JPS6036967A - Method and reagent for measuring atlv antibody - Google Patents

Abstract

PURPOSE:To enable exact decision of the presence of an ATLV antibody by using the result obtd. by using the antigen obtd. by treating an ATLV negative cell strain and making measurement with respect to a sample as contrast and making decision by comparison. CONSTITUTION:The constituting elements for the entire part of a measuring system are a solid phase, an antigen associated with ATL, the antigen of this invention, the same antibody, antihuman IgG antibody, enzyme and substrate. The cup of a microtiter plate for enzyme immonoassay or glass beads may be used for the solid phase. The antigen associated with ATL and the antigen are respectively dissolved in a physiological buffer salt soln. of 0.15M phosphoric acid and the soln. is put into, for example, a polystyrene cup for enzyme immunoassay and is allowed to stand for one hour at <=4 deg.C, then the surface of the solid phase is coated by the antigen associated to ATL and the antigen. The serum to be inspected may be added straightly into the measuring system but may be more preferably added thereto after diluting the same to, for example, 100 times by a normal rabbit serum (NRS). The measurement may be accomplished in accordance with the ordinary procedure in an enzyme immunoassay method. Overall decision is made finally.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and reagent for measuring ATLV antibodies. What is ATLV?
kemia virus.

That is, it is an abbreviation for adult T-cell leukemia virus.

ATLV and its antibody, the ATLV antibody, are described in detail in, for example, the following literature.

Reference is made to those descriptions in the present invention.

1) Miyoshi, 1. et al: Ga
nn, 71:155.19802) Miyos
Hi, 1. et al: Jap, J, C1
jn, 0nco1. .

9 (Suppl, ): 485.19793)
Hinuma, Y, et al: Proc, N
at, Acad, Sci.

78:6476, 1981 4) Miyoshi, 1. et al: G
ann, 73:399.19825) Miyo
Shi, 1. et al: Lancet: 6
83. 19826) Shimoyama, M.
et al: Jap, J, Cnn, 0nc
ol.

12: 109.1982 The necessity of measuring ATLV antibodies, the method for producing the ATL-related antigen necessary for the measurement, the method for measuring ATLV antibodies using the antigen, and the measuring reagents are described in Japanese Patent Application No. 169670/1982. Since they are described in detail in the publications, reference is also made to those descriptions in the present invention.

According to the description in the above literature, a considerable number of A.
Since there are TLV carriers and blood transfusions may play an important role in the transmission of ATLV, it is important to search for the presence or absence of ATLV in transfused blood donors and thereby prevent infection from antibody-positive individuals.

It is known that there is a need to provide highly sensitive measurement methods and measurement reagents for this purpose. And, the patent application 1986-169
The invention described in Publication No. 670 discloses a measurement method and a measurement reagent that produce an ATL-related antigen by processing to achieve the above object, and use the antigen.

After that, the present inventor investigated conventional ATL-related antigens in general, including the antigen according to the above-mentioned invention, and found that in addition to the ATL V antibody, other non-specific antibody components present in the sample, such as antinuclear It was learned that this antigen also recognizes antibodies or anti-T cell membrane antibodies, and therefore, measurement results obtained using this antigen do not necessarily accurately represent the presence of ATLV antibodies. That is, 2
For example, when measuring the serum of a patient with systemic lupus erythematosus as a sample, the sample has ATLV antibodies.

A positive measurement result may be obtained even though the test result is originally negative. This is thought to be the result of a reaction between ATL-related antigens and non-specific antibody components produced in conjunction with systemic lupus erythematosus.

In view of these circumstances, the present inventors9 investigated a method that does not involve the above-mentioned measurement error and can accurately determine the presence of ATLV antibodies.

As a result, an antigen obtained by treating the ATLV-negative cell line (a) (hereinafter referred to as the antigen according to the present invention) was separately prepared, and the measurement results using this antigen were used as a control. The present invention was completed based on this knowledge.

