NL8301464A - IMMOBILIZED ADULT T-CEL LEUKEMIA ANTIGEN, METHOD FOR PREPARING THE SAME AND METHOD FOR DETERMINING AN ANTIBODY THEREOF - Google Patents

Description

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Immobilized adult T-cell leukemia antigen, method of preparing it and method of determining an antibody therewith *

The invention relates to an immobilized or insolubilized adult T-cell leukemia antigen and a method of preparing it. And further to a method of determining an antibody associated with adult T-cell leukemia, in particular by indirect fluorescence immuno-engineering or enzyme immuno-technique using an antigen that reacts specifically with sera from patients with adult T-cell leukemia .

Adult T-cell leukemia (ATL) is a malignant disease in adults, the cause of which has not yet been fully elucidated (see Takatsuki, K., Uchiyama, T., Sagawa, K. and Yodoi, J., (1977) in "Topics in Hematology," eds. Seno, S.,

Takaku, F., and Irino, S. (Excepta Medica, Amsterdam), pp.

73-77 and Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K. and Uchino, H., Blood 50, 481-492 (1977)).

Cultured ATL cells or ATL virus associated cell lines are known to contain an ATL specific antigen (ATLA) that is not detected in the fresh ATL cells in peripheral blood of ATL patients, and that sera from ATL patients or from some healthy adult in ATL endemic regions contain an antibody that reacts specifically with the ATLA (ATLA antibody).

On this basis, a method has recently been proposed for determining ATLA antibody by the fluorescence immuno technique and by which ATL can be determined (Proc. Natl. Acad. Sci., Vol. 78, no. 10, 1981, pp. 6476 - 6480).

This method includes applying ATL positive cells as an antigen to a glass slide, reacting the antigen with a test serum and fluorescently labeled anti-human immunoglobulin 8301464 * 2 buline G, and microscopically locating the fluorescent marker to determine ATLA antibody.

However, this method has several shortcomings because live cells are used as an antigen and each 5 test sample (test serum) must be viewed under the microscope which is not only cumbersome but also prevents the easy and fast determination of a large number of test samples for diagnosis . Furthermore, this method has the drawback that it is difficult to avoid fluctuations in antigen positivity because intact cells are used so that it is difficult to obtain antigen of the desired quality all the time otherwise errors in the assay and misdiagnosis could be made asked.

Therefore, this method is not suitable for determination under rigid nature conditions as required in connection with the techniques used.

The object of the invention is to overcome the outlined drawbacks of the conventional method for determining ATLA antibody.

It has been found that soluble cytoplasmic protein from cultured ATL cells (SCP) and solubilized protein from ATL virus (VAP) can be used as an antigen that reacts specifically with ATLA antibody, and that a large number of test samples are simple and rapid as well as under standard conditions. It can be determined today when using an insolubilized antigen prepared by immobilizing SCP or VAP on an insoluble support.

The invention has led to a method of determining ATLA antibody according to the fluorescence immunoassay technique or enzyme immunoassay technique using an insolubilized antigen consisting of an insoluble carrier on which a soluble cytoplasmic protein from ATL cells and / or a solubilized protein from ATL virus is immobilized. .

With the method according to the invention it is possible to determine a large number of test samples 830 1 46 4 * * 3 simply and quickly. For example, about 10 times more samples can be determined than by the conventional microscopic method or the same number of samples can be determined more than about 10 times faster than by the conventional method.

The culture of ATL cells already established as a cell line can be used as a source of SCP in the method according to the invention (Miyoshi, I., Kubonishi, I.,

Sumida, M., Hiraki, S., Tsubota, T., Kirnura, I., Miyamoto,

10 K. and Sato, J., Gann 71, 155-156 (1980)), the culture of ATI

cells conventionally secreted from peripheral blood or lymph node of patients with ATL, and ATL virus associated cells obtained by co-culturing the above ATL cells and human T cells (Nature, 294 (1981), pp. 770-). 15 771). For example, the human T cells used in the co-culture may come from peripheral blood, bone marrow, lymph node, bile, tonsilla or thymus.

Cultivation of the above ATL cells or co-cultivation of the ATL cells and human T cells can be carried out in a conventional manner. The medium can contain the nutrients usual for normal cell cultures. Preferred media are, for example, KPMI 1640 medium (Flow Laboratories) and minimum Eagle essential medium (MEM medium) modified with a supplemental serum such as fetal bovine serum (FCS) or calf serum. The culture temperature is generally about 36-38 ° C and the pH usually about 6.4-7.6. If desired, growth promoters can be added such as T cell growth factor (TCGF) or chemical compounds known to induce mammalian retrovirus such as 5-iodo-2'-deoxyuridine (Idürd), cyclohexylimide (CH), puromycin, phorbol-12 myristate-13-acetate (TPA) and n-butyrate (sodium n-butyrate). To promote the growth of the cells, it is recommended to change the solution every 3-5 days.

