JPH01123152A - Diagnostic method using diagnostic drug derived from bovine leukemia virus - Google Patents

Abstract

PURPOSE:To enhance sensitivity, by measuring the antibody titer of an immunized cattle or bovine leukemia virus (BLV) infected cattle by enzymatic antibody immunoassay using a plate to which protein derived from BLV is bonded as an antigen. CONSTITUTION:Inert protein occurring from BLV is preliminarily bonded to an antibody titer measuring plate in enzymatic antibody immunoassay as an antigen and the enzymatic antibody immunoassay is used to assay an antibody titer. Herein, the inert protein derived from BLV adsorbed by the plate is prepared by a method wherein crude protein containing BLV is prepared from a cultured cell of a BLV infected host cell or a culture solution thereof and, after the BLV of the crude protein is deactivated, a solution containing deactivated BLV-containing crude protein is treated with ultrafiltration to obtain a conc. fraction and the protein in said conc. fraction is adsorbed by the plate. As the host cell, a sheep cell or goat cell of FLK is used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a retrovirus that causes bovine leukemia.

[Prior art] Bovine leukemia virus (nt, ■) is known as a type of retrovirus that causes bovine leukemia, and as a preventive measure against bovine leukemia, which is becoming widespread, BLV vaccine and BL
There is a strong demand for the practical application of antiserum or antiviral agents against V.

LV antigens, which are necessary for the development of BLV vaccines, have been studied in sheep, goats, cows, etc., and in cells derived from these animals.

As for how to obtain BLV itself, for example, culturing established cell lines such as Fetal lamb kidney cells (hereinafter abbreviated as FLK-BLV) persistently infected with BLV, which can be obtained from the Livestock Hygiene Laboratory of the Ministry of Agriculture, Forestry and Fisheries. , a method for isolating IILV from cultured cells or culture supernatant is disclosed in JP-A-61-200471.

[Problem to be solved by the invention] Cattle or BL suffering from the increasingly widespread bovine leukemia
There is an increasing demand for highly sensitive diagnostic agents to identify the whereabouts of V-infected cattle.

In addition, as mentioned above, as the demand for lLV vaccines increases, the demand for highly accurate assay methods to confirm the effectiveness of the developed BLV vaccines is also increasing.

That is, immunization tests using IILV inactivated antigens have been conducted on various animal models, and various assay methods have been developed, but there are few studies on cows, which are the original host.

In addition, BL such as inactivated BLV, which is necessary for diagnosis of BLV-infected cattle and as an antigen component for old and vaccine assays, is also available.
A method for separating V-infected cells from cultured cells or culture supernatants has not yet been established.

For example, in the method disclosed in JP-A-61-200471 mentioned above, BLV is separated using ultracentrifugal density gradient method using sucrose, etc., but this is costly and very complicated. However, if BLV is damaged in the process of BLVIA release, a large amount of the ring protein constituting the envelope is likely to be lost, making it impractical.

In addition, it is sufficient if the BLV host cell is a living cell, but p+
In the case of sheep cells such as g-BLv, when the sheep cells are violently destroyed, the sheep cell antigens are mixed with BLV antigens and fetal bovine serum to form a vaccine, and when cattle are immunized,
There is a risk that the whole seed antigen will appear on the whole surface and cause a stronger allergic reaction. Therefore, it was necessary to devise an operating method that does not include a process of treatment with surfactants or the like.

Furthermore, concentration methods such as ammonium sulfate salting out may denature and deactivate the BLV protein.

The present invention was made in order to solve the technical problems necessary for the practical application of a new diagnostic method during infection and a BLV vaccine. The object of the present invention is to provide a more sensitive method for diagnosing BLV-infected cattle and a method for testing BLV vaccines with higher accuracy.

[Means for Solving the Problems] The assay method of the present invention that achieves the above-mentioned purpose is to determine the antibody titer of immunized cattle by enzyme-antibody immunoassay using a plate to which a protein derived from bovine leukemia virus is attached as an antigen. It is characterized by measuring.

