JPH032664A - Cattle-leukemia diagnostic medicine - Google Patents

Abstract

PURPOSE:To perform quick, sharp judgment by using the floating liquid of latex particles to which only a specified antigen is bonded. CONSTITUTION:As the latex of a carrier for diagnosis medicine, it is desirable that the diameter of a particle is 0.8 - 1.5mum and the specific gravity is about 1.05 - 1.50. As a BLV antigen which is bonded to the particle of the latex, there are the following antigens: a gp60 antigen which is envelope glycoprotein; a p24 antigen which is structural protein; a p12 antigen which is nucleic-acid protein; a p15 antigen which is phosphorylated protein; p30 antigen which is transmembrane protein; and the like. As the respective BLV antigen, only the substantially purified antigen is bonded. As the first diagnosis medicine, only the gp60 antigen is bonded. Only the p24 antigen is bonded as the second diagnosis medicine. Only the gp60 antigen and the p24 antigen are substantially bonded as the third diagnosis medicine. In this way, the diagnosis for the holding of the antigen for the leukemia of cattle can be performed quickly and sharply.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a diagnostic agent for bovine leukemia.

[Prior Art] Bovine leukemia is a type of lymphoma that affects cattle, and it has been confirmed that bovine leukemia virus (hereinafter sometimes referred to as BLV) is the etiological agent. Although the incidence of BLV-infected cattle is low, some infected cattle with bovine leukemia definitely turn into tumors and take a fatal course. People appear to be healthy during asymptomatic infections, but their blood remains infectious throughout their lives and continues to be a source of infection. As a result, the number of infected cattle gradually increases, and the number of infected cattle increases accordingly. When the number of infected animals increases to such an extent that it becomes a social problem, the number of infected animals at the base of the disease becomes extremely large, and countermeasures require huge amounts of money. Therefore, diagnosis of bovine leukemia is extremely important for the dairy industry.

Currently, in-gel precipitation reaction using an agar plate is used as a diagnostic method for bovine leukemia. This in-gel precipitation reaction first requires preparing an agar plate, making holes in it, and adding BLV antigen solution and bovine serum to each hole, and it takes 24 to 96 hours for determination. The drawback is that detection sensitivity is low. That is, due to the low detection sensitivity, it is impossible to diagnose the initial stage of infection, which is the most important step for bovine leukemia control, and currently it is only possible to diagnose when the infection has progressed to a certain point.

[Problem to be Solved by the Invention 1] Therefore, the object of the present invention is to overcome the drawbacks of the conventional in-gel precipitation reaction method using an agar plate, to provide sensitivity, convenience, and rapidity, and to prevent infection. To provide a diagnostic agent for bovine leukemia for use in a serological diagnosis method for bovine leukemia, which can diagnose even the early stage and furthermore, depending on the method of use, can diagnose the degree of progress of infection. That's true.

c. Means for Solving the Problems] As a result of intensive research, the inventors of the present application discovered that latex particles contain B.
When a suspension of LV antigen bound to a specific antigen component is mixed with test serum, anti-BLV is present in the test serum.
The agglutination of latex particles only in the presence of antibodies can be used to sensitively diagnose whether a cow is suffering from leukemia, and this test uses the traditional in-gel precipitation method. The present invention was completed based on the discovery that the process can be performed much more sensitively, quickly, and easily than in the case of conventional methods.

That is, the present invention provides a bovine leukemia diagnostic agent comprising a suspension of latex particles to which substantially only gp60 antigen is bound as a bovine leukemia virus antigen, and a suspension of latex particles to which substantially only p24 antigen is bound as a bovine leukemia virus antigen. To provide a bovine leukemia diagnostic agent consisting of a suspension of latex particles to which substantially only gp60 antigen and p24 antigen are bound as bovine leukemia virus antigens.

[Detailed Description of the Invention] The latex used as a carrier for the diagnostic agent of the present invention is
Any latex may be used, but the particle size of the latex particles is preferably 0.8 μm to 1.5 μm, and the specific gravity is preferably about 105 to 1.50.

Furthermore, it is preferable to use particle aggregates that are uniform in particle size and specific gravity.

The BLV antigen contains the envelope glycoprotein gp.
60 antigen, p24 antigen, which is a structural protein, and p12 antigen, which is a nucleic acid binding protein.

Examples include p15 antigen, which is a phosphorylated protein, and p30 antigen, which is a transmembrane protein. The antigen component bound to the latex particles in the first diagnostic agent of the present invention is substantially only the gp60 antigen among the antigen components described above. That is, only substantially purified gp60 antigen is bound as the BLV antigen component. In the second diagnostic agent of the present invention, substantially only p24 antigen is bound as a BLV antigen component. Furthermore, in the third diagnostic agent of the present invention, substantially only gp60 antigen and 924 antigen are combined as BLV antigen components.

When only gp60 antigen and/or 1)24 antigen is bound on latex particles, false positives are less likely to occur in the measurement results compared to when the whole virus or a mixture with other antigen components is bound. . Another advantage is that it is easier to produce a diagnostic agent of constant quality than when the entire virus or a mixture with other antigenic components is bound to latex particles.