Furthermore, as SCP, a preparation can be used which has been obtained 8301464 -J4 by homogenizing a culture of the above ATL cells in a buffer such as physiological saline or phosphate buffered saline and the supernatant containing the desired substance, eg 5 spin off.

As a source of VAP, for example, ATL virus can be used which has been isolated from the culture fluid of the above-mentioned ATL cells in a usual manner, for example by centrifugation. Preferably, the ATL virus is then further purified by the density gradient method.

VAP can be obtained by solubilizing the ATL virus with conventional means eg surfactants such as non-ionic surfactants eg Triton X-100 (from Wako Pure Chemical Co.), 15 NP-40 (from Shell) , digitonin or urea, and anionic surfactants, for example, sodium dodecyl sulfate (SDS). The amount of solubilizing agent is preferably 0.01% of the amount to form a micelle at a temperature of about 0-100 ° C in about a few minutes 20 to 6 hours. Preferably, the VAP thus obtained is further purified, for example, by centrifugation or dialysis and then subjected to anion exchange column chromatography using, for example, DEAE cellulose or DEAE-Sephadex to remove viral nucleic acid remaining in the VAP by absorption.

The insolubilized antigen of the invention used in the assay method of the invention can be prepared by absorbing the SCP and / or VAP on an insoluble carrier. Suitable carriers are porous materials such as polystyrene, glass, polycarbonate or polypropylene.

Fixation of SCP and / or VAP on the insoluble support can be carried out in the usual manner. For example, the SCP and / or VAP is or are added together with an insoluble carrier to a solution such as a physiological saline solution or phosphate buffer, after which the mixture is about 2-24 8301464

* V

Held at about 0-37 ° C for 5 hours. Preferably, reduced pressure is used. Afterwards, unoccupied sites on the support are saturated in the usual manner with, for example, 0.2% gelatin or 0.2% bovine serum albumin (BSA).

The insolubilized antigen thus obtained is washed with water. The washed preparation is stored in the dry state or in the above buffer solution.

The method of determining an antibody for adult T-cell leukemia can be performed by determining the activity of a labeled antigen-antibody complex according to the conventional fluorescence immunoassay technique or enzyme immunoassay technique using the insoluble antigen. In particular, the method can be performed by adding an optional diluted sample of test serum to the insoluble antigen to effect the immune response, wash the antigen-antibody complex formed, label the complex, and the activity of the labeled complex in a conventional manner. to decide. As mentioned, the use of the insoluble antigen makes it possible to determine a large number of test sample serum quickly and very accurately under standard conditions. For example, the test sample may be serum secreted from adult blood that requires the ATLA anti-body assay and optionally diluted with a buffer, preferably a phosphate buffer of pH 7.4.

The immune reaction between the serum test sample and the insoluble antigen can be easily performed by mixing. Usually, the reaction proceeds at about 4 - 37 ° C in about 0.5 - 16 hours. The reaction product is optionally washed with a suitable buffer solution, preferably a physiological saline solution or the buffer solution used to dilute the test sample.

Labeling of the formed antigen-antibody complex 35 can be performed by mixing the complex with a marker 8301464 6 diluted with said buffer solution and leaving the mixture at about 4 - 37 ° C for about 0.5 - 16 hours. The labeled complex is preferably washed as indicated above.

A composite material consisting of a substance capable of specifically binding an antibody and an enzyme or fluorescent compound may be used for marking. Examples of labeling enzymes are peroxidase (POX), chromotrypsinogen, pro-10 carboxypeptidase, glyceroaldehyde-3-phosphoric acid dehydrogenase, amylase, phosphorylase, D-nase. and P-nase.

Suitable labeling fluorescent compounds are the conventional fluorescent dyes such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITTC) substituted rhodamine isothiocyanate (XRITC),

Rhodamine B isothiocyanate and dichlorotriazine fluorescein (DTAF).

Examples of compounds that can bind specifically with an antibody are Protein A (Pro A from 20 Pharmacia Co.) and anti-human immunoglobulin G (IgG) antibody such as sheep anti-human IgG antibody, rabbit anti-human IgG antibody, anti-human IgG antibody, mouse anti-human IgG antibody and rat anti-human IgG antibody.

Various complexes of a substance which can bind specifically with an antibody and the marker and which are suitable for use in the method according to the invention are commercially available. However, such complexes can also be freshly prepared, for example, as described by B.F. Erlanger et al in Acta Endocrinol.

