KR20170105997A - Composition and method for detecting Shigella spp. belonging to 4 serotype groups - Google Patents

Abstract

The present invention relates to a composition for detecting bacterial heterogeneous bacteria of four serotype groups and a detection method thereof and, more specifically, to a composition, a kit, a bacterial binary detection method, and a method for preparing the composition, wherein the composition contains polyantibodies against six different types of bacterial serotypes, which do not have an antigen-antibody bond when being in contact with a specific Escherichia coli serotype fungus body, as antibodies against bacterial heterogeneous bacteria, thereby effectively detecting bacterial heterogeneous bacteria of four serotype groups. When a composition for detecting bacterial heterogeneous bacteria, a method for detecting bacterial heterogeneous bacteria, or a kit for detecting bacterial heterogeneous bacteria of the present invention is used, heterogeneous bacteria can be directly separated from a specimen, and such a performance can be very efficiently performed. Particularly, the present invention can separate heterogeneous bacteria even from a specimen containing many kinds of microorganisms, including various kinds of E. coli of different serotypes, such as feces or the like. Thus, compared to a conventional method in which it is difficult to detect heterogeneous bacteria under the same conditions or much time is needed, more effective detection can be ensured in the present invention. In addition, it is possible to more rapidly and accurately perform the diagnosis of bacterial differences which can be finally confirmed when microbial bacteria are directly separated.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for detecting bacterial pathogenic bacteria in four serotypes and a method for detecting the same. belonging to 4 serotype groups}

The present invention relates to a composition and method for detecting bacterial climacteric bacteria in four serotypes, and more particularly, to a composition and method for detecting bacterial clusters of four serotypes, A kit, a method for detecting a bacterial heterogeneity, and a method for producing the composition. The present invention relates to a method for detecting a bacterial heterogeneous bacteria in a blood group.

Bacterial dysentery is a waterborne infectious disease caused by Shigella spp., Which causes acute or pandemic inflammatory bowel disease only in humans and monkeys. Dysentery germs are characterized by gram negative bacilli, non-swinging, and tuberous anaerobes. In 1898, Shiga first reported Shigella dysenteriae in a large number of heterozygous feces, followed by S. flexneri (group B), S. boydii (group C) and S. sonnei (group D) . It is divided into S. dysenteriae (group A), S. flexneri (group B), S. boydii (group C) and S. sonnei (group D) by the cell O antigen. Physiologically similar to Groups A to C, S. sonnei (group D) can be distinguished by biochemical metabolic processes.

The infectious dose of Alzheimer's disease differs from species to species. For example, S. sonnei can induce infection with only 5 × 10 ² CFUs, and S. dysenteriae has an infectious dose of 10 ¹ CFUs. The low infectious capacity of this pathogen is a major cause of human-to-human transmission. Therefore, rapid and sensitive diagnostic methods are urgently needed to prevent the spread of infectious diseases.

However, according to FDA's method of isolating heterozygous bacteria, it takes four days to isolate the pathogen (see FIG. 1). Furthermore, this confirmation time is a case where a large amount of foreign bacteria exists in a specimen of a patient, and in the case of an initial or ameliorated patient specimen in which a small amount of foreign bacteria exists, more confirmation period is required.

With the development of genetic diagnostic methods, the diagnosis of many pathogens has recently been improved by genetic diagnosis. However, in the case of stool specimens of patients, genetic diagnosis errors may occur due to many inhibitory substances. In addition, since the gene is present in some Escherichia coli even though the ipaH target gene exists in the case of bacterial quality, Genetic diagnosis is used only as screening method. Serologic test and biochemical test are essential for pathologic confirmation for final confirmation. For this reason, according to the Act on the Prevention and Management of Infectious Diseases, the criteria for confirmation of bacterial heterogeneity is limited to the case of isolating the pathogen, and separation of the pathogen is necessary since it is possible to take measures such as isolation to prevent diffusion only in confirmed patients.

Recently, a method of selectively diagnosing only pathogens using a magnetic bead-antibody conjugate against various bacteria and viruses has been developed. Although there is a method developed by Islam et al. In 1992 for S. dysenteriae type 1, only a genetic diagnosis (Polymerase Chain Reaction, PCR) is possible, not a method for selectively isolating S. dysenteriae type 1, The time required for diagnosis is also 7 hours. In addition, a monoclonal antibody was prepared and adhered to a magnetic bead. However, because of the cross reaction with non-pathogenic Escherichia coli in a specimen (feces), it is not possible to isolate a pathogen.

As described above, the conventional method for confirming the bacterial heterogeneity has a problem that the inspection period is long and the accuracy is low. Especially, infection of these infectious pathogens requires isolation of the infected person in order to prevent further transmission. If the person is misdiagnosed, there is a problem that it is possible to isolate healthy person, not the infected person, and development of rapid and accurate diagnosis method is required.

The present inventors have made efforts to develop a novel method capable of detecting foreign bacteria more rapidly and accurately by solving the problems of the conventional diagnostic methods as described above. As a result, the polyvalent antibody against the six serotypes of the heterozygous fungus was identified as a specific E. coli serotype- To obtain an antigen-antibody-free binding. Using the antibody thus obtained as an antibody against foreign bacteria, it was confirmed that foreign bacteria can be detected quickly and accurately from various kinds of bacterial cells present in the specimen. .

Dilara Islam and Alf A. Lindberg. 1992. Detection of Shigella dysenteriae Type 1 and Shigella flexneri in Feces by Immunomagnetic Isolation and Polymerase Chain Reaction. J. Clin. Microbiol. 30 (11): 2801-6. Kothary, M., and Babu, u. 2001. Infective Dose of foodborne pathogen in Volunteers: A Review. Journal of Food Safety, 21: 49-73. Sasakawa, C. 2010. A New paradigm of bacterial gut interplay brought to the study of Shigella. Proc. Jpn. Acad. 86: 229-243. BAM, Chap.6 http://www.cfsan.fda.gov/~ebam/bam-6.html. 2001. Multiplex real-time PCR for detection of Campylobacter, Salmonella, and Shigella. Barletta F, Mercado EH, Lluquee, Ruiz J, Cleary TG, Ochoa TJ. J Clin Microbiol. 2013 Sep; 51 (9): 2822-9. 2014. Infectious Disease Diagnosis and Reporting Standards for Courts. Publication registration number: 11-1352109-000021-01.

Accordingly, it is a main object of the present invention to provide a rapid and accurate detection method capable of directly isolating a heterogeneous bacterial cell.