That is, the purpose of the present invention is to make it possible to accurately determine the presence or absence of ATLV antibodies, and 2 to achieve the purpose, the present invention uses antigens obtained by treating an appropriate ATLV-negative cell line. Prepare separately.

The present invention discloses a measurement method and a measurement reagent, which are characterized in that a measurement result measured using an ATL-related antigen is judged by comparison with a measurement result measured on a sample using the antigen. .

The present invention will be explained below.

The ATL-related antigen according to the present invention is a so-called adult T-cell leukemia-related antigen, and more specifically.

It exists as an antigen protein or C-type virus particle in ATL-related antigen-producing cells.

If an ATLV antibody exists, a specific antigen-antibody reaction will occur with it. For production, for example, an antigen-producing cell line called MT-2 derived from human debris leukocytes, which was established by mixed culture with leukemia cells collected from the peripheral blood of an adult T-cell leukemia patient, is grown using a surfactant. Just process it. For a more detailed description of ATL-related antigens and treatment with MT-2 and detergents, reference is made to the following references:

7) Yoshida M, et al: P
roc, Nat, Acad, Sci,.

79:2031.1982 8) Miyoshi, 1. et al: Ga
nn, 72:97B, 19819) Miyo
Shi, 1. et al: Nature, 2
94: 7701198110) Patent application 1986-169
ATLV antibody No. 670 exhibits a specific antigen-antibody reaction with ATL-related antigens, and specifically, it is contained in the test serum to be measured by the measurement method and measurement reagent of the present invention. Ru.

Next, 2 ATLV-negative cell lines according to the present invention are cell lines that do not contain ALT-related antigens, and therefore 1, for example, B cells,
Examples include nonT nonB cells, He1a cells, and cell lines derived from acute lymphoblastic leukemia patients. A T cell line called TALL-1, which is a cell line established from an acute lymphoblastic leukemia patient, is a preferred example in the present invention. In addition.

Regarding TALL-1, the description of the following document is cited.

11) Nature, Vol. 267, No.
5614. pp843844 (June 30.1
977) Treatment of ATL V-negative cell lines may be accomplished by any method that processes antigen-producing cells to obtain antigen. Therefore, the treatment may be carried out using a surfactant such as sodium deoxycholate, octylphenocylate polyethoxyethanol, or polyoxyethylene (10) octylphenyl ether. A preferred example of treating TALL-1 cells with sodium deoxycholate is as follows.

107-108 TALL-1 cells were injected with 0.00% for washing.
Add 15M phosphate saline solution (pH 7.2) and centrifuge to pack the cells. This was added to a veronal buffer containing 0.2% sodium deoxycholate (pI (7
, 5°1t, -0,145) and stir for 4 hours at 4°C, for example. Next, centrifuge, collect the supernatant, pack it into a dialysis tube, and save 0.15M! For example, at 4°C,
Dialyze for days. This dialysis is performed for the purpose of removing surfactant contained in the supernatant. After dialysis, centrifugation
When the supernatant is taken, the antigen according to the present invention is contained in the supernatant.

For details of the treatment using a surfactant, reference may be made to the description in Japanese Patent Application No. 169670/1983.

Next, the measuring method of the present invention will be explained.

The measurement method of the present invention includes enzyme immunoassay, radioimmunoassay,
It utilizes one of the passive hemagglutination methods. The usual procedures in these methods are already generally known per se. Therefore, the measurement method of the present invention will be explained as follows when using, for example, an enzyme immunoassay.

The components of the entire measurement system are a solid phase and an ATL-related antigen.

Antigen, antibody (test serum), antihuman) Ig according to the present invention
G antibody (labeling antibody), enzyme and substrate. As the solid phase, a cup of a microtiter plate for enzyme immunoassay or glass beads may be used. Prior to measurement, the ATL-related antigen and the antigen according to the present invention are each dissolved in 0.15 MIJ acid-buffered saline, placed in, for example, a polystyrene enzyme assay cup, and left at 4°C for 1 hour. , the solid phase surface is coated with an ATL-related antigen and an antigen according to the invention.

