JP2012122921A - Method for detecting specific substance in milk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunochromatography method and an immunochromatography apparatus for detecting a substance in milk of a domestic animal by using an antigen-antibody reaction.SOLUTION: An immunochromatography method for detecting a specific substance in milk, which includes: (1) a step of, with respect to a specimen having a first portion in which a labeled first antibody to the specific substance or the labeled specific substance is held, a second portion which is connected to downstream of the first portion and to which a second antibody to the specific substance is immobilized, and a third portion which is connected to upstream of the first portion or the second portion and has a gap through which milkfat globule in the milk can be removed, allowing the milk to contact the third portion or an upper stream portion than the third portion; and (2) a step of allowing the milk to flow to the second portion or a lower stream portion of the second portion and acquiring a signal detectable by the label in the second portion or the lower stream portion of the second portion.

Description

  The present invention relates to an immunochromatographic method and an immunochromatographic apparatus for detecting substances in milk using an antigen-antibody reaction.

  Livestock milk represented by cattle, sheep and goats is not aseptic and may contain some microorganisms due to disease or the environment. Particularly in diseases caused by microbial infection, it is often known that many microorganisms are eliminated in milk. Mastitis is a typical livestock disease caused by microbial infection.

  Mastitis is inflammation of the ductal system and mammary gland tissue, and is mainly caused by microorganisms invading, colonizing and proliferating in the breast. Mastitis affects many animals, but bovine mastitis, particularly in dairy cows, is said to affect 15-40% of all dairy cows and is one of the most important diseases for dairymen. Dairy cows suffer from mastitis, which impedes milk synthesis and reduces milk yield and, in some cases, halts lactation, as well as significant economic losses such as treatment costs and milk price penalties to dairymen. give. In addition, labor burdens on dairy farmers will increase, such as individually milking mastitis-affected quarters to prevent infection.

  Mastitis is caused by infection with various microorganisms, but the antibiotics that are effective differ depending on the causative bacteria, and because certain microorganisms are transmitted to other quarters and individuals, their properties and subsequent responses differ. It is extremely important to identify causative bacteria present in milk quickly and easily.

  Known methods for identifying the causative bacteria of an infectious disease include culture methods, gene identification methods, and antigen-antibody reaction identification methods. Currently, the method for identifying the causative bacteria of livestock mastitis is mainly a culture method, but there are problems that the operation is complicated and it takes several days to obtain the result. An identification method based on detection of a specific gene by a gene amplification method (PCR method) has also been reported (Non-patent Document 1). Although this method can produce results in about a day, it still requires special equipment and procedures. In recent years, an apparatus for detecting Staphylococcus aureus and Escherichia coli, which are one of the causative bacteria of mastitis, has been developed by surface plasmon resonance (SPR) based on an antigen-antibody reaction (Non-patent Document 2). In this method, an antibody against an antigen specific to each bacterium is prepared, and this antibody is immobilized on an SPR chip. Although this method can quickly detect a specific causative bacterium, it requires a special device and is difficult to measure at a dairy site or the like.

  On the other hand, a simple measurement device (immunochromatography device) using an immunochromatography method that can quickly and easily measure biological samples such as blood and urine at home or in an examination room is widespread (for example, see Patent Document 1). ). This method is a test in which a test paper containing an antibody specific to a measurement target substance (antigen) labeled with colored particles such as colloidal gold is connected to a porous membrane on which an antibody that captures the measurement target substance is immobilized. Use a piece. When a specimen containing a measurement target substance is dropped, the measurement target substance binds to an antibody labeled with colored particles or an antibody immobilized on a porous membrane, and a visually discernible line or the like appears on the membrane. Therefore, the presence or absence of the measurement substance can be detected by confirming the presence or absence of this line or the like. However, a simple measuring device using an immunochromatographic method capable of measuring livestock milk quickly and simply at a dairy farm has not been proposed.

JP-A-1-244370

J.Dairy.Sci. 84: 74-83 JRA Advanced Livestock Management System Practical Report (FY2006-20) p58-65

  An object of the present invention is to provide an immunochromatographic method and an immunochromatographic apparatus for detecting substances in milk of livestock using an antigen-antibody reaction.