Suppl., 168, 206 (1972) and M.H. Karol et al in Proc. Wet.

Acad. Sci., 57, 713 (1967).

When the marker is an enzyme, the coupling agent is Pro A or anti-human IgG antibody and the coupling is carried out in a buffer solution with a pH

t 8301464 * '* 7 4-6 in the presence of an oxidizing agent such as NalO 2 at room temperature for 2-5 hours after which it is reduced with, for example, NaBH 2.

Preferably, about 1-3 moles of enzymatic marker and about 100-300 moles of oxidizing agent per mole of Pro A or anti-human IgG antibody are used. The reducing agent is preferably used in an amount of about 1 to 2 moles per mole of oxidizing agent.

The above coupling agents are also used for labeling with a fluorescent compound. In that case, the coupling is carried out by a mixture of the fluorescent compound and Pro A or anti-human IgG antibody in water or physiological saline with a pH 6-8 for about 0.5-3 hours at a temperature between about 0 ° C and room temperature (see Keiko Kotai Ho (Fluorescent Antibody Method) in Ikagaku Jikkenho Koza, no. 4, 263-270 (1972), first edition, Nakayama Shoten Co.,

Ltd., Tokyo). Preferably, the amount of the fluorescent marker is about 1/50 part by weight per part by weight of Pro A or anti-human IgG antibody.

Depending on whether the marker is an enzyme or a fluorescent compound, the determination of the complex of insoluble antigen, ATLA antibody in the serum test sample and marker is performed according to conventional enzyme immunoassay technique or fluorescence immunoassay technique. As stated above, the assay can be performed very quickly and easily so that a large number of serum samples (ATLA antibody) can be accurately determined in a short time.

Example I

Preparation of insoluble antigen

Blood (20 ml) from a 50 year old Nagasaki man with ATL was centrifuged with heparin in Ficoll Pack (from Pharmacia Japan Co.) to obtain 5.10 'cells of peripheral blood lymphocytes.

The cells were grown in RPMI at 37 ° C for 3 days

830 1 46 4 8 - 1640/10% calf serum (Flow Laboratories Co.) at a cell concentration of 3.10 cells / ml. A portion of the resulting cells (6.10 cells) were homogenized in a 0.05 M phosphate buffer solution (PBS containing 0.14 M NaCl 5 and pH 7.4) followed by centrifugation at 105,000g for 1 hour. The supernatant was collected and the protein content was adjusted to 120 µg / ml with PBS (the protein content was determined colorimetrically using Tonein-TP, a commercial reagent for quantitative determination of total protein from Otsuka Assay Laboratories).

To the SCP solution (140 ml) thus obtained, 700 polystyrene beads (6.4 mm in diameter), a product of

Precision Plastic, America) previously washed successively with 0.1N hydrochloric acid, an aqueous 0.1N sodium added hydroxide, and ethanol. The mixture was left under reduced pressure at room temperature for 6 hours after which it was filtered off. The resulting insolubilized antigen was stored in a 0.2% gelatin containing 0.05M phosphate buffer with a pH 7.4 at 4 ° C.

Example II

Preparation of 'insoluble' antigen.

1) ATL cells (Kyo-Ya cells from the Kyoto University Virus Research Institute) were cultured in RPMI 1640/10% fig calf serum + 50 µg / ml IdUrd (from Flow Laboratories) at 37 ° C for 4 days at a cell concentration of 3.10J cells / ml. The culture fluid was centrifuged at 1500 rpm for 10 minutes. the cells and fluid being collected separately.

q 2) To a portion of the cultured ATL cells (5.10 cells), 30 ml of physiological saline was added and the mixture was homogenized and then centrifuged at 105,000g for 1 hour. The supernatant was collected and the protein content was adjusted in the manner indicated in Example I to obtain an SCP solution with a protein content of 120 µg / ml. An insoluble 8301464 * 'Mil 4 9 antigen was prepared therefrom by adsorption on polystyrene beads as described in Example 1. After overnight in a 0.2% gelatin containing 0.05M phosphate buffer with a pH 7.4, the antigen was washed with water, dried and stored before use.

3) The culture liquid (500 ml) obtained under 1) was centrifuged at 40,000g for 1 hour after which the formed granules were collected. These were then subjected to density gradient centrifugation on 25-60% sugar and the 10 fractions with a density of 1.15 to 1.16 were collected.

These fractions were solubilized with 1.5 ml aqueous 0.8M sodium chloride solution containing 0.5% Triton X-100 at 4 ° C for 60 minutes and then centrifuged at 35,000 rpm for 1 hour.

