JP6387063B2 - Method for detecting specific substances in milk - Google Patents

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 immobilizes a test paper containing a first antibody specific to a substance to be measured (antigen) labeled with colored particles such as colloidal gold and a second antibody that captures the substance to be measured. A test piece connected to a porous membrane is used. When a specimen containing a measurement target substance is dropped, the measurement target substance binds to an antibody labeled with colored particles and / or an antibody immobilized on a porous membrane, and a line or the like that can be visually discerned 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.

  Patent Document 1 teaches that the above method can be applied to blood (whole blood), plasma, serum, urine, saliva, sputum, sweat, etc., but the above method is applied to the milk of livestock. There is no teaching, and there is no teaching or suggestion of problems that arise in its application. Further, methods for applying the immunochromatography method using biological samples such as blood (whole blood) as specimens are also disclosed in Patent Documents 2 to 4, but the immunochromatography method is also applied to livestock milk in these patent documents. There is no suggestion or teaching to do.

  As a simple measuring device that can quickly and easily measure on the dairy farm by using immunochromatographic method using livestock milk as a specimen, Patent Document 5 describes an immunochromatographic method for the purpose of examining the causative bacteria of livestock mastitis using milk as a specimen. A method of using is proposed. This document teaches that milk fat globules and casein contained in milk inhibit detection by immunochromatography, and that it is preferable to remove them prior to testing. This document discloses a technique in which milk is allowed to stand at the time of removing milk fat globules and casein, and the cream layer is separated and treated with a surfactant. However, the milk fat globules are removed using a filter. There is no teaching.

  In applying the immunochromatography method, methods for removing contaminants with a physical filter or a film having chemical affinity are taught in Patent Documents 2 to 4, but as described above, these techniques are described in these documents. This method is a method of applying the immunochromatography method to a biological sample such as whole blood, and not a method of applying it to livestock milk. In Patent Documents 1 to 4, there is no teaching or suggestion about problems that occur when the immunochromatography method is applied to the milk of livestock, and there is no teaching at all about means for solving the problems.

JP-A-1-244370 Special table 2003-512624 gazette Japanese National Patent Publication No. 11-505327 JP 2002-214236 A International Publication WO02 / 075310 Pamphlet

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 and developed in the test piece of the immunochromatography apparatus in which the porous membrane was connected. It was thought that this was because the liquid did not flow sufficiently. 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. There are many milk fat globules with a particle size of around 1 to 10 μm in milk immediately after milking (in commercially available milk etc., the milk fat globules in raw milk are homogenized to reduce the particle size to 1 μm or less. However, milk fat globules with a wide range of particle sizes are present in milk that has not been homogenized.) Since the particle size distribution of milk fat globules is much wider than that of red blood cells, It was thought that measurement would be difficult due to clogging.

As a result of intensive studies to solve the above problems, the present inventor has identified a part of milk fat globules in milk by size processing on the upstream side of the test piece used in the immunochromatography method, thereby identifying the milk. The present inventors have found that it is possible to measure this substance by immunochromatography, 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 immunochromatographic method is a lateral flow type.
[3] The method according to [1] or [2], wherein the first part retains a labeled first antibody against the specific substance.
[4] The method according to any one of [1] to [3], wherein the specific substance is a bacterial component or a substance secreted by the bacteria.
[5] The method according to any one of [1] to [4], wherein at least one of the first antibody and the second antibody is a monoclonal antibody.

[6] The method according to [5], wherein each of the first antibody and the second antibody is a monoclonal antibody.

[7] The method according to any one of [1] to [6], wherein the retained particle size of the voids in the third portion is 1 to 3.5 μm.
[8] The method according to any one of [1] to [7], wherein the third part is constituted by two or more members having voids capable of removing milk fat globules having different particle sizes.
[9] The third portion is configured by the first member disposed downstream and the second member disposed upstream, and the retained particle size of the second member is larger than the retained particle size of the first member. The method according to [8].
[10] The method according to [9], wherein the retained particle size of the first member is 1.0 to 2.0 μm, and the retained particle size of the second member is 3.0 to 3.5 μm.