When a cow is infected with BLV, among the various BLV antigens mentioned above, antibodies against gp60 antigen appear first after 3 to 4 weeks, and then antibodies against p24 antigen appear. Therefore, gpso determines whether a cow is suffering from leukemia.
and p24, both antigenic components of the present invention are combined.
The progress after infection can be determined using diagnostic drugs such as gp
This can be determined by testing in combination with a first diagnostic agent to which only p24 antigen is bound and a second diagnostic agent to which only p24 antigen is bound.

The above-mentioned BLV antigen component is bound to the latex particles, but the binding mode is not particularly limited, and may be bound by physical adsorption or chemical bonding using a coupling agent such as glutaraldehyde or carbodiimide. be able to. Alternatively, a BLV antigen component and a carrier such as bovine serum albumin (BSA) bound by carbodiimide or the like can be bound to latex particles by physical adsorption. Physical adsorption is convenient and preferable because it occurs simply by mixing a BLV antigen (or BLV antigen-BSA conjugate) solution and a latex suspension.

The latex particles bound to the BLV antigen may be, for example, 0.1
Glycine buffer containing wt% bovine serum albumin (0,1
Mglycine-0.15M sodium chloride-sodium hydroxide, pH 8.2).

The concentration of latex particles in the suspension is not particularly limited, but is usually about 0.5% to 5% by weight, preferably about 2% by weight.

The diagnostic agent of the present invention can be prepared, for example, as follows. However, the preparation method is not limited to the following method.
J. and J. M. Miller, “Replica
tion of bovineleukemia v
iris in monolayer cell
culturesComparativeLe
ukemia research 1975. Bi
bleHaemat,, No, 43.360-36
2 (19761). The FLK strain can be obtained, for example, from the Livestock Hygiene Laboratory of the Ministry of Agriculture, Forestry and Fisheries. FLK cells are cultured, and then BLV in the FLK strain culture solution is inactivated. Inactivation can be performed by centrifuging the culture to remove cells and adding 3-propion lactone to the supernatant to a final concentration of 0.4% by weight.

Next, salt out by adding ammonium sulfate (30 g/100 ml), collect the precipitate by centrifugation, dissolve it in a buffer such as phosphate buffer, and remove ammonium sulfate by dialysis. Then, concentrate by ultrafiltration.

Next, the concentrated solution is applied to Concanavalin A-Sepharose (manufactured by Pharmacia). Then, BLV antigen components having sugar chains such as gp60 antigen are adsorbed, and BLV antigen components without sugar chains such as p24 antigen pass through.

Concanavalin A-Sepharose column with 0, IMα-
When the methyl D-mannoside solution is passed through, the adsorbed antigen component is eluted, and this is eluted with Sephadex G-150 (
The gp60 antigen can be purified by performing gel filtration chromatography using a column such as Pharmacia Co., Ltd.) and collecting a fraction with a molecular weight of 60,000.

On the other hand, the p24 antigen can be purified by chromatography of the liquid that has passed through a concanavalin A-Sepharose column through a column such as Sephadex G-150, and collecting both components with a molecular weight of 24,000.

The purified antigenic component may be bound to a carrier such as BSA using a carbodiimide or the like in a conventional manner.

On the other hand, the suspension of latex particles suspended in a glycine buffer is centrifuged to precipitate the latex particles, suspended in a glycine buffer again, and centrifuged. After repeating this operation twice, for example, the suspension is diluted with a glycine buffer. The suspension is preferably 1% by weight at a time.

Next, the BLV antigen-BSA solution prepared above (BLV antigen-BSA conjugate antigen concentration 0) was added to this latex suspension.
．． Add an equal volume of 3 mg/ml), mix, and incubate at 37°C for 2 hours.
Shake slowly for an hour. After shaking, add 30% by weight bovine serum albumin (BSA) to this mixture to a final concentration of 1% by weight.
Add it so that it becomes like this, and leave it at 4°C overnight. -After overnight incubation, centrifugation and washing twice with glycine buffer, 0.1
% sodium azide to obtain a diagnostic agent of the present invention consisting of a 2% sensitized latex suspension.

The bovine leukemia diagnostic agent of the present invention can be used, for example, as follows.

(1) Slide method The back side is painted black, and bovine leukemia positive serum, negative serum, and test serum (diluted as necessary) are separately dropped, for example, 50 ul each, onto a partitioned glass plate. For example, 50 ul of diagnostic reagents are added to each of these sera, and after mixing,
Support the glass plate with both hands and mix for, for example, 5 minutes while slowly moving it from side to side. Formation of aggregates was observed for positive serum, whereas almost no change was observed for negative serum.

(2) Microtiter method Positive serum,
Serial dilution of negative serum and test serum, e.g. 50 μm and 0
．． Add 25 μm of diagnostic reagent diluted 6 times with 1% BSA-glycine buffer solution and mix (final latex concentration 0.1
weight%). After reacting at room temperature for 2 hours to overnight, the potency is determined by observing the latex precipitate image.

