JPH01500367A - Treatment and diagnosis using soluble T cell surface molecules - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Treatment and diagnosis using soluble T cell surface molecules 1, Introduction The present invention relates to soluble T cell growth factor receptors and T cell differentiation antigens or fragments thereof. Measurement of soluble T cell surface molecules such as T-cell fragments and diagnosis of diseases and disorders and the application of such measurements in health and therapy. Measurement of such molecules is to test the effectiveness of medical treatments, detect differential diagnoses of diseases or physiological conditions, and/or can be used to stage.

Lord - Ban Alliance Evil Sentai 2.1. T-thin and receptor T cells secrete various polypeptides that influence the immune regulation of hematopoietic cells, and The hormone peptides themselves interact with specific receptors on the surface of these cells. It is adjusted by using Interlo, the first named T-cell growth factor In the presence of macrophage-induced interleukin-1, Ikin-2 It is synthesized and secreted by activated T lymphocytes and also exerts its biological effects. must interact with specific high-affinity membrane receptors in order to exert its effects. [Sumi S, K., A., 1980, Imrol.

Shiv. 51: pp. 337-357; Leonard, Daburi 21, Jay et al. 1983 Year, Bros, Natl, Akado, Sai, Ni, Nis, Ei 80; No. 6957-6 961 pages (Smith, K. ^,, 1980. Immunol, Rev. 51:337-357; Leonard, W. J., et al., 198 3. Proc, Natl, Acad, Sci, U, S, A, 80:6 January 957-696. Interleukin 2 receptor (IL2R, Tac antigen) is a specific antigen or mitogen that is not present on the surface of resting T or B lymphocytes. Upon activation by JEN, T cell proliferation is dependent on the autourin mechanism (alJ tOcrine mechanism) and thereby activate cells. secretes interleukin 2 (IL-2) and also secretes IL-2 (IL2R) expressing cell surface receptors for [Mueller, J.I.

Daburi, 21, and Gallo, R., C., 1980, Bros.

N) ~ Le, Akad, Sai, Ni, Nis, A, 77: p. 6134; Rob, Ar. Lu, J. et al., 1981, J.E., Exbo.

Met, 154: p. 1455; Leonard, W. J. et al.

1982, Nature 300: p. 267; Muller, Nis.

C. et al., 1984, Bross, Natl., Akad, C., N.I.

Niss, A. 81: p. 1509; Tsudo, M. et al., 1984.

J, Exp, Met, 160: pp. 612-617; Waldman, T. -, Ei, et al., 1984, G.E., Exp., Met. 160: No. 1450 ~1466 pages (Metr, J.W., and Ga1lo, R.C., 1 980, proC, Natl, ACad, Sci, tl, s, A, 77 :6134; Robb, R.J., et al., 1981. J, E Xp, Med, 154:1455; Leoriard.

W, J, et at, 1982. Nature 300; 267; H euer, S.C., etal, 1984. Proc, Natl, Acad, Sci, U, S, A, 81:1509; Tsudo, M. et at, 1984. J, Exp, ) led 160:612- 617; Waldmann, T.A., et at, 1984. J,E xp, Med, 160: 1450-1466) 1.

Interaction of IL-2 with its cell surface receptor causes continuous T cell proliferation. Kosushi Green, W, C and Leonard, W, J. 1986, Ann, Shiv. Imtsur, 4: pp. 69-95; Smith, K., E. Lee, 1984, Ann, Shiv. Imtsuru, 2: pp. 319-333 (Gree n, W, C, and Leonard, W, J, 1986. Ann, Rev, Immunof, 4:69-95; Sm1th, A, 19 84. Ann, Rev. Immunot, 2:319-333)l. I Measurement of L-2R provides information on the status of immune activation of the lymphoid population. this is, Cell analysis using flow cytometry or fluorescence microscopy This is achieved by measuring IL2R on the surface. restricts IL-2 receptors Modified IL-2 receptor expression (expr ession> has been reported in some immunopathologies [Green et al. and Leonard; Dever, J. E., M. et al., 1984, Imzuru, J. .

133: pages 1691-1695 (Greene and Leonard, 5upra; oepper, J, M, et at, 1984. J. , rmmunol, 133:1691-1695)l.

Membrane IL2R is associated with Burkitt's lymphoma [Waldman, T., E. et al., 198 4th year, J, Exp, Met, 160: pp. 1450-1466 (Wal Dmann, Ding, A., at al., 1984. J, Exp, ) led.

160:1450-1466)], Hairy Cell Respa Il! [Waldman , T., A., etc., said: Korsmeyer, Nis., J.A.

et al., 1983, Bros, Natl, Akado, Sai, Ni, Nis.

A, 802, pp. 4522-452B (Waldmann et al.) Culm 1; K.

rsmeyer, S. J., et al., 1983. Proc, W att, Acad, Sci.

U,S,A,80:4522-4526)3 and human T-cell leukemia virus (HTLV)-I-associated adult T-cell leukemia [Dever, Jay.

M et al., 1984, J.I., Imil, 133: pp. 1691-1695 ( Depper, J. tl., et al., 1984. J, Immun ol, 133:1691-1695) in certain B- or T-cell malignancies, including It has been found that The function of cellular IL2H in lymphoid malignancies is still unclear. It has not been fully elucidated. Many cases have common Blastoblastic leukemia (ALL) has been shown to express activated f&IL2R in vitro. [Tou, Ai et al., 1985]. Blood 66; pages 556-561 ; Tou, Ai et al., 1986, Blood 682, pp. 1088-1094: Pine Oka, M. et al., 1986, Leuk, Res., 10: pp. 597-603. (Dour, 1., et al, 19B5. 131ood 66:556 -567; Touw, 1. , et al., 1986. Blood 6 8:1088-1094;) latsuoka,)1. , et al,, 1986. Leuk, Res. 10:597-603), also In some cases, interleukin 2 is expressed in neoplastic progenitor cells (ne continuous colony formation of plastic progenitor cells) Stimulated "The Tow, 1985; The Tow, 1986 (Ding OUW, 19 85. 1986. −≦”Lp”=? ,)].

Leukemic cells from patients with chronic lymphocytic leukemia have receptors and are susceptible to external factors. “Uchama,” which was shown to exhibit a good proliferative response to interleukin-2, T. et al., 1985, Tallinn, J., Invest. 76: No. 446-45. Page 3; Grapes. M, 1986, Blood 67: pp. 316-321 (U chiyama, T, et at, 1985. J, Cl1n, Invest, 76:446-453; Tsudo, M., t986. Blood 67:361-321) Ii, however, HTLV-1 Associated adult T-cell leukemias inherently express high levels of cell surface IL2R, but Shows no or only a weak proliferative response to interleukin-2. No 1 Said Uchiyama, 1985; Aria, Nis, Kei et al., 1984, Sci. Ens 223: pp. 1086-1087 (Uchiyama, 1985.5u pra; Arya, S, et at, 1984.5cienc e223:108B-1087)]. Ever 1~ etc. (Ebert et a l,) shows that T cells from patients with the AIDS virus are activated. reported that they lack the ability to express IL2R on these surfaces even when isolated. [1985, Tarino.

Imtsuru, Imtsubasor, 37: pp. 283-297 (1985, Cl1n, Immunol, In! Tlunopatho1.37:283-297) ].

] Secondary signals in T cell activation, such as interleukin-1IL-1, are Provided by monocytes or other accessory cells and also requires IL-2 secretion [Siemidek, J., Earle and Hatfield, Niss, 1976, J.I., Imuil, 116: 357th page: Maitzel, A.

L et al., 1981, J. Exp. Met., 153: p. 470; Liams, J., M., et al., 1985, J. E., Imtsuru, 135: No. 2 249 pages (Schmidtke, J.R., and 1iatfield, S.

1976, J. Immunol, 116:357; Maizel, A.L. , et al, 1984, J, Ext) Med, 153:47 0; William S, J, H, et al, 1985゜J, Immu nol, 135:2249)].

2, 2. T silk surface Human leukocyte differentiation antigen-limiting differentiation strain clusters (CD) are induced against differentiation antigens. It was established by comparing the reactivity of monoclonal antibodies, and and Baumswell, 1984, Huyum, Imzur, 11. Pages 1 to 10 ( Bernard and Boumsell, 1984. llum, Tm munol, 11:1-10) 1° T cell surface antigen, epitope-restricted subgroup Their classification and their distribution on T cells are modeled on human T cells. have been investigated by the use of noclonal antibodies [Clark et al., 1983, Munogenetics 18: pp. 559-615; Hansen et al., 1984, In Leucoseito Typing, edited by Panert 9ei, etc., Sbringer Bell Rag, New York, pp. 195-212 (C1ark et at, 19 83.　ImmLlno(lenetics　18:559-615;　Han sen et al., 1984. in Leucocyte Dingyping , Bernard, A., et at, , eds, , Springer-Ver. lag, New York, pp, 195-212)], I<)Kanobu T cell strain groups and other T cell surface molecules are shown in Table 1.

These T cell surface markers include markers of cell diameter (lineage); The identity of the functional T cell subset to which the T cell belongs and the activation state of the T and 1B cells. and work. Some: FBi surface molecules have been studied in great detail and important for initiating and regulating immune function and for communication processes between immune cells. has been found to be important. T cell antigen receptor, i.e. 90 kg A surface molecule consisting of disulfide-bonded dimers of Dalton (kd) order has a specific The biochemical events that recognize antigens and constitute the T cell activation process t) is capable of initiating a series of complexes such as 1984, Ann, Lev.

Imil, 2, pp. 23-50, Acute 1 Oh et al., 1983.

Cell 34; 1 pages 7179-7268 () leuer, S, C, et a l,, 1984゜Ann, Rev. Immunol, 2:23-50; Acuto, O, et at, 1983. .

Ce1l 34 Near 17-728)] CD3tfl construction is T! fJ cell receptor [Cannelobouras, J., M. et al., 198 3rd year, EMBL J, 2: p. 1807; Prost, J, et al. 1983, Yoro, J., Ino, Nol 13; p. 576; Ruthen, Pe et al., 1984, Nature 312. Page 413; Mueller, Niss, C. et al., 1983, J. Exp., Met. 157: p. 705 (Kannellopoulos, J, M, et al, 19B3. EMBOJ, 2:1807; Borst, J., et al., 19 83. Eur, J, Immunol 13:57B, Van Dan EISen, P. et al., 1984. tvature 312: 413; Heuer, S.C., et al., 1983. J, EX I),) led, 157:705)], for example, interleukin 2 (IL -2) Receptors and interleukin 1 (IL-1 > receptor-like receptors) The protein receptor is essential for T cell activation and proliferation [Smith, et al. Kay, A. 1984, Ann, Shiv. Im Tsuru.

2: pp. 319-333; Dower, Niss, K. et al., 1985, J. Exp, Met, 162: pp. 501-515 (Smith, K.A., 1984. Ann, Rev. Immunol, 2:319-333; DoWer, S., et al., 1985. J, EXp, Med, 162:501-515)], CD8 is present in approximately 30% of T lymphocytes. A T cell-specific surface molecule expressed on the surface and suppressive (5uppression) and associated with cytotoxic functional groups. CD4 (OKT4 antigen) is a total T lymphocyte "Reinharts et al.", which is expressed on the surface of about 60% of the , 1979, Pross, Natl, Akad, Sai, Ni, Nis, Ei, 76: No. pp. 4061-4065 (Reinherz et al., 1979. pr oc, Natl, Acad, Sci, U, S.