The resulting supernatant was dialyzed against 0.3M sodium chloride containing 0.02M Tris-HCl buffer solution of pH 7.5 using a cellophane membrane for 5 hours to remove solubilizing agent and adjust the concentration of salts. The VAP solution thus obtained was passed through one with the same

Tris-HCl buffer solution equilibrated DEAE cellulose column and the fraction passing through the column was collected.

This was diluted with distilled water to adjust the protein content to 2.8 µg / ml. 80 pre-washed polystyrene beads as described in Example 1 were added to 40 ml of the dilution and the mixture was allowed to stand at room temperature for 6 hours. The resulting insoluble antigen was washed with water overnight, in a 0.2 gelatin containing 0.05M phosphate buffer, pH 7.4, dried, and stored for use.

Example III

Preparation of a serum sample

Blood samples were collected from a patient with ATL and a healthy adult using heparin. After standing at room temperature for 3 hours, the samples were 8301464 for 10 minutes

JO

centrifuged at 2000 rpm. The supernatants thus collected were used as test samples in the following examples.

Example IV

5 Determination of ATLA antibody

Two series of dilute sera (80 x, 160 x, 320 x, 640 x and 1280 x) were prepared by diluting the test samples from Example III with 0.2% gelatin containing 0.05 microcosphate buffer, pH 7.4. One piece of insoluble antigen (antigen adsorbed on a bead of polystyrene as prepared in Example I) was added to 0.5 ml of each dilution. After standing at 37 ° C for 2 hours, the liquid was aspirated and the bead was washed with 2 ml of physiological saline which was also removed by suction. This procedure was repeated three times.

After this, 0.55 ml of peroxidase labeled Protein A (from E.Y. Laboratories) diluted with the aforementioned buffer solution to 44000 x was added to the treated antigen particle. After standing at 37 ° C for 2 hours, washing was done in the manner indicated above.

Hydrogen peroxide was added with stirring to 20 ml 0.2M McLevin buffer solution of pH 5.8 and containing 60mg o-phenylenediamine to bring the final concentration to 0.02 vol / vol to prepare a color reagent. Then 2 ml of physiological saline, 0.1 ml of the aforementioned color reagent and one bead of treated antigen were placed in a test tube. After standing at room temperature for 30 minutes, 1 ml of 3N hydrochloric acid was added to terminate the enzymatic reaction. The optical density at 492 nm was determined on the mixture. The results are shown in Table A below.

8301 464 11 0 * Λ V%

Table A

Dilution of test serum sample x 80 x 160 x 320 x 640 x1280 5 ATL patient A 0.789 0.748 0.646 0.550 0.410 B 0.701 0.624 0.550 0.423 0.356 C 0.558 0.449 0.367 0.291 0.222

Healthy person 10 D 0.166 0.114 0.081 0.047 0.028 E 0.114 0.089 0.054 0.037 0.027 F 0.142 0.089 0.063 0.037 0.023

The procedures described above were repeated using instead of the insoluble antigen prepared in Example I the insoluble antigens prepared in Example II (antigen adsorbed on polystyrene beads) for 80 or 100-fold diluted sera. The results of the optical density measurement are shown in Tables B and 20 C below.

Table B

Insoluble antigen prepared in Example 11 (2) optical density at 492 nm 100-fold diluted serum_ 25 ATL patient G 1.154 H 1.012 I 0.879

Healthy person 30 J 0.108 K 0.094 L 0.011 8301464 el 12

Table C

Insoluble antigen prepared in Example II (3)

Optical density at 492 nm 80-fold diluted serum_ 5 ATL patient M 0.826 N 0.722 O 0.611

Healthy person 10 P 0.234 Q 0.329

Example V

Example IV was repeated but with the difference that instead of Protein A goat anti-human IgG (from E.Y.

Laboratories) was used as the coupling agent and that the coupling product had been diluted 600-fold with peroxidase.

The results of the optical density measurement at 492 nm on each diluted serum test sample are shown graphically in the figure. In it, curve 1 refers to the ATLA antibody content for an ATL patient and curve 2 refers to that of a healthy person. From this and from Tables A, B and C it appears that the method according to the invention for determining ATLA antibody can be carried out simply by measuring the optical density and is therefore suitable for diagnosis and screening of ATL patients.