[11] The method according to any one of [1] to [10], including a step of flowing the milk that has been treated with the lytic enzyme to the second portion or the downstream portion thereof.
[12] The method according to [11], wherein the lytic enzyme is an autolytic enzyme.
[13] The method according to [11], wherein staphylococci in the milk are detected using lysostaphin as a lytic enzyme.
[14] A method for diagnosing whether a causative bacterium of mastitis in livestock is staphylococci,
Obtaining milk containing causative bacteria from livestock suffering from mastitis, releasing specific substances present in the causative bacteria by mixing a lytic enzyme in the milk, and the identification A diagnostic method comprising a step of measuring the presence or amount of an external specific substance by an immunological method using an antibody whose substance is an antigen.
[15] The method according to [14], wherein lysostaphin is used as a lytic enzyme to diagnose whether the causative bacterium is staphylococci.
[16] A labeled first antibody against the specific substance or a first part in which the labeled specific substance is held, 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.
[17] The immunochromatographic apparatus according to [16], wherein a lytic enzyme or surfactant is held in the first part or the second part [18] an additive solution containing the lytic enzyme or surfactant, and [16] A detection kit comprising the immunochromatography apparatus described.

  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., and a cream layer separation process is not necessary. It is possible to quickly identify the causative bacteria before the worsening, and 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 Example 1 is shown. 1: Labeled antibody-impregnated member (first part), 2: Chromatographic development membrane carrier (second part), 3: Capture site, 4: Sample addition member, 5: Absorption member, 6: Substrate, and 7 : Fat globule removal member (third part) 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. The detection sensitivity improvement effect at the time of using an autolytic enzyme is shown. The measurement result by ELISA of Staphylococcus aureus added to milk using achromopeptidase and lysostaphin is shown. The cross-sectional schematic diagram of the test piece of the immunochromatography apparatus used in Example 6 is shown. 1: Labeled antibody-impregnated part (first part) 2: Chromatographic development membrane carrier (second part) 3: Supplementary part 4: Sample addition member 5: Absorption member 6: Substrate The result of having detected Staphylococcus aureus in milk by immunochromatography using lysostaphin as a lytic enzyme 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. The retained particle size is the particle size of milk fat globules that are retained in the third portion without allowing milk fat globules having a particle size larger than that size to pass through the void, and the average pore size of the voids in the third portion However, milk fat globules having a particle size equal to or larger than 50%, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more. % Or more, and most preferably 98% or more cannot be passed through the gap and is held in the third portion. The percentage of milk fat globules retained can be measured by methods well known to those skilled in the art. For example, GF / B provided by GE Healthcare Biosciences has been shown in the product catalog to have a retained particle size of 1.0 μm. It is possible to confirm.

  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. . In the case where the third part is composed of two or more members having voids that can remove milk fat globules of different particle sizes, it is a preferred aspect of the present invention, and the third part is a first part disposed downstream. It is a further preferred embodiment of the present invention when it is constituted by a member and a second member disposed upstream, and the retained particle size of the second member is larger than the retained particle size of the first member. When the third portion is constituted by two types of members, the holding particle size of the first member disposed downstream is 1.0 to 2.0 μm, and the second member disposed upstream is retained. The particle size is preferably 3.0 to 3.5 μm. In order to detect a specific substance with high sensitivity from milk containing milk fat globules having a high concentration and a wide particle size distribution, preferably undiluted milk after milking, the third part is composed of a member having a small retaining particle size and retaining particles. It is preferable to configure a combination with a member having a large size.