A, 76:4061-4065) Ko. CD4 is also HT　V　V　-III Identified as a receptor for virus-associated acquired immunodeficiency syndrome (AIDS) McDougal, Jay, Nis et al., 1986, Science 2 31; pp. 382-385 ()lcDougal, J.S., et at. , 1986.5science 231:382-385)], these Wi-Fi Rus cell surface markers are useful for the identification of lymphocyte populations and their functional status. [Krensky, A., M. and Kreiba] Gar, C., 1985, Transplant, 39(4): No. 339~ 348 pages; Kang, B., C., et al., 1984, Monoclonal Antibodies Zu in Clinical Investigations, Clinical Biochemistry Tory - Contemporary Theories and Technics, Volume 3, Akade Mitsuku Press, pp. 89-115; Kanta, B., C., et al.

1983, India, J.A., Dermatol, 22(2): pp. 67-73. (Krensky, A.) 1. and Clayberger, C,, 1 985, TranspIant, 39(4): 339-348; Kung , P.C., et al., 1984. )Ion.

clonal Antibodies in C11nical InVeSt igatiOnS, C11nical Biochemistry-Cont emporary dingheories and techniques, vo l, 3. Academic Press, pp, 89-115; Kun g, P, C, etal, 1983. Int, J, Dermato I, 22(2):67-73):.

Current medical methods of T cell typing are based on T cell surface T cell surface markers to detect the presence of specific cell surface markers on including the use of monoclonal antibodies. Measure the total number of cells by surface markers. determination is useful for the characterization and classification of lymphoid malignancies [Greaves et al. , M. et al., 1981, India, J. Immunopharmac., 3(3): No. pp. 283-300 (GreaVes, H, et at, 1981. I nt-J, Immunopharmac, 3(3):263-300) l. Changes in the relative percentages of T helper and T suppressor/cytotoxic cells Immunological events in liver and organ transplantation due to sexually transmitted infections [Colvin, Earl, Bee et al.

1981, Pros, Eighth, India, Congress, Nephrol.

Athens, pp. 990-99B (Colvin, R, B, et al, 1 9131. Proc, 8th Int, Congr, Nephrol, Athens, pi), 990-996)l, autoimmune diseases ``Bayesian, -, M et al., 1981, India.

J. Immunopharmac. 3(3): pp. 313-319 (veVs, E. M., et al., 1981. Ir1t, J, Immunopharma c, 3(3):313-319)] and AIDS [Gupta, Nis, 19 1986, Tarif. Immutsuru, Immunoasor. 38: pages 93-100; Eve T., E., C., et al., 1985, Tariff. Immutsuru, Immunoasor.

37: pp. 283-297 (Gupta, S., 198B, Cl1n, Immunol, ImmUnOpathO+, 38:93-100; Eb ert, E. C. et al., 1985. Cl1n, Immunol , Immunopathol, 37: 283-29 July is relevant. I started it.

Expression of T cell surface markers has also been used to assess a patient's immune status.

Relative numbers of recognized, functional T cell subsets, and/or in peripheral blood or tissues It is possible to assess the immunological status of a patient by measuring the relative number of active cells. It has been proven that Activating antigens (such as IL-2 receptors) , appears to be involved in T cell growth and differentiation processes.

2.3. III゛・Detailed table (Some immune cell surface markers have been detected in serum. Complex molecules (HLA molecules) are a series of cell surface sugar proteins involved in immune recognition. be. These macromolecular antigens have also been found to be present in body fluids such as serum. [Pellegrino, M., E. et al., 1984.

, p<su, xenzymol, 108: pp. 614-624 (Pel Iegri No.

M.A., et al., 1984. Heth, Enzymol, 10 8:614-624)l.

Serum levels of type 1 HLA-A and HLA-B determine the type of HLA in the serum. It has been found that there are sufficient amounts to achieve this. [Uh, noso, C. et al., 1983, Transplant, Pros., 15(1): No. 66- 68 pages; Pellegrino, M., A. et al., 1981, Transplant, Pros. , 13(4): pp. 1935-1938 (Russo, C., et al. ,, 1983, Dingransplant, Proc, 15(1): 66-6 8: Pellegrino, H.A., et al., 1981. T ransplant, Proc: 13(4): 1935-1938)] . The presence of the first IUHLA-DR in serum has also been detected [Sandlin, E. Mu, Nis et al., 1981, G.I., Natl.

Cancer Instrument, 66(2): 279-283: U-no-so, C- et al., 1983, Transplant, Pros., 15 (1): 57-59) ]. Serum HLA-DR (Ia) was found to be significantly attenuated in tumor patients. I was taken out. However, HAL molecules are present in numerous cell types; Limited effectiveness for disease diagnosis.

Using monoclonal antibodies against different nibtopes of human IL2H, Rubin et al. [1985, Jay, Imzuru, 135: pp. 3172-3177, 1985, Feth, Pross, 44: No. 946 pages (1985, J, Immunol, 135: 3172-3177 : 1985. Fed, Proc, 44:94B)] is a normal peripheral blood molecular cell released by activating cells and by HTLV-I infected leukemia cell lines. We detected a soluble form of IL2R that is synthesized in large amounts in vitro. sandwich A enzyme immunoassay was used to quantify soluble IL2R.

Soluble immunoactive molecules are derived from the 55-65 kilodalton cell surface form of IL2R. It is somewhat small and also contains an autolysable free form or an intramembranous site as much as possible. It may be either a secreted species [Traeger, B., F. et al., 1986.

Jay, Imtsuru, 136: p. 4099 (Treigen, B, F,... et at, 1986. J, Immunol, 136:4099)] , about the functional significance of soluble IL2H (51gn1t1cance). Little known.

Because soluble IL2R is able to bind interleukin 2 [the Lupi N, L., A. et al., 1985 (Rubin.

L, A, et al, 1985.5 upra) 1. It is the ligand exhibits an immunomodulatory role by competing with cellular IL2R against , down-regulate the immune response. Regarding this soluble IL2R is produced by malignant cells that suppresses the host's immune response to the tumor. It has been proposed that it is a "blocking factor" that can be used to 1985, supra, Green, W. C. et al., 1986, Ann, In. Turn, Met, 105: pp. 560-572 (Rubin, L, ^,, et al., 1985. 5upra; Greerie, W, C,, eta! , , 198B, Ann, Interr+, Med, 105:560-5 72), and children with otitis media compared with that reported for healthy adults. It has been found that soluble IL2R levels are increased [the Greene, W, C, et al. 1986 ,, 198B, 5upra)l, Continued research will focus on parallel studies from healthy adults. disclose comparable levels of soluble IL2H in or peripheral blood [Gree Ng, Dabrini, Cee et al., 1986, Anne, Intern, Met.

105: pp. 560-572: Nelson, D., L. et al., 1986, Ped. Yateley, Rhys, 20; pp. 136-139 (Greene, W, C,, e tal,, t98B, Anri, Intern,)! ed, 105 :560-720; Nelson, D. L., et al., 1986.　 ediatr, Res, 20:136-139)].

Increased serum levels of IL2H are associated with HTLV-I-associated adult T-cell leukemia, Sézary disease syndrome, Hodgkin's disease, chronic lymphocytic leukemia, hairy cell leukemia certain B or T-cell malignancies, including leukemia) and T-cell lymphomas. [Green, Daburi 21, C+, 1] 986; Nelson, D. C., 1986, Feto.

Pross, 45; p. 377; Search. Seay et al., 1986, Fett, Pross , 45: p. 378, Matskeen, L. et al., 1986.

Fet, Pross, 45; p. 454; Lupine, L., A. et al.

1986, in Leucosites and Host Defense, Oppen Heim, J.A., J.A. and D.

M, Jacob, ed., Alan Earl, Rhys, Incorporated, New York. Greene, W.C., pp. 95-102.

, 1986. Namiari, Ne1sori, D, C,, 1986, Fed. Proc, 45:377; 5aadeh, C, et al, 1986 ．． Fed, Proc, 45; 33f3; MacKeen.

L, et at, 1986. Fed, Proc, 45:454; Rubin, L.A., et al., 1986. in Leuko cytes and Ho5t Defense, 0pper+heim, J., J., and D. H. Jacobs, eds8. Alan R, Li5S, Inc.

New York, pp. 95-102], and the elevated level of soluble IL2R. Bell is an AIDS patient [said search]. Saadeh 1st Prize, 1986 , C., et al., 1986.5 upra)'] in serum (ibid. ) and T-cell activity in vivo, such as in rheumatoid arthritis and erythematous lupus. present in the serum of other therapeutic conditions characterized by increased oxidation [nerlin, Dee.

L., 1986, Feto, Pros., 45: p. 311: Matskeen, L. et al. , 1986, Feth, Pros., 45: p. 454 (NeIson, D.L. ,, 1986. Fed, Proc, 45:377; Mackeeri, L, etal, 1986. Fed, Proc, 45:45 April It has been reported. The inventors also have David Nerlin et al. Filed on April 19, 1985 in the name of Elson et al. It was transferred to the government and “Soluble Interleukin-2 Level as an Indicator” Scepter and its assay method 5olable Interleukir-2R ceptor As An Indicator and Method U.S. Patent Application No. 72 entitled Assaying the Same) J. No. 4,897 is pending. Rubin et al. et al, ) [1985, Jay, Imzuru, 135(5),: No. pp. 3172-3177 (1985, J. Immunol, 135(5)l: 3172-3177)], this patent application contains data identical to that of 1 regarding the sexual IL-2 receptor assay method, and methods for diagnosing and testing diseases. The purpose is to post.

However, Lupin et al. A method of assessing the severity of the disease or two or more implied classifications according to the classification Teach a method for specifically diagnosing one disease or condition from among other diseases or conditions. Or not suggested. Additionally, U.S. Patent Application No. 724,987 provides a To test the effectiveness of a therapeutic treatment, assess the severity of a disease, or treat two or more diseases or conditions. It does not allow for specific diagnosis of one disease or condition among the symptoms.

Several other cell surface markers present primarily on T cells are also present in soluble form. It has been discovered. CD2, i.e. for all normal T cells and sheep red blood cells T cell surface molecules present on the receptor than found in normal comparison patients. High levels have been detected in the serum of certain cancer patients [Falcao, R. Bee et al., 1984, Tarino.

Love, Imzuru, 13; pp. 141-143; Oh, Niss, K. et al., 198 5th year, Sukyant, J.A. Imtur, 22:5l-60 (FalCaO,R , Pl, et at, 1984. Cl1n, Lab, Immuno l, 13:141-143; Oh, S, -ni, et at, 19 85.5cand, J, Immnol.