It is another object of the present invention to provide an antibody for detection, a composition for detection, and a detection kit which enable such detection.

It is still another object of the present invention to provide a method for producing a detection composition capable of such detection.

In accordance with one aspect of the present invention, the present invention provides a method for the detection of a multivalent antibody against a Shigella dysenteriae type 4 serotype by contacting a cell of Escherichia coli serotype O42 and O159 with antigen- An antibody obtained that does not bind to an antibody; b) an antibody obtained by contacting a polyvalent antibody against the Shigella flexneri 2b serotype with the cells of E. coli serotypes O14, O89 and O114, thereby preventing antigen-antibody binding; c) an antibody obtained by contacting a polyvalent antibody against a serotype of Shigella flexneri 5b (5b) with a cell of Escherichia coli serotype O99 to prevent antigen-antibody binding; d) an antibody obtained by contacting a polyvalent antibody against a Shigella flexneri variant Y serotype to a colony of Escherichia coli serotype O87 to prevent antigen-antibody binding; e) an antibody obtained by contacting a polyvalent antibody against Shigella boydii type 7 serotype to a cell of E. coli serotype O96 to prevent antigen-antibody binding; And f) an antibody obtained by contacting a polyvalent antibody against Shigella sonnei with cells of E. coli serotypes O70, O87, O90, O128 and O164 to prevent antigen-antibody binding; And a composition for detecting a bacterial pathogen.

The present invention produces a highly sensitive antibody by reacting the produced antibody with E. coli in advance and removing the antibody bound to E. coli. A variety of microorganisms may be present in a specimen used for detecting foreign bacteria. Escherichia coli is mainly used for detection of fungus, and there are many kinds of E. coli. The use of various types of Escherichia coli so far known to react with antibodies to all E. coli and then to screen for antibodies that have not reacted with them is best for specificity, but it is virtually impossible to do so. The present invention provides the most suitable type of Escherichia coli that can be used for the production of high specificity antibodies.

In the composition for detecting a bacterial foreign body of the present invention, the antibody is preferably an antibody derived from a rabbit.

In the composition for detecting a bacterial pathogenic bacterium of the present invention, the antibody of a) to f) is preferably a synthetic antibody in a form bound to magnetic particles.

In the composition for detecting a bacterial pathogenic bacterium of the present invention, it is preferable that the synthetic antibody is bound to only one kind of antibody among the antibodies of a) to f) per one molecule of the magnetic particles.

The composition for detecting a bactericidal foreign body of the present invention may further include an additive that can be included in a conventional antibody reagent such as an auxiliary substance necessary for maintaining the activity of an antibody in addition to the above-mentioned antibody.

According to another aspect of the present invention, the present invention provides a method of preparing a composition comprising: a) contacting the composition with a sample; And b) obtaining a conjugate of the antibody and the Shigella cells contained in the composition.

In the method of the present invention for detecting a bactericidal fungus, it is preferable that the method further comprises: c) inoculating and culturing the complex obtained in the step b) in TSA (Tryptic Soy Agar) medium. In general, the TSA medium contains pancreatic digest of casein, casein peptone (pancreatic), papainic digest of soybean, soy peptone (papainic), sodium chloride (NaCl) Means an agar-containing medium, and the content of each component is adjustable in some cases. In the present invention, a culture medium containing 5-20 g / l of pancreatic enzyme degradation products of casein, 1 to 10 g / l of seafood of soybean papaya, 1 to 10 g / l of sodium chloride and 5 to 20 g / More preferably, a culture medium containing about 15 g / l of pancreatic enzyme degradation products of casein, about 5 g / l of seafood of soybean papaya, about 5 g / l of sodium chloride and about 15 g / l of agar is used It is good to do.

According to another aspect of the present invention, the present invention provides a method for producing a polyclonal antibody against a Shigella dysenteriae type 4 serotype of Escherichia coli serotypes O42 and O159, To obtain an antibody that is not antigen-antibody bound; b) contacting a polyvalent antibody against the Shigella flexneri 2b serotype to cells of E. coli serotypes O14, O89 and O114 to obtain an antibody that does not bind antigen-antibody; c) contacting a polyvalent antibody against the Shigella flexneri 5b serotype to the cells of Escherichia coli serotype O99 to obtain an antibody that does not bind antigen-antibody; d) contacting a polyvalent antibody against Shigella flexneri variant Y serotype with a cell of E. coli serotype O87 to obtain an antibody that does not bind antigen-antibody; e) contacting a polyvalent antibody against Shigella boydii type 7 serotype to a cell of E. coli serotype O96 to obtain an antibody that does not bind antigen-antibody; And f) contacting a polyvalent antibody against Shigella sonnei with cells of E. coli serotypes O70, O87, O90, O128 and O164 to obtain an antibody that does not bind antigen-antibody, A method for producing a composition for detection is provided.

In the method of the present invention for producing a composition for detecting a bacterial pathogenic bacterium, a multivalent antibody to each of the above-mentioned Shigella strains is obtained by inoculating the rabbits with the above-described Shigella strain to induce an immune response and to obtain a polyvalent antibody produced by an immunological reaction .

In the method for producing a composition for detecting a bacterial pathogenic bacterium of the present invention, it is preferable that the method further comprises the step of preparing a synthesized antibody by binding magnetic particles to the antibody obtained in each of the steps a) to f).

In the method of the present invention for producing a composition for detecting a bactericidal fungus, it is preferable to synthesize a synthesized antibody by binding only one antibody among the antibodies obtained in the respective steps a) to f) per molecule of magnetic particles.

In accordance with another aspect of the present invention, the present invention provides a method for the detection of a multivalent antibody against a Shigella dysenteriae type 4 serotype by contacting a bacterial strain of Escherichia coli serotype O42 and O159, - an antibody obtained that does not bind to an antibody; b) an antibody obtained by contacting a polyvalent antibody against the Shigella flexneri 2b serotype with the cells of E. coli serotypes O14, O89 and O114, thereby preventing antigen-antibody binding; c) an antibody obtained by contacting a polyvalent antibody against a serotype of Shigella flexneri 5b (5b) with a cell of Escherichia coli serotype O99 to prevent antigen-antibody binding; d) an antibody obtained by contacting a polyvalent antibody against a Shigella flexneri variant Y serotype to a colony of Escherichia coli serotype O87 to prevent antigen-antibody binding; e) an antibody obtained by contacting a polyvalent antibody against Shigella boydii type 7 serotype to a cell of E. coli serotype O96 to prevent antigen-antibody binding; And f) an antibody obtained by contacting a polyvalent antibody against Shigella sonnei with cells of E. coli serotypes O70, O87, O90, O128 and O164 to prevent antigen-antibody binding; And a kit for detecting a bacterial pathogen.