  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 kind of bacteria is not particularly limited, and may be either a gram positive bacterium or a gram negative bacterium. Examples include staphylococci (bacteria belonging to the genus Staphylococcus), preferably Gram-positive bacteria such as Staphylococcus aureus, or bacteria belonging to the genus Escherichia coli and Klebsiella, but are not limited thereto. .

  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 it is preferable to prepare the full-length protein as the antigen. This partial peptide or full-length protein is directly or after cross-linking with a carrier protein, and then inoculated into an animal together with an adjuvant as necessary, and an anti-recognition containing an antibody (polyclonal antibody) that recognizes the L7 / L12 ribosomal protein by recovering the serum. Serum 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, even an antibody that recognizes other than ribosomal protein L7 / L12 as an antigen can be used.

  As the monoclonal antibody, it is preferable to use a monoclonal antibody in which the antigen-antibody reaction is not inhibited by a contaminant other than the specific substance contained in the milk. For example, milk contains a large amount of protein such as casein, which may inhibit the reaction between a specific substance and a monoclonal antibody. When a monoclonal antibody against a specific substance is produced by a conventional method, for example, it may be preferable to appropriately select and use a monoclonal antibody in which the antigen-antibody reaction is not inhibited by casein or the like, or a monoclonal antibody that has little influence on the antigen-antibody reaction. is there. After manufacturing a monoclonal antibody that specifically reacts with an antigen according to a conventional method, such a monoclonal antibody is examined whether the antigen-antibody reaction is inhibited in the presence of contaminants such as casein, It can be easily obtained by selecting a monoclonal antibody that does not substantially inhibit the reaction.

  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 is a combination of a container, for example, a microtube, and an additive solution, for example, an additive solution containing a lytic enzyme or a surfactant for lysing bacteria and eluting the ribosomal protein L7 / L12 into the solution. It can also be provided as a kit.

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 added directly to the milk, or is added to the additive solution, or upstream from the second part, preferably the first part. The bacterium can be lysed by holding the surfactant component upstream of the portion (see JP-A-61-111464 for lysis treatment with a surfactant). You may combine these methods suitably. 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.

  In another embodiment, the lytic enzyme is added directly to the milk, or contained in the additive solution, or is immobilized upstream of the second part, preferably upstream of the first part, The milk that the specific substance contained in the bacterial body is exposed outside the bacterial body through the treatment with the lytic enzyme can flow to the second part or the downstream part thereof, and the specific substance existing in the bacterial cell is highly sensitive. Can be detected. You may combine these methods suitably. In a preferred embodiment, the lytic enzyme can be added directly to the milk and / or contained in the additive solution, and in a particularly preferred embodiment, the additive solution containing the lytic enzyme can be added to the milk.

  The type of lytic enzyme is not particularly limited, and any two or more lytic enzymes may be used in appropriate combination. Escherichia coli, a bacterium belonging to the genus Klebsiella, or Staphylococcus aureus as a mastitis-causing fungus. It is also preferable to use one or a combination of two or more enzymes. For example, one or more lytic enzymes selected from lysozyme, lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, β-N-acetylglucosaminidase and the like can be used. For example, a method using lysozyme and a cell membrane lysing agent as a lytic enzyme has been proposed (Japanese Patent Laid-Open No. 63-167799).

  As the lytic enzyme, an autolytic enzyme having a high lytic effect is preferred (see FIG. 4). Autolytic enzyme refers to an enzyme that is produced by bacteria and that melts itself. The details of why such autolysis occurs are unknown, but it is thought to play a role in dissolving and separating the cells when the cells grow and divide. Known bacterial autolytic enzymes include lysostaphin, acetylglucotaminidase, achromopeptidase, and the like.

  When Staphylococcus aureus is particularly suspected as a causative agent of mastitis, the lysis rate of Staphylococcus aureus is low because of the surfactant lysis. It may be preferred to use the lysostaphin that exerts alone or in combination with other lytic enzymes. A method for lysing Staphylococcus aureus with lysostaphin is disclosed in JP-A-11-28099, and those skilled in the art can easily obtain lysostaphin as a lytic enzyme. The entire disclosure of the above patent publication is incorporated herein by reference.