22: 5l-60)]. Found on the surface of suppressor/cytotoxic T cells CD8 (Leu2,0KT8) is a surface marker and it is included in cellular identification. > has been reported to be at highly elevated levels in the serum of T-cell leukemia patients [Fujimoto , Jay et al., 1983, Jay, Exbo, Met, 159: No. 752] ~ 166 pages (Fujimoto, J, et al, 1983. J, E xp, Hed, 159 Near 52-766) 1.　L　e　u　-1、So However, other T cell surface markers were detected following anti-Leu-1 monoclonal antibody treatment. Measured in serum [Miller, Earl, Ei et al., 1982, , J., Met., 306, pp. 517-520 (Miller, F<, A,, et al, t982. Nev Engl, J,) led, 30 6:517-520)]. Oh et al. (1985, supra. ) found that less than half of the malignant patients in their study had soluble K in their serum. reported the presence of elevated levels of TII receptors.

However, not all T cell molecules are released into the serum [Bushimoto , Jay 1st Class, 1983, Jay.

Exp, Met, 159: pp. 752-766 (FujimOtO, Jl, etat,, J, Exp, Red, 159 Near 52-766>1. L eU1 antibodies are present in the serum of normal or leukemic patients who have not received antibody therapy. and not detectable. Lev3 was also detected in soluble form in the T cell lysis supernatant. (ibid.)

In addition, “a detection system for cell-free detection of T-cell antibody receptor-related molecules and “Assay Systems for Detection” and its clinical use (“Assay Systems for Detection”) ng Ce1l Free T Ce1l Antiger+ Recepto r　Re1ated　)Iolecu! es and C11nica! tl assets of the Assays”). No. 804 filed December 3, 1985 ．． No. 289, pending U.S. patent application No. 289, discloses methods for diagnosing diseases and Test for disease status by measuring the amount of soluble T cell antibody receptors in Regarding the method.

In short, it examines the patient's immune status in response to therapeutic treatments and assesses the severity of the disease. specific for one disease or condition among a variety of diseases or conditions; The use of measured levels of non-disease specific soluble markers along with other tests to diagnose There are no known methods for using this method.

1-Kinsara Kyousara Show The present invention relates to soluble T cell growth factor receptors, soluble T cell differentiation antigens or related Measuring soluble molecules or their fragments and diagnosing diseases and disorders guide the use of such measurements in health and therapy. Measurement of the molecule is performed during therapeutic treatment of the subject. to test the effectiveness of treatment, detect and/or stage disease in subjects, and It is useful for the diagnosis of specific physiological conditions in patients. These measurements also It can help predict outcome and assess and test a patient's immune status.

In certain embodiments, serum or plasma interleukin-2 receptor levels Measuring leukemia can be done to detect or stage leukemia or lymphoma.

In other embodiments, IL2R levels or CD8 levels are To specifically diagnose renal allograft rejection as distinguished from porin A nephrotoxicity. It can be used to In another embodiment, CD8 levels are determined from other combined diseases. It can be measured to specifically diagnose chronic rheumatoid arthritis as differentiated.

In a particular embodiment, the measurement of soluble T cell surface molecules is performed using a sandwich enzyme. This can be accomplished by immunoassay.

1, 1, 1, 1, 1, 1, 1, 1, 1 As used herein, the following abbreviations have the following meanings: Staging a disease = assessing the degree of severity using standard classifications.

IL-2 = interleukin-2° IL-1 = interleukin-1° IL2R = interleukin-2 receptor.

mAb = monoclonal antibody.

NHL = non-Hodgkin's lymphoma.

B cells ALL=B! tI! J cell acute lymphoblastic leukemia.

HTLVIII/LAV/HIV = Human T-cell leukemia type virus/ Lymphatic joint disease related items rus/immunodeficiency virus.

PHA = phytohemageltin.

PBMC = peripheral blood mononuclear cells.

Raw-ku plate Ωkanho Figure 1. In vitro experiments on two T cell activation.

Figure 1A: Media supernatant, cell lysate or PHA stimulated human peripheral blood in media. Plot of the concentration of soluble IL2H assayed at the cell surface.

Figure 1B: Plot of the concentration of soluble 1.2R in the medium versus the percentage of activated T cells. to.

Figure 2: Soluble (serum) IL-2 receptors versus cell surface IL-2 in vivo. 2 receptors.

Figure 3: Observation of therapeutic treatment in cancer patients. Patient serum concentration of soluble IL2R Plot against the interval. Indicates duration, treatment and recovery of active disease.

Figure 4: Otitis media (OM); stage III or II non-Hodgkin lymphoma (NHL) : Stage III or ■v lymphoblastic (LB) NHL; poorly diffuse, undivided Cell (diffuse smallr+oncleaved-cell) ( SNC) NHL; and among children with Bffl follicular acute lymphoblastic leukemia Distribution of serum interleukin-2 receptor (iL2R) levels. The dashed line is 1 Separate IL2R values above or below 00 u/ml.

Figure 5: Logarithm of serum interleukin-2 receptor (IR2R) levels and blood Comparison with logarithm of serum lactate dehydrogenase levels. A straight line is the minimum value that fits the data. Represents square regression.

Figure 6 2 (A) All cases of non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia and (B) stage III or 1v non-Hodgki's lymphoma or Bm Serum interleukin-2 receptor levels in patients with acute lymphoblastic leukemia Comparison of time and decay by Bell (IL2R). Significantly worse treatment when comparing both The results are evident in patients with higher levels (>1,000 U/ml).

Figure 7: Soluble (serum) in samples obtained from patients treated with IL-2. IL-2 receptor levels.

Figure 8: Serum levels in lung cancer patients treated with IL-2. All patients received recombinant IL-2 was continuously infused for days 0-14. Patients 1 and 2 received 3 patients 3.4 and 5 received 2X106 Received U/ml.

Figure 9: Transplant examination and specific diagnosis of immune rejection from cyclosporin A poisoning (Δ・・・Δ=patient with rejection: ○・・・○=patient with C5A poisoning;・・・・=safety) standard liver transplant patients).

10th N: Serum IL2R measurement overlaid with non-linear regression plot (dashed curve) (triangle) Scatter plot of opposing histological classes with opposing endomyocardial biopsies. Regression equation The equation is shown in the upper left of the plot below the linear correlation coefficient (R>).

Figure 11: Graph for patient A shows normal site transplantation. Serum IL2R level (open triangle) and biopsy grade (open square) versus time ) is shown.

The patient was treated with additional stetoids due to medical indications of early rejection. Patient B The plot shows the correlation between IL2R levels and biopsy grade. This patient is An episode of recurrent myocarditis (ep isode). During treatment of rejection with additional steroids, biopsy grade remained at 2.0 (resulting in a rejection reaction), but IL2R remained normal, i.e. 5. It dropped to 45 units/ML. At 60 days after site transplantation, the biopsy grade had dropped to 1.0. However, IL2R levels increased while the patient was receiving ATG. 11 days later biopsy was 2.5 and IL2R was 2.298 units/m indicating recurrent rejection. It was l.

SM = solumedrol, ATG = anti-cell line cell group Roblin. 0KT-3 = orthoclone 0KT3゜Figure 12: Viral infection grading. Soluble IL2H in medically diagnosed AIDS patients as indicated Plot of serum levels of .

Figure 13: Soluble IL2H using the CELLFREE assay. Representative standard curve for measurements of.

Section 14N: Addition of recombinant IL-2 to a series of CellFree™ assays effect.

Figure 15: Normal healthy host and measurements using the IL2R Cellfree™ assay. Levels of serum IL2H in patients with defined diseases.

Figure 16: Rheumatoid arthritis (RA), degenerative joint disease (DJ) and unclassified Distribution of CD8 levels in serum and synovial fluid among patients with combined disease (UJ) .

Figure 17: Testing of kidney transplant patients for serum CD8 and serum IL2R. soluble Plot serum levels of either rL2R or soluble CD8 against time. Ru. An episode of CsA toxicity and rejection is shown.

Figure 18: Non-Hodgkin lymphoma (NHL) or acute lymphoblastic leukemia (ALL) ) Distribution of serum CD8 in children.

Figure 19: Distribution of serum CD8 levels in patients with infectious diseases.

i engineering north ox detailed l friend The present invention provides soluble Tffi follicular growth factor receptor, soluble T cell differentiation antigen, and for the measurement of relevant soluble molecules or their fragments and the diagnosis of diseases and disorders. This guides the use of the measurements in diagnosis and therapy. The measurement of this molecule is testing the effects of therapeutic treatments on, detecting and/or staging the disease in a subject It is useful for specific diagnosis of physiological conditions in subjects. These measurements The test may also help predict the outcome of treatment and assess and test the patient's immune status. . Soluble molecules include, but are not limited to, serum, plasma, urine and saliva. can be measured in any body fluid of a person.

Protein-like macromolecules derived from the surface of T cells, or fragments thereof, and on the surface of T cells or activated T cells, and present in body fluids and on the surface of T cells. Protein-like macromolecules with immunologically similar replication moieties that are not bound to It is called a soluble T cell surface molecule. These h5 polymers can be glycosylated or non-glycosylated. can be either cosylated and soluble in itself or other highly soluble Molecules may be considered soluble due to their potency associated with them.

Furthermore, polymers present in body fluids and not associated or bound to the surface of cells. The molecules are called soluble polymers.

5.1. SL-Treatment 1 5 no Tomo The present invention provides a method for testing the effects of a therapeutic treatment on a subject who has received the treatment. provide This method consists of a T cell growth factor receptor or a T cell differentiation antigen. or the amount of immunologically relevant soluble molecules or soluble fragments thereof. It consists of taking measurements at suitable time intervals. Any change in one of the soluble molecules change or unchanged can be equated and correlated with the effect of therapeutic treatment on the subject1 In certain embodiments of the invention, interleukin-2 receptor (IL2R) Soluble molecules that are immunologically related to Immune modulators such as crossporin A and monoclonal antibody 0KT3 sandwich enzyme immunoassay (e.g. (see Section 15, below). In other embodiments Soluble molecules related to interleukin receptors can be measured.

Therapeutic treatments that may be evaluated according to the present invention include, but are not limited to, radiotherapy, drug therapy, This includes drug administration, immunosuppressive or immunostimulatory regimens, etc. Immunosuppressive regimen is limited to this but not cyclosporin A, chlorambucil, cyclophosphamide or Drugs like zathioprine, and anti-T3 monoclonal antibodies and antithymocytes This includes the administration of anti-T cell antibodies such as glypurin. Immunity promotion regimen is limited to this However, interleukin-1, interleukin-2 and other T cell Including the administration of long-term factors.

5.2. Detection and treatment of diseases in subjects In other embodiments of the invention, the T cell growth factor receptor or T cell differentiation antibody Measurement of soluble molecules of body or immunological relevance in a subject It may be used to detect and/or stage a disease or disorder.

Measured amounts of soluble molecules are known to be normally present in patients with varying degrees of disease or disorder. It is compared with Standard 1, which is certified by Subject during certain stages of disease or disorder Body fluids of a subject that are similar to the standard amount established to be normally present in a subject's body fluids. The amount present in is an indication of the stage of the disease in the subject. Subject according to the present invention Diseases or disorders that can be detected and/or staged in but includes those listed in Table 1N below.