In the kit for detecting a bactericidal foreign body of the present invention, the antibody is preferably an antibody derived from a rabbit.

In the kit for detecting a bacterial pathogenic fungus of the present invention, the antibody of a) to f) is preferably a synthetic antibody in a form bound to magnetic particles.

In the kit for detecting a bacterial pathogen, it is preferable that the synthetic antibody is bound to only one kind of antibody among the antibodies of a) to f) per one molecule of the magnetic particles.

The kit for detecting a bactericidal foreign body of the present invention may further comprise a tool, an apparatus, or a reagent which can be included in a conventional kit for detecting a microorganism from a biological sample using an antibody in addition to the above-mentioned antibody. For example, a tool for collecting a specimen to be inspected, a buffer solution, and the like.

By using the composition for detecting a bactericidal foreign body bacteria of the present invention, the method for detecting a bactericidal foreign body bacteria, or the kit for detecting a bacterial foreign body bacteria, the foreign body cells can be directly separated from the specimen and can be performed very efficiently. In particular, it is possible to isolate heterozygous bacteria from specimens containing a large amount of various microorganisms including various E. coli strains of different serotypes, such as stool, compared with the conventional method which requires difficult time or detection of heterozygous bacteria under the same conditions There is an advantage that effective detection is possible. In addition, it is necessary to directly isolate heterogeneous bacterial cells so that diagnosis of bacterial heterogeneity which can be finally confirmed can be performed more quickly, accurately, and easily.

Figure 1 is a flow chart of the Bacteriological Analytical Manual (BAM).
Fig. 2 shows a process for producing a synthetic antibody in a form bound to magnetic particles in the antibody of the present invention.
Fig. 3 shows a method for selectively isolating foreign bacteria from a specimen using the synthetic antibody (magnetic bead-antibody conjugate) of the present invention.
4 is a flowchart illustrating a method for detecting a bacterial pathogen according to an embodiment of the present invention.
FIG. 5 is a graph showing the number of heterozygotes and their incidences that have been isolated in Korea between 2010 and 2014. FIG.
FIG. 6 shows the cross reaction of 38 serotypes of antibodies against 38 heterozygous antigens of each serotype.
FIGS. 7 to 12 show the results of the cross-reactions (A, C, E, G, I, K) between the antiserum (containing the antibody) and the serotype-specific E. coli and the antiserum to the cross- (B, D, F, H, J, and L) between the obtained antibody and the serotype of E. coli were analyzed by ELISA. The serologic type of six selected serotypes ( S. dysenteriae type 4, S. flexneri 2b, S. flexneri 5b, S. flexneri variant Y, S. boydii type 7 , S. sonnei ) and 158 E. coli serotypes reaction. The yellow part is the result for the E. coli serotype used for absorption.
Fig. 13 is a graph showing the cross reaction of an antibody against E. coli before (blue) and after (red) absorption of E. coli.
FIG. 14 is a graph showing a separation rate according to the kind of medium (TSA, MacConky, XLD) used in the process of selectively isolating foreign bacteria with a magnetic bead-antibody complex according to an embodiment of the present invention.
FIGS. 15 to 24 show the results of direct PCR and IMS PCR for 10 major strains of dominant dengue sterilized genotype in Korea. Direct PCR: PCR for spiking samples, IMS PCR: A PCR for the selective isolation of foreign bacteria with the magnetic bead-antibody complex of the present invention.
25 shows the colonies of alien bacteria according to the kind of medium (TSA, MacConky, XLD). Yellow arrow: Diphtherium, red arrow: unknown microorganism.
FIGS. 26 to 35 show results of PCR of 94 colonies identified on TSA medium by selectively isolating foreign bacteria from the magnetic bead-antibody complex of the present invention. M: a molecular marker of 100 bp, N: a negative control, and P: a positive control.
FIG. 36 shows the results of direct detection and IMS detection on the medium. Direct Detection: Conventional Detection Method, IMS Detection: A method for selectively isolating foreign bacteria by the magnetic bead-antibody complex of the present invention.
FIG. 37 is a graph showing the results of selective separation of heterologous bacteria using the magnetic bead-antibody complex of the present invention from specimens spiked with 10 major sera of dominant outbreaks of dengue virus in Korea, and then counting the number of bacterial colonies in each medium (TSA, MacConky, XLD) Fig. (A): S. dysenteriae type2, (B): S. dysenteriae type4, (C): S. flexneri 1a, (D): S. flexneri 2a, (E): S. flexneri 2b, (F): S flexneri 3a, (G): S. flexneri 4a, (H): S. flexneri type 6, (I): S. size type4, (J): S. sonnei , blue graph: number of clods counted from specimens spiked with 10 ⁵ CFU of each heterozygous serotype, red graph: counts from samples spiked with 10 ⁴ CFU of each heterologous serotype Number of contaminants counted from the spiked sample of 10 ³ CFU of each heterozygous serotype, blue hatched graph: Bacterium other than bacteria counted from specimens spiked with 10 ⁵ CFU of each heterozygous serotype Number of bacterium other than alien bacteria counted from specimens spiked with 10 ⁴ CFUs, green hatching graph: other than alien bacteria counted from specimens spiked with 10 ³ CFU of each heterozygous serotype Of bacteria.
38 is a result of slide aggregation test of colonies produced in the TSA medium after selectively isolating foreign bacteria using the magnetic bead-antibody complex of the present invention. Yellow arrow: Disease-positive, red arrow: Disease negative.

In the present invention, each serotype of alien bacteria can be confirmed by the coagulation reaction with antiserum for each. For example, when coagulation occurs by reacting a heterogeneous bacterial cell with a corresponding antiserum, it can be classified into the corresponding serotype. Each antiserum can be purchased from Denka Seiken Co., Ltd. Anticancer contains a monoclonal antibody for each serotype and an antibody against several serotypes separately. When these are appropriately selected and applied, Can be easily classified. Escherichia coli serotype can also be classified by purchasing commercially available antiserum and confirming the coagulation reaction with Escherichia coli cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

<Method>

1. Production of multivalent antibody against heterozygous bacteria by serotype

1-1. Antigen production

38 serotypes belonging to the genus Shigella were used as antigens for the preparation of polyclonal antibodies.

