JP4554472B2 - Parvovirus antigen detection kit - Google Patents

Description

  The present invention relates to an immunodetection method for detecting a parvovirus antigen, which is a causative virus of animals such as dogs and cats, with high sensitivity and specificity, in particular, a sandwich immunoassay method, particularly immunochromatography. The present invention relates to a measurement method and an immunochromatographic test strip, and more particularly to a detection method useful for diagnosing parvovirus infection quickly and easily.

In the late 1970s, unexplained infections with dehydration such as severe vomiting and diarrhea attacked dogs around the world. This is the canine parvovirus was the beginning of the spread of infectious diseases by (hereinafter sometimes referred to as a "CPV" (C anine P arvo v irus stands for)).

  Later, the causative virus was identified, and the number of cases of canine parvovirus infection decreased with the spread of vaccination. However, puppies and old dogs that have not been vaccinated and have weak physical strength and immunity often die from infection with a dog parvovirus.

In addition, the cat of parvovirus infection (feline panleukopenia) are also known, this infection, feline panleukopenia virus (hereinafter referred to as "FPLV" (F eline p an l eukopenia v irus stands for) It is specified that it is based on the above. This infectious disease is sometimes called “cat infectious enteritis” or “cat distemper” because of clinical symptoms, all of which mean this disease. Symptoms are stillbirth when pregnant cats are infected like dog parvovirus, infection of cats older than 2 to 3 months of age, and organs with active cell division such as bone marrow and intestinal mucosa are targeted and leukopenia and diarrhea occur, Fever, appetite, loss of energy, dehydration, vomiting, bloody stool, etc. are characteristic.

  Dog parvovirus and cat parvovirus are antigenically related, and it has recently been pointed out that cats are infected with 2a or 2b serotype of CPV-2.

  Diagnosis of canine parvovirus infection has traditionally been done by testing dog parvovirus antigens at veterinary hospitals or by collecting blood from dogs under a veterinarian and entrusting a test to a specialized laboratory. All of them required time for examinations and expensive treatment costs.

  In recent years, canine parvovirus test kits employing immunochromatography have become commercially available, but are limited to those using polyclonal antibodies or IgG monoclonal antibodies, and further improvements in detection sensitivity are desired. Yes.

Takeshi Sanekata, Tatsuya Sugimoto, Shinobu Ueda, Misao, Tsubokura, Yoshihisa Yamane and Megumi Senda, `` Latex agglutination test for canine parvovirus '', Australian Veterinary Journal, Vol. 73, No. 6, June 1996. Rimmelzwaan GF, Groen J, Juntti N, Teppema JS, UytdeHaag FG, Osterhaus AD., `` Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies '', J. Virol.Methods, 1987, Mar; 15 (4) : 313-22. A Paul Reed, Elaine V. Jones and Timothy J. Miller, “Nucleotide Sequence and Genome Organization of Canine Parvovirus”, Journal of Virology, Jan. 1988, 266-276. JP 2004-219346 A Japanese Patent Laid-Open No. 11-311625 Japanese Laid-Open Patent Publication No. 8-136545

  Due to the recent trend of the pet boom, many people have kept animals, but the owners are burdened with expensive treatment costs, and viral infections can spread rapidly over a wide area if discovery is delayed Therefore, a rapid, simple and highly accurate detection method is desired for early detection and prevention of such viral infections.

  An object of the present invention is to provide a method and a measuring instrument that can detect parvovirus that infects animals such as dogs and cats with high sensitivity, specificity, and speed and convenience.

  The inventors have succeeded in obtaining an IgM monoclonal antibody against canine parvovirus, and using it in immunoassays, particularly sandwich immunoassays, in particular immunochromatographic assays, The present inventors have found that the detection can be performed quickly and easily with sensitivity and specificity, and the present invention has been completed.

  That is, according to one aspect of the present invention, there is provided a method for detecting a parvovirus antigen comprising an immunoassay using an IgM monoclonal antibody against canine parvovirus.