  The present inventor has examined identification of causative bacteria of mastitis by antigen-antibody reaction using an immunochromatographic apparatus. However, when the present inventors actually examined whether or not microorganisms could be detected by immunochromatography using milk as a sample, it was found that the antigen-antibody reaction did not proceed on the test piece of the immunochromatography apparatus. As a result of investigating the reason why the antigen-antibody reaction does not proceed smoothly on the test piece, the present inventor found that a large amount of milk fat globules contained in the milk was clogged in the test piece of the immunochromatography apparatus in which the porous membrane was connected. I thought that it might be caused by insufficient flow. Usually, in an immunochromatography apparatus, a test piece having a pore diameter of about several tens to several hundreds of nanometers is used in order to make the transport by the developing solution and the speed of the antigen-antibody reaction appropriate. On the other hand, in the case of raw milk, there are a large number of milk fat globules having a particle size of about 1 to 10 μm (in commercially available milk or the like, milk fat globules in raw milk are homogenized to 1 μm or less).

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have trapped milk fat globules in milk on the upstream side of the test piece used in the immunochromatographic method, thereby immunochromatographically analyzing a specific substance in the milk. The inventors have found that it is possible to perform measurement with the above, and have completed the present invention. That is, the present invention is as follows.

[1] An immunochromatographic method for detecting a specific substance in milk,
(1) a labeled first antibody against the specific substance or a first part in which the labeled specific substance is retained, a second antibody linked downstream of the first part and against the specific substance For a test strip having an immobilized second part and a third part connected to the first part or upstream of the second part and having a cavity capable of removing milk fat globules in the milk, Contacting the milk with the third portion or upstream thereof; and (2) flowing the milk to the second portion or downstream thereof, and detecting with the label at the second portion or downstream thereof. Obtaining possible signals:
Including the above method.
[2] The method according to [1], wherein the retained particle size of the voids in the third portion is 1 to 3.5 μm.
[3] The method according to [1] or [2], wherein the immunochromatographic method is a lateral flow type.
[4] The method according to any one of [1] to [3], wherein the first part retains a labeled first antibody against the specific substance.
[5] The method according to any one of [1] to [4], wherein the specific substance is a bacterial component or a substance secreted by the bacteria.
[6] The method according to [5], wherein the specific substance is ribosomal protein L7 / L12.
[7] The method according to [6], wherein the first antibody and the second antibody are monoclonal antibodies against ribosomal protein L7 / L12.
[8] The first antibody and the second antibody react with a component of a bacterium of a specific species or genus causing mastitis or a substance secreted by the bacterium to cause mastitis other than the specific bacterium. The method according to [5], which is a monoclonal antibody that does not react with a component of the bacterium or a substance secreted by the bacterium.
[9] The monoclonal antibody reacts with ribosomal protein L7 / L12 of bacteria of a specific species or genus causing mastitis, and ribosomal protein L7 / L12 of bacteria causing mastitis other than the specific bacteria [8] is a monoclonal antibody that does not react.
[10] A labeled first antibody against the specific substance or a first part in which the labeled specific substance is retained, a second antibody linked downstream of the first part and against the specific substance Milk comprising a test piece having an immobilized second part and a third part connected to the first part or upstream of the second part and having a cavity capable of removing milk fat globules in the milk An immunochromatographic device for detecting a specific substance in the inside.

  According to the present invention, the presence or absence of a specific substance in milk can be detected quickly and easily on the spot. In particular, when diagnosing bovine mastitis, if the sign is visible, it can be carried out on a dairy farm without using a device, etc., so the causative bacteria must be identified before the disease worsens further. It is possible to determine an appropriate treatment policy such as selection of appropriate antibiotics and measures for preventing the spread of infection at an early stage.

The cross-sectional schematic diagram of the test piece of the immunochromatography apparatus used in the Example is shown. The cross-sectional schematic diagram of another example of the test piece of an immunochromatography apparatus is shown. The cross-sectional schematic diagram of another example of the test piece of an immunochromatography apparatus is shown.

Hereinafter, the present invention will be described in more detail.
The immunochromatography apparatus of the present invention is an apparatus for detecting a specific substance in milk by an immunochromatography method, wherein the first antibody labeled with the specific substance or the labeled specific substance is retained. A first part, a second part connected downstream of the first part, and a second part against which the second antibody against the specific substance is immobilized, connected to the first part or upstream of the second part, and It is an apparatus containing the test piece which has the 3rd part which has the space | gap which can remove the milk fat globule in milk. Specific examples of the configuration of the test piece include the test pieces whose cross-sectional schematic diagrams are shown in FIGS. 1, 2, and 3. In FIG. 1, the sample addition member 4 and the fat globule removal member (third portion) 7 are integrally installed upstream of the labeled antibody-impregnated member (first portion) 1. In FIG. 2, the fat globule removing member (third portion) 7 is installed downstream of the labeled antibody-impregnated member (first portion) 1 and upstream of the chromatographic development membrane carrier (second portion) 2. . In FIG. 3, the fat globule removing member (third part) 7 is installed downstream of the sample addition member 4 and upstream of the labeled antibody impregnated member (first part) 1.