Each Shigella strain was inoculated in 10 ml of Tryptic Soy Broth (TSB) and cultured for 18 hours in a shaking incubator at 37 ° C.

② 10 μl of the preculture was added to 10 ml of fresh TSB medium and cultured for 8 hours with shaking.

③ The culture was diluted to OD600 = 1.0, and 10 ml of the solution was centrifuged to precipitate the cells.

④ The cells were suspended in 10 ml of 4% formalin solution and fixed at room temperature for 30 minutes.

⑤ After fixation, the formalin solution was discarded by centrifugation, and the cells were suspended in 10 ml of PBS and used in refrigerated storage.

Strain used for antigen production number Strain name NCCP No. number Strain name NCCP No. One S. dysenteriae type 2 10097 21 S. size 2 10554 2 S. dysenteriae type 3 10100 22 S. size 3 10616 3 S. dysenteriae type4 10101 23 S. size 4 10854 4 S. dysenteriae type 5 10105 24 S. size 5 10614 5 S. dysenteriae type6 10104 25 S. size 6 10098 6 S. dysenteriae type7 10103 26 S. size 7 10099 7 S. dysenteriae type8 10341 27 S. size 8 10245 8 S. dysenteriae type9 Wild type 28 S. size 9 Wild type 9 S. dysenteriae type 10 Wild type 29 S. size 10 10342 10 S. flexneri 1a 11251 30 S. size 11 10343 11 S. flexneri 1b 10108 31 S. size 12 Wild type 12 S. flexneri 2a 10612 32 S. size 13 Wild type 13 S. flexneri 2b 10852 33 S. size 14 Wild type 14 S. flexneri 3a 10111 34 S. size 15 10876 15 S. flexneri 4a 10114 35 S. size 16 Wild type 16 S. flexneri 5b 10853 36 S. size 17 Wild type 17 S. flexneri 6 11252 37 S. size 18 Wild type 18 S. flexneri variant X 10116 38 S. sonnei 11180 19 S. flexneri variant Y 10117 20 S. size 1 11190

1-2. immune

In order to use as a control group, 5 ml of blood was collected from the ear vein of the preimmune rabbits, and the serum was separated and stored at 4 ° C.

(2) The vein behind the ear of the rabbit was disinfected with 70% ethanol, and the antigen (Shigella cells) (100 μl / mouse) prepared in the above method 1-1 was inoculated to induce a primary immune response.

③ Two weeks later, antigen (200 ㎕ / mouse) was inoculated to induce secondary immune response.

④ After 13 days, 1 ml of blood was collected and the antibody titer was measured.

⑤ If the antibody level is not high, the antigen (400 μl / mouse) was inoculated two weeks after the second immunization to induce a third immunity reaction.

⑥ 13 days after the third immunization, 1 ml of blood was collected and the antibody was detected.

⑦ When it was judged that the antibody against Schizelacus was sufficiently generated, whole blood was collected from the rabbits and the serum (antiserum) was separated and refrigerated.

1-3. Immunity evaluation

Each of the strains of Shigella used as an antigen was coated on a 96-well ELISA plate at a concentration of 10 ⁷ CFU / well and stored frozen.

② The plate was left at room temperature for 30 minutes, then 100 μl of 10% Bovine serum albumin (BSA) was added and the mixture was blocked at room temperature for 30 minutes.

③ The antiserum and the control serum were diluted 2,048 times and added to the plate in an amount of 50, and reacted for 30 minutes.

④ It was washed 5 times with 100 μl of 0.01% PBST.

⑤ HRP-conjugated anti-rabbit IgG antibody was diluted 10,000 times and added to each well in an amount of 50 μl, followed by reaction for 30 minutes.

⑥ It was washed five times with 100 μl of 0.01% PBST.

⑦ Add 50 ㎕ of Ultra TMB solution (Thermo, cat No. 34028) to each well and react for 2 minutes.

⑧ The reaction was stopped by adding 50 0.2 of 0.2NH ₂ SO ₄ solution.

⑨ Analyzed at 540nm with an ELISA reader.

⑩ The control group was confirmed to not react with the antigen, and when the antiserum diluted 2,048 times showed a result of 0.5 or more, it was judged to be immune and blood was collected.

2. Identification of cross-reactivity by heterozygous serotype

Cross - reactivity of different serotypes among antibodies produced by serogroups of Shigella were confirmed by ELISA.

(1) 38 serotypes of antigen prepared in Method 1-1 were attached to an ELISA plate at 10 ⁵ CFU / well.

② Each antiserum prepared in Method 1-2 was diluted 2,048 times in PBS buffer and added at 50 μl / well, followed by reaction for 30 minutes.

③ Wash with 100 ㎕ PBST 5 times.

④ HRP-conjugated anti-Rabbit IgG antibody was diluted 10,000 times, added 50 ㎕ / well each, and reacted for 30 minutes.

⑤ It was washed 5 times with 100 μl PBST.

⑥ Add 50 ㎕ of Ultra-TMB coloring solution to each well and react for 2 minutes.

After the reaction, 50 μl of H ₂ SO ₄ solution was added to stop the reaction.

⑧ Analyzed at 450nm wavelength using an ELISA reader.

3. Absorption of Shigella antibody

3-1. Selection of absorbing Escherichia coli

(1) 158 strains of E. coli with different serotypes were cultured in TSB for 18 hours, diluted to a concentration of OD600 = 1.2, coated with 50 μl each in a 96-well plate, and stored frozen (see Table 2).

② The plate was left at room temperature for 30 minutes, then 100 μl of 10% Bovine serum albumin (BSA) was added and the mixture was blocked at room temperature for 30 minutes.

③ The antiserum and the control serum were diluted 2,048 times and added to the plate in an amount of 50, and reacted for 30 minutes.

④ It was washed 5 times with 100 μl of 0.01% PBST.

⑤ HRP-conjugated anti-rabbit IgG antibody was diluted 10,000 times and added to each well in an amount of 50 μl, followed by reaction for 30 minutes.

⑥ It was washed five times with 100 μl of 0.01% PBST.

⑦ Add 50 ㎕ of Ultra TMB solution (Thermo, cat No. 34028) to each well and react for 2 minutes.

⑧ The reaction was stopped by adding 50 0.2 of 0.2NH ₂ SO ₄ solution.