  The immunoassay method in this detection method is not particularly limited, but a sandwich immunoassay method, particularly an ELISA (Enzyme-linked immunosorbent assay) method, an immunochromatographic assay method and the like are preferable. Examples of parvovirus antigens to be detected include parvovirus itself, parvovirus surface proteins and fragments thereof.

  Therefore, according to another aspect of the present invention, there is provided a sandwich immunoassay method using a first antibody and a second antibody against canine parvovirus, wherein the first antibody and the second antibody are used. Provided is a method for detecting a parvovirus antigen, wherein at least one of the antibodies is an IgM monoclonal antibody.

  According to a preferred embodiment of the present invention, a membrane carrier comprising a capture site formed by preliminarily fixing a first antibody against a dog parvovirus in a predetermined position is prepared, and a second carrier against a dog parvovirus is prepared. A mixed solution of the antibody and a predetermined amount of the test sample is chromatographed on the membrane carrier toward the capture site, and the complex of the parvovirus and the second antibody contained in the test sample is captured. An immunochromatographic assay characterized in that it is captured at a site, wherein at least one of the first antibody and the second antibody is an IgM monoclonal antibody. .

  According to a preferred embodiment of the present invention, at least a first antibody against a canine parvovirus, a second antibody, and a membrane carrier are provided, and the first antibody is immobilized in advance on a predetermined position of the membrane carrier. For detection of parvovirus, wherein the second antibody is labeled with an appropriate labeling substance and arranged so as to be chromatographed on the membrane carrier at a position separated from the capture site. There is provided an immunochromatographic test strip for detecting parvovirus, wherein at least one of the first antibody and the second antibody is an IgM monoclonal antibody.

  In the case of a sandwich immunoassay such as an immunochromatography assay, at least one of the first antibody and the second antibody used there may be an IgM monoclonal antibody against canine parvovirus, and the other antibody is a polyclonal antibody. However, from the viewpoint of reaction specificity, it is generally preferable to use both antibodies as IgM monoclonal antibodies.

According to the present invention, since an IgM monoclonal antibody is used in an immunoassay for detecting a parvovirus antigen in dogs, it is more sensitive and specific than an immunoassay using a conventional polyclonal antibody or IgG monoclonal antibody. Viral antigens can be detected. The IgM monoclonal antibody has 10 antigen-binding sites and can bind to more antigens than IgG monoclonal antibodies and the like, so that the sensitivity is improved and non-specific reactions can be prevented. In addition, dog parvovirus and cat parvovirus are all belonging to the genus Parvoviridae, Parvovirinae, and parvovirus, and are closely related antigenically, and it has been reported that dog parvovirus actually infects cats. Thus, the present invention can be used to detect parvovirus antigens that infect cats and other animals in addition to canine parvovirus antigens.
In addition, according to the immunochromatographic measurement method and immunochromatographic test strip of the present invention, parvovirus antigens can be detected easily and quickly with high accuracy at the site of sample collection such as feces without the need for special techniques, equipment and facilities. It becomes possible to diagnose an infection caused by the virus.

In the present invention, each step in the production of an antibody and the detection method and measurement method using the antibody is performed according to a known immunological technique.
In the present invention, the IgM monoclonal antibody was obtained, for example, by immunizing an animal such as a mouse using the extracted and purified canine parvovirus as an antigen, and cell fusion of the spleen cells and myeloma cells of the immunized animal. The fused cells are allowed to grow after selection with HAT-containing medium. Using the canine parvovirus used in the above-mentioned growth, the anti-canine parvovirus antibody-producing strain is selected by, for example, enzyme-labeled immunization. A strain producing an IgM antibody can be easily obtained by collecting spleen cells with high titer obtained in the early stage after immunization and cell fusion.