That is, the method of the present invention uses an enzyme antibody immunoassay (El
In this method, an inactive protein derived from bovine leukemia virus is attached as an antigen to a plate for measuring the antibody titer in advance (ISA method), and the antibody titer is assayed by enzyme-antibody immunoassay.

The bovine leukemia virus-derived inactive protein to be adsorbed onto the plate was prepared as follows.

A crude protein containing bovine leukemia virus is prepared from cultured host cells infected with bovine leukemia virus or its culture fluid, and after inactivating the bovine leukemia virus in the crude protein, the inactivated bovine leukemia virus is prepared. A solution containing crude protein containing
sette pore 5ize 10. (100 (manufactured by Millipore) or the like to obtain a concentrated fraction. The protein in this concentrated fraction is adsorbed onto a plate and used.

Host cells that can be used in this method include:
Examples include sheep cells such as FLK, goat cells, bovine cells, and Kollamori cells, and FL is preferred because it is commonly used.

The medium used for culturing these host cells may be appropriately selected depending on the host cells to be cultured.

For example, in the case of FLK, RPM116 containing 5 to 20 tons of purified fetal calf serum (hereinafter abbreviated as FC5)
40 (manufactured by Lasui), Dulbecco's
MEM (manufactured by Lasui) etc. can be used.

In general, infection of host cells with BLV and cultivation of BLV-infected host cells may be carried out according to conventional methods.

For example, in the case of culturing FL-BLV, it is cultured for 2 to 4 days at 37° C. in a carbon dioxide atmosphere in RPMI 1640 containing funerary FC5 in 5 to 205. The cell concentration is, for example, 105 NIO'/ml.

After completion of the culture, cells are removed from the culture medium by low-speed centrifugation at, for example, about 2000 rpm to obtain a culture supernatant.

Next, inactivation treatment of [lLV] contained in the culture supernatant is performed.

For this inactivation treatment, treatment using an inactivating agent such as formalin, glutaraldehyde, rose formaldehyde, etc. can be used.

When using formalin, add the culture supernatant obtained as above to a final concentration of 0.05 to 0.5, preferably 0.
．． Nermarin solution (manufactured by Wako Pure Chemical Industries, Ltd.) is added to the solution so as to give one solution, and the mixture is gently shaken and treated at 4° C. for 24 to 72 hours.

In addition, Tween 100.80, Triton X-
The feature of this method is that it does not include the step of strongly destroying the cell components of FLK-BLV using a surfactant such as 100.

When the inactivation treatment is completed, the treated solution is immediately subjected to ultrafiltration without going through any other steps.

The operating conditions for this ultrafiltration are such that at least cell-derived components with a molecular weight of 10,000 or less and components such as the drugs used in the above-mentioned inactivation treatment can be removed from a solution containing inactivated BLV, and the BLV vaccine can be removed. The conditions are set to obtain a concentrated fraction containing inactivated BLV that can be used directly as an antigen component.

As the filtration A membrane used here, for example, Petlicon
Lab-cassette, Pore 5ize to
, 000 NMWL (manufactured by Millibore, PT filte
r), Amicon YM to (manufactured by Amicon), and the like.

When carrying out ultrafiltration, for example, 5 to 5000 mg per treatment solution! B is concentrated 50 to 90 times over θ time.
A concentrated fraction from which S^, formalin, etc. are completely removed can be obtained. Furthermore, BL was determined by sucrose gradient centrifugation.
V protein can be purified.

This concentrated fraction can be lyophilized to obtain a powder preparation containing the BLV protein, and a solution of this powder preparation at an appropriate concentration is prepared and brought into contact with a plate used in the Elisa method.

The above example uses a BLV-infected cell culture supernatant, but is not limited to this, and instead of the culture supernatant, an antigen solution containing BLV, such as a crude BLV antigen manually available from Kitasato Research Institute, may be used as appropriate. be able to.

The obtained powder sample (BLV protein) was assayed by preparing a standard solution (1 mg/ml protein concentration) of the concentrated fraction after formalin treatment, and adding it to the anti-bovine leukemia virus envelope glycovelast. The O
, D, (410nm) of 0.3 or more is determined to be positive, and it can be done by determining to what extent the serum dilution shows positivity, and the serum dilution of 1:16 or more is positive. It is desirable to have a standard item that shows the following.