⑨ Analyzed at 450nm with an ELISA reader.

⑩ Escherichia coli serotype showing the highest value among ELISA results was selected.

3-2. Induce absorption reaction

After incubation for 18 hours, 100 ml of TSB was inoculated with E. coli serotypes O14, O42, O70, O87, O89, O90, O96, O99, O114, O128, O159 and O164 10%, and stored at -80 ° C.

② The stored E. coli was dissolved in a constant temperature water bath at 37 ° C and then diluted continuously to measure the number of bacteria.

③ Each Escherichia coli was adjusted to a concentration of 10 ¹¹ CFU and centrifuged to obtain cells.

④ Antibodies and E. coli serotypes with high cross-reactivity values were suspended in 400 μl of antiserum containing each antibody, followed by induction of absorption reaction at 4 ° C for 2 hours. If there is only one type of E. coli serotype to be used for absorption, follow the procedure in step 7 below. If there is a large number of E. coli serotype in step 5, follow steps ⑤ through ⑥.

⑤ After the absorption reaction, the supernatant was recovered by centrifugation (6,000 × g, 10 minutes), and the new coliform bacteria were suspended in the supernatant, and the secondary absorption reaction was induced overnight at 4 ° C.

⑥ After the second absorption reaction, the supernatant was recovered by centrifugation (6,000 × g, 10 minutes), and new coliform bacteria were suspended in the supernatant, and the third absorption reaction was induced at 4 ° C. for 2 hours.

⑦ After all the E. coli absorption reaction was completed, the supernatant was recovered by centrifugation (8,000 × g, 10 min). The supernatant was placed in a new tube, and sodium azide was added to a final concentration of 0.05%. The supernatant was stored at 4 ° C and then used to adhere to magnetic beads.

4. Manufacture of magnetic bead-antibody complex

① 100 μl of magnetic beads (Pureproteome ™ protein A / G Mix Magnetic bead, Millipore, Cat No. LSKMAGAG10) was placed in an E-tube and mounted on a magnetic stand.

② When the beads adhered to the wall of the E-tube, discard the supernatant, add 1 mL of PBST (Phosphate Buffered Saline in 0.01% Tween 20), close the lid of the E-tube and remove it from the magnetic stand.

③ After washing, put E-tube back on magnetic stand and discard supernatant.

④ To the washed beads, 200 μl of Escherichia coli absorbing antibody solution prepared in Method 3 was added and inverted.

⑤ E-tube mixed with beads and antibody was reacted for 4 hours or more with stirring at 4 캜.

After the reaction, the E-tube was mounted on a magnetic stand, and the supernatant was discarded and washed once with 1 ml of PBS.

⑦ After washing, 100 ㎕ PBS containing 0.02% sodium azide was added and stored at 4 ℃.

5. Preparation of major isolates of Schizera strain in Korea

① 10 weeks of major Shigella strains isolated in Korea from 2010 to 2014 (see Fig. 5) were each inoculated into 10 ml of TSB medium and cultured in a shaking incubator at 37 ° C for 18 hours.

(2) 50 μl of the culture was inoculated into 50 ml of each fresh medium, and then cultured for 8 hours in a shaking incubator at 37 ° C.

③ Glycerol was added to 50 ml of the culture solution to a final concentration of 10%, and the mixture was dispensed into cryogenic vials in an amount of 1 ml each. The mixture was frozen at -80 캜 and stored.

④ The frozen strain was taken out and dissolved in a constant temperature water bath at 37 ° C, and then diluted stepwise by 10 times with PBS.

⑤ Stair dilution was added to TSA medium in an amount of 100 μl each, and the mixture was spread and incubated in a thermostat at 37 ° C for 18 hours.

⑥ The colonies of the plate grown on the bacteria were calculated and the concentration of frozen bacteria at -80 ℃ was calculated and used in the experiment.

6. Fungal isolates from stool specimens and Shigella strains

(1) Stool of diarrhea symptom patient (causative pathogen diagnosis negative) 1 g of specimen was suspended in 10 ml of PBS, and then 10 ^{5 to 1} CFU / 100 μl of each Shigella strain was spiked into 400 μl of the suspension.

② 100 μl of the magnetic bead-antibody complex was washed with 1 ml of 0.01% PBST and suspended in 100 μl of PBS.

③ A single suspension of stool specimen and a mixture of Shigella strains and stool specimen were added to a 4 ml tube and mixed with a magnetic bead-antibody complex, and then 400 μl of TSB medium was added to give a final volume of 1 ml.

④ The tubes were incubated in a shaking incubator at 37 ℃ for 30 minutes.

⑤ After the reaction, the mixed solution contained in the tube was transferred to the E-tube, attached to the magnetic stand, and the bead was attached to the wall, and the supernatant was discarded.

⑥ After adding 1 ml of 0.05% PBST to the E-tube, the lid of the E-tube was closed, and it was removed from the magnetic stand and washed by inverting.

⑦ After washing, the E-tube was attached to the magnetic stand and the supernatant was discarded.

⑧ Repeat steps ⑤ to ⑦ four more times, then wash one more time with 1 ml of PBS.

⑨ Finally, 150 μl of the solution was suspended in sterilized distilled water. 50 μl of the solution was used for PCR (IMS PCR), 100 μl was stair-diluted and spread on TSA, Mac and XLD selective medium.

(10) For the PCR control (Direct PCR), the mixed spiking sample was centrifuged in step (1), and the pellet was separated and suspended in 50 μl of sterilized distilled water.

7. Identification of isolated foreign body bacteria

In order to confirm whether or not the cells isolated according to the method of the present invention are actually heterogeneous bacteria, the PCR method and slide aggregation test, which have been conventionally used, were performed.

7-1. PCR diagnosis

1) Direct PCR sample and IMS PCR sample prepared in Method 6 were used as the PCR template.

② Prepared samples were boiled at 100 ℃ for 5 minutes, centrifuged (12,000 × g, 10 minutes) and 5 ㎕ of supernatant was used as a template.

(3) PCR was performed according to the reaction liquid composition shown in Table 3 and the PCR reaction conditions shown in Table 4. Primer set IPA / 1 & 2 and primer set INV / 1 & 2, products of TaKaRa, were used as primers (sequence unknown).

(4) The reaction solution after completion of the PCR reaction was loaded on 2% agarose gel (containing shine green) and electrophoresed at 100 V for 30 minutes. Then, the gene product amplified with UV was confirmed in the gel Documentation system.