The immunochromatographic measurement method of the present invention for detecting parvovirus in a test sample can be easily carried out according to the configuration of a known immunochromatographic test strip.
In general, such an immunochromatographic test strip is labeled with a first antibody capable of reacting with an antibody at the first antigenic determinant of the antigen and an antibody antigen reactive with the second antigenic determinant of the antigen. At least a second antibody and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody is separated from the capture site. The membrane carrier is arranged and configured to be chromatographed. At least one of the first antibody and the second antibody may be an IgM monoclonal antibody. Usually, the first antibody and the second antibody are used in a “hetero” combination, that is, the first antibody and the second antibody, each recognizing each antigenic determinant differing in position and structure on the antigen. These antibodies are used in combination. However, the first antigenic determinant and the second antigenic determinant may be structurally the same as long as the positions on the antigen are different. It may be a “homo” combination monoclonal antibody, ie the same IgM monoclonal antibody can be used for both the first antibody and the second antibody.

A specific example of the immunochromatographic test strip is, for example, the test strip shown in FIG. In FIG. 1, numeral 1 is an adhesive sheet, 2 is an impregnation member, 3 is a membrane carrier, 31 is a capture site, 4 is an absorption member, and 5 is a sample addition member.
In the example shown in the figure, the membrane carrier 3 is made of a strip-like nitrocellulose membrane filter having a width of 5 mm and a length of 36 mm.
The first antibody is fixed to the membrane carrier 3 at a position 7.5 mm from the end on the side of the chromatographic expansion start point, and a specimen capture site 31 is formed.
In the illustrated example, the membrane carrier 3 uses a nitrocellulose membrane filter. However, the membrane carrier 3 should be capable of chromatographic development of the specimen contained in the test sample and immobilize the antibody forming the capture site 31. Any cellulose membrane, nylon membrane, glass fiber membrane, etc. can be used.

The impregnated member 2 is a member impregnated with a second antibody that reacts with the antigen with the second antigen determinant located at a site different from the first antigen determinant to which the first antibody binds. Become. The second antibody is previously labeled with an appropriate labeling substance.
In the illustrated example, a 5 mm × 15 mm band-shaped glass fiber nonwoven fabric is used as the impregnating member 2, but is not limited thereto, and examples thereof include cellulose cloth (filter paper, nitrocellulose membrane, etc.), polyethylene, Porous plastic cloths such as polypropylene can also be used.

The labeling substance for the second antibody may be any substance as long as it can be used, and examples thereof include a coloring labeling substance, an enzyme labeling substance, and a radiation labeling substance.
Among these, it is preferable to use a color labeling substance from the viewpoint that the color change at the capturing site 31 can be determined quickly and easily by observing with the naked eye.
Examples of the color labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with respective pigments such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloid are particularly preferred.
The impregnated member 2 can be produced by impregnating the labeled second antibody suspension into a member such as the glass fiber non-woven fabric and drying it.

As shown in FIG. 1, the membrane carrier 3 is stuck in the middle of the pressure-sensitive adhesive sheet 1 and the chromatographic development start side of the membrane carrier 3 (that is, the left side of FIG. 1, hereinafter referred to as “upstream side”, The downstream side of the impregnation member 2 is overlapped and connected to the opposite end, that is, the right side of FIG. Can be attached to the pressure-sensitive adhesive sheet 1 to produce the immunochromatographic test strip of the present invention.
Further, if necessary, the downstream portion of the sample addition member 5 may be placed on the upper surface of the impregnation member 2, and the upstream portion of the sample addition member 5 may be attached to the adhesive sheet 1. Further, the upstream portion of the absorbing member 4 can be placed on the upper surface of the downstream portion of the membrane carrier 3, and the downstream portion of the absorbing member 4 can be attached to the adhesive sheet 1.