  Natural lysostaphin is a zinc protease produced by Staphylococcus simulans and is known to lyse these bacteria by hydrolyzing the glycylglycine bond in the glycopeptide chain of the cell wall peptidoglycan of Staphylococcus aureus or its congeners. It has been. In the present specification, the term lysostaphin includes, in addition to the above-mentioned natural lysostaphin, addition, deletion, and / or addition to one or more amino acids in the amino acid sequence of natural lysostaphin, as long as the hydrolysis activity is not lost. Mutant lysostaphin into which mutations such as substitution have been introduced is included. In addition, modified lysostaphin in which other compounds such as saccharides and polyethylene glycol are bound to the natural lysostaphin or mutant lysostaphin is also included as long as the hydrolysis activity is not lost. These lysostaphins can be obtained by the culture method or genetic engineering method described in JP-A No. 11-28099, or can be obtained by purchasing commercially available products. When lysostaphin is used as a lytic enzyme, for example, an antigen comprising the ribosome L7 / L12 protein of Staphylococcus aureus can be detected by an immunochromatographic method, or together with or in place of the immunochromatographic method. It is also possible to detect the above antigens by a physiologic measurement method. Examples of immunological measurement methods other than immunochromatography include agglutination reaction method, enzyme immunoassay method (ELISA method), radioimmunoassay method (RIA method), or fluorescent immunoassay method (FIA method). However, it is not limited to these.

  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 produced 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.

  The combination of monoclonal antibodies SA-1 and SA-2 reacts with S. aureus ribosomal protein L7 / L12 and does not react with bacterial ribosomal proteins L7 / L12 that cause mastitis other than S. aureus, And it confirmed that it was an antibody which does not receive reaction inhibition by the component of milk as follows.

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.

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, wash it several times with a washing solution (0.02% Tween20, PBS), 20 μl of S. aureus (approximately 1 × 10 ⁸ cells / ml) diluted 10-fold with 0.5% TritonX-100, PBS, and milk (Commercial drinking milk) 80 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 positive control (solution excluding milk) signal, and it was confirmed that there was no decrease in reactivity due to milk.

(B) Colloidal gold label impregnated member
BBInternational's colloidal gold solution (particle size 60nm) 0.9mL is mixed with 0.1M potassium phosphate pH7.5, gold colloid-labeled monoclonal antibody SA-2 is added at 100μg / mL and allowed to stand at room temperature for 5 minutes. After binding 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. Then, 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 1.5 mg / mL of the monoclonal antibody SA-1 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 is used as a member serving as a sample addition member 4 and a fat globule removal member (third portion) 7 A filter paper was prepared as the absorbing 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 developing solution would flow to the absorption member 5.

  The results are shown in Table 3. + Indicates that the developing solution has flowed to the absorbing member 5, and-indicates that it has stopped halfway. The liquid flowed to the absorbing member 5 only in the member having a retained particle size of up to 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 (1) Production of immunochromatographic apparatus The immunochromatographic apparatus shown in Fig. 1 was produced by the method described in Example 1. As a member serving as both the sample addition member 4 and the fat globule removal member (third portion) 7, GF / DVA described in Table 2 was used.

(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 captured at the capture 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. In the culture results, + indicates that a S. aureus colony was observed, and-indicates that it was not observed. Moreover, in the kit determination result, + indicates that a reddish purple line can be visually observed, and − indicates that it cannot be visually observed.

  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. .

Example 3: Measurement of high-concentration milk by immunochromatography The immunochromatography apparatus shown in Fig. 1 was prepared by the method described in Example 1, and the liquid flow when the ratio of milk was changed was confirmed. The sample addition members used were those listed in Table 3. Mix raw milk sample and additive solution at different ratios (final concentration of additive solution 1% Tween20, 0.05M NaCl, 0.1M MOPSO pH7.5, total solution volume 250μl), and immerse immunochromatograph device from sample addition member Chromatographic development was performed using the lateral flow method, and it was examined whether or not the entire liquid was developed.