Furthermore, a crude protein containing bovine leukemia virus is prepared from a host cell infected with bovine leukemia virus or its culture solution, and the crude protein is brought into contact with a surfactant to degrade the protein, and ions are extracted from the degradation mixture obtained. Bovine leukemia virus core protein and bovine leukemia virus envelope protein obtained by fractionation by the exchange method can be used as the protein for adsorption onto the plate.

That is, by this method, the envelope protein (gp51) and core protein (p2) of bovine leukemia virus were isolated.
4) and can be obtained by separating E.
It becomes possible to accurately test the antibody titer of BLV vaccine using the lisa method.

The loading and separation method for gp51 and p24 will be described in detail below.

First, as in the case of obtaining the concentrated fraction above, a solution containing BLV, such as a culture supernatant, is treated with a surfactant to decompose the BLV contained in the solution into envelope protein and core protein.

For this decomposition treatment, one or more surfactants such as Nonidet P-40 and Triton X-100 can be used.

The amount of surfactant used or processing conditions can be selected as appropriate.
For example, when Triton X-100 is used, the final concentration is about 1%, and the treatment can be carried out at 4° C. for about 3 hours.

Note that before and after the treatment with the surfactant, a step of concentrating the solution containing B1 and (2) by dialysis, ultrafiltration, 'IIt concentration gradient method, etc. or a preliminary separation and purification step may be incorporated.

For example, the solution containing BLV is 0.1! Nino Tween
10 nM Tris-HCI containing 80 p) l 7
．． 2 and then concentrated using an 11 m gradient using sucrose.

In addition, after treatment with a surfactant, ultrafiltration is performed using a filter with a molecular weight of 100,000 to 1,000,000, such as Labocassette (manufactured by Millibore) or Vericon (manufactured by Millibore), and the concentrated fraction is used for gp51 preparation. , and the filtrate fraction may also be used for the preparation of 924.

The protein mixture obtained by surfactant treatment is
Next, it is treated by an ion exchange method using an anion exchanger.

The anion exchanger used here is DE8-5e.
phacel (manufactured by Pharmacia), DE Hachihe-T
oyopearl (manufactured by Toso Corporation) or the like can be used, and the treatment may be performed multiple times as necessary.

The protein mixture obtained by surfactant treatment can be contacted with an anion exchanger to obtain both components that are not adsorbed to the ion exchanger. This non-adsorbed fraction can be subjected to various purification steps to obtain a preparation containing core protein p24.

Purification of this non-adsorbed fraction includes a concentration step by ultrafiltration using Amicon YMIO, etc. excluding molecules with a molecular weight of 10,000 or less;
Triton X-100 removal treatment by gel filtration using Uperose 6 or 12 (Pharmacia), anti-p24 monoclonal antibody binding-5uper
One or more methods such as affinity chromatography by ose can be incorporated.

On the other hand, the protein mixture obtained by surfactant treatment is brought into contact with an anion exchanger, and both components containing gp51 are eluted from the components adsorbed on the anion exchanger, and a sample containing gp51 is obtained by further purification. Can be done.

A salt concentration gradient method can be used to elute both components containing gp5 from the anion exchanger.

For example, using a stepwise salt concentration gradient with a combination of 0.1 M and 0.5 M Na(:I) solutions, 0.5 M Na1
gp51 can be obtained in one fraction, and O −+0
．． Using a linear salt concentration gradient of 5M NaCl, 0.2
gp51 can be obtained in the M~0.3M fraction.

For purification of a sample containing gp51 from the elution fraction containing gp51, ultrafiltration using a filter such as Amicon YMIO, 5uperose 6 (manufactured by Pharmacia),
TSK: Gel filtration using 1000SW (manufactured by Toso Corporation), affinity chromatography using Con 8-5 Epharose (manufactured by Pharmacia Corporation) and α-methyl-mannoside as the eluent, anti-gp
One or more methods such as affinity chromatography using 51 monoclonal antibody binding and 5epharose can be incorporated.

Representative examples of operations in the special method for separating gp51 and p24 of the present invention are shown in FIGS. 1 to 3.