The test serum may be added directly into the measurement system, but
Preferably, it is added after being diluted 1, for example, 100 times with normal rabbit serum (NR3).

Examples of anti-human IgG antibodies (labeling antibodies) include:

A goat anti-human IgG antibody or a mouse anti-human Fc monoclonal antibody may be used. Further, as the enzyme, for example, alkaline phosphatase, glucose oxidase, peroxidase, beta-galactosidase, etc. can be used. Prior to measurement, a binding agent such as geltaraldehyde (i) is used to bind an enzyme to a labeling antibody to form a conjugate, which is prepared in advance as part of a reagent for carrying out the measurement method of the present invention. I can do it.

The substrate may be used as appropriate depending on the enzyme selected. For example, when alkaline phosphatase is selected as the enzyme, p-nitrophenyl phosphate or the like may be used.

Among the components of the entire measurement system, the selections illustrated here for other elements other than the ATL-related antigen and the antigen according to the present invention are examples of preferred embodiments, and are not intended to particularly limit the measurement method of the present invention. do not have.

The measurement may be carried out according to the usual procedure for enzyme immunoassay. Therefore, as shown in the Examples below, test serum was added to a cup coated with an ATL-related antigen and an antigen according to the present invention, respectively, and incubated, followed by an enzyme-labeled antibody.

For example, goat anti-human IgG-alkaline phosphatase conjugate is added and incubated;
For example, tbp-nitrophenyl phosphate may be added and incubated, and the amount of substrate degradation may be measured using a spectrophotometer.

Finally, a comprehensive judgment is made by comparing the measurement results obtained using the cup coated with the ATL-related antigen and the measurement results (B) obtained using the cup coated with the antigen according to the present invention. . Comprehensive judgment based on this comparison may be performed as follows.

First, certain criteria are set for the measurement results. For example, if the measurement result is 0.07 or more, give it a ten.

Also, if it is less than 0.07, it is set as -. Next, the criteria +
or - judgment (B judgment), A judgment and B judgment.
According to the overall judgment table in Table 1 for the combination of judgments, A
An overall judgment is made regarding the presence of TLV antibodies.

Table 1 In the overall evaluation, 〇 means that the test serum is positive for ATLV antibody, or means that it is negative.

According to the measurement method of the present invention, it is possible to accurately discriminate between positive serum and negative serum for ATLV antibodies (as shown in the experimental examples described later).

Next, one measurement reagent of the present invention is a reagent containing an ATL-related antigen and an antigen according to the present invention as essential components.

The measurement reagent of the present invention is a reagent that is directly used in carrying out the measurement method of the present invention, and achieves the same purpose as in the measurement method.

The measurement method related to the measurement reagent of the present invention utilizes any one of enzyme immunoassay, radioimmunoassay, and passive hemagglutination. Therefore, specific embodiments of the assay reagent of the present invention when using enzyme immunoassay, for example, are as follows.

The measurement reagent of the present invention is a combination of an ATL-related antigen and an antigen according to the present invention itself, or a combination arbitrarily selected from the group consisting of an ATL-related antigen and an antigen according to the present invention, a solid phase, a labeling antibody, an enzyme, and a substrate. This is a set that combines 4 to 4. Here, when the set includes a solid phase, the solid phase is provided in a state coated with an ATL-related antigen and an antigen according to the present invention, or a Sera! - When a labeling antibody and an enzyme are contained therein, it is free to provide a conjugate in which both are bound, that is, a labeled antibody, and these are also included in the aspect of the measurement reagent of the present invention. Furthermore, for the convenience of measurement operations, a suitable buffer solution, normal rabbit serum (NR8), etc. may be included in the set, and these do not limit the present invention.

The present invention will be explained in detail using the experimental examples described below.

Experimental Example 1 Samples: 1 adult T-cell leukemia patient serum (2 samples in total, 80-fold diluted and 320-fold diluted), 3 normal human serum
For example, 11 cases in total: 4 cases of serum from patients with systemic lupus erythematosus, 1 case of serum from patients with primary biliary cirrhosis, and 1 case of serum from patients with rheumatoid arthritis.
A sample was prepared. In addition, one blank sample was prepared.

