JPS58187861A - Assay of antibody related to adult type t leukemia - Google Patents

Abstract

PURPOSE:To assay an adult type T leukemia (ATL) antibody quickly and accurately employing a non-soluble antigen in which one or more kinds of a soluble cytoplasmic protein (SCP) of an ATL cell and a solubly treated protein (VAP) are fixed to a non-soluble support body. CONSTITUTION:An ATL cell is cultivated and homogenated and then, centrifuged to obtain SCP from the supernatant thereof. On the other hand, the sediments obtained by centrifugal separation are treated to produce a VAP of the ATL virus. One or both of the SCP and VCP are fixed to a porous bead-like carrier made of polystylene and glass to make a non-soluble antigen. Serum of a patient which was cultivated and diluted is added to the non-soluble antigen. This enables an accurate assay of an antibody related to ATL for a number of serums to be inspected at a time easily and quickly by use of a fluorescent or enzymatic immunoassay as widely accepted method. Thus, this serves for the diagnosis and screening of ATL patients.

Description

[Detailed description of the invention] Adult T-cell leukemia (ADULTT-CE)
LL LEUKEMIA, hereinafter referred to as "ATLJ"
For details on how to measure K-related antibodies, please refer to ATL K-related antibodies.
L-related IA source 1,4TL-assocLattd an
ti, qtn, J'J lower 1.4 TLA) using an insoluble antigen immobilized on an insoluble support by fluorescence or enzyme immunoassay, the present invention relates to a new method for easily and rapidly measuring antibodies to ATLA. .

ATL is a malignant disease that affects adults, but its etiology is currently not clear [: Takatruk
i.

K., Uchiyama, T., Sagawa
, f, and Yodoi, /, .

(1977) 1rLTopics iyLHema
toLoay, , etLv.

5erLo, S., Takaku, F.
, and IrLno, S, (Excepta
Medicα, Arn5terdern), pp73
77 and Ucktyctrua.

T, , Yodoi, /, , ScLgawa
, K, , Takatsakt, K, and
Uchino, H., B1oocL50.481-49
2 (1977) etc.].

In recent years, ATLA anti-P64 has made it possible to diagnose the above-mentioned ATL.
76-6480 (1981)']. This method is cultured AT
Using the L cell itself as an antigen, place it on a glass slide, and test the test serum and fluorescently labeled anti-human immune antibody J'1', X under a microscope on the slide. However, the above method requires the use of living cells themselves as antigens and observation under a microscope for each sample (test serum). Because it is mandatory, it not only requires frequent operations, but also has the disadvantage of not being able to easily and quickly measure and diagnose a large number of samples.In addition, if the measurement method is to be widely used, it is necessary to perform measurements under certain conditions. However, since the method proposed above uses living cells themselves, it is possible to avoid fluctuations in the antigen positivity rate, and to ensure that a certain antigen remains stable in the cell. It was difficult to obtain, and there was a risk of errors in measurement and diagnosis.

The present invention provides a new method for measuring ATLA antibodies that eliminates all the problems found in the above methods for measuring ATLA antibodies.

That is, the present invention provides soluble cytoplasmic proteins of ATL cells and ATL cells.
A by a fluorescence or enzyme immunoassay method characterized by using an insolubilized antigen obtained by immobilizing at least one virus solubilized protein on an insoluble support.
This relates to a method for measuring TLA antibodies.

As a result of intensive research, the present inventors found that soluble cytoplasmic protein (hereinafter referred to as -,5CPJ) and A
It was discovered that the solubilized protein of the TL virus (hereinafter referred to as "VAP") can serve as an antigen that specifically reacts with ATLA antibodies, and when using an insoluble antigen obtained by immobilizing this on an insoluble support, a large amount It has been discovered that a specimen can be measured easily, quickly, and always under constant conditions with high precision. The present invention was completed based on this new knowledge.

According to the method of the present invention, a large amount of specimen can be measured quickly and with simple operations, for example, at a volume and speed about 10 times or more compared to the above-mentioned conventional method using a microscope.