Reaction solution composition for PCR reaction reagent volume Template DNA 5 μl primer Inv-F 0.5 μl Inv-R 0.5 μl Ipa-F 0.5 μl Ipa-R 0.5 μl Distilled water 3 μl Taq polymerase 10 μl Total reaction liquid 20 μl

PCR reaction conditions Reaction step Temperature (℃) time Number of reaction cycles Initial PCR activation 94 5 minutes One Denaturation 94 1 minute
35 Annealing 55 1 minute Extension 72 1 minute Final extension 72 7 minutes One

7-2. Slide agglutination test

① 30 ㎕ of antiviral (polyvalent or monovalent) (Denka Seiken) solution was dripped onto a clean slide.

② The colonies cultured on TSA plate were tipped and spread on the slides and mixed evenly with the reaction solution.

③ The slides were tilted so that the cells reacted with the solution.

④ The agglutination was confirmed within 1 minute.

<Result>

1. Antibody Selection for Separation of Diseases

In order to minimize the types of antibodies to be attached to the magnetic beads, cross - reactions among 38 serogroups of Shigella were confirmed. Each of the antigens was coated on a 96-well plate, and 38 kinds of prepared serotypes were reacted. Antibodies with higher autoantibodies were selected when the autoantibody to autoantigen was 100%.

But many are cross-reactive antibody is an antibody selected for a high paper 9, and finally, after attachment to the beads of low detection efficiency antibody (S. dysenteriae type2, S. dysenteriae type3, S. boydii type3) the 6 kinds of antibodies other than (S dysenteriae type 4 , S. flexneri 2b, S. flexneri 5b, S. flexneri variant Y, S. boydii type 7 , S. sonnei ) (Fig.

2. Cross-reactivity and antibody uptake of 156 selected serogroups of Shigella antibodies

2-1. Cross-reactivity with E. coli

It is most important to minimize cross-reactivity with Escherichia coli for the selective isolation of foreign bacteria. As reported in many documents, it is presumed that E. coli was differentiated from E. coli. Escherichia coli is known to have about 180 serotypes so far.

Cross-reactivity of the 158 species of E. coli bacteria in the laboratory to the six selected foreign body antibacterial antibodies in the above-mentioned result 1 was observed, and the cross-reactivity with the respective antibodies and Escherichia coli serotypes was found to vary Reference).

As shown in Fig. 13, the S. dysenteriae type 4 antibody was cross-reacted as compared to the pre-absorption state as shown in Fig. 13. As a result, the cross-reacted E. coli serotype was selected to reduce the cross- 98% was reduced, 84% S. flexneri 2b, S. flexneri 5b, S. flexneri Y variant, S. boydi i type7, S. sonnei antibodies, each cross-reactivity, 87%, 95%, 98%, 89% Respectively.

2-2. Selection of Escherichia coli for Absorption of Disease Antibody

Escherichia coli was selected for which the cross reaction with E. coli was totally reduced as a result of absorption by ELISA titer E. coli with each heterologous antibody.

As shown in FIG. 7 (A), the S. dysenteriae type 4 antibody showed the highest cross-reactivity with O159 serotype E. coli, indicating that the E. coli was able to undergo an absorption reaction with the E. coli, And the second absorption was further performed with the O42 serotype E. coli, thereby reducing the overall cross-reaction as shown in Fig. 7 (B). However, in the case of S. flexneri 2b antibody, the cross-reaction of O123 serotype was the highest, but the absorption of O123 serotype did not decrease the overall cross-reactivity. Thus, an absorption reaction with O89 and O114 sera was performed, followed by a secondary absorption reaction with O14 serotype E. coli to reduce the overall cross-reaction (see FIG. 8). A total of 12 serotypes were determined from E. coli serotypes, which will eventually carry out the absorption reaction with six heterologous bacterial antibodies through these methods. Table 5 summarizes the results of selecting E. coli for each antibody.

Escherichia coli serotypes selected for the absorption reaction with heterologous antibodies Heterogeneous antibody Absorption E. coli serotype S. dysenteriae type4 O42, O159 S. flexneri 2b O89, O14, O114 S. flexneri 5b O99 S. flexneri variant Y O87 S. Heavy type7 O96 S. sonnei O90, O87, O70, O128, O164

3. Magnetic bead-antibody complex

The efficiency of the two methods for the attachment of magnetic beads and antibodies was analyzed. In the first method, six selected antibodies are attached to magnetic beads at the same time. In the second method, six selected antibodies are attached to magnetic beads, and then mixed before use.

In order to analyze the efficiency of these two magnetic bead - antibody complexes, we measured the number of strains detected after reacting 10 ¹ - 10 ⁵ CFU of S. sonnei with each complex. As a result, as shown in Table 6, the detection efficiency of the second method was increased by 26% when cultured in TSA and 29% when cultured in XLD, compared with the first method. The same results were obtained when cultured in MacConky.

Detection efficiency of S. sonnei by magnetic beads and antibody attachment method Magnetic bead - antibody attachment method TSA MacConky XLD Six selected absorbed antibodies
Attach simultaneously to magnetic bead 180% * 167% * 157% * Each of the 6 selected absorbed antibodies was attached to a magnetic bead and mixed before use 206% * 167% * 186% *

* Assuming that 100% cultured in 10 ¹ to 10 ⁵ CFU was separated into each magnetic bead-antibody complex, the number of cultured colonies was measured and converted to%.

4. Selection of badge

It was confirmed that when XLD selection medium was used in the process of selectively isolating foreign bacteria with magnetic bead-antibody complex (hereinafter referred to as 'IMS'), culture of foreign bacteria was inhibited. In order to identify the medium suitable for the isolation of foreign bacteria in IMS, the efficiency of isolating the pathogen was measured in a MacConky, XLD agar known as a heterogeneous selection medium and TSA medium as a general bacterial growth medium.

For a selective medium selected for domestic separation 10 serotypes (S. dysenteriae type2, S. dysenteriae type4 , S. flexneri 1a, S. flexneri 2a, S. flexneri 2b, S. flexneri 3a, S. flexneri 4a, S. flexneri type6 , S. boydii type4, S. sonnei ).

Ten colonies of 10 ¹ to 10 ⁵ CFU / sample were reacted with magnetic bead-antibody complexes, respectively, and then cultured in TSA, Mac, XLD to determine the number of colonies produced. When the number of bacteria in the sample was 100%, the number of bacteria measured in each medium was calculated as a percentage. As a result, an average of 156.1% was confirmed in TSA, 105.9% in MacConky, and 96.9% in XLD (see Table 7). The reason for the low segregation rate in MacConky and XLD is not clear, but it is presumably due to the characteristics of the medium selected by the biochemical characteristics of the strain. And more than 100% is due to the growth of the cells during the separation process.