GP51 and p24 obtained as described above are adsorbed onto a plate.

An example of the method of the present invention will be described in detail below.

First, 50 μl of a solution of one of the inactive BLV proteins, gp51 and p24 obtained as described above at an appropriate concentration is brought into contact with a 96-well microplate for Elisa method.

To obtain solutions or diluted solutions of these proteins, for example, 50 mM carbonate buffer, pH 9.6, etc. can be used.

The protein concentration of the protein solution to be adsorbed is:
It is appropriately selected from about 1ng/ml to 100 μg/ml.

These proteins and the plate are heated at 4-37°C, for example.
Sufficient adsorption can be achieved by contacting for about 1 to 24 hours.

As a 96-well microplate for Elisa method,
Any material that can be used in the Elisa method can be used, such as those manufactured by Flow, Nunc, and Greiner.

When the adsorption operation is completed, insert Dulbecco PBS (-)
Wash each well with (0,05!Ii containing Tween 8020, hereinafter abbreviated as PT).

Next, 100 μl of PT containing ovalbumin (OVA), casein, and gelatin is added to each well, left at room temperature for 6 hours, and each well is further washed with PT.

The concentration of OVA or the like at this time may be about 1%.

The antiserum obtained from the cow immunized with the vaccine was 0.1! kO
The sample is diluted with PT containing v^ to an appropriate concentration, for example, 1:1OO, and used as a sample for analysis.

After adding 50 μm of this analytical sample to the wells of the plate prepared as described above, it was incubated at 4-37°C.
Leave it for about 1 to 24 hours and wash with PT.

Next, 0.1! k 50 μl of rabbit anti-bovine IgG(II+L)-peroxidase diluted to an appropriate concentration in PT containing OVA is added to the wells, left for 2 hours at room temperature, and washed with PT.

The concentration of rabbit anti-bovine IgG (H+L)-peroxidase at this time is appropriately selected depending on the level of antibody titer of the immunized cow. Also, as an enzyme, peroxidase is β-
For galactosidase etc., rabbit anti-cow antibodies may be substituted with sheep anti-cow antibodies, etc., and IgG (H+L) may be substituted with IgG (Fc).

After washing with PT, the wells were injected with 50 mM citrate buffer containing 0.02 μl of 2,2′-azinobis(3-ethylbenzthiazolinesulfonate diammonium salt and O,+5% hydrogen peroxide, 100μ at pH 4.0
m was added to each well, reacted for 15 minutes at room temperature, and immediately measured the absorbance at 414 nm using an Immunoreader NJ-2000.
(manufactured by Nippon Inter Med Co., Ltd.).

[Example] Hereinafter, the present invention will be explained in more detail using experimental examples and examples.

Experimental example! FKL-BLV (established by Dr. Van der Maaten) was cultured in T-75f at a culture cell concentration of 10 cells/ml.
alcon plastic flask (50II1
1) in a RPMI 1640 culture solution containing 10'4 Fe2 (manufactured by Lasui) at 37°C under a carbon dioxide atmosphere for 2 hours.
Cultured for ~4 days.

The obtained culture solution was centrifuged at 200 rpm and F
KL-BLV cultured cells were precipitated and the supernatant was collected.

Next, the final concentration of the culture supernatant (in) was 0.1! A formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the mixture so as to give the same amount, and the mixture was gently shaken at 4°C for 48 hours.

This formalin-treated culture supernatant was then transferred to Pc1li.
con Lab-cassette Pore 5iz
e It was subjected to ultrafiltration using 10,000 NMWL (manufactured by Millibore, PT filter).

Filtration took 5 to IO hours per 12 formalin-treated culture supernatants and concentrated 50 to 90 times. Further, during the filtration operation, distilled water was added to the 200101 concentrate and the ultrafiltration was repeated several times.

The solution concentrated to about 20ml was freeze-dried to obtain a powder sample (BLV protein).

Experimental Example 2 The powder preparation obtained in Experimental Example 1 was dissolved in distilled water to prepare a solution with a protein concentration of 1 mg/ml. It was assayed by enzyme antibody immunoassay (ELISA method) using a monoclonal antibody (GPA-11, manufactured by Serotik), and O, D, (410?+111) of 0.3 or more was judged to have antigenic activity. , showed positivity up to a serum dilution of 1:256.