Cultured ATL used as raw material for SCP in the present invention
The cells include ATL cells, which have already been established as a cell line [Miyosht, 1. , Kaho
nizhi, /,.

, SILmtda + M, + Htrakt +
S, +1'5vbota, T, , Kimur
a, /, .

Miyarnoto, I (,arLd 5ato,
/,, Gann 71.155-156 (1980)
), as well as cultured ATL cells isolated from the peripheral blood or lymph nodes of ATL patients according to conventional methods, and cultured cells obtained by separately culturing the above-mentioned ATL cells in combination with human T cells [for example, Naturt, 294].

770-771 (1981)]. There are no particular limitations on the T cells used in the above-mentioned mixed culture, and various T cells derived from peripheral blood, bone marrow, lymph nodes, spleen, tonsils, thymus, etc. can be exemplified. Further, the above-mentioned culture of 4TL#I cells and the mixed culture of the ATL cells and intact T cells can be carried out by a conventional method. As the medium, any of the various nutrient media commonly used for cell culture can be used. A preferred medium is, for example, RPMI 1640 medium (Flow J-
aboratories), Eagle Minimum Essential Medium (
HEM medium) Examples include a medium modified using a serum supplement such as isotopic fetal bovine serum (Fe2) or calf serum. Culture conditions 1 are not particularly different from those used for normal cell culture, and are generally at a temperature of about 36 to 38°C and a temperature of about 6°C.
．． A pH of 4 to 7.6 can be employed. In addition, the above medium contains T.
Various drugs known as growth promoters such as cell growth factor (TCGF) or as inducing agents for mammalian toroviruses, such as 5-3-do-2'-dehydrogenase" (IdUrd
), Schiff D Heshijiimido (CH), ZuD Mai 5) (p
uromycin), phosphor 12-tristate 13-acetate (TPA), ruzutitate (sodium ruux acetate), etc. can be added and used as appropriate. Culture medium # is usually advantageously replaced every 3 to 5 days, and the desired cells can thereby be grown well.

Further, the above SCP is, for example, F culture, 4 T 1. Cells were soaked in physiological saline or lysinate buffered saline (/l).
After hotting in a soaking solution, the supernatant can be collected by appropriate means such as centrifugation.

ATL used as a raw material for V/IP in the present invention
Examples of the virus include those isolated from the above-mentioned ATL cell culture solution by conventional methods. The above-mentioned separation of the ATL virus is carried out by a conventional centrifugation method, and in the present invention, it is particularly preferable to use an ATL virus that has been further purified by a density gradient method or the like. VAp
can be obtained by solubilizing the above-mentioned /iTL virus. The solubilization can be easily carried out using conventional solubilizing agents. As the solubilizing agent, various surfactants such as "Torito υX-100J (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), 1#/'-40j
(manufactured by Shell), non-iosic surfactants such as urea, anionic surfactants such as sodium dodecyl sulfate (,5/)S), and the like. There is no particular restriction on the solubilization method, but it is more preferable to use 0'-J generalizing agent.
．． It takes several minutes to about 6 hours under 0.01% to critical MiWi.

The VAP thus obtained is preferably further purified according to a conventional method such as centrifugation or dialysis, and then subjected to an iodine exchanger such as DEAE-cellulose or DEAE-Sephadex to remove residual It is preferable to adsorb and remove viral nucleic acids that have or are likely to do so.

The insolubilized antigen used in the present invention is produced by immobilizing the above SCI) and/or VAP on an insoluble support. The insoluble support to be used is not particularly limited, and examples thereof include those commonly used in physical adsorption methods, specifically porous supports such as polyethylene, glass, polycarbonate, and polyurethane. sep and ('1Vi) to the non-bathable support-F
VA/'IAj>i can be determined according to a conventional method. For example, in each supplementary fluid such as physiological saline, lysinate buffer, etc.
may be added and allowed to react at about 0 to 37°C for about 2 to 24 hours. The above-mentioned anti-Lr; is preferably carried out under reduced pressure conditions. After the reaction is completed, the adsorption sites remaining on the insoluble support used are saturated using, for example, 0.2% bovine serum albumin (BSA) or the like in a conventional manner.