Table 1I Detected and tracked in subjects according to the present invention - and 1. Infectious diseases induced by viruses - lepe, virus cytomegalovirus Epstein-Barr virus HTLV-III/LAV/HIV (AIDS) HTLV-I related adult T cells Shirokumi disease 'rB murine lymphoma Burkitt lymphoma hairy cell lymphoma sézary syndrome B-cell acute lymphoblastic leukemia solid tumor III. Autoimmune diseases sumamanoid arthritis Diabetes multiple burden syndrome Systemic erythematous lupus lv, organ allograft rejection In certain embodiments of this aspect of the invention, IL2R or related molecules in plasma or measurement of serum levels to detect disease, or to determine disease stage and identify hazards. can be used to define For example, non-Hodgkin lymphoma or B-cell acute lymphoma Cancers such as lymphocytic diseases such as lymphocytic leukemia or adult T-cell leukemia , can be tested by measuring soluble IL2R serum levels; i.e. Elevated serum IL2R correlates primarily with the severity of the disease state and also with minimal response to treatment. It has a poor response and poor prognosis. In other cases, patients with non-lymphocytic cancers The response to treatment to IL-2 can be tested: in this case, an increase in soluble IL2H Serum levels indicate a positive response to IL-2 treatment. The response to viral infection is also It can also be tested by measuring the patient's soluble IL2R levels. for example , patients infected with herpesvirus or AIDS virus have soluble IL2 Gives elevated serum levels of H. In other embodiments, plasma or serum levels are can be measured in transplant patients; ie, elevated serum levels of soluble IL2H It is a diagnostic indication of allogeneic graft rejection. In another specific embodiment, the T-line CDS-like molecules can be measured: i.e., detection of increased levels of soluble CD8 Associated with various diseases or disorders such as rheumatoid arthritis. on CDS-like antigen Detection of elevated levels is distinct from immune activation as measured by elevated cell release. and specific pathological episodes and markedly elevated numbers of suppressor/cytotoxic T cells. It is possible to show the relationship between

5.3. In other embodiments of the present invention, it is possible to Measurement of soluble T cell growth factor receptors, T cell surface antigens or immunologically related molecules is a specific biological condition that can be differentiated between two or more physiological states in a subject. It can be used to specifically diagnose physical conditions. For this purpose, soluble The measured amount of the molecule is the soluble amount normally present in a patient with one of the putative physiological conditions. compared to the amount of children. Normally present in subjects with one of the physiological conditions and one Measurements of soluble molecules similar to those abnormally present in subjects with physiological conditions such as The quantification represents the physiological state of the subject.

In certain embodiments of this aspect of the invention, the measurement of soluble molecules is performed in renal allografts. Used for the specific diagnosis of graft rejection, especially for the differentiation of cyclosporin A renal toxicity It is possible. In a particular embodiment of this aspect of the invention, the soluble molecule is IL2R or related molecules thereof. Allograft rejection using the methods of the invention Similar specific diagnoses include, but are not limited to, the liver, heart, and pancreas. may be applied to other organ allografts.

In other specific embodiments, measuring serum CD8 levels is used to distinguish from other comorbidities. It can be used for the specific diagnosis of equine arthritis.

In yet another specific embodiment of the invention, the lymphocytic leukemia can be specifically diagnosed by identification and detection, distinguishing it from other leukemias. .

5.4. Soluble T, growth factor receptor, T cells (and present in soluble form in body fluids at levels that correlate with the disease state or disorder or its stage; Any T cell surface molecule or immunologically relevant molecule present in the practice of the present invention It can be used for administration. T cell surface markers that can potentially be used include, but are not limited to: This includes those listed in the above schedule.

Some of the markers listed in Table 1 above are already known to exist in soluble form. It is being CD2, a receptor for sheep red blood cells, was found in normal control patients. [Falcao , R., P. et al., 1984, Tariff. love.

Imtsur 13: pp. 141-143, Oh, Nis, K. et al.

1985, Skyrondo, J., Imtsur, 22: pp. 51-60 (Falc. ao, R, P, et at, 1984. Cl1n, Lab, Im munol 13:141-143. Oh, S, -ni,, et al,, 1985.5cand, J. Immunol 22:5l-60)l. Thin Surface markers found on suppressor/cytotoxic T cells that may be involved in cellular recognition Leu2 (OKT8), which is a cancer cell, shows highly elevated levels in the serum of patients with T-cell leukemia. [Fujimoto, J. E. et al., 1983; J. E., Exp., Me. 159: pp. 752-766 (Fuj imoto.

J., et al., 1983. J, EXD, Hed, 159 Near 52-786)]. Miller et al. [1982, F. Ingle, J.A.

Met, 306. pp. 517-520 (1982, N, Enpt, J, H ed, 3.06:517-520)] is a monoclonal anti-antibody laboratory. The release of Leul from inside was reported. However, Leul antigen is normal or cannot be detected in the serum of leukemic patients who have not received antibody treatment.

Other T cell surface molecules whose soluble forms can be measured according to the invention include, but are not limited to: However, for example, interleukin-2 receptor and interleukin-2 receptor Contains T-cell growth factor receptors such as receptors. In certain embodiments, Serum IL2R measurement predicts treatment outcome and is useful in cancer, immunodeficiency, autoimmune diseases, and can be used to test the immune status of patients suffering from allograft rejection.

5.4.1. ’　　　・　ゞゞ Any known method for measuring soluble molecules can be used in the practice of the present invention.

Such methods include, but are not limited to, radioimmunoassays to name but a few. , ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassay, precipitation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay Imm [Jnoradiometric assay] fluorescence immunoassay, Competitive or non-competitive testing using techniques such as protein A assays and immunoelectrophoresis Includes competitive assay systems.

In a preferred embodiment, a sandwich enzyme immunoassay can be used. like this A description of one such embodiment is as follows. induced against soluble antigen A monoclonal antibody (capture antibody, mAbl>) is adsorbed onto a solid substrate.

Soluble antigen present in the sample binds to antibodies and unreacted sample composition is removed by washing. Remove it. Oxygen-binding monochrome directed against the second epitope of the antibody the null antibody (detection antibody mAb2>) binds to the antibody captured by mAbl; Complete the sandwich. After removing unbound mAb2 by washing, the substrate solution was Add to well. A colored product is formed in proportion to the amount of antigen present in the sample. anti The reaction is stopped by the addition of a stop solution and the absorbance is measured spectrophotometrically. standard song A line is generated from a known concentration of soluble antibody from which unknown sample values can be determined. special implementation In many cases, such assays can detect soluble IL2R levels or soluble T cell anti- It can be used to determine the original level. Preferred embodiments for measuring IL2H In this study, anti-IL2R mAbs 2R12 and 7G7 were tested in a sandwich immunoassay ( odor (such as the CELLFREE™ test described in Section 15 below) can be used as capture and detection antibodies, respectively. Regarding measurement of anti-CD8 antigen levels In a preferred embodiment, anti-CD8 mAbs 4C9 and 5F4 are each capture in a dewy oxygen immunoassay (as described in Section 17 below). can be used as a detection and detection antibody.

6. 16 IL2Rf7) Detection in patients described in Sections 7 to 15 below Examples include detection of soluble or cell-free IL2R in patients and treatment of various diseases and The use of such detection to stage calls and conditions is illustrated.

The results show that: (a) Serum IL2R levels are associated with leukemia and lymphoma. Elevated in patients with active lymphocytic diseases such as. in the patient , serum IL2R levels create a primary relationship between disease severity and poor prognosis. vinegar. (b) Serum I L2R levels are usually elevated in patients with non-lymphocytic cancers. However, IL-2 patients receiving the treatment who respond to treatment have lower serum IL2H indicates an increasing level of (C) Serum It,2R levels reject allografts. Serum II, 2R is elevated in transplanted patients, but serum II, 2R is elevated in Not elevated in patients experiencing intoxication caused by suppressive drugs does not show true allograft rejection.

The methods used in these examples are described in the following subsections.

6.1 monoclonal Monoclonal antibodies directed against IL2H were produced as previously described. Jita [Uchama, T. et al., 1981, Jiei, Imtsuru, 12B (4) : pp. 1393-1397 Nirubin, L., A. et al., 1985, Hybrid 4: pp. 91-102; Jiang, L., K., L. et al., 1984, Jie. stomach.

Exbu, Met, 160: p. 1951 (Uchiyama, etc. At.

, 1984. J, ImmUnol, 128(4) J393-1397; RUb in, L, A,, et al.

1985, Hybridoma 4:91-102; Jung, L., L. , et al., 1984. J.

EXD, ) led, 160:1957) co. In addition, induction against IL2H Monoclonal antibodies can be purchased commercially [Becton-Dickenson, Cocounter, California Diagonostec, Florida (Becto n-Dickenson, Calif.

Counter Diagnostic, Florida) The announced monoclonal antibodies [Uchama; Lupin, Jean] are , It, and 2R, respectively, but each of these monoclonal antibodies recognizes a different epitope. Recognize taupe. Monoclonal antibodies were purified according to established standard methods. . [Kossayer, G. et al., 1984, J., Immuno, Method 66 Cortheir, G., et at., 1984. J, Imn+uno, Method 66:75-79)l. wasabidai Conherence oxidase conjugation was performed according to published methods [Wilson, M. .

B and Nakane, B, K. 1987 “Recent Developments” In the Bar Iodating Method of Conjuncting Horsla Ditsch Peroxide (HRPO) To Antibodies”, in im No Florescence and Related Steining Techniques, Kuna Pu, Duburi, K. Holber and Gee, Wick, eds., Elsevier/ North Holland Biochemical Press, pp. 215-224 (WilSO IIIM, B. and Nakane, P., 1978. ”ReCent　 DE! VelOpmer)tS　in extraction　Periodate　)tetho d of Conjugating Horesradish Peroxid e (HRPO) to Antibodies'', tn Immunoflu orescence Re1ated Staining Technique s, KnalX), W,, K, No! uber, andG, Wic k, eds,, Elsevier/North-Holland Biomed icat Press, PP, 215-224).

6.20 mouths I L2R2R In the examples described in Sections 7-14, the methods described below or Cellfree™ interleukin-2 receptor described in Section 15 below -Test Kit [Teesel Science, Incoholated.

Cambridge, Mass. c, Cambridge, HA) of soluble IL2R in medical samples. used to measure the amount. Each of these assay methods detects differences on the target antibody. Two monoclonal antibodies that recognize epitopes were used.

6.2.1. ” Soluble IL2R was detected in the samples using the method outlined below.

(a) Polystyrene microtiter well [Flow Lab ] in phosphate buffered saline (PBS) at 100 uJ! anti-IL2R It was coated with mouse monoclonal antibody overnight at room temperature.

(b) Discard the coating solution and coat the wells with 0.05% Tween 20 and 0.15M Tris-buffered saline (25mM in sodium chloride) containing Lis pH 7.4 Tween buffer) 300u, l! of 1% bovine serum albumin (BSA) at room temperature. It was shut off for 1 to 2 hours.

(C) Wash wells three times with Tris-Tween buffer.

(d) Add 50 ufI sample per well followed by in Tris-Tween buffer. A 10011 dilution containing fetal bovine serum (Fe2) was added. Wells at 37°C Incubated for 2 hours.

(e) Wells were washed three times with Tris-Tween buffer.

(f) 100uJ per well! Wasabi radish peroxidase [Sigma] Manufactured by Sigma Chemical Co., Ltd. 1 Junction Anti-oxidant A PBS solution containing 50% FC3 of IL2R monoclonal antibody was added. well Incubate for 2 hours at 37°C.