  The results are shown in Table 5. + Indicates that the developing solution has flowed to the absorbing member 5, and-indicates that it has stopped halfway. When the milk ratio was 50%, the liquid flowed all the way to the absorption member 5 with the sample addition member other than VF1. When the milk ratio is 70%, the liquid flow stops in the middle of the membrane carrier 2 for chromatographic development regardless of which sample addition member is used, or even when the absorption member 5 is reached. As a result, it was impossible to measure correctly.

  Next, two pieces of sample addition members were overlapped, and the same examination was performed. As shown in FIG. 3, two sample addition members were overlapped. GF / AVA and GF / DVA were used as members for sample addition. GF / DVA was placed upstream (sample addition member 4) and GF / AVA was placed downstream (fat globule removal member 7), and the length used was examined. The milk ratio was 80% and the liquid volume was 250 μl. The results are shown in Table 6 (refer to FIG. 3 for “length” and “overlap” in the table). + Indicates that the developing solution has flowed to the absorbing member 5. Even when the milk ratio was 80%, the liquid flow could not be stopped and the measurement could be performed correctly. Furthermore, it was possible to measure correctly even if the lengths of the two sample addition members were changed.

  As described above, by using a combination of two types of sample addition members with different retention particle sizes, milk fat globules can be removed even when high-concentration milk is used, and the milk sample without clogging the membrane It has become possible to measure with high sensitivity and accuracy.

Example 4: Effect of lytic enzyme in detection of Staphylococcus aureus by enzyme immunoassay (ELISA) (a) Preparation of monoclonal antibody against ribosomal protein L7 / L12 Monoclonal antibodies SA-1 and SA-2 described in Example 1 It was used.
(B) Confirmation of effect of lytic enzyme by ELISA The effect of lytic enzyme was confirmed as follows.
S. aureus was adjusted with 10 mM Tris-HCl (pH 8.0) using the surfactants or lytic enzymes shown in Table 7, treated at 37 ° C. for 20 minutes, and then the same ELISA as in Example 1 (a). Absorbance was measured at. Staphylococcus aureus was adjusted with physiological saline, and the final concentration was 2.5 × 10 ⁵ (cells / ml). As a negative control, phosphate physiological buffer solution (PBS) was used. As a positive control, a nonionic surfactant Triton X-100 was used. The final concentration was 1% and treated overnight at room temperature.

The results are shown in FIG.
The absorbance of the surfactant and any of the lytic enzymes was higher than that of PBS used as a negative control. Compared with the surfactant Triton X-100, which is used as a general lysing agent, the absorbance increased approximately twice when the autolytic enzymes achromopeptidase and lysostaphin were used.

Example 5: Effect of lytic enzyme using milk Using achromopeptidase (Wako Pure Chemical Industries) and lysostaphin (Wako Pure Chemical Industries) which showed high lytic effect in Example 4, they were added to milk. Measurement by Staphylococcus aureus ELISA was performed. For milk milk, commercially available drinks are used, and the ratio is 0 to 80% (the balance is 10 mM Tris-HCl (pH 8.0)). Staphylococcus aureus is adjusted with physiological saline to a final concentration of 1 × 10 ^5. (Cells / ml). Achromopeptidase and lysostaphin were prepared using 10 mM Tris-HCl (pH 8.0) and mixed with the S. aureus solution to a final concentration of 5 (μg / ml). The measurement by ELISA was in accordance with Example 4.

  The results are shown in FIG. When achromopeptidase was used, the absorbance decreased as the milk ratio increased. On the other hand, when lysostaphin was used, the absorbance was constant regardless of the proportion of milk, and achromopeptidase did not show a sufficient effect in the presence of a high concentration of milk.