The insoluble antigen thus obtained is stored in a dry state after washing with water or - in the above-mentioned buffer.

The method of the present invention is carried out by measuring the labeling activity of a labeled antigen-antibody complex according to a conventional fluorescence or enzyme immunoassay using the intolerable IA source obtained as described above. Ru. More specifically, (“6I cine-soluble antigen is subjected to an immune reaction by adding the necessary diluted test serum; this is then washed with preferential treatment)”! The original antibody reaction product is reacted with a labeling agent and labeled [2].The labeling activity of this labeled compound is determined according to the standard procedure. In particular, according to the present invention, by using the imageable antigen described in -F, it is possible to measure the test serum of the large operculum at one time easily, quickly, and accurately under certain conditions. In the above, as the test serum, for example, serum separated from blood collected according to a conventional method from a test subject who wishes to measure 4TLA antibodies, or serum diluted with an appropriate mild solution (II) can be used. The buffer used here is not particularly limited, but it is usually appropriate to use a mild phosphate solution (pH 7.4).

Furthermore, an immunological reaction between the above-mentioned test +fn serum and an insoluble antigen can be carried out simply by bringing the two into contact with each other in a mixed manner. The reaction usually completes in about 0.5 to 16 hours at a temperature of about 4 to 37°C, and after completion of the reaction, an appropriate buffer solution, preferably physiological saline or the buffer used for diluting the test serum, is sufficient. It is recommended to wash the

Following the reaction described in F, labeling of the obtained antigen-antibody reaction product with a labeling agent; -4'
Mix with the reactants and heat to about 4 to 37 degrees Fahrenheit.
This is carried out by reacting for 6 hours, and after the end of the reaction, it is desirable to thoroughly wash the resonant body obtained in the same manner as above. - In the above, any conjugate of a labeling agent (for example, a substance capable of specifically binding to an antibody, and various enzyme labeling substances or fluorescent labeling substances) can be used.

Typical enzyme labeling substances include, for example, Pero+Jittert.
! (POX), +t Torizusinogeshi, Zurocarposhisipezu titase, D-aldehyde 3-lysicate dehydrogenase, amylase, phosphorylase, D-Na5t
Examples include various enzyme reagents such as 1P-Nase. Examples of fluorescent labels include fluorescein isothiocyanate (FITC), tetramethylrotamisioisothiocyanate (TRITC), B
- Isothiocyanate, dichlorotriazine fluorescein (DTAF), etc. can be exemplified.

° Examples of substances that have the ability to specifically bind to antibodies and become labeling agents when combined with such substances include, for example,
CpγoA (Pharmacia), sheep anti-human immunoglobulin G antibody, rabbit anti-human immunoglobulin G antibody, cef anti-human immunoglobulin G antibody, mouse anti-human immunoglobulin G antibody Anti-human immunoglobulin G antibodies such as globulin G antibodies and rat anti-human immunoglobulin G antibodies can be mentioned.

A number of conjugates of a substance capable of specifically binding to the antibody and a labeling substance have already been prepared and commercially available, and in the present invention, various commercially available conjugates may be used as the labeling agent. Also commonly known methods CB, F, ERLANG
ER et al. 1.4cta, Endocrtnol.

5Ltpp1. , 168.206 (1972), and MH8KAROL et al., proc. Hat, Acad.
, Sci., US, 4, 57°713 (19
67)), a new conjugate may be created and used. For example, when using an enzyme-labeled substance in the above preparation method, this and 7self1)ro,4V-1'1γ1toimmun&-diDrinG antibody are mixed with an appropriate oxidizing agent, for example, Nal0
2 at around room temperature in a mild solution of pH 4 to 6 in the presence of L, etc.
This is carried out by allowing the reaction to react for up to 5 hours, followed by reduction with a suitable reducing agent such as 7VaBHu.

The ratio of each reagent to be used is approximately 1 part of the enzyme labeling substance per 1 volume of Pγ oil or anti-human immunoglobulin antibody.
Appropriately, the amount of the oxidizing agent is about 100 to 300 tons, and the amount of the reducing agent is about 1 to 3 tons of the oxidizing agent.