830 1 46 4

Claims (26)

1. Immobilized antigen associated with adult T-cell leukemia, characterized in that the antigen consists of a soluble cytoplasmic protein from an adult T-leukemia cell 5 and / or solubilized protein from an adult T-cell leukemia virus immobilized on an insoluble carrier. Immobilized antigen according to claim 1, characterized in that the antigen consists of soluble cytoplasmic protein immobilized on a carrier of polystyrene, glass, polycarbonate or polypropylene. Immobilized antigen according to claim 2, characterized in that the soluble cytoplasmic protein is immobilized on polystyrene beads. Immobilized antigen according to claim 2 or 3, characterized in that the soluble cytoplasmic protein consists of the centrifuged supernatant of homogenate of the adult T leukemia cells. Immobilized antigen according to claim 2, characterized in that the adult T leukemia cells are from peripheral blood or lymph node of a patient with adult T cell leukemia. Immobilized antigen according to claim 5, characterized in that the adult T leukemia cell is an established adult T leukemia cell line. Immobilized antigen according to claim 2, characterized in that the adult T leukemia cell is transformed by culturing an adult T leukemia cell from peripheral blood or lymph node of a patient with adult T cell leukemia together with human T cell . Immobilized antigen according to claim 7, characterized in that the human T cell is from peripheral blood, bone marrow, lymph node, bile, tonsilla or thymus. Immobilized antigen according to claim 7, 35, characterized in that the co-cultivation is carried out in EPMI 8301464 ^. 14 1640 medium or minimal Eagle essential medium modified with fetal bovine serum or calf serum. Immobilized antigen according to claim 7, characterized in that the adult T leukemia cell is an established adult T leukemia cell line. Immobilized antigen according to claim 1, characterized in that the antigen consists of solubilized protein from an adult T-cell leukemia virus immobilized on a carrier consisting of polystyrene, glass, polycarbonate or polypropylene. Immobilized antigen according to claim 11, characterized in that the solubilized protein is immobilized on polystyrene beads. Immobilized antigen according to claim 11, characterized in that, the adult T cell leukemia virus is purified by density gradient centrifugation. Immobilized antigen according to claim 13, characterized in that the solubilized protein is obtained by solubilizing adult T-cell leukemia virus with a solubilizing agent. Immobilized antigen according to claim 14, characterized in that the solubilized protein is obtained by solubilizing adult T-cell leukemia virus with a non-ionic surfactant such as Triton Immobilized antigen according to claim 15, characterized in that the solubilized protein is obtained by solubilizing adult T-eukememia virus with Triton X-100 as solubilizing agent. A method of preparing an immobilized adult T cell leukemia associated antigen on an insoluble carrier, characterized in that it is a soluble cytoplasmic protein from an adult T leukemia cell and / or a solubilized protein from an adult T cell leukemia virus or are mixed with an 8301464 onop • 15 insoluble support in an aqueous medium, after which the non-adsorbed sites on the support are saturated and the immobilized antigen is washed with water. A method according to claim 17, characterized in that soluble cytoplasmic protein is immobilized on polystyrene, glass, polycarbonate or polypropylene. A method according to claim 17, characterized in that solubilized protein is immobilized on polystyrene, glass, polycarbonate or polypropylene. 20. Method for determining an antibody for adult T cell leukemia associated antigen by the fluorescence immunoassay technique or enzyme immunoassay technique, characterized in that an immobilized antigen according to claims 1-16 is reacted with a serum test sample and the antigen formed antibody complex is determined. 21. Method according to claim 20, characterized in that the antigen-antibody complex is reacted with a coupling agent which can specifically bind to the antibody. 22. Method according to claim 21, characterized in that the coupling agent is an enzyme or fluorescent compound-labeled substance which can bind specifically with the antibody and consists of Protein A, sheep human immunoglobulin G antibody, rabbit anti-human immunoglobulin G antibody, goat anti-human immunoglobulin G antibody, mouse anti-human immunoglobulin G antibody, or rat anti-human immunoglobulin G antibody. 23. A method according to claim 22, characterized in that the enzyme marker consists of peroxidase, chymotrypsinogen, procarboxypeptidase, glyceroaldehyde-3-phosphoric acid dehydrogenase, amylase, phosphorylase, D-nase or β-nase. Method according to claim 22, characterized in that the labeling fluorescent compound consists of fluorescein 35 isothiocyanate, tetramethylrhodamine isothiocyanate, 8301464 * 16 alpha substituted rhodamine isocyanocyanate, rhodamine B isothiocyanate or dichlorotriazine fluorescel . Method according to claim 23, characterized in that the enzyme marker is peroxidase and is coupled with Protein A. X-100, NP-40, digitonin or urea, or with an anionic surfactant such as sodium dodecyl sulfate. 26. Products, methods and the products used, obtainable and obtained products as well as methods and products used therein as described in the description and examples. 8301464