(g) Wells were washed four times with Tris-Tween M buffer.

(h) 17 rriM citric acid and 0.015% H2C, phosphoric acid per well. Sodium dihydrogen (citric acid-phosphate buffer) 100 ujcr) 0.2% 0 - Added fluorinated diamine (OPD). Ink the plate for 30 minutes at room temperature. It was incubated.

(i) Add 2N H2SO4 of 5OLIJ2 to each well and measure the absorbance of each well. Small titer plate reader [Guinatec HR600, Dynatic Corporation Alexandria Virginia River (Dynatech) lR600 , Dynatech Corp.

, AIeXandria, νA)] at 400 mm.

6.2,2 IL2R・Sho-゛ The immunoassay results were based on the supernatant medium defined as 1,000 units/ml. Expressed in units. A supernatant medium solution was produced by the following method.

(a) Ficoll-ha is summarized in producing mononuclear leukocytes and package inserts. Ibark method [Sigma Chemical Company] [Co, )] was isolated from 500 ml of whole blood.

(b) About 1xlO6 cell/ml of white blood cells were mixed with 10% FC3 and 100 units. Penicillin-Streptomycin [Flow Labo The mixture was suspended in PDM I-1640 medium containing [Ratory)].

(c) 2ug/ml phytohemageltin [PHA-Welcome Diagono Manufactured by Stix (PHA-Wellcome Diagnostics)] Added to nutrient solution.

(d) After 3-6 days of culture, the supernatant was removed by centrifugation at 200×g and collected. .

(e) The supernatant was pooled and a relevant value of 1,000 units/ml was determined.

6.2.6’l Motel day III Tongue ten arm 2 Peripheral blood lymphocytes and tissue culture cells Flow cytometry assays were performed as previously described by E. Hoffmann, R.

Ai et al., 1980, 10th, Natl, Akado, Sai, Ni.

Nis, stingray, 71. Pages 4914-4917 Group Offman, R, A,, etc. al.

1980, Proc, Natl, Acad, Sci, lJ, s, A, 77:4914-4917) J.

7. Soluble IL2R levels and interaction with IL2R in vitro Yes As shown in Figures 1A and 1B, serum (soluble) IL2R levels; The number of T lymphocytes retaining cell surface IL2R refers to the number of T lymphocytes that retain cell surface IL2R. well correlated in muageltin (PHA)-stimulated human peripheral blood T11 cell media solution. . However, in vivo, the number of IL2Rs that retain TMA vacuoles in the circulation had little correlation with serum IL2R levels. Therefore, soluble T cells Determining from in vitro experiments whether markers correlate with in vivo observations I can't. Clinical use of serum T cell markers can only be determined from direct patient studies. It can be done.

Levels of soluble IL2R are found to be increased in a wide spectrum of diseases. It's being served. The nonspecificity of elevated soluble IL2R levels is shown in Table 1II.

Table 1II Nail mouth I [-2 receptor average level test sample I-2-2-7! =(lffl/j)-ヱλsuL-Healthy control 250 50÷ AIDS related 960 41 Herpes virus infection 1500 + 9 Other virus infections 1000 5 Systemic erythematous lupus healing 620 50 Rheumatoid arthritis 500’20 + stands for “excess” Specific medical applications of the measurements are summarized in the extensively described examples.

8. Serum measurement of soluble IL2H can be used to stage or investigate leukemia. Human T-cell leukemia, a subgroup of human TM cell leukemia/lymphoma that contributes to Disease virus (human T-lymphocytic virus) type I (HTLV-I) is a human Closely related to adult T-cell leukemia (ATL). Possible etiological factors of ATL and The identification of HTLV-I as a virus is based on antibodies induced against the virus in patient serum. have enabled the development of assay systems for the detection of Ro, R 6 C.

1983, Love, Invest, 49; pp. 371-377 (Sax i ng er, C, and Ga1io, R, C,, 1983, Lab, Inve st, 49:37ff-377) J, antibodies to HTLV-I are virtually all It has been found in all ATL patients. However, HT without proof of ATL LV-I seropositive healthy individuals are common in ATL endemic areas of Japan. Is it an asymptomatic individual? Identification of patients with active leukemia has significant diagnostic and prognostic value. At present No reliable test is available for staging patients with active leukemia.

Impairments in host immune surveillance may influence the development of various cancers in humans and animals. It is known that I Serum levels of L2R-related macromolecules are It provides important identification of different stages of the disease. Serum levels of IL2R are shown in Section 6.2. The described assay was used to measure in patients exhibiting different stages of leukemia. Below As shown in Table 1V below, patients with activated T-cell disease have normal health and HT LV-■ Elevated levels of soluble IL2R compared to seropositive healthy individuals. similar In addition, T-cell deficient patients in recovery state exhibit decreased levels of soluble IL2R.

Table 1V Soluble in leukemia patients 2nd level of J[-2 receptor Soluble IL-2 receptor Blood LP! '(Ie-Kou 1 lost) Active ATL (Japan) 1765 3 Activated T-cell leukemia (USA > 1050 7 seropositive healthy patients 230 17 Recovery Shirahaku group patient 2304 Figure 3 shows that IL2R 1 in serum corresponds to the medical condition of lymphocytic cancer patients. Show that. Elevated levels of IL2R in serum indicate failure of therapeutic treatment. effect After therapeutic treatment, serum levels of IL2R return to the normal range.

9. High serum IL2R levels are associated with advanced disease and early childhood non-Hodgkin lymphomas. Associated with inadequate results of □− The results presented in the following subsections are as measured by the Self-Free™ test (Section 15). Elevated levels of serum IL2H such as showed a primary correlation with the advanced stage of Kin's lymphoma and the effects of selected chemical treatments. Shows poor response to therapy and poor prognosis.

9.1. Disease and treatment Lord - Yoyu 1 Thought From 1979 to 1986, non-Hodgkin's lymphoma (NHL) or B-cell acute lymphoma A series of 99 infants with lymphoblastic leukemia (ALL) had histological features and Depending on the stage of their disease, St.

Mitsuno medical examination at Jude's Research Ho5pital) [Link, 211 etc., 1984 Plos, Am, Sos, Klin, Oncor, 3: p. 251 1 Maafi, Nis, Bee et al., 1986, Jie, Tallinn, Oncol, 4: pp. 1732-1739; Dahl, G., Bui et al., 1985 Blood 66: pages 1110-1114 ink, H, et al, 1984. Proc, Al11. SOC, Cl1n, 0ncof, 3:251:) Lurphy, S. B., et al., 1986. J. , Cl1n, 0nco1, 4:1732-1739; Dahl, G.V. ,, et at,, t985. Blood 66: 1110-1114 )] was given. Serum samples taken before the start of chemotherapy were collected from 65 patients. It was effective in these patients. 41 boys and 18 girls with NHL The age range was 1.8 to 17.9 years (median, 10.7 years). BIB cell A The three boys and three girls with LL were tested in 2.3. ~10.2 years old (median , 5.8 years old).

In each case, the prognosis depends on medical, cell culture, histological, and cytological criteria. In addition, several cases were based on a combination of immunological and cytogenetic studies. operation formula (Ding he Working Formulation) [National Ca Sur Institute Sponsored Study on Class Figure tions on non-hodgkins lymphoma: thermal and di Script John on a Walking Formulation Fork Linical Knee Sage, The Non-Hodgkins Lymphoma Fatology Classification Project, 1982, Camp-49, No. Pages 2112-35 (National Cancer In5 position 5 ponsored 5 studies of C1assfications of Non-)1odgkin’s Lymphoma:Summary and Description of a Working Formula For clinlca+ USAQe, The Non-Hodgkin’ s Lymphoma Pathologic C1assfication Project, 1982. [Cancer49:2112-35)] Small undivided cells, lymphoblastic or giant cells (undivided, dividing and lymphoblastic) ) classified according to type. The first category includes not only Purkitoma but also Also includes lymphoid tumors designated as non-Burkitt type. B Cells A and L are determined according to France-America-British (FAB) criteria. The L3 morphology rate according to Nnet, J., M. et al., 1976, Bri.

Jay. Haematl, 33; pp. 451-458 (Bennet, J, H.

, et at, 1976, Br, J, Haenatol, 33;4 51-458)].

The stages were given in each case of NHL with the use of the system previously described. B., Nis., B., 1980. Semin.

Oncol, 7: pp. 332-339 () Lurphy, S. B., 1980. S em1n, 0nco! , 7:332-339)]. Locally and in appropriate locations Children with uncontrolled NHL present with stage 1 or 11 disease.

! The first floor III is disease disseminated to both sides of the septum, intra-abdominal disease that cannot be resected at high cost. disease, and all major epidurals without bone marrow or central nervous system (CNS) involvement. or involving tumors of the ventral mediastinum. Stage IV involves early CN in addition to other tumor sites. Identified by S and/or bone marrow involvement (25% blasts or less). to the bone marrow If more than 25% of cases are accompanied by malignant B-cell tumors, the leukemia has progressed to the stage of leukemia progression. It is classified as B-cell ALL, which stands for B-cell NHL. In this study, 3 0 patients had diffuse microundivided cell NHL (stage 18, 1110, Irll 22 patients had lymphoblastic NHL (stage 11 patients, stage II 2 people, 114 people Il, and 5 people IV), and 7 people had giant cell NHL (stage N1). 3 and 114 patients, and 6 patients showed B cell ALL.

In addition, those with otherwise normal soluble IL2R and in the age range of 1 to 6 years (median, 3 years) Measurements were made in infants with otitis media.

9.1.2. Placement Cyclophosphamide, doxorubicin (d.

X0rUbiCin), vincristine (VinCriStille) and Rednisone and 6-months of 6-mercaptoppri for combined and sequential treatment Pediatric Oncology Group [Ped. iatric Oncology Gr.

up (, POG)] Step ■ or II according to the protocol I Treating Children with NHL, Link, M. et al., 1984, Press, Am. , Sys, Clin, Oncol, 3: p. 251 (Link, ) 1. , et al. , 1984. Proc, Am, Soc, Cl1n, 0nco1. 3:2 51) J, stage III or small terminally dividing NHL with B cells ALL children with high doses of isolated cyclophosphamide followed by vincristi and doxorubicin or equivalent high doses of methotrexate and citara underwent 6 months of intensive treatment with either method of changing bottles [Murphy, Varnish, Vin -et al., 1986, Jie, Tallinn.

Oncol, 4: pp. 1732-1739 (Murphy, S. B., et al. al,, 198B, J, Cl1n, 0nco1.4: 1732-1 739)], children with stage III or IV lymphoblastic NHL are Another conventional regimen of treatment is teniposide plus cytarabine. [Dahl, G., Bui et al., 1985, Blood 66: No. 1110-111 4 pages (Dahl G, V, et al, t985. Blood 66 :1110-1114) fSe informed consent Results were obtained for all patients and the study was approved by the Society's Medical Examinations Committee.

9.1.3 0 sucrose I L2R level 2 (commercial) interleukin 2 receptor – Test kits (T Cell Sciences, Inc., Cambridge) The measurements were taken using the following: The serum level of I L2H is in units of (U) /ml is displayed. Phytohemageltin stimulated peripheral blood lymphocytes A control sample of 1000 tJ/ml supernatant was used as a standard. In healthy adult host Normal serum IL2R value is 50-500 U/mI (median, 260 U/n +I).