Thus, the biggest problem with using MacConky or XLD in comparison to TSA medium is that some serotypes are not cultured in MacConky or XLD medium or cultured below 50% in comparison with TSA medium (see FIG. 14). Especially, S. flexnri 2a was not cultured on MacConky agar but TSA showed 179.5% isolation. The results were obtained after culturing for 18 hours. After incubation for 36 hours on MacConky agar, S. flexnri 2a was cultured. This means that the culture rate of some alien bacteria is slowed by the medium component. Therefore, it is considered that TSA is the most suitable medium for the isolation of heterogeneous bacteria.

TSA, MacConky, and XLD agar Heterozygous TSA Mac XLD S. dysenteriae type 2 121% 75.9% 89.7% S. dysenteriae type4 128.8% 116.2% 40.2% S. flexneri 1a 128% 77.5% 71.4% S. flexneri 2a 179.5% 0% 112.5% S. flexneri 2b 134.6% 61.5% 91.2% S. flexneri 3a 133.8% 69% 80.5% S. flexneri 4a 172% 112.6% 103.1% S. flexneri type6 195.2% 164% 101% S. Heavy type4 142.2% 108.2% 93.9% S. sonnei 206% 167% 186% Average 156.1% 105.9% 96.9%

5. Comparison of direct PCR and IMS PCR of heterozygous spiking samples

As a result of direct PCR, 9 spiking samples except S. dysenteriae type 4 samples were negative, and all spiked samples were spiked. In the case of IMS PCR, 9 specimens were positive up to 10 ² CFU except for the S. sonnei sample (positive to 10 ³ CFU) (see FIGS. 15 to 24). In the case of S. dysenteriae type 4, only a small amount of PCR inhibitor was present in the spiked stool specimen, suggesting that a PCR positive result was obtained even at a low spiking sample. However, except for this, IMS PCR showed much better results. This result implies that many PCR inhibitors present in stool specimens can be efficiently removed by the IMS method.

6. Comparison of properties of different bacteria and Shigella strains on TSA plate

Generally, the colonies of MacConky and XLD plates used as selection media for Shigella strains are shown in Fig. The characteristics of the Shigella strain on the TSA plate are as shown by the yellow arrow in FIG. 25, the colony area is rugged and shows a light milk light color.

Based on these characteristics, detection was carried out by the IMS method of the present invention in a sample in which 10 serotypes were spiked on a specimen, and 94 colonies considered as Shigella strains were identified by PCR by culturing on a TSA plate. A result of this, in the case of S. flexneri 2b Eight colonies of the 94 colonies were found to be not the S. flexneri 2b was found to be lowest in 91.5% positive rate. On the other hand, S. dysenteriae type 4 , S. flexneri 1a, S. flexneri 2a, S. flexneri 3a, S. flexneri 4a and S. flexneri 6 were 100% positive. Finally, a colony suspected to be a shigella by a colony-like image (a light, oily, and rugged borderline) on the TSA plate was detected by PCR using the IMS method of the present invention, and it was confirmed that an average of 98.5% of the colonies was positive Reference). This means that it is possible to use the TSA medium as a selective medium by selecting only the Shigella strain from other bacteria present in the sample through the method of the present invention.

7. Comparison of direct and IMS detection in spiking samples

Comparison of IMS detection using the magnetic bead-antibody complex of the present invention was compared with detection of the conventional method (Direct detection) after spiking S. flexneri 1a 10 ⁵ CFU in the stool sample. As a result, colonies were observed in TSA, MacConky and XLD plates diluted 10 ^-4 in direct detection, but colony of S. flexneri 1a was not confirmed. There is a possibility that there is a probability of more than 10 ¹ CFU of S. flexneri 1a in the plate diluted to 10 ^-4 , but this could not be confirmed. On the other hand, in the case of the IMS detection of the present invention, S. flexneri 1a could be detected in all three kinds of plates as shown in Table 8 and FIG.

IMS detection and direct detection Colony count Sample Detection method ^* TSA Mac XLD Shigella ^*** Unknown Shigella ^*** Unknown Shigella ^*** Unknown Stool + Shergella ^***
(10 ⁵ CFU) IMS ≒ 2700 ≒ 570 260 318 27 153 Direct Not available ^** 5200 Not available ^** ≒ 3100 Not available ^** 5100

* IMS detection: 10 ^-1 dilution plate, Direct detection: 10 ^-4 dilution plate

** Indivisible : Probably 10 ¹ CFU of S. flexneri colonies are expected but not identifiable

*** Shigella: S. flexneri 1a (10 ⁵ CFU)

8. IMS detection of major isolates in Korea

The sputum of the normal type fecal specimen was spiked with 10 ³ to 10 ⁵ CFU of each serotype of the Schlegeli strain, and the cells were separated using the IMS method of the present invention. The number of shigella and other normal flora on the plate was counted, The isolation of Shigella strains from TSA was much higher in all of the serotype (FIG. 37). In addition, the isolation of bacteria other than Schlegeli was significantly lower in the eight serotypes except S. flexneri 2b and S. sonnei . In S. flexneri 2b and S. sonnei , growth of other bacteria was high, but there was no problem in screening.

9. Limitations of detection of Shigella strains in IMS method

Table 10 shows the results of measuring the minimum limit that can be detected by the IMS method of the present invention after spiking 10 ¹ to 10 ⁵ CFUs on the stool specimens of major domestic isolates.