  The immunochromatographic apparatus can be produced using a commercially available material by a known method.

  The material used for the first part is not particularly limited as long as it can perform immunochromatography, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, cotton or the like.

  The material used for the second part is not particularly limited as long as it can perform immunochromatography, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon or the like.

  It is preferable that the material used for the third part has a void that can remove milk fat globules having a diameter of 1 to 10 μm contained in the milk. The third part needs to be arranged on the upstream side of the second part made of a porous membrane having a pore size of several 10 to several 100 nm, and is a position where the sample solution can first contact and pass through. It is preferable that the first portion is disposed on the upstream side.

  The space | gap which a 3rd part has should just be a magnitude | size which can isolate | separate a milk fat globule, Preferably holding | maintenance particle | grain size is 0.1-10 micrometers, More preferably, it is 1-3.5 micrometers. The material is not particularly limited as long as it has voids in the above range, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, cotton or the like.

  For the third part, only a material having a specific retention particle size may be used, and in order to increase the separation efficiency of milk fat globules, particles having a large retention particle size may be used in a stepwise manner. .

  The first part holds the labeled first antibody against the specific substance or the labeled specific substance. In the case where the first antibody labeled with the specific substance is retained in the first part, the specific substance can be detected by a sandwich assay method. In addition, when the specific substance labeled in the first portion is retained, the specific substance can be detected by the competition method. In the present invention, since the sandwich assay method in which detection sensitivity is high and positive and an antibody detection line appears is preferable, the first portion should contain the first antibody labeled with a specific substance. Is preferred.

  When a first antibody labeled with a specific substance is held in the first part, two types of antibodies are used: a first antibody against the specific substance and a second antibody against the specific substance. The first antibody and the second antibody are antibodies that can simultaneously bind to the specific substance so that the specific substance can be detected by sandwich assay, and the first antibody recognizes the first antibody. The epitope of the specific substance is preferably different from the epitope of the specific substance recognized by the second antibody.

  In the present invention, in order to obtain a detectable signal, the first antibody or specific substance held in the first portion is labeled. Examples of the label used in the present invention include colored particles, enzymes, radioisotopes, and the like, but it is preferable to use colored particles that can be detected visually without the need for special equipment. Examples of the colored particles include metal fine particles such as gold and platinum, non-metal particles, and latex particles, but are not limited thereto. The colored particles may have any size as long as it can be transported downstream through the voids of the test piece, but the diameter is preferably 1 nm to 10 μm. More preferably, it is 5 nm to 1 μm, and further preferably 10 nm to 100 nm.

  The specific substance measured in the present invention may be any substance that can be measured by immunochromatography, but is preferably a bacterial component or a substance secreted by bacteria. More preferably, it is a bacterial L7 / L12 ribosomal protein. The L7 / L12 ribosomal protein has high detection sensitivity due to the presence of multiple copies in the cell. Further, as shown in the following examples, an antibody capable of distinguishing a specific bacterium causing mastitis from another bacterium by species or genus can be actually obtained by a known method described below.

  The above antibody can be prepared by the method described in International Publication No. 00/06603 pamphlet. When the bacterial ribosomal protein L7 / L12 is used as an antigen, it can be prepared using the full-length protein of the bacterial ribosomal protein L7 / L12 or a partial peptide thereof as the antigen, but is preferably a full-length protein. Including this partial peptide or full-length protein as it is or after cross-linking with a carrier protein, inoculate an animal with an adjuvant if necessary, and collect an antibody (polyclonal antibody) that recognizes Ribosomal Protein L7 / L12 protein by collecting its serum Antisera can be obtained. Further, the antibody can be purified from the antiserum and used. The animals to be inoculated include sheep, horses, goats, rabbits, mice, rats and the like, and sheep, rabbits and the like are particularly preferable for producing polyclonal antibodies. Further, as the antibody, it is more preferable to apply a monoclonal antibody obtained by a known method for producing a hybridoma cell. In this case, a mouse is preferable. As the monoclonal antibody, a monoclonal antibody that reacts with ribosomal protein L7 / L12 of a specific bacterium that causes mastitis and does not react with ribosomal protein L7 / L12 of a bacterium that causes mastitis other than the specific bacteria. By screening, it can be used for diagnosis of whether or not the bacterium has an infection.