9.1.4 Serum lactate dehydrogenase (LDH) ) was monitored using the Kinetic AMB-610 test on the KDA analyzer. [American Monitor Corporation 1. indianapolis, indiana Anna State (American Montor Corp.

, Indianapolis, IN).

Samples with enzyme activity greater than 700 U/L were diluted and reassayed; The obtained value was then multiplied by the dilution factor. Typical values for the inventor's laboratory are 30~ The range is 300 tl/L.

9.1゜5″, tongue・7 The Ruskal-Wallis study was conducted in NHL patients. Comparing soluble IL2R or serum LDH levels between different subgroups of people. Do not use it to Human production-moment correlation (Ding he Person pro duct-Illoment correlation) (r> and t - Test assay used to determine the association between soluble IL2R and serum LDH levels Shina. Log-rank test (!ogrank) of time vs. decay (fai1ure) curve test) [Beto, R., and Beto, J.A., 1972, J.A. .

R, Stato, Sis, 135A: pp. 185-216 (peto, f?.

and Peto, J., 1972. J, R, 5tat, Soc, 1 35A:185-216)] using the Kablan-Tyre method (Ka plan - Meier procedure) [Kablan, E, L, O Mayer.

B., 1958, J.A., Am., Stat., ASIS, 53: No. 457-4. (page 81)]. Achieving recovery and relapse or something that causes decline versus time This was determined as the interval between death and death. Patients who did not achieve recovery had a It was established that The impact of potentially significant prognostic factors on decline versus time is While allowing comparison of treatment outcomes for two or more subsets, Covariates that simultaneously adjust for the effects of factors Cox proportional-hazard model [Cox, D., R., 1972; J.A., R., Statt, S. .

Bee, 34: pp. 187-220 (Cox, D, R, 1972, J, R , 5tat, sOc, B, 34:187-220)]. With P value The final selection of factors for models that depend on rward 5tepWiSe procedure) is less than 0.10 after adjustment for other variables. 100OLJ/ml IL2R values are well within reach of medically distinct groups of diseases and treatment outcomes. is chosen as the dividing point between high and low receptor levels.

Lord - Suuu - Sustainment The most reliable prognostic factors in early childhood NHL are diagnosis and serum LDH levels. [Murphy, Niss, Bee et al. 1986, Jie et al. I, Tallinn, Oncol, 4: pp. 1732-1739; Murphy, Nis, B. , 1980, Semin, Oncol, 7: pp. 322-339; Arecenew.

J. E., C. et al., 1975, Am. J. E., Met., 58; No. 314-321. Pages; McGrath, I, T. et al. 1984, Blood 63; No. 1102-11 p. 11 (Hurphy, S. B., et al., 1986. J.

Cl1n, 0nco1.4:1732-1739;) Iurphy, S, B ,, 1980. Sem1.0nco1.7:332-339; Ar5ene au, J.C., et al., 1975. 8m, J.

Med, 58-314-321; Hagrath, 1. T,, et a l,, 1984. Blood63:1102-1111)]. The inventor is Highly soluble IL2R levels indicate insufficient treatment even after adjustment for these two factors. We report here the expected results of the installation.

9.2.1 Soluble IL2R levels have a strong relationship to disease stage All patients were 12 children with otitis media (404-9421/m1; median 8 t51J/　m! > showed detectable soluble I L2R levels (fourth figure). The highest value was 1030 to 77251/+ml for patients with B cell ALL. (median, 3283υ/m1) was found in serum. for stage or IV Children with minimally dividing cell NHL may have stage III or IV lymphoblastoid NHL. 376-3390υ/ml (median 1808I) regarding L (p・0.02) J/rnl> significantly higher than 329-53350/ml (median, 1 showed a soluble IL2R level of 832 υ/ml). The latter concentration is the step FRI or is II NHL, 1000-11431/ml (median, 4771/ml > cp = o, ooi). have lower stage disease Only one of the 28 subjects tested had soluble IL2R levels above 1000υ/+nl. showed.

9.2.2 Soluble IL2R levels show a linear relationship with serum LDH levels Serum LDH level was ALL [Puy, C, H, et al.

1985, Blood 66: pp. 778-782 (Pui, C, -H, et al. al., 1985. Blood 66:778-782)] and NHL [? - Fee, Nis, Bee et al., 1986, Jie, Tallinn, Onkor.

4: No. 1732-1739; Arseigne, Jay, Seay et al. 1975, Am, Jay, Met, 58. Pages 314-321; McGrath.

I, T. et al., 1984, Blood 63; pp. 1102-1111 ( Hurphy, S, B, et al, 198B, J, Cl1n, 0n co1゜4:1732-1739; Ar5eneau, J.C., et al. ,, 1975. Am, J, Hed, 58:314-321; Hagr. ath, 1. T. et al., 1984. Blood 63: 11 02-1111) Reliable indication of the total body burden of malignant cells in the therapy of J. be. The distribution of serum LDH levels among different subgroups of patients shows that IL2H The pattern was similar to that observed for B cells: Larger than stage I-II NHL and thicker than stage III-IV lymphoblastic NHL Thicker than stage 11I-IV diffuse microundivided cells NHL. In this study serum There is a strong positive correlation between IL2R and LDH levels (Figure 5).

9.2.3 Highly soluble IL2R level is 1t!・ High soluble IL2R levels were associated with poor treatment outcome. 47 people at low level 18 patients with levels above 10,000/ml compared to only 6 of the patients Nine of the patients failed treatment (Figure 6A, p=0.001). Stage I also soluble 11. even when excluding patients with disease II. ．． 2R high level Figure 6B is associated with still unsatisfactory results.

1) = 0.02) Possible correlation between medical and physiological risk factors in this patient Due to interactions, other covariates (COVariate) were determined using Cox regression analysis. After adjusting for the effects of , other relatively important factors were assessed. As shown in Table 7 , both soluble IL2R levels and serum LDH levels have been shown to have independent prognostic value. was discovered.

Table 7 Blood over time versus decline in 65 children with NHL Manaha B@cystis ALL Sei IL2R and Noru' Category - 111” Temple-m-′ Good Bad Ai Pi.

- Original change analysis IL2R level (U/ml) 1000-, 1000- 0.004 Kano level Le (U/L) 300-300+ 0.007 steps I, IS III, IV　O,017 or all B Histological type: lymphoblastoid! Small unmolecule flI O,65 multidimensional change model le 9 IL2R level (Ll/ml) 1000-1000+ 0.004LDH level Bell (U/L) 300-3004 0.065 steps I, II, III , IV O, 62 or all B Histological type Lymphomonocytic mineral ■ Small undivided all 0.488 Probability ratio B cox propo from likelihood ratio test In a national-hazard model, the displayed category is calculated without adjustment for the effects of other variables. Decay versus time between patients with good versus worsening risk characteristics for recurrence in Golly connection of.

Considering the competing effects of the hardware variables added to the Cox model, the treatment outcome step*ise regre analysis to identify the best prediction of results. Same as above except that ssiori analysis) was used.

Elevated serum IL2R levels in this patient may be associated with malignant cells or activated normal lymphocytes. may lead to greater release of receptors from either. The inventors believe that soluble IL2R levels not only correlate with disease stage but also provide a reliable table of malignant cell burden. This supports the first explanation as it shows a linear relationship with serum LDH levels.

The present invention shows that soluble IL2R levels in children with NHL are an independent predictor. later in life, higher levels associated with more advanced disease, larger tumor burdens and We conclude that the results show poor performance.

10. Upper Cl-soluble IL2R in blood of patients under therapeutic treatment with IL-2 Generally, patients with non-lymphocytic cancers have elevated levels of serum IL2R. Not shown. However, the following subsections indicate that IL-2 responds favorably to IL-2 treatment. 2 shows that such patients receiving treatment exhibit elevated levels of serum IL2H.

10.1 I L2R levels in cancer patients not treated with 1L-2 As shown in Figure 7, serum levels of soluble IL2R were significantly increased after treatment with IL-2 inoculation. It is highly elevated in a variety of patients. Serum level of measurable IL2R in serum The bell remains elevated during the treatment procedure. Serum levels of IL2H are important for patient treatment. can be used to check the response of

10.2 Blood IL2R levels in cancer patients responding to IL-2 treatment IL-2 treatment using the Cell-Free IL2R test (Section 15, described below) Serum IL2R levels were measured in patients with lung cancer who had undergone cancer. patient 1 and 2, 3 × 1061 J/ml recombinant IL-2 continuously from 0 to 14 days. The patient showed a good response to the treatment. These patients have elevated serum IL2H levels. (See Figure 8).

Patients 3.4 and 5 received 2 x 106 U/ml recombinant IL-2 for days 0-14. The patient was injected continuously until 2000 and showed little or no response to treatment. these patients showed no increase in serum IL2R (see Figure 8).

11. Serum levels of soluble IL2H differentially differentiate between transplant rejection and pretreatment. Diagnose Also, ゛゛゛゛゛゛゛゛゛゛ The immune system plays an important role in tissue transplantation. Graft rejection is mainly It is a cell-mediated phenomenon, and hypolymphocytes are primarily responsible for the response to graft rejection. It is Noh.

Therefore, soluble T cell surface molecules such as IL2H are important for immune status and graft rejection. Useful for testing reactions. The following study demonstrates this utility. serum IL2 R was measured in kidney transplant patients using the assay described in Section 6.2.

Today, a more effective approach to prolonging the existence of transplanted organs is to use human T cells. , antilymphocyte antisera, and a number of monoclonal antibodies against immunomodulatory drugs. The goal is to control cell-mediated immune responses using multiple therapeutic reagents. many organ transplants One of the immunosuppressive drugs currently prescribed to recipients is a Matomoma-derived cyclic peptide. Cyclosporin A (CsA). CsA shows specificity for lymphoid cells. It also effectively suppresses IL-2 production at an early point in T cell activation. deer However, the medical use of CsA in transplant patients is hampered by the presence of significant toxicity. have difficult problems.

Currently, the most effective diagnostic test for functional impairment in transplant patients is is to measure the serum level of tinin. However, this testing method Distinguish between true kidney rejection and toxicity caused by immunosuppressive drugs such as sA. (Figure 9). As shown in Table V1, IL2R-like in the patient's serum Measurement of macromolecules is advantageous in distinguishing between CsA poisoning and liver rejection.

Table VI Oral It-2 Blood S Label in Kidney Transplant Patients Measured in Units/HL le -1" 1" Ll-Mo Jun Shusei ■Sha Ekomi Before transplant (dialysis> 721 434 18 normal 150 105 20 Stable transplant 270 206 89 C3A poisoning 493 241 12 Rejection reaction (before treatment > 1032 584 13 Viral infection 1420 55 5. Immunotherapy with 4 antibody 0KT3 or ATG 1758 724 23 In this experiment, serum levels of IL2R remained low in response to CsA intoxication. IL2R levels were up to or slightly elevated in response to renal rejection. increased in response to Therefore, in combination with serum creatinine measurements, I Measurement of the immune response, as shown by serum levels of L2R, tests the effectiveness of therapeutic treatments. It is effective for Furthermore, measurement of JL2R levels is important for CsA toxicity and renal rejection. It is effective as a method for specific diagnosis between reactions. This non-invasive reaction Existing specificity of CsA poisoning and renal rejection including experiments on biopsy specimens from kidneys Preferred across diagnostic methods.