EXAMPLES Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to the following Examples.

[Example LLLL (Preparation of sensitized latex particle suspension) FLK strain of fetal sheep kidney cells persistently infected with bovine leukemia virus was inoculated into Eagle MEM medium containing 2% fetal bovine serum, and cultured at 37°C for 4 days. did. 13000 rp of this culture solution 20 times
The cells were removed by centrifugation for 10 minutes at m, and a culture supernatant was obtained.

BLV was then inactivated by adding β-propion lactone to the supernatant to a final concentration of 0.4% by weight.

Next, ammonium sulfate was added to the solution at a concentration of 30 g/100 ml for salting out, and the precipitate was collected by centrifugation, dissolved in a phosphate buffer, and then dialyzed against the same buffer to remove ammonium 5-sulfate. .

After concentrating by ultrafiltration, it was applied to a concanavalin A-Sepharose column (manufactured by Pharmacia). The adsorbed components were eluted with 0.1 M α-methyl D-mannoside and subjected to gel filtration chromatography over Sephadex G-150 (manufactured by Pharmacia) to obtain a molecular weight of 6.
The gp60 antigen was purified by collecting both portions of 0000.

On the other hand, both fractions that passed through the Concanavalin A-Sepharose column were applied to Sephadex G-1,50 (manufactured by Pharmacia) and subjected to gel filtration chromatography.
The p24 antigen was purified by collecting fractions of % ii 24,000.

Each of the purified BLV antigen components was bound to BSA by a conventional carbodiimide method.

On the other hand, a suspension of commercially available high-density polystyrene latex (average particle size 0.93 tLm, specific gravity 1.14) was centrifuged to precipitate latex particles, suspended in glycine buffer again, and centrifuged twice. After repeating this, it was diluted with glycine buffer to obtain a 1% by weight latex suspension.

gp60 prepared as above was added to this latex suspension.
-BSA conjugate and/or p24-BSA conjugate solution (0.3 mg/ml glycine buffer) was placed in a passenger ship, mixed, and slowly shaken at 37°C for 2 hours. After shaking, 30% by weight bovine serum albumin was added to the mixture to give a final concentration of 1% by weight, and the mixture was allowed to stand overnight at 4°C. -After overnight incubation, centrifugation and washing twice with glycine buffer,
The first, second, and third diagnostic agents of the present invention, each consisting of a 2% sensitized latex suspension, were obtained by suspending them in a 0.1% BSA-glycine buffer containing 0.1% sodium azide.

Slide method 50 ul of bovine leukemia positive bovine serum and negative bovine serum were separately dropped on a glass plate with black-painted partitions.6 The positive serum was serially diluted 2 times from 80 times to 2560 times. did. Negative serum was diluted 4 times.
The third diagnostic agent obtained in Example 1 was added in an amount of 50 μm each, and mixed for 5 minutes while supporting the slope of the glass with both hands and slowly moving it from side to side. As a result, agglutination was observed in the positive serum up to a 640-fold dilution, whereas no agglutination was observed in the negative serum.

ml Microticou method bovine leukemia positive bovine serum and negative serum dilutions each 50 μl
was added to each formula in a 96-well microtiter plate. The positive particles were serially diluted 2 times from 80 times to 81,820 times.Next, the latex particle suspension, which is the third diagnostic agent obtained in Example 1, was diluted with 0.1% by weight BSA-glycine buffer. 25LLl of the diluted product was added to each formula (final latex concentration 0.1% by weight), and after reacting at room temperature for 2 hours to overnight, the presence or absence of aggregation of latex particles in each well was observed. The results are shown in the figure.

As is clear from the figure, no agglutination was observed in the negative serum regardless of the dilution ratio, but agglutination was observed in the positive serum.

[Effects of the Invention] By using the diagnostic agent of the present invention, it is possible to quickly diagnose whether or not a person has antibodies against bovine leukemia virus using a sensitive and simple method. Furthermore, it is possible to diagnose whether the patient is in the initial stage of infection or in the intermediate stage or later.

[Brief explanation of drawings]

The figure uses the bovine leukemia diagnostic agent of the present invention. This is a diagram showing the test results using the microtiter method.

Claims (4)

[Claims] (1) A diagnostic agent for bovine leukemia consisting of a suspension of latex particles to which substantially only gp60 antigen is bound as a bovine leukemia virus antigen. (2) A diagnostic agent for bovine leukemia comprising a suspension of latex particles to which substantially only p24 antigen is bound as a bovine leukemia virus antigen. (3) A diagnostic agent for bovine leukemia comprising a suspension of latex particles to which substantially only gp60 antigen and p24 antigen are bound as bovine leukemia virus antigens. (4) The average particle size of latex particles is 0.8 μm to 1
．． The bovine leukemia diagnostic agent according to any one of claims 1 to 3, which has a diameter of 5 μm and a specific gravity of 1.05 to 1.50.