As the sample addition member 5, for example, a porous synthetic resin sheet or film such as porous polyethylene or porous polypropylene, and a paper or woven or non-woven fabric made of cellulose such as filter paper and cotton cloth are used. be able to.
The absorbing member 4 may be of any material that can absorb and hold liquid quickly, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., and filter paper is particularly suitable. is there.

  Further, in the case of a commercially available product, the immunochromatographic test strip of FIG. 1 is accommodated in a suitable plastic case in which a test sample injection part and a judgment part are opened above the sample addition member 5 and the capture part 31, respectively. Provided.

Thus, after mixing a test sample composed of a biological sample with an appropriate developing solvent as necessary to obtain a mixed solution that can be chromatographed, the mixed solution is added to the sample of the immunochromatographic test strip shown in FIG. When injected onto the member 5, the mixed solution passes through the sample addition member 5 and is mixed with the labeled second antibody in the impregnating member 2.
At that time, if a sample is present in the mixed solution, a complex of the sample and the second antibody is formed by the antigen-antibody reaction.
This complex is chromatographed in the membrane carrier 3 to reach the capture site 31, where it is captured by an antigen-antibody reaction with the first antibody immobilized thereon.
At this time, if a colored labeling substance such as colloidal gold is used as the labeling substance, the capture site 31 is colored due to the accumulation of the colored labeling substance. Therefore, the sample should be measured qualitatively or quantitatively immediately. Can do.

Although there is no restriction | limiting in particular as a test sample, For example, feces, a vomit, blood (it may be whole blood, serum, or plasma), saliva, urine, an organ emulsion, etc. are mentioned. The test sample may be diluted with an appropriate diluent such as a developing solvent and injected into the membrane carrier.
When whole blood is used as a test sample, and particularly when a colored labeling substance such as gold colloid is used as the labeling substance of the labeled antibody, it is preferable to dispose a blood cell capturing membrane member on the sample addition member. . The blood cell trapping membrane member is preferably laminated between the impregnation member and the sample addition member. This prevents red blood cells from being developed on the membrane carrier, so that it is easy to confirm the accumulation of the colored label at the capture site of the membrane carrier. As the blood cell capturing membrane member, a carboxymethylcellulose membrane is used. Specifically, ion exchange filter paper CM (trade name) sold by Advantech Toyo Co., Ltd. or ion exchange cellulose sold by Whatman Japan Co., Ltd. Paper or the like can be used.

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

Example 1 (Preparation of anti-CPV monoclonal IgM antibody)
CPV as an immunogen was prepared as follows. A crude composition CPV was obtained from the culture supernatant of CPFK-infected CPFK cells by centrifugation, and a purified CPV fraction was further prepared therefrom by the metrizamide density gradient centrifugation method.
100 μg of purified CPV prepared per mouse was boosted subcutaneously with Freund's complete adjuvant, and further boosted twice with Freund's incomplete adjuvant twice at three-week intervals. Thereafter, purified CPV was inoculated into the abdominal cavity of the mouse at intervals of 3 weeks or more, and the spleen was removed 3 days later. The splenocytes were fused with mouse myeloma cells, and anti-CPV antibody-producing hybridomas were obtained by screening and cloning. The obtained hybridoma supernatant was subjected to ImmunoPure (trade name) Monoclonal Antibody Isotyping Kit 1 (Pierce). As a result, the isotype of the anti-CPV antibody produced by the hybridoma was determined to be IgM. This anti-CPV antibody-producing hybridoma was administered into the abdominal cavity of BALB / c mice on the 10th day after administration of Prisitan (manufactured by Sigma), and ascites was collected about 10 days later. 2 ml of the obtained immune ascites was centrifuged at 4 ° C. and 10,000 rpm for 15 minutes. The resulting supernatant was filtered using a 0.45 μl filter. Thereafter, it was subjected to gel filtration using a Sephacryl S-200 column, and 1 ml each was collected. Fractions in which the reaction was observed by the octerony reaction were collected to obtain an anti-CPV monoclonal IgM antibody.