Method Each sample was measured using the following enzyme immunoassay method.

That is, the ATL-related antigen-containing supernatant obtained in Example 1 and the antigen-containing supernatant according to the present invention were each mixed at 0.
OD2. with 15M +7 phosphate buffered saline. . It was diluted to a value of 0.4, and 150 μL each was added to polystyrene enzyme immunoassay cups.

Leave at 4°C for 1 hour, then wash the cup with deionized water and add 100μ of test serum diluted 100 times with NR8.
Added t. Leave at 37°C for 1 hour, then wash the cup with deionized water and add 100u, l of anti-human gG-alkaline phosphatase conjugate (diluted to a concentration of 50% in 25% NR8). added. Leave at 37°C for 1 hour, then rinse with deionized water.
17L4'1 (p-nitrophenyl phosphate 4
Carbonate buffer pH 9.5°4 to give 75g/ml
rrbM (dissolved in MgCl2) 100 μL
added.

Leave at 37°C for 1 hour, then I N NaOI-11
00 gL was added to stop the reaction, and the absorbance 0D405 value at 405 nm was measured.

Results The results are shown in Table 2. The values for A4Frl in the table are the measurement results obtained using a cup coated with ATL-related antigen.
The value of B41i indicates the measurement results obtained using the cup coated with the antigen according to the present invention. Also.

ATL is adult T-cell leukemia patient serum, NH8 is normal human serum, and SLE is systemic lupus erythematosus patient serum.

PBC represents primary biliary cirrhosis patient serum, REU represents rheumatism patient serum, and Blank represents a blank sample.

Moreover, each numerical value shows the average value of two measurements.

Next, set 0.07 as the judgment standard for each numerical value, and if the numerical value is 0.07 or more, it will be judged as +, and if it is less than 0.07, it will be judged as -. A B rating was given for the numerical value on the packaging.

Furthermore, for the combination of A judgment and B judgment.

Comprehensive evaluation was made according to the above-mentioned Table 1 comprehensive evaluation table.

The results are shown in Table 3. Samples other than ATL were found to be negative for ATLV antibodies. Therefore, as shown in Table 3, two measurements were made according to the measurement method of the present invention.

It turns out that it is possible to accurately determine whether the ATLV antibody in the test serum is positive or negative.

Table 2 Table 3 The present invention will be explained in more detail with reference to the following examples.

Example I 2.4 each of MT-2 cells and TALL-1 cells
x 107 cells with 0.15M phosphate buffered saline (pi-
I7.2) was added and centrifuged to pack the cells.

To this, add 5 ml of veronal buffer containing 0.2% sodium deoxycholate (pH 7.5, μm 0.145).
added. Stir with a stirrer for 4 hours at 4°C,
Thereafter, centrifugation was performed at 10.00 rpm for 30 minutes. The supernatant was collected, packed into dialysis tubes, and dialyzed against 0.15M phosphate buffered saline at 48C for 2 days, followed by centrifugation at 10.000 rpm for 30 minutes. The supernatants were taken as ATL-related antigen-containing supernatant and antigen-containing supernatant according to the present invention, respectively. The 0D280 value of the supernatant is 1
It was 5/mL.

Example 2 0.15 M in the ATL f9J d antigen-containing supernatant obtained in Example 1 and the antigen-containing supernatant according to the present invention!
The mixture was diluted with J-phosphate buffered saline so that the 0D2AO value was 0.4, and 150 μL of the mixture was poured into polystyrene enzyme immunoassay cups.

It was left to stand at 4°C for 1 hour, then the liquid was drained and the cup was washed with deionized water. A set was prepared by combining goat anti-human IgG antibody-alkaline phosphatase conjugate and P-nitroenyl phosphate in the cup prepared as follows.

This set is a measurement reagent that uses enzyme immunoassay. In addition, normal rabbit serum (NR
8) and 0.05M carbonate buffer (pH 9.5).

4mM MgC12) was added during the setting.