Similar observations have been made in liver transplant patients. As shown in Table Vl1, liver Patients with organ transplant rejection exhibit elevated levels of soluble IL2R in their serum. . Decreased serum levels of IL2R indicate that patients who did not show any signs of rejection after transplantation was discovered by someone.

Table Vl1 unit/) I[-2 receptors in liver transplant patients measured in Tar's blood "Level I" Before transplantation 6474 Stable transplant 600 1 Rejection reaction 1500+3 + indicates "excess".

12. The effects described in the subsection below plasma IL2R levels in liver graft recipients In the examples, the Cellfree™ Interleukin-2 Receptor Kit was used. Serum levels of IL2H were measured in kidney allograft patients using . These patients who experienced true allograft rejection were Elevated serum levels were expressed.

Upper λ-upper-imaginary hill meng y direction Thirty-two patients received HLA non-isogenic renal allografts from deceased or living related hosts. studied patients. Patients should be treated with steroids and cyclosporine (CsA) i or Maintained with zathioprine. A rejection episode (i.e. decreased urine output) Progressive rise in serum creatine caused by one or more of the following: diagnosis based on standard medical or pathological fields. CsA level, radiation Measured in plasma by immunoassay. CsA intoxication in response to decreasing dosage of CsA 700 ng/m, serum creatinine levels associated with plasma levels of 1 or higher. This was identified as an increase in bell progressivity. Rejection episodes plus steroids, anti T3 monoclonal antibody (mAb) OKT3 [Ortho Pharmaceutical Manufactured by Kano Pharmaceuticals or ATGAM (trademark) Treated with [Equine antithymocyte globulin, manufactured by Abjohn (tJpjohn)] .

Plasma (citrate) for IL2R assay was administered once or twice a week before transplantation and for the first few weeks. After that, the samples were taken out at intervals of 1 week to 1 month and stored at -70°C. X The L2R assay can be performed using the Cellfree™ Interleukin-2 Receptor Kit. Cell Science, Inc., Cambridge, Massachusetts (see Section 15 below). The test starts with the first antibody (mAb 2R) into plastic microwells, washed 1, blocked, or 50u Two non-competing monomers applied to human JL2R at a dilution of 100u of jJ sample and Q. Use clonal antibodies. After 2 hours of incubation, wash the wells and The indicated antibodies conjugated to Radish peroxidase were added. Two hours later, wow The well was washed and orthophenylenediamine (OPD) was added. The reaction was carried out with 1N sulfur After adding the acid, the mixture was rapidly cooled for 30 minutes, and the absorbance was measured at 490 nm. 1000 units/ml Phytohemageltin (PHA)-stimulated peripheral blood nucleated cells defined as values Clear sequential diluents were used to generate a standard curve. The summary data (Table ■Ili) is Exclude the first 4 patients studied before converting values to PHA units.

12.2 Plasma contains soluble IL during episodes of renal allograft rejection Including 2H rising level - Data regarding plasma IL2R levels in study patients are summarized in Table Vll1 .

Table Vll1 Good encounter uni≦loliko hell Sample unit / ml Dansko D” 20 150 ± 105 Before transplantation (dialysis) 14800±450 0.001 ~ vs. normal stable transplantation 63 26 6 out 205 0.02 pair normal rejection 15 t050±637 0.001 -Kiding OK Ding 3/A Ding G under treatment 20 1788±726 0.004 pair rejected Reactive viral infection 6 1076±654 0.001 pair of stable cyclostomes Lin-13430±218 0.003-M Rejection kidney toxicity 'P defined as 1,000 υ/ml Arbitrary value based on HA supernatant standard; Data on 28 patients and 20 normal laboratory personnel, mean above standard deviation .

- indicates "less than".

Patients on long-term dialysis (pre-transplant samples) have significantly elevated levels over normal controls. (po, less than 001).

These values fell to normal levels during the first week after transplantation.

Patients with stable renal function had values slightly greater than 5 points (po, (less than 02). In contrast, patients with acute rejection episodes are less likely to be treated as stable patients. showed a significantly higher average value. Serum creatinine level increased but anti-rejection These samples, taken before the initiation of reactive treatment, showed that 13 patients had a rejection reaction. Ten patients with episodes showed elevated IL2R levels. anti-T cell monochrome Immediately after treatment with natural antibodies (OKT3 or ATG), I L2R levels It rose sharply. This increase subsided approximately 1 hour after the first dose of mAb 0KT3. was evident in the samples submitted (average increase of 36% in 5 sets of samples, po, less than 05). mAbs for which activated T cells were probably not previously present An exception was seen in patients given ATG prophylactically. have CsA kidney toxicity Patients showed significantly lower levels (po, less than 003) than patients with rejection.

Three episodes of nitrogen overload (no biopsy) could not be classified (decreased CsA and (improved with increase in other immunosuppression) and were not used in data analysis. 2 patients people are susceptible to difficult viral infections (cytomegalovirus, herpes simplex virus). others contracted a viral syndrome with diarrhea; all showed significantly elevated levels of L2H. One patient with renal failure due to renal vein stenosis (Cr3-4%) and one patient with hemolytic uremic syndrome (Cr5-6%). ■%> did not show increased IL2R levels seen in patients with rejection. Ta.

In allograft rejection, activated T cells are found in the blood and in the graft. [Fan] Enis is brought out.

A. et al., 1984, Transplant, 37. Page 65; Pankok. da Brew, W. et al., 1985, Transplant, 39; p. 430 (Van ES, A., et al., 1984. TralSplant , 37:65; Hancock, W.W., et al., 1985. Transplant.

39:430)], while the soluble/L An increase in the amount of free 14JL2H was observed in vitro in PHA-stimulated media. This was not expected with an observation level equal to that which occurred in the previous year. The inventors also found elevated levels of IL2H in certain infections.

Administration of 0KT3 or ATG anti-T cell mAbs likely inhibits antibody-induced cell damage. inducing an increase in IL2R levels, likely due to the resulting release of cellular IL2H. I woke you up.

An exception to this observation occurred when ATG was given prophylactically. Therefore, IL2R Progressive rise in Bell may be a useful confirmatory test during anti-T cell antibody therapy.

Long-term dialysis patients showed elevated concentrations of IL2R in serum. These patients Elevated T cells are not known to exhibit cell activation and, in fact, are not known to exhibit impaired T cell function. has not been found at all. The nature of damage in chronic renal failure is evidenced by urinary toxicity immunosuppressive factors in the serum or plasma of patients, but remain puzzling [fermen et al., 1980, J. Clin. Ne7yr7-R. 14: p. hrman et at, 1980 J, Cl1n, Nephrol, 11:183)], because I L2R can bind IL-2, It is possible that soluble IL2R itself is an immunosuppressant; Yi et al.

1986L Shei, 4 M/L, 137: No. 3841-384431r (Rubiri.

L,A,,et,at,,198B,J,Immunot,137:3841 -3844) and therefore can compete with IL2R on the surface of T cells. Chronic uremia The reason for the elevation of IL2H in this disease is unknown. The simplest explanation is that soluble I L2R is normally secreted by the kidneys. Further research will address this layer. However, certain other causes (renal vein stenosis, hemolytic uremic syndrome) In principle, acute hypernitricemia due to Our data do not show a significant increase in renal function and IL2R levels. Shows the opposite conclusion to a simple relationship with Bell. This could be due to acute renal failure or another factor ( It is not known whether it is related to either dialysis or immunosuppression (eg dialysis or immunosuppression). in addition Despite the struggle, elevated levels in acute uremia occur before levels fall to normal. This makes interpretation difficult, especially in the early post-transplant period.

Our data indicate that plasma is associated with an episode of marked renal allograft rejection. plasma/serum T L2 The test for R is specific for the diagnosis of renal allografts, especially for CsA renal toxicity. We propose that it may be pharmaceutically effective in identifying

13. IL2R levels and levels in cardiac allograft rejection testing The current clinical success of V heart transplantation with of allogeneic grafts using immunosuppression and sequential endomyocardial biopsies based on the use of A. Related to testing [Austin, W., G. and Koshimi, A., B. et al. , 1984, N. Ingle, J., Met.] 311: No. 1436-143. Page 8 (Austen, W, G, and Co51m1. A, B,, 1 984. N, Engl.

J, Med, 311:1436-1438)], intracardial pericardial biopsy Although relatively stable and reliable in allogeneic graft recipients, is an invasive technique associated with some pathological conditions that is not easily performed in children. . Furthermore, biopsy resolution is subjective and this is an expensive detection technique. . However, intracardial pericardial biopsy currently does not lead to potentially irreversible rejection. is the only accepted means of diagnosing cardiac allograft rejection prior to initiation. Ru.

Heart graft rejection is a cell-mediated immune response that primarily involves activation of T lymphocytes.

The process of T cell activation involves the expression of interleukin 2 receptor (IL2R) achieved by. The experiments described below demonstrate that cardiac allografts compared to endocardial pericardial organisms Intended to test the diagnostic value of serum IL2H for allograft rejection. Ru. 56 sera from 6 heart transplant patients obtained at the time of intracardial pericardial biopsy. IL2R levels did not correlate with histological grade of rejection (r=0.56).

For biopsy grades less than 2°, the mean serum level was 387 and 41 units/mI. For biopsies diagnosed as rejection (2,0 or more), the average IL2R levels were 1017 ± 178 units/ml (p less than 0.0001). full). The starting serum level for rejection was calculated to be 546 units/ml. It was done. In patient serum with IL2R levels below 545 units/ml, Corresponding biopsies showed no evidence of rejection in up to 40 of 42 samples (specificity = 95%). IL2H levels above 545 units/ml indicate that the corresponding biopsy is significantly It was present in 8 of the 14 cases that showed a strong rejection reaction (sensitivity = 57%). These de data indicate that normal IL2R levels indicate the absence of cardiac allograft rejection. In contrast, elevated IL2R levels imply a strong rejection response.

13.1”- Forty-eight right ventricular endopericardial biopsies were performed on six adult orthostatic transplant recipients using standard techniques. Obtained between 3 and 752 days (average SEM, 159 ± 22) after transplantation from [Deck, G., W. et al., 1985, F. Ingle, J.A. , Met, 313: pp. 885-890 (DeC, G, W,, et at .

, 1985. N, Engt, Red, 312:885-89 0)], at least 5 endocardial 7 fragments were subjected to cardiac allograft rejection. tested by light microscopy, immunoperoxidase and immunofluorescence for evidence of did. Biopsy according to Stanford (5tanford) criteria [margin, J. , W. and Billingham, M., E., 1980, 2. Progress I Curve 4 o C 9; pp. 113-146 (MaSOn, J, W, an d BillBllin, ) f, E, 1980. Progress iri Cardiology 9:113-1!6) I L2R using J Expected grade 0.0 normal without knowledge of level 0.5 Few foci of periintestinal lymphocytic infiltration 1°0 Periintestinal and/or or occasional foci of interstitial lymphocytic infiltrate 1.5 Numerous diseases of periintestinal and interstitial lymphocytic infiltrates without myocyte necrosis nest Multiple foci of lymphocytic infiltration associated with foci of myocyte necrosis >2.01 2.5 Scattered lymphocytic infiltrates associated with occasional eosinophil content and numerous foci of myocyte necrosis. Jun 3.0 Eosinophils and neutrophils, periintestinal hemorrhage, edema, and multiple myocyte necrosis All biopsies containing foci of diffuse lymphocytic infiltration were graded for their most severe component. Ta. A histological grade of 2° or higher is considered an indication of rejection requiring additional treatment. did.