Example 2 (Immunochromatography kit using anti-CPV monoclonal IgM antibody)
(1) Preparation of anti-CPV monoclonal IgM antibody The anti-CPV monoclonal IgM antibody obtained in Example 1 was used.

(2) Preparation of colloidal gold solution Add 1 ml of 1% (v / w) chloroauric acid aqueous solution to 99 ml of ultrapure water boiled by heating, and 1 minute later 1% (v / w) sodium citrate After 1.5 ml of an aqueous solution was added and heated to boil for 5 minutes, it was allowed to cool to room temperature. Subsequently, 200 mM potassium carbonate aqueous solution was added to this solution to adjust to pH 9.0, and ultrapure water was added thereto to make a total volume of 100 ml to obtain a gold colloid solution.

(3) Preparation of colloidal gold labeled anti-CPV monoclonal IgM antibody solution The antibody obtained in (1) above was used as the anti-CPV monoclonal IgM antibody to be labeled with gold colloid.
1 μg protein weight of the above antibody (hereinafter, when the protein protein weight of the antibody is shown, simply indicate the weight of the purified protein by weight analysis) and 1 ml of the gold colloid solution (2) above are mixed at room temperature. After leaving for 2 minutes to bind all of these antibodies to the surface of the gold colloid particles, a 10% bovine serum albumin (hereinafter referred to as “BSA”) aqueous solution is used so that the final concentration in the gold colloid solution is 1%. In addition, all the remaining surfaces of the gold colloid particles were blocked with this BSA to prepare a gold colloid-labeled anti-CPV monoclonal IgM antibody (hereinafter referred to as “gold colloid-labeled antibody”) solution. This solution was centrifuged (5600 × G, 30 minutes) to precipitate colloidal gold labeled antibody, and the supernatant was removed to obtain colloidal gold labeled antibody. The colloidal gold labeled antibody was suspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, 0.5% Triton-X100 to obtain a colloidal gold labeled antibody solution.

(4) Preparation of immunochromatographic test strip for measuring parvovirus antigen The immunochromatographic test strip shown in FIG. 1 was prepared by the following procedure.
(4-1) A strip-like nitrocellulose membrane having a capture site width of 5 mm and a length of 36 mm for a complex of a parvovirus antigen and a colloidal gold labeled antibody was prepared as a membrane carrier 3 for chromatographic development of a chromatographic medium.
0.5 μl of a solution containing 1.0 mg / ml of anti-CPV monoclonal IgM antibody was applied in a line at a position 7.5 mm from the end of the chromatographic development start side of the membrane carrier 3 for chromatographic development. To obtain a capture site 31 of a complex of parvovirus antigen and colloidal gold labeled antibody. As the anti-CPV monoclonal IgM antibody, the antibody obtained in (1) above was used.

(4-2) Gold colloid-labeled antibody-impregnated member
A 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric was impregnated with 37.5 μl of a colloidal gold labeled antibody solution, which was dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 2.
(4-3) Preparation of immunochromatographic test strip In addition to the membrane carrier 3 for chromatographic development and the labeled antibody impregnated member 2, a cotton cloth and a filter paper as an absorbing member 4 were prepared as the sample adding member 5. Then, using these members, a chromatographic test strip similar to that shown in FIG. 1 was prepared.

(5) Test canine parvovirus broth was diluted with the sample dilution solution was adjusted to each concentration (16,32,64,128,256,512,1024 fold dilution) to obtain a test sample. Then, 100 μl of the test sample is dropped with a micropipette onto the sample addition member 5 of the test strip obtained in (4) above, chromatographed, left for 15 minutes at room temperature, and then parvovirus captured at the capture site 31. The amount of captured complex of the gold-colloid-labeled antibody was observed with the naked eye. The amount of capture was determined by classifying the degree of reddish purple color increasing or decreasing in proportion to the amount into six stages with the naked eye. The results are shown in Table 1.

