bound to antigen was detected by a chromogenic system using alkaline phosphatase-labeled anti-mouse IgG and pNPP, which are secondary antibodies (Reference: WHO Laboratory manual for the diagnosis of whooping cough caused Bordetella pertussis / Bordetella parapertussis. WHO, WHO / IVB / 04/14 (http://www.who.int/immunization/documents/WHO_IVB_04.14/en/index.html)). The results are shown in FIG. 3A shows the measurement result of anti-PT IgG antibody, FIG. 3B shows the measurement result of anti-FHA IgG antibody, FIG. 3C shows the measurement result of anti-Prn IgG antibody, and FIG. 3D shows the measurement result of anti-Vag8 IgG antibody. As shown in Fig. 3D, no anti-Vag8 IgG induction was observed with the 4 factory DTaP vaccine, so the current vaccine does not contain Vag8, and serodiagnosis using Vag8 is not affected by vaccination It was judged.

Example 4 Distribution of Anti-Vag8 Antibody in Healthy Adults Anti-Vag8 IgG antibody and anti-Vag8 IgM antibody were measured by the conventional indirect ELISA method for sera of 80 healthy individuals. The results are shown in FIG. FIG. 4A shows the measurement results of anti-Vag8 IgG antibody, and FIG. 4B shows the measurement results of anti-Vag8 IgM antibody. As shown in FIGS. 4A and 4B, many anti-Vag8 IgG antibodies showed a high value even in healthy subjects, whereas few anti-Vag8 IgM antibodies showed a high value. From this, when Vag8 was used as a diagnostic antigen, it was considered necessary to measure an anti-Vag8 IgM antibody.

Example 5 Induction of anti-Vag8 antibody in children with pertussis Serum was collected over time for 6 infant patients from whom Bordetella pertussis was isolated, and anti-Vag8 IgM and anti-Vag8 IgG in the serum were measured. Anti-Vag8 IgM antibody was measured by IgM capture ELISA, and anti-Vag8 IgG antibody was measured by conventional indirect ELISA. The results are shown in FIG. FIG. 5A shows the measurement result of anti-Vag8 IgM antibody, and FIG. 5B shows the measurement result of anti-Vag8 IgG antibody. As shown in FIGS. 5A and B, it is shown that anti-Vag8 IgM antibody is induced on an earlier date than anti-Vag8 IgG antibody, and diagnosis using anti-Vag8 IgM antibody is considered useful for patients in the early stage of infection. It was.

Example 6 Distribution of anti-Vag8 IgM antibody titer in pertussis patients Anti-Vag8 IgM antibody in serum was measured in 21 pertussis patients diagnosed by genetic testing (LAMP method) (IgM capture ELISA method). The results are shown in FIG. As shown in FIG. 6, it was shown that pertussis patients possess significantly higher anti-Vag8 IgM antibody compared to healthy subjects (P <0.001). Healthy human serum (n = 29) was used as a control.

Example 7 Evaluation of diagnostic accuracy of anti-Vag8 IgM capture ELISA method Using the serum of 21 pertussis patients and 29 healthy subjects who were diagnosed by genetic testing (LAMP method), the diagnostic accuracy of anti-Vag8 IgM capture ELISA method was calculated using ROC. It was evaluated by analysis. The results are shown in FIG. As shown in FIG. 7, the sensitivity of the anti-Vag8 IgM capture ELISA is 90%, the specificity is 83% (cut-off value A450 = 0.291), and the area under the curve (AUC) is 0.92 (95% confidence interval: 0.84-1.00 ) And was shown to have high diagnostic accuracy.

  The serum diagnostic method according to the present invention can be expected to be put into practical use as an in vitro diagnostic agent for pertussis infection.

Claims (10)

To detect the infection of Bordetella pertussis, how you measure the IgM antibody to Vag8 of B. pertussis in the blood, serum or plasma samples taken from a subject. Measuring the IgM antibody against fragment consisting of the 36 to 636 amino acid of Vag8 comprising the amino acid sequence represented by SEQ ID NO: 2, Method who claim 1. Contacting a carrier on which an anti-human IgM antibody is immobilized with a blood, serum or plasma sample collected from a subject to capture the IgM antibody in the sample on the carrier, the captured antibody comprising a tag protein and Vag8 or a fragment thereof step of binding the fusion protein, and include the step of bringing into association the labeled anti-tag antibody to the tag protein, methods who according to claim 1 or 2. Contacting a carrier on which an anti-human IgM antibody is immobilized with a blood, serum or plasma sample collected from a subject to capture the IgM antibody in the sample on the carrier, the captured antibody comprising a tag protein and Vag8 or a fragment thereof step of binding the fusion protein, comprising the step of attaching a biotin-labeled anti-tag protein antibody to the tag protein, and the step of binding the labeled streptavidin or avidin to biotin method who according to claim 1 or 2. Tag is a protein maltose binding protein, methods who according to any one of claims 1 to 4. Sensitivity 85%, detected with specificity 75% or more, methods who according to any one of claims 1 to 5. Based on the cut-off value determined by ROC analysis to determine the presence or absence of infection with B. pertussis, methods who according to any one of claims 1-6.   An anti-human IgM antibody-immobilized carrier, a fusion protein of a tag protein and Vag8 or a fragment thereof, a labeled anti-tag protein antibody or a biotinylated anti-tag antibody and a labeled streptavidin (avidin). Kit for detecting IgM antibody against Vag8.   The kit according to claim 8, wherein Vag8 or a fragment thereof is a fragment comprising the 36th to 636th amino acids of Vag8 comprising the amino acid sequence represented by SEQ ID NO: 2.   The kit according to claim 8 or 9, wherein the tag protein is a maltose binding protein.

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