Next, the powder sample was dissolved in distilled water to prepare a solution with a protein concentration of 300 mg/ffl, and the same volume of Freund's complete adjuvant (manufactured by Difco) was added to obtain the old vaccine.

Experimental Example 3 A solution of the powder sample obtained in Experimental Example 1 (protein concentration,
300 mg/ml) and the same volume of aluminum hydroxide gel solution (containing 300 mg aluminum hydroxide) BLV vaccine was obtained.

Example 1 The BLV vaccine obtained in Experimental Example 2 was injected into two cows (Holstein, 21 months old) that were kept isolated from others and were proven not to be infected with BLV.
The antibody titer against V was assayed over time to examine vaccine efficacy. The results are shown in FIGS. 4 and 5.

In addition, for the assay of antibody titer, E shown in Examples 2 to 4 below
A method such as the lisa method was used. FIG. 4 also shows Example 2, which will be described later, in which a microplate to which a BLV protein preparation obtained from the concentrated fraction of Experimental Example 1 was adsorbed was used to assay the antibody titer.
Figure 5 shows the results when using the Elisa method shown in Figure 5, and Figure 5 shows the results when using the EIiSa method shown in Example 3 below using a microplate to which gp51 was adsorbed.

In addition, in combination with the Elisa method, Western blotting using serum from immunized cows, and BLV ferritin antibody method (immunoferritin) were used to detect BL.
The production of antibodies specific to gp51 and p24 (described below), which are infectious antigens of V, was examined.

As is clear from the results shown in Figures 4 and 5, at the time of the first immunization, the protein concentration of the above preparation was 60Qa.
When immunized with +g, the antibody titer increases two weeks after immunization,
Three weeks later, all were given a booster dose of 300 mg without adjuvant, and two weeks later, the antibody titer further increased. The antibody titer continued thereafter, and when the mice were boosted again without adjuvant at week 15, the antibody titer clearly increased again.

On the other hand, when blood was collected from grazing cows and the obtained serum was measured by the Elisa method shown in Examples 2 to 4 below, antibody-positive cows were found. In addition, in the method of Example 2, 0. D, 0.3 or more was considered positive.

buffer, pH 7,2 (0,196Twe, en
80) at 4° C. for 1 to 2 days and nights.

After dialysis, take out the dialysis fluid and add it to a final concentration of 1.
% of Triton X and dissolve it at 4°C.
It was left for 15 minutes.

Next, it was filtered through a 0.45 gm membrane (manufactured by Millibore), and the filtrate was equilibrated with the above dialysis buffer.
It was applied to a Toyopearl 650S (manufactured by Toso Corporation) column (I x IO cm). The effluent fraction (pass-through fraction) obtained here was used in the specially prepared method for separating p24 in Experimental Example 5.

Furthermore, after thoroughly washing the column with the above dialysis buffer, the protein adsorbed on the column was eluted using a linear gradient with a sodium chloride concentration of 0 to 0.5M.

This elution operation was performed at 0.6 ml/min, and 2 ml aliquots were collected.

The absorbance at 280 nm of each portion was measured, and the elution pattern of the antigen was further analyzed by the Elisa method shown in Reference Example 1 below.

As a result, it was confirmed that the target gp51 was contained in the 0.2 M - 0.3 M NaCl fraction.

Collect both parts containing gp51 and transfer them to Amicon YMIO
Menpuran (molecular weight cutoff, 10,000, manufactured by Amicon)
It was concentrated by ultrafiltration using .

The solution with a volume of about 1 ml was added to 0.1M phosphate buffer, pH 7.4 (containing 0.3M sodium chloride).
Dialysis was performed for one day and one night.

After the dialysis, the dialysate was transferred to a 5uperose 6 column (I
m.

(manufactured by Pharmacia).

Furthermore, the column was treated with the same buffer to obtain an eluate. The elution rate at that time was 0.1 ml/min, and the elution rate was 0.5 m
1 fractions were collected.