  The antibody reacts with a component of a specific bacterium causing mastitis or a substance secreted by the bacterium, and does not react with a component of a bacterium causing mastitis other than the specific bacterium or a substance secreted by the bacterium. As long as it is a monoclonal antibody, an antibody other than ribosomal protein L7 / L12 can be used.

  In this invention, it is good also as an immunochromatograph apparatus using the test piece mentioned above as it is, and accommodating in a case as an immunochromatograph apparatus. In the former case, when the amount of milk serving as a sample is large, it is preferable that one end of the test piece is directly immersed in the sample contained in the container. In the latter case, when the amount of milk serving as a sample is small, it is preferable to measure a predetermined amount with a pipette or the like and drop it onto a test piece. The shape of the case in the latter case may be any shape as long as the test piece can be accommodated. The material of the case may be any material, and preferred examples include polypropylene and polycarbonate.

  The immunochromatography apparatus of the present invention can also be provided as a kit in which a container, for example, a microtube, and an additive solution, for example, a solution for lysing bacteria to elute the ribosomal protein L7 / L12 into the solution are combined.

The immunochromatographic method according to the present invention is an immunochromatographic method for detecting a specific substance in milk,
(1) a labeled first antibody against the specific substance or a first part in which the labeled specific substance is retained, a second antibody linked downstream of the first part and against the specific substance For a test strip having an immobilized second part and a third part connected to the first part or upstream of the second part and having a cavity capable of removing milk fat globules in the milk, Contacting the milk with the third portion or upstream thereof; and (2) flowing the milk to the second portion or downstream thereof, and detecting with the label at the second portion or downstream thereof. Obtaining possible signals:
It is a method including.

  The milk is brought into contact with the sample addition member located in the third part or upstream thereof as it is or as a mixed solution with an additive solution added. When the specific substance is a substance present in bacterial cells, the surfactant component is contained in the additive solution, or the surfactant component is retained upstream of the first part, Bacteria can be lysed. The other component of the additive solution is not particularly limited as long as it does not inhibit the antigen-antibody reaction, but a suitable buffer solution (for example, MOPSO) can be used.

  The antigen-antibody reaction is performed by sandwich assay using “labeled first antibody against a specific substance” retained in the first part and “second antibody against the specific substance” immobilized on the second part. It can be detected by the method. Alternatively, the antigen-antibody reaction may be detected by a competition method using a labeled specific substance held in the first part and an antibody against the specific substance immobilized in the second part. However, in the present invention, a sandwich assay method in which the detection sensitivity is high and the antibody detection line appears positive is preferable.

  The immunochromatography method has a lateral flow type that utilizes the fact that the test piece is placed in the horizontal direction and the liquid moves by capillary action, and a flow-through that is arranged in the vertical direction and passes the liquid from top to bottom mainly by gravity. Broadly divided into types. In the present invention, either a lateral flow type or a flow-through type may be used, but a lateral flow type is more preferable.

  The following examples further illustrate the invention, but the invention is not limited by the examples.

Example 1: Confirmation of liquid flow of milk sample by immunochromatography method (1) Production of immunochromatography apparatus An immunochromatography apparatus shown in a schematic cross-sectional view in Fig. 1 was prepared as follows.
(A) Production of ribosomal protein L7 / L12 antibody Staphylococcus aureus (Staphylococcus aureus) ribosomal protein L7 / L12 monoclonal antibody was used as the colloidal gold labeled antibody. According to the method described in Example 5 of International Publication WO00 / 06603, an L7 / L12 ribosomal protein of Staphylococcus aureus was obtained, and a monoclonal antibody was produced using the protein. As the monoclonal antibody, a combination of two kinds (SA-1 and SA-2) capable of simultaneously binding to another site of the L7 / L12 ribosomal protein was selected.

  Monoclonal antibodies SA-1 and SA-2 react with ribosomal protein L7 / L12 of S. aureus and do not react with ribosomal protein L7 / L12 of bacteria that cause mastitis other than S. aureus. As confirmed.