Limit of detection of Shigella strain using IMS method TSA Mac XLD S. dysenteriae type 2 10 ² CFU 10 ⁵ CFU 10 ⁵ CFU S. dysenteriae type4 10 ¹ CFU 10 ⁴ CFU 10 ⁵ CFU or more * S. flexneri 1a 10 ³ CFU 10 ³ CFU 10 ⁴ CFU S. flexneri 2a 10 ¹ CFU 10 ⁴ CFU 10 ⁵ CFU S. flexneri 2b 10 ² CFU 10 ⁴ CFU 10 ⁵ CFU S. flexneri 3a 10 ³ CFU 10 ⁵ CFU 10 ⁵ CFU S. flexneri 4a 10 ¹ CFU 10 ⁵ CFU 10 ⁵ CFU S. flexneri type6 10 ² CFU 10 ⁴ CFU 10 ⁵ CFU or more * S. Heavy type4 10 ² CFU 10 ³ CFU 10 ⁵ CFU or more * S. sonnei 10 ³ CFU 10 ⁵ CFU 10 ⁵ CFU or more *

* 10 ⁵ CFU or more: 10 ⁵ CFU spiked sample means that the bacteria were not separated

Except for S. flexneri 1a, 3a, and S. sonnei (10 ³ CFU), the detection limits were below 10 ² CFU. In the case of the symptomatic person, it is known that more than 10 ⁵ CFU / g of pathogen exists in the specimen, so that the IMS method of the present invention is considered to be sufficient for the method of isolating bacteria in the patient.

10. Identification of isolated colonies

After the detection of IMS as described above, the selective separation efficiency of Shigella strain was verified on the TSA plate. The advantage of screening the Shigella strains from the TSA plate is that the colonies can be used for direct serological identification, which can shorten the final confirmation diagnosis by more than 24 hours (see Figures 4 and 38). On the other hand, in the case of the conventional FAD method, there is a disadvantage in that it is necessary to subculture the Schizelas strain using a selective medium such as the MacConky or XLD selection medium, and to subculture the strain with TSA medium for biochemical tests (see FIG. 1) .

Claims (14)

(a) an antibody obtained by contacting a polyvalent antibody against Shigella dysenteriae type 4 serotype to Escherichia coli serotypes O42 and O159 to prevent antigen-antibody binding;
b) an antibody obtained by contacting a polyvalent antibody against the Shigella flexneri 2b serotype with the cells of E. coli serotypes O14, O89 and O114, thereby preventing antigen-antibody binding;
c) an antibody obtained by contacting a polyvalent antibody against a serotype of Shigella flexneri 5b (5b) with a cell of Escherichia coli serotype O99 to prevent antigen-antibody binding;
d) an antibody obtained by contacting a polyvalent antibody against a Shigella flexneri variant Y serotype to a colony of Escherichia coli serotype O87 to prevent antigen-antibody binding;
e) an antibody obtained by contacting a polyvalent antibody against Shigella boydii type 7 serotype to a cell of E. coli serotype O96 to prevent antigen-antibody binding; And
f) an antibody obtained by contacting a polyvalent antibody against Shigella sonnei with cells of Escherichia coli serotypes O70, O87, O90, O128 and O164 to prevent antigen-antibody binding; as an antibody against bacterial fungus Or a pharmaceutically acceptable salt thereof. The method according to claim 1,
Wherein the antibody is a rabbit-derived antibody. The method according to claim 1,
Wherein the antibody of a) to f) is a synthetic antibody in a form bound to magnetic particles. The method of claim 3,
Wherein the synthetic antibody is one in which only one antibody of the antibodies of a) to f) is bound per one molecule of the magnetic particles. a) contacting the composition of any one of claims 1 to 4 with a sample; And
b) obtaining a conjugate of the antibody and the Shigella cells contained in the composition. 6. The method of claim 5,
c) culturing the complex obtained in step b) in a TSA (Tryptic Soy Agar) medium and inoculating it. a) contacting a polyvalent antibody against the Shigella dysenteriae type 4 serotype to Escherichia coli serotypes O42 and O159 to obtain an antibody that does not bind antigen-antibody step;
b) contacting a polyvalent antibody against the Shigella flexneri 2b serotype to cells of E. coli serotypes O14, O89 and O114 to obtain an antibody that does not bind antigen-antibody;
c) contacting a polyvalent antibody against the Shigella flexneri 5b serotype to the cells of Escherichia coli serotype O99 to obtain an antibody that does not bind antigen-antibody;
d) contacting a polyvalent antibody against Shigella flexneri variant Y serotype with a cell of E. coli serotype O87 to obtain an antibody that does not bind antigen-antibody;
e) contacting a polyvalent antibody against Shigella boydii type 7 serotype to a cell of E. coli serotype O96 to obtain an antibody that does not bind antigen-antibody; And
f) contacting a polyvalent antibody against Shigella sonnei with cells of E. coli serotypes O70, O87, O90, O128 and O164 to obtain an antibody that does not bind to the antigen-antibody, &Lt; / RTI &gt; 8. The method of claim 7,
A method for producing a composition for detecting a bacterial pathogen, comprising the step of inoculating a rabbit with the above-mentioned Shigella strain to induce an immune response and obtaining a polyvalent antibody produced by an immunological reaction, . 8. The method of claim 7,
A method for preparing a composition for detecting a bacterial pathogenic fungus, which comprises the steps of: a) preparing a synthetic antibody by binding magnetic particles to the antibody obtained in each of the steps a) to f). 10. The method of claim 9,
Characterized in that only one of the antibodies obtained in each of the steps a) to f) above is bound per molecule of the magnetic particles to produce a synthetic antibody. (a) an antibody obtained by contacting a polyvalent antibody against Shigella dysenteriae type 4 serotype to Escherichia coli serotypes O42 and O159 to prevent antigen-antibody binding;
b) an antibody obtained by contacting a polyvalent antibody against the Shigella flexneri 2b serotype with the cells of E. coli serotypes O14, O89 and O114, thereby preventing antigen-antibody binding;
c) an antibody obtained by contacting a polyvalent antibody against a serotype of Shigella flexneri 5b (5b) with a cell of Escherichia coli serotype O99 to prevent antigen-antibody binding;
d) an antibody obtained by contacting a polyvalent antibody against a Shigella flexneri variant Y serotype to a colony of Escherichia coli serotype O87 to prevent antigen-antibody binding;
e) an antibody obtained by contacting a polyvalent antibody against Shigella boydii type 7 serotype to a cell of E. coli serotype O96 to prevent antigen-antibody binding; And
f) an antibody obtained by contacting a polyvalent antibody against Shigella sonnei with cells of Escherichia coli serotypes O70, O87, O90, O128 and O164 to prevent antigen-antibody binding; as an antibody against bacterial fungus A kit for detecting a bacterial foreign body. 12. The method of claim 11,
Wherein the antibody is a rabbit-derived antibody. 12. The method of claim 11,
Wherein the antibody of a) to f) is a synthetic antibody in a form bound to magnetic particles. 14. The method of claim 13,
Wherein the synthetic antibody comprises only one antibody of the antibodies a) to f) bound to one molecule of the magnetic particles.

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