When each of the obtained fractions was analyzed by the Elisa method shown in Reference Example 1 below, it was found that gp was found in the void volume.
51 activity was observed. Therefore, fractions in which gp51 activity was observed were collected and designated as a fraction containing gl)ri1.

Experimental Example 5 DEAE-Toyopearl 650 obtained in Experimental Example 4
The effluent fraction from No. 5 was concentrated by ultrafiltration using Amicon YMIO (molecular weight 10,000 cutoff, manufactured by Amicon).

The solution with a volume of about 1 ml was added to 0.1M phosphate buffer, pH 7.4 (containing 0.3M sodium chloride).
Dialyzed against.

Next, after the end of dialysis, the dialyzed fluid was transferred to a 5uperose B column (1x3
0 cm, manufactured by Pharmacia).

The column was treated with the same buffer to obtain an eluate. The elution rate at that time was 0.1 ml/min, and fractions of 0.5o+1 were collected.

As a result, Tri eluted near the total volume.
ton X 100 were removed.

In addition, each of the obtained fractions was analyzed using Elis
It was analyzed by method a, and both fractions in which p24 activity was observed were collected, and these were designated as both fractions containing p24.

Experimental Example 6 10mg of both gp51-containing doses obtained in Experimental Example 4 and 10mg of aluminum hydroxide were mixed to obtain a gp51 vaccine.

Experimental Example 7 1 mg of both p24-containing portions obtained in Experimental Example 5 were mixed with aluminum hydroxide IB to obtain a p24 vaccine.

Experimental Example 8 1 lOn+g/ml of gl)ri obtained in Experimental Example 4 and 1 mg/ll11 of serum thymus factor (FTS) were added to l iposome and mixed, and gl)! i
1 vaccine was obtained.

Example 2 Eli analysis performed by attaching BLV protein to a plate
Measurement of the immune antibody titer by the sa method was carried out as follows.

First, 20 to 60% of the BLV protein obtained in Experimental Example 1 was
BLV protein was collected at a density of 1.16 g/ml by centrifugation at 80.000×g using a sucrose gradient method. This BLV protein was placed in a 96-well Nunk microplate at 400 to 800 ng/well.
After that, 3% glutaraldehyde solution was added to the wells and treated at 4°C for 15 minutes to fix the BLV protein.

Next, it was washed several times with PBS-Tween 20 solution.

Before adding the serum for which the antibody titer is to be measured, the plate is washed with 1% BS8, and then the diluted bovine serum Example 3 gp51 is attached to the plate. The antibody titer is measured by the Elisa method as follows. It was carried out as follows.

Both portions containing gp51 obtained in Experimental Example 1 were diluted with 50 mM carbonate buffer, pH 9.6, to give a solution with a protein concentration of 1 μg/+ml.

50 μm of this diluted solution was added to each well of a 96-well microplate for Elisa method (manufactured by Flow), and incubated at 4°C.
After leaving for 18 hours, Dulbecco PBS (-) (0,
05! Each well was washed with K Tween 8020 (hereinafter abbreviated as PT).

Next, 100 μm of PT containing 1% of ovalbumin (Ov^, Sigma) was added to each well and incubated for 6 hours at room temperature.
After standing for a while, each well was further washed with PT.

Antiserum obtained from a cow immunized with the vaccine was administered at 0. OVA
The sample was diluted 1:100 with PT containing PT and used as a sample for analysis.

After adding 50 μm of this analytical sample to the wells of the plate prepared as described above, it was left at 4° C. for 18 hours and washed with PT.

Next, PTTe1 containing 0.14k OV8:
Rabbit anti-bovine IgG diluted to 1000 (H+L,
)-peroxidase was added to the wells, and the wells were further left at room temperature for 2 hours and washed with PT.

After washing with PT, the wells were filled with 0.02596 2.2'
-Azinobis(3-ethylbenzthiazoline sulfonic acid diammonium salt and O, 50mM citrate buffer containing 15% hydrogen peroxide, pl+ 4.0+
7) to.

μm was added to each well and allowed to react for 15 minutes at room temperature.
00 (manufactured by Nihon Intermed Co., Ltd.) to determine the antibody titer.