10 μg / ml monoclonal antibody SA-1 and 100 μl of PBS solution were dispensed into a 96-well ELISA plate (Nunc Maxsorp ELISA plate) and adsorbed overnight at 4 ° C. After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, it was washed several times with a washing solution (0.02% Tween20, PBS), and various bacteria shown in Table 1 (about 1 × 10 ⁸ cells / ml) diluted 10-fold with 0.5% TritonX-100, PBS 100 μl was added and reacted at room temperature for 1 hour. Further, after removing the supernatant, the peroxidase-labeled monoclonal antibody SA-2 was diluted with 0.02% Tween20 and PBS to a final concentration of 1 μg / ml, added 100 μl each, and allowed to react at room temperature for 1 hour. After removing the supernatant and further washing with washing solution several times, add 100 µl of TMB solution (KPL), react at room temperature for 10 minutes, add 100 µl of 1 mol / l hydrochloric acid, stop the reaction, and then measure the absorbance at 450 nm The reactivity was evaluated by the difference from the negative control (solution excluding bacteria) signal. The results are shown in Table 1. Here, positive (+) is at least 0.5 or more difference in absorbance between ELISA and negative control, and negative (−) is 0.1 or less difference in absorbance between negative and negative control.

(B) Colloidal gold label impregnated member
BBInternational's colloidal gold solution (particle size 60 nm) 0.9 mL is mixed with 0.1 M potassium phosphate pH 7.5, added with gold colloid-labeled monoclonal antibody SA-2 100 μg / mL, and allowed to stand at room temperature for 5 minutes. After binding to the gold colloid particle surface, add 10% bovine serum albumin (BSA) aqueous solution so that the final concentration in the gold colloid solution is 1%, and block the remaining surface of the gold colloid particles with BSA, A colloidal gold labeled monoclonal antibody SA-2 (hereinafter referred to as “gold colloid labeled antibody”) solution was prepared. This solution was centrifuged (15000 × rpm, 5 minutes) to precipitate the colloidal gold labeled antibody, and the supernatant was removed to obtain a colloidal gold labeled antibody. The colloidal gold labeled antibody was suspended in 20 mm Tris-HCl buffer (pH 8.2) containing 0.25% BSA, 2.5% sucrose and 35 mm NaCl to obtain a colloidal gold labeled antibody solution. A 10 mm × 300 mm belt-shaped glass fiber pad was impregnated with 2 mL of the colloidal gold labeled antibody solution, which was dried under reduced pressure at room temperature to obtain a colloidal gold labeled antibody impregnated member 1 (first part).

(C) Capture site of complex of antigen and colloidal gold labeled antibody A nitrocellulose membrane having a width of 25 mm and a length of 300 mm was prepared as a membrane carrier 2 (second part) for chromatographic development of a chromatographic medium.

  A solution containing the monoclonal antibody SA-1 1.5 mg / mL was applied in a line at 1 μL / cm at a position 10 mm from the end of the chromatographic development start side of the membrane carrier 2 for chromatographic development. It was dried at 50 ° C. for 30 minutes, then immersed in 0.5% sucrose solution for 30 minutes and dried overnight at room temperature. Staphylococcus aureus ribosomal protein It was designated as capture site 3 for the complex of L7 / L12 antigen and colloidal gold-labeled antibody.

(D) Production of immunochromatography apparatus In addition to the labeled antibody-impregnated member 1 and the membrane carrier 2 for chromatographic development, a cotton cloth was prepared as a sample addition member (third portion) 4 and a filter paper was prepared as an absorption member 5. And after bonding these members to the base material 6 (thickness 254 μm, made of polystyrene and having an adhesive for bonding the members), it was cut to a width of 5 mm, and a cross-sectional view is shown in FIG. An immunochromatographic apparatus was created. The member shown in Table 2 was used for the sample addition member 4.

(2) Test The measurement of cow's milk using an immunochromatographic apparatus was performed by taking 100 μl of a raw milk sample in which S. aureus was detected in a microtube and adding the solution (final concentration 1% Tween 20, 0.05 M NaCl, 0.1 M). 150 μl of MOPSO pH 7.5) was added and mixed. The immunochromatograph obtained in (1) above was immersed in the mixed solution from the sample addition member 4 and chromatographed by the lateral flow method, and it was examined whether the liquid would flow to the absorption member 5.