Comparative Example 1
The same test as (5) of Example 2 was performed using Checkman CPV (trade name; distributor Adtech Co., Ltd.), which is a commercially available reagent for testing canine parvovirus. This commercially available test reagent uses a polyclonal antibody as an antibody to be immobilized at a site corresponding to the capture site in FIG. The results are shown in Table 1.

  In Table 1,-indicates no coloring, means that the degree of coloring is strong in the order of ±, +, ++, ++++, +++++, an upward arrow indicates a stronger, a downward arrow indicates a weaker, ~ Means between the two.

  From Table 1, it can be seen that according to the present invention, 10 times or more sensitivity can be obtained compared with a commercially available test reagent.

Example 3 (Reactivity specificity of immunochromatography kit using anti-CPV monoclonal IgM antibody)
Ten normal dog stool specimens were subjected to the reaction. 80 mg of stool was collected and dissolved in 3.2 ml of buffer (final stool concentration: 2.5%) to prepare a test sample. A test similar to (5) of Example 2 was conducted except that 3 drops of this test sample were dropped on the sample addition member. The results are shown in Table 2.

  As shown in Table 2, a negative result was confirmed in all 10 samples, and no non-specific reaction was confirmed.

  The present invention provides an immunoassay using an IgM monoclonal antibody against canine parvovirus, particularly a sandwich immunoassay, particularly an immunochromatographic assay and an immunochromatographic test strip. Since animal parvovirus antigens can be specifically detected quickly and with high accuracy by a simple method, it is useful for quickly and easily diagnosing diseases caused by the virus.

a is a plan view of an immunochromatographic test strip, and b is a longitudinal sectional view of the immunochromatographic test strip indicated by a.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 31 Capture part 4 Absorption member 5 Sample addition member

Claims (13)

  A method for detecting a parvovirus antigen comprising an immunoassay using an IgM monoclonal antibody against canine parvovirus.   The detection method according to claim 1, which is used for detection of a parvovirus antigen of a dog or cat.   A sandwich immunoassay using a first antibody and a second antibody against canine parvovirus, wherein at least one of the first antibody and the second antibody is an IgM monoclonal antibody A method for detecting a parvovirus antigen.   The detection method according to claim 3, wherein either one of the first antibody and the second antibody is immobilized on a carrier.   The detection method according to claim 3 or 4, which is used for detecting a dog or cat parvovirus antigen.   Prepare a membrane carrier with a capture site formed by preliminarily fixing a first antibody against canine parvovirus at a predetermined position, and a mixture of a second antibody against canine parvovirus and a predetermined amount of test sample. And immunochromatographic measurement, wherein the membrane is chromatographed on the membrane carrier toward the capture site, and the complex of the parvovirus and the second antibody contained in the test sample is captured at the capture site. In the method, an immunochromatographic assay method, wherein at least one of the first antibody and the second antibody is an IgM monoclonal antibody.   The method according to claim 6, wherein the second antibody is labeled with a metal colloid or latex.   The measurement method according to claim 6 or 7, wherein the membrane carrier is a nitrocellulose membrane.   The measurement method according to any one of claims 6 to 8, which is used for detecting a dog or cat parvovirus antigen.   At least a first antibody against a canine parvovirus, a second antibody, and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody Is an immunochromatographic test strip for detecting parvovirus, which is labeled with an appropriate labeling substance and arranged so that it can be chromatographed on the membrane carrier at a position separated from the capture site, An immunochromatographic test strip for detecting parvovirus, wherein at least one of the antibody and the second antibody is an IgM monoclonal antibody.   The immunochromatographic test strip according to claim 10, wherein the second antibody is labeled with a metal colloid or latex.   The immunochromatographic test strip according to claim 10 or 11, wherein the membrane carrier is a nitrocellulose membrane. The detection method according to any one of claims 10 to 12, which is used for detecting a dog or cat parvovirus antigen.

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