Example 4 Both samples containing p24 obtained in Experimental Example 5 were treated with 50 mM carbonate to detect the antibody titer.

Reference example 1 BL by Elisa method in each fractionation operation in the above experimental example
Detection of V protein, BLV core protein, and envelope protein was performed as follows.

Transfer 5 μl of both fractions obtained at each fractionation step to Elisa method 9
After adding it to each well of a 6-well microplate (manufactured by Flow) and leaving it at 4°C for 18 hours, add Dulbecco's PBS (
-) (0,05!ei Tween 8020 included,
Each well was washed with PT (hereinafter abbreviated as PT).

Next, 100 μl of PT containing 1% to OV was added to each well, left at room temperature for 6 hours, and each well was further washed with PT.

Here, Nakakui BLV polyclonal antibody (manufactured by Kitasato Institute)
or mouse anti-BLV gp69 monoclonal antibody (
After adding 50 .mu.l of Celtic (manufactured by Serotik) to the wells of the plate prepared as described above, it was left at 4.degree. C. for 18 hours and washed with PT.

Next, PTTE1 containing 0.1960VA: 1o
50 μl of rabbit anti-bovine IgG (H+L)-peroxidase or anti-mouse IgG (Fc)-peroxidase diluted with 0.00 dilution was added to the wells, left at room temperature for 2 hours, and washed with PT.

After washing with PT, wells were treated with 0.025% 2.2'-
Azinobis (50mM citric acid buffer containing 3-ethylbenzthiazolinesulfonic acid diammonium salt and O,1,596 hydrogen peroxide solution, pH 4.0)
00 μm was added to each well, reacted for 15 minutes at room temperature, and immediately measured the absorbance at 414 nm using Immunoreader NJ-
2000 (manufactured by Nippon Intermed Co., Ltd.).

[Effects of the Invention] The present invention is useful for diagnosing BLV-infected cattle, and
An accurate assay method for a vaccine useful for preventing bovine leukemia has been provided.

In particular, the protein adsorbed to the Elisa plate used in the method of the present invention can be isolated and purified without using particularly complicated methods, maintains high activity, and is useful for diagnosing BLV-infected cattle according to the present invention. In addition, a simple method for assaying a vaccine useful for preventing bovine leukemia was provided.

[Brief explanation of the drawing]

Figures 1 to 3 are gl)! ] FIG. 4 and FIG. 5 are graphs showing changes in antibody titers of two cows in Example 1. Patent applicant: Mitsui Toatsu Chemical Co., Ltd. Mitsui Pharmaceutical Industries, Ltd. Agent: Tadashi Wakabayashi GL)! ] I and P24 purification method Figure 1 Figure 2 Virus culture solution ↓ 5ucro*e gradient ↓ Virus concentrate ↓ Triton X-100 treatment ↓ DEAE treatment Figure 3 Virus culture solution (Triton X-10
1) After treatment) f liquid (p24)
Concentrate (gp51) DEAE column
DEAE column passing fraction (p24)
gp51 gel filter (excluding Triton
-100) Gelu over p24
gp51 fraction Fig. 4 Fig. 5

Claims (1)

[Claims] 1) The antibody titer of immunized cows or BLV-infected cows is measured by enzyme-antibody immunoassay using a plate to which a protein derived from bovine leukemia virus (BLV) is attached as an antigen. A diagnostic method for bovine leukemia. 2) After the protein inactivates the bovine leukemia virus in the crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or its culture solution, the crude protein containing the inactivated bovine leukemia virus The diagnostic method according to claim 1, wherein the diagnostic method is contained in a concentrated fraction obtained by ultrafiltrating a protein solution. 3) The protein is obtained from a mixture of dissociated products obtained by contacting a crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or a culture solution thereof with a surfactant to dissociate the protein. The diagnostic method according to claim 1, wherein the core protein of bovine leukemia virus is selectively isolated by an ion exchange method. 4) The protein is obtained from a mixture of dissociated products obtained by contacting a crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or a culture solution thereof with a surfactant to dissociate the protein. The diagnostic method according to claim 1, which is an envelope protein of bovine leukemia virus selectively separated by an ion exchange method.