Example 3 Goat anti-human IgG antibody or mouse anti-human IgG (
Fc) Dissolve the antibody in pH 7,5,0,05M') acid buffer and adjust the protein concentration to 1 mg/rrtL. This liquid 10/7. 12'l of 1mm curie in L
-Add 50 μL of Na, and add 1.5 mg/mL of chloramine T dissolved in the phosphate buffer.
Add 0μ and stir for 30 seconds. Next, 100 μt of a solution prepared by dissolving sodium metabisulfite in the phosphate buffer at a ratio of 2 m, g/rnL is added to stop the reaction. Add potassium iodide to the phosphate buffer at 10 rng/7.
1 oolLl of a solution dissolved in the proportion of rLL was added to this reaction solution, and immediately the 12'■ labeled substance and 12'1 were separated by gel filtration using Sephadex G-50.

Got 12! The specific radioactivity of the II labeled substance is approximately 5-20t
LCi/μg.

This 125-labeled substance is combined with a cup prepared as in Example 2 to form a set and used as a measurement reagent using radioimmunoassay.

Example 4 Sheep blood was taken into a centrifuge tube and diluted to 2.0% using physiological saline.
The red blood cells were washed by repeating centrifugation five times at 0 rpm for 10 minutes. Phosphate buffered saline solutions of pH 7, 5, 0, and 15M were added to the red blood cells to adjust the concentration to 5%. Add one-fifth volume of geltaraldehyde solution adjusted to 2.5% concentration with the same phosphate buffered saline to the red blood cell suspension, and react at room temperature with stirring for about 5 hours to fix red blood cells. did. This solution was then centrifuged to obtain fixed red blood cells, which were further washed several times with physiological saline by centrifugation. The fixed red blood cells were adjusted to a 5% suspension with the phosphate buffer, and 5 mg of the same phosphate buffered saline was added to the suspension.
/di by adding an equal amount of tannic acid solution adjusted to 3.
Stirred for 0 minutes. This solution was centrifuged to obtain tannic acid-treated fixed red blood cells, which were then washed several times with physiological saline by centrifugation. The phosphate buffer solution was added to the obtained tannic acid-treated fixed red blood cells to obtain a 5% red blood cell suspension.

This red blood cell suspension and each of the ATL-related antigen-containing supernatant obtained in Example 1 and the antigen-containing supernatant according to the present invention were mixed with the above-mentioned phosphate buffered saline until the protein concentration was about 5.
The same amount of the solution diluted to mg/ml was mixed and sensitized by stirring at room temperature for 60 minutes.

This solution was centrifuged to obtain sensitized blood cells, which were further washed several times by centrifugation using physiological saline. Phosphate buffered saline containing 2% normal rabbit serum was added to the obtained sensitized red blood cells to make a 7% suspension. This sensitized red blood cell is used as a measurement reagent using the hemagglutination method.

patent applicant Ko-zai Co., Ltd.

Claims (1)

[Claims] K) AT by ATL-related antigen using any of enzyme immunoassay, radioimmunoassay, and passive hemagglutination method.
A method for measuring an ATL V antibody (2) characterized in that, when measuring an L V antibody, the measurement result is subjected to a determination using an antigen obtained by treating an ATLV-negative cell line (2) ATL-related antigen is MT -2 cells (31A)
'The method for measuring an ATL V antibody according to claim 1 or 2, wherein the rLV-negative cell line is a cell line derived from an acute lymphoblastic leukemia patient (4) Enzyme immunoassay, radioimmunoassay, A using either passive hemagglutination method
A measurement reagent for measuring ATLV antibodies using a TL-related antigen, characterized in that the measurement reagent includes a determination reagent containing an antigen obtained by treating an ATLV-negative cell line as a component. Reagent (5) AT
A reagent for measuring ATL V antibody according to claim 4, wherein the L-related antigen is MT-2 cells (claim 4, wherein the 51ATLV-negative cell line is a cell line derived from an acute lymphoblastic leukemia patient) or ATLV antibody measurement reagent according to item 5.