゛When preparing a fluorescent labeling agent using a fluorescent labeling substance, place the fluorescent labeling substance in water or physiological saline with a pH of 6 to 8, and add it to /'roA or at around 0°C to room temperature. All you need to do is react with anti-immune scylodulin G antibody for about 0.5 to 3 hours [Fluorescent antibody method, medical chemistry experiment method course, 4.26
3-270 pages, first printing published in 1972, Nakayama Shoten]. p
Generally, it is appropriate to use a fluorescent labeling substance for roA or anti-immune dioxidase G antibody at around 1150 times the fluorescent label.

In the present invention, a complex consisting of an insoluble antigen, a test serum (ATL, 4 antibodies), and a labeling agent is measured according to a conventional method depending on the labeling substance in the labeling agent used. This is done by determining labeling activity (enzyme activity or fluorescent activity). Thus, according to the present invention, ATLA antibodies in a large amount of test serum can be measured easily and quickly.

Examples will be given below to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 (Manufacture of insolubilized antigen) Blood 20+1117 obtained by heparesis from an ATL patient (50 years old, male, resident in Nagasaki City) was centrifuged using Ficoll Pack-1 (manufactured by Pharmacia Jisepaci Co., Ltd.). Peripheral blood lysipocyte cells "5 X l O'
Get sH.

10% calf RPMI 1640M ground (Flow Laboratories, Inc.)
Culture at 37° C.-FrC for 3 days at a cell concentration of /ml.

These 6 × 10 cells were purified in 3Qml of 0.05M phosphoric acid solution (containing 0.14M NaCl, pH 7.4, hereinafter referred to as "PBsJ"), and then centrifuged for 1 hour. Separate (105,000x). - Collect the supernatant, and 1) measure the protein amount at 120 μf/mJ with BS (this protein II is a total protein quantitative reagent made by Inuzuka Assay Institute, "Toneishi ■-TP"). 611 depending on the coloring method used.
) to obtain scP solution.

Next item FF=QS Ct' liquid (7J:) 140H1
l ni, preliminary 0. I#-HC1 aqueous solution, 0. l #-N
Hollystyrene lenses (diameter 0.4, precision
1Jla! tic Co., Ltd., USA
), 700 cells were added, and the mixture was removed for several hours at room temperature under reduced pressure using an aspirator, and passed through fF to obtain an insolubilized antigen.

It is stored at 4°C in a 0.05M iM lysic acid solution (7)/77.4) containing 0.2% gelatin.

Example 2 (Production of insolubilized antigen) +1) ATL cells (Kyo-Yace 11,
(obtained from Kyoto University Virus Research Institute), 10% calf serum (Fe2) and 50fi? /rrt15-iodo-
2'-Io + Urd (Iei:Urd) RPM
The cells are cultured in I1640 medium at 37° C. for 4 days at a cell concentration of 3×15 cells/1 IIl. Centrifuge the culture solution (+
5001pry.

10 minutes) and then separate and collect the cells and the culture medium.

(2) To 5 x 10 cultured ATL cells obtained in (1) above,
Add 30 g of physiological saline, hot and dry 7, then 1
The supernatant was collected by centrifugation (105,000 x f ) for a period of time, and 120 μram of protein was collected in the same manner as in Example 1.
A 10SCP solution is prepared, and an insolubilized antigen (antigen-adsorbed polystyrene beads) is obtained in the same manner. This is 0.2
0.05M lysinate buffer (PH7,
4) After being left overnight, it is washed with water, dried and stored.

+31 Centrifuge 500 i/ml of the culture solution obtained in (1) above (40,000 x y, 1 hour) (~ to collect precipitated components. This is subjected to 25-60% sucrose density gradient ultracentrifugation for 4
1 and collect both portions with a density of 1.15 to 1.16. Add 5% of this to 0.8 M
NctC1 water bath liquid 1. Solubilization treatment (4℃, 60℃)
After centrifugation (3500 rpm, 1 hour),
Collect the supernatant.