  The results are shown in Table 3. The liquid flowed to the absorbing member 5 only in the member having a retained particle diameter of 3.5 μm. Otherwise, the liquid flow stopped in the middle of the chromatographic development membrane carrier 2.

Example 2 Detection of Staphylococcus aureus in Staphylococcus aureus in Milk by Immunochromatography Method (1) Production of Immunochromatography Device An immunochromatography device was prepared by the method described in Example 1. GF / DVA described in Table 2 was used for the sample addition member 4.

(2) Test For measuring milk milk using an immunochromatograph apparatus, 100 μl of raw milk samples 1 to 6 are collected in a microtube and added (final concentration 1% Tween 20, 0.05 M NaCl, 0.1 M MOPSO pH 7.5). 150 μl was added and mixed. The immunochromatograph apparatus is immersed in the mixed solution from the sample addition member 4 and allowed to stand at room temperature for 15 minutes in a lateral flow manner for development. Then, the ribosomal protein L7 / L12 antigen supplemented at the supplementary site 3 and gold The presence or absence of capture of the complex with the colloid-labeled antibody was judged visually by the presence or absence of a red-purple line that increased or decreased in proportion to the capture amount.

  On the other hand, in order to confirm the presence or absence of Staphylococcus aureus in milk, detection was performed by a culture method. Appeared after 100 μl of milk was seeded on Trypticase Soi II 5% sheep blood agar medium (Nippon Becton Decktonson) and Staphylococcus aureus selection medium X-SA agar medium (Nissui), and cultured for 24 hours at 37 ° C. Colonies were confirmed.

  The visual judgment results and the culture results are shown in Table 4.

  Lines did not appear in 2 specimens where no bacteria were detected in the milk. In addition, no line appeared for two specimens in which streptococci were detected. On the other hand, a line appeared for the specimen in which Staphylococcus aureus was detected, and S. aureus could be detected.

  As described above, by using the immunochromatography apparatus of the present invention, it is possible to develop a milk sample without causing clogging of the milk and accurately measure the milk in which milk fat globules are present. .

  The present invention can be used for diagnosis of infectious diseases in livestock.

1. Labeled antibody impregnated member (first part)
2. Chromatographic membrane support (second part)
3. Capture site 4. 4. Sample addition member 5. Absorbing member Base material 7. Fat globule removal member (third part)

Claims (10)

An immunochromatographic method for detecting specific substances in milk,
(1) a labeled first antibody against the specific substance or a first part in which the labeled specific substance is retained, a second antibody linked downstream of the first part and against the specific substance For a test strip having an immobilized second part and a third part connected to the first part or upstream of the second part and having a cavity capable of removing milk fat globules in the milk, Contacting the milk with the third portion or upstream thereof; and (2) flowing the milk to the second portion or downstream thereof, and detecting with the label at the second portion or downstream thereof. Obtaining possible signals:
Including the above method. The method according to claim 1, wherein the retained particle size of the voids in the third portion is 1 to 3.5 μm. The method according to claim 1 or 2, wherein the immunochromatographic method is a lateral flow type. The method according to any one of claims 1 to 3, wherein a labeled first antibody against the specific substance is retained in the first portion. The method according to any one of claims 1 to 4, wherein the specific substance is a bacterial component or a substance secreted by the bacteria. The method according to claim 5, wherein the specific substance is ribosomal protein L7 / L12. The method according to claim 6, wherein the first antibody and the second antibody are monoclonal antibodies against ribosomal protein L7 / L12. Bacteria causing mastitis other than the specific bacteria, wherein the first antibody and the second antibody react with a component of a specific species or genus bacterium causing mastitis or a substance secreted by the bacterium. The method according to claim 5, wherein the antibody is a monoclonal antibody that does not react with any of the above components or a substance secreted by the bacterium. The monoclonal antibody reacts with ribosomal protein L7 / L12 of bacteria of a specific species or genus that causes mastitis, and does not react with ribosomal protein L7 / L12 of bacteria that cause mastitis other than the specific bacteria. The method according to claim 8, which is a monoclonal antibody. A first antibody labeled with the specific substance or a first part holding the labeled specific substance, and a second antibody linked to the downstream of the first part and immobilized with the specific substance are immobilized. A test piece having a second part and a third part connected upstream of the first part or the second part and having a void capable of removing milk fat globules in the milk. An immunochromatographic device for detecting substances.

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