Example 6: Detection of Staphylococcus aureus in milk by immunochromatography (1) Production of immunochromatography apparatus An immunochromatography apparatus was produced as follows.
(A) Gold colloid label impregnated member The gold colloid label impregnated member described in Example 1 was used.
(B) Capture site of complex of antigen and colloidal gold labeled antibody It was prepared in the same manner as in Example 1.

(C) Production of immunochromatography apparatus In addition to the labeled antibody-impregnated member 1 and the membrane carrier 2 for chromatographic development, GF / Filter paper was prepared as DVA (manufactured by GE Healthcare Bioscience: a filter member having a thickness of 776 μm and a holding particle size of 3.5 μm made of glass fiber) and the absorbing 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 FIG. 6 shows a cross-sectional view. An immunochromatographic apparatus was produced.

(2) Test Milk milk was measured with an immunochromatograph apparatus as follows.
100 μl of milk containing a known amount of Staphylococcus aureus added to a microtube, 150 μl of an additive solution (final concentration 1% Triton X-100, 5 μg / ml lysostaphin, 0.1 M MOPSO pH 7.5) is added and mixed at room temperature did. Commercial milk drinks were used for milk juice. For comparison, an additive solution not mixed with lysostaphin (final concentration: 1% Triton X-100, 0.1 M MOPSO pH 7.5) was also used for the study. The immunochromatograph apparatus is immersed in the mixed solution from the sample addition member 4 and allowed to stand at room temperature for 30 minutes in a lateral flow manner for development. Then, the ribosomal protein L7 / L12 antigen supplemented at the supplementary site 3 and gold A reddish purple line that increases or decreases the capture of the complex with the colloid-labeled antibody in proportion to the capture amount was quantified by the apparatus FASTKIT immunochromatography reader DiaScan 20-A (manufactured by Becton Decktonson, Japan).

  The results are shown in FIG. When an additive solution with lysostaphin added is used, the curve shifts to a lower concentration side compared to the additive solution with Triton X-100 added instead of lysostaphin, and S. aureus can be detected about 10 times more sensitively. Became clear.

Example 7: Measurement of milk sample by immunochromatographic method An immunochromatographic apparatus was prepared according to Example 6.
100 μl of a raw milk sample in which Staphylococcus aureus was detected was collected in a microtube, and 150 μl of an additive solution was added and mixed at room temperature. Two types of additive solutions described in Example 6 were used. After the measurement according to Example 6, the reddish purple line that appeared was visually judged (positive +, negative−).

  On the other hand, in order to confirm the number of Staphylococcus aureus in milk, it quantified by the culture method. 100 μl of milk was seeded on Trypticase Soy II 5% sheep blood agar medium (Nippon Becton Decktonson) and Staphylococcus aureus selective medium X-SA agar medium (Nissui), and cultured for 24 hours at 37 ° C. Colonies were counted.

Table 8 shows the number of Staphylococcus aureus calculated by the culture method and the determination result by immunochromatography. When lysostaphin was not added, S. aureus could not be detected at 1 × 10 ⁴ (cfu / ml) or less, but by adding lysostaphin, it was possible to detect up to 5 × 10 ³ .

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

Claims (2)

A method for diagnosing whether the causative agent of bovine mastitis is staphylococci,
A step of obtaining milk containing a causative bacterium from a cow suffering from mastitis, a step of releasing a protein present in the causative microbial cell body by mixing a lytic enzyme and Triton X-100 in the milk, And measuring the presence or absence of the protein by an immunological method using an antibody having the protein as an antigen,
A diagnostic method wherein the lytic enzyme is lysostaphin. The immunological method is a method selected from the group consisting of an agglutination reaction method, an enzyme immunoassay method (ELISA method), a radioimmunoassay method (RIA method), and a fluorescent immunoassay method (FIA method). The diagnostic method according to claim 1 .

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