0.0% of the obtained supernatant containing 0.3 M NaCl
Dialysis (Celofasi membrane) with 2M Tris-HCl buffer (CpHL5) for 5 hours is performed to remove the solubilizing agent and adjust the degree of salt fik. The VAP solution J44 thus obtained is subjected to an I) EAE-p lulose force ram equilibrated with the above buffer solution to obtain a flow-through fraction. Add distilled water to both parts to make the protein amount 2.8.
Prepare ttf/ml K and add +i to this 4Qml in advance.
80 sterile beads (same as in Example 1) washed in the same manner as described in J.I. are added and left at room temperature for 6 hours to obtain insolubilized antigen. This was left overnight in 0.05 A1: J salt solution (Pll74) containing 0.2% gelatin, washed with water, dried and stored.

'Example 3 (Preparation of test serum) Blood was collected from ATL patients and healthy individuals, left to stand at room temperature for 3 hours, the supernatant was collected, centrifuged at 2000 γ pm for 10 minutes, and the supernatant was and use it as the test serum.

Example 4 (Measurement of ATLA antibody) Each test serum prepared in Example 3 was mixed with 0.2% gelatin strength no.
, 05Af lysinate buffer (PH7,4), 80.1
601320.640.1280 times to prepare diluted serum. An example of this diluted serum with 0.5 go! One piece of the insolubilized antigen (antigen-adsorbed polystyrene, i) obtained in step 1 is added, and the mixture is left at 37°C for 2 hours. The reaction solution is removed by suction using an asgorator, the beads are washed by adding 2 rrrl of physiological saline, and the washing solution is removed by suction. Repeat this operation three times.

44,000 times diluted with the same buffer as above and 44,000-fold diluted with the same buffer solution as above.
Add 1 bead at f', rt+ to 55 ml of O-55 (manufactured by Radley's), leave at 37°C for 2 hours, and wash thoroughly in the same manner.

On the other hand, 0.26 offspring of o-phenylene diamicillo 0■'?
A coloring reagent is prepared by stirring and mixing 20 tnl solution of '71y Bishi Relaxing Solution (7'H5, 8) KlI202 to a final concentration of 0102 V/V%.

2ml of physiological saline in a test tube! and 0.5 m of the above coloring reagent
1 ml of 3N hydrochloric acid, then add 1 bead prepared above, leave it at room temperature for 30 minutes, and add 1 ml of 3N hydrochloric acid.
was added to stop the enzyme reaction, and the absorbance of the reaction solution at 4924 m was measured. The results are shown in Table 1 below.

Table 1 In addition, in place of the insolubilized antigen obtained in Example 1, each of the insolubilized antigens (antigen-adsorbed polystyrene) obtained in Example 2-+2+ and Example 2-I31 was used.
The results of repeating the same test with 0-fold or 100-fold diluted serum are shown in Table 2 (Example 2-+2+).
(using insolubilized antigen) and Table 3 (using insolubilized antigen in Example 2-13i).

Table 2 Table 3 Example 5 As the insolubilized antigen, the 1
゛ηηNaturally aspirated lysti-recipes, - also labeling agent r+j
Bar oxidase labeled total 4 diluted 600 times
Anti-inflammatory drug Dzurin GCE, manufactured by Y Laboratories)
ATL,
4FA integrated was measured. Absorbance at each serum dilution factor (4
9S+nn) is shown in FIG. In the diagram (1)
(2) indicates an ATL patient, and (2) indicates a fi patient.

As is clear from the following Tables 1 to 3 and Figure 1, the method of the present invention allows 4TL4 antibodies to be easily measured by absorbance measurement. Published to be extremely effective.

[Brief explanation of the drawing]

;, +'G 1 Figure shows the method of the present invention (Example 5) according to VC 1
．． I T1. ．． Graph C shows the results of measuring 4 antibodies.

Claims (1)

[Claims] ■ It is characterized by using an insoluble antigen obtained by immobilizing at least one selected from soluble cytoplasmic protein of adult T-cell leukemia cells and solubilized protein of adult T-cell leukemia virus on an insoluble support. A method for measuring antibodies associated with adult T-cell leukemia using fluorescent or enzyme-linked immunosorbent assay.