Freeze the serum obtained from each biopsy at -20°C for immunofluorescence studies. A of each sample Recoat with double monoclonal antibody using a non-competing monoclonal antibody to human IL2R. Analyze with null antibody sandwich technology (Cellfree (trademark), Teacell) Science, Inc., Cambridge, Massachusetts, below. (see Section 15). Normal IL2R as determined from 157 blood host samples Levels were 267 and 119 (± standard deviation) units/ml.

All patients were treated with maintenance immunosuppressants consisting of prednisone and cyclosporin A. did. Five patients also received azathioprine. first rejection episode were treated with increasing doses of steroids. ATG (antithymus cell globulin) or 0KT-3 [orthoclone 0KT3 monoclonal antibody, orthoclone Pharmaceutical Corporation, Raritan, New Chassis (Or thOCIOne oniding3 monoclonal antibody, 0r tho Pharmaceutical C.

RP, Raritan, NJ) antilymphocyte treatment with additional steroids. given to patients with rejection who were non-responsive. Statistical analysis suitable for analysis Nonparametric, linear regression and nonlinear regression For regression analysis, R8/1 software [Bolt, Perlanek and Newman] was used. .

Cambridge, Massachusetts (Bolt, Beranek and Ne Perform this using the following procedures: wman, Cambridge, HA). The average value Their standard errors are shown.

13.2. Normal IL2R indicates absence of cardiac allograft rejection whereas And it rises, 2R level is six in six All results are shown graphically in Figure 10, and the completed data for two patients are shown in Figure 11. As shown in the figure. For a pathology grade of rejection less than 2.0, the average IL2R level is 387±41 units/m1 (n=46, range 156–1386) The average level for grades 2.0 and above was 1017±178 units/m1 (n=1 0. range 328-2298). The difference is very large with p less than 0°0001. It was remarkable. Histological grade of rejection based on biopsy corresponding to serum It, 2R level All showed a linear correlation of 0.56 (p=0.0001) with however The data were analyzed using the nonlinear regression shown in Figure 10 to determine the best fit (r=o.44.f =21. p=o,,oool). against the rejection response calculated from this analysis. The starting level of IL'2R was 545 units/ml.

IL2H levels below 545 units/m1 were below 2.0 in 40 out of 42 measurements. specificity associated with a high rejection grade (95%). Average biopsy for this group The score was 0.5±0.1 with a range of 0-2.0. Associated with biopsy grade of 2.0 Two normal IL2R measurements indicate rejection under treatment with additional steroids. occurred continuously in patient B during the episode (Figure 11). accompanying both Biopsy showed resolution of rejection, i.e. myocyte necrosis was still present but lymphocytic Infiltration is significantly less than in the initial biopsy, which was diagnosed as a rejection grade of 2.0. It showed that there was no.

Serum TL2R levels in excess of 545 units/rr+i give a specificity of 57%. Eight out of four measurements showed a corresponding biopsy score of 2.0 or higher. average biopsy The score was 1.6±0.2 on a scale of 0 to 2°5. This is normal ■L Significantly different from the mean biopsy score of the 2R group (p=0.0001). 2.0 less 6 cases associated with a complete biopsy score of IL2R serum levels exceeding 545 units/ml In 5 of these, elevated levels precede episodes of treatment rejection (r+ = 2: 2d and 11d) or successively ((n = 3: 9d, 12d and 35d). The sixth case with elevated levels showed that the corresponding biopsy histology Initial transplantation of patient B, whose symptoms were grade 0 but whose transplanted heart showed an active lymphocytic infiltrate. From post-implantation serum samples.

There is no significant correlation between serum levels and cyclosporine A or days post-transplant. It was.

The data show that normal levels of circulating IL2R are present in allogeneic patients diagnosed on the basis of endocardial biopsy. whereas elevated serum IL2R levels indicate that cardiac transplant rejection is excluded. demonstrated to be a strong indicator of visceral allograft rejection. Obvious hyporesponsiveness ( 57%), the intracardial pericardium and its resolution rather than the IL2R measurement in this analysis. This is probably due to a misunderstanding of the interpretation.

Intracardial pericardial biopsy may remove the focus of rejection. In addition, false positives reduce the measured attenuation. Differences in timing in low numbers, i.e., increases in self-deprecation IL2H, may be due to actual rejection. This can occur before a complete reaction can be diagnosed by biopsy (tissues >2.0 (Specific by class) In fact, elevated levels of IL2R are and elevated IL2R, levels histological evidence of rejection in 6 of the pts is associated with rejection in the two preceding instances. IL2R levels were treated This may be helpful in determining whether the rejection continues or has resolved. It may be possible to prove that. Kidney with AT G or ○K T-3 antibody The procedure of organ transplantation causes a significant increase in IL2H. Similarly, in patient B Following ATG treatment, IL2R levels increased dramatically but with a corresponding biopsy-grade was only 1.0 (Figure 11). However, after 11 days, this The biopsy showed grade 2.5 rejection and the corresponding IL2R level was 2298. year/ml.

Elevated IL2R levels are also associated with severe viral infections, certain autoimmune diseases, [Nelzin. D, L., 1986, Feto, Bros., 45: p. 317. (Abstract) (Nelson, D. L., 1986. Fed, Pro c, 45:377 (abstract)], T-cell leukemia (McKean, E. Le et al., 1986, Feto, Bros., 45; p. 454 (abstract) (Hackeen, L, et at, '1986, Fed, Pr oc, 45:454) (abstract)] and occur during chronic renal failure. . Patient B, who was transplanted during an episode of recurrent lymphocytic pericarditis, had showed elevated serum IL2R levels soon after transplantation without evidence of rejection. follow Therefore, the presence of one of the above conditions and possible other conditions are not associated with rejection. IL, which can cause elevation of 2H in heart transplant patients.

The data show that serial measurements of IL2R in serum in cardiac allograft recipients were rejected. This shows that it is confirmed to be effective in detecting and testing absolute reactions. ascending collapse IL2R level is 1. Rejection may also occur in the presence of other diseases that increase L2R levels. Indicates the need for intracardial pericardium to confirm or rule out a complete reaction. Normal I L2R level Le's findings represent the elimination of rejection and thus obviate the performance of intracardial pericardial biopsies. It can be used for. 1. Measuring the L2R level is important because intracardial pericardial biopsy has a higher risk. Useful in infants and children with cardiac allografts associated with risks and technical difficulties. prove that it is effective. This approach reduces mortality and expensive cardiac allografts. Transplants have proven effective in both handling patients.

14. Serum measurement of soluble IL2R was performed on viral-related --'''ru　--Acquired immunodeficiency syndrome (AIDS) is a difficult disorder of cell-mediated immunity. All are characterized by increased opportunistic infections and certain malignancies. human T cells White group disease ET lymphocyte affinity (T-1ymphotropic)] Virus tie III (HTLV-III) is closely related to the disease and can cause active disease. The associated patient exhibits a difficult T, I cell disorder. Clinically, AIDS is caused by persistent phosphorus It is manifested by pacytopenia and a marked decrease in T cell function. However, H.T.L. V-III virus (HTL V-III seropositive) infected patients must It does not show any medical abnormality. Therefore, exposing the virus to HT　V　V　-Hr clinical subgroups of patients, such as AIDS-related complexes (ARC). It is essential to distinguish these from those indicative of AIDS-related diseases.

As shown in Figure 12, serum IL2R levels correlated with disease activation. So Therefore, in conjunction with other medical tests, measurements of soluble IL2H in serum are provides an indicator of disease difficulty in

15. IL2H for 2R test for detection of soluble, free IL2H Two different mouse monoclonal antibodies directed against different epitopes of To describe a serum immunoassay test kit that uses Self-Free (trademark) Inter Leukin 2 Receptor Test Kit (T Cell Science, Incoho) (Rated, Cambridge, MA) is free and soluble in human serum. A direct enzyme immunoassay for the quantitative detection of IL2H [Lupin et al., 198 5th year, Jay, Imtsuru, 135: pp. 3172-3177 (Rubin et al., 1985. J, Immunol, 135:3172-3 177)]. Accurate serum quantification of IL2H helps characterize immune disorders and disease. important for testing activity and evaluating the efficacy of immunotherapeutic treatments for such disorders. be.

The Cellfree™ Interleukin-2 Receptor Test Kit was administered to various patients. and used to detect cell migration IJL2R in control serum. I　L2H possible The soluble or free form is present in the serum of patients with leukemia and certain immunological diseases. was found to increase. Serum IL in active adult T-cell leukemia patients 2H levels were highly elevated, but not in the HTLV-I seropositive asymptomatic patient group. The average price remained low.

15.1. 'Rizu's 5, The Cellfree™ Interleukin-2 Receptor Test Kit uses human serum or in a sandwich enzyme immunoassay for determination of levels of IL2R in plasma. be. Antibody-coated anti-IL2R was first adsorbed onto polystyrene microtiter wells. be done. IL2R present in the sample or standard will bind to the antibody-coated wells and Wash and remove reaction sample components. directed against a second epitope on the IL2R molecule The enzyme-conjugated anti-I L2R monoclonal antibody captured with the first antibody Binds to L2R and completes the sandwich.

After removing unbound enzyme-conjugated anti-IL2R by washing, substrate solution is added to the wells. Yes Color products are formed in proportion to the amount of IL2R present in the sample. Stop the reaction. Stop with liquid and measure absorbance at 490 nm. Four 1L2 standard curves Prepared from R standard. Determine the unknown value from the standard curve.

15.2 Cellfree(TM) Test Kit Components and Suggestions Each Cellfree™ Interleukin-2 Receptor Test Kit contains 96 Have enough reagents in the cell. The expiry date of the completed kit is determined by the external measurement box level. The recommended storage temperature is 2-8°C.

15.2.1 Supply reagents (a) Antibody coated anti-IL2R-1 bottle Each bottle contains a mouse monoclonal antibody to human interleukin-2 receptor. include. Store at 2-8℃.

(b) HRR-conjugated anti-IL2R antibody-1 bottle Each bottle contains human interleukin-2 Horseradish herboxidase (HRP) complex mouse monochrome to receptor Contains null antibodies. Store at 2-8℃.

(C) Sample diluent - 1 bottle Each bottle contains a buffered solution of serum proteins. At 2-8℃ Each bottle contains free or soluble human interleukin-2 receptor (IL2R) Contains a buffer solution. Store at 2-8℃.

(e) OPD tablets - 1 bottle Each bottle contains 14 OPD tablets consisting of O-phenylenediamine in an inert binder. Contains agents. Store at 2-8℃.

Also supplied: (96-hole microtiter plate with f > holders. Each plate has 12 holes. It consists of a small piece of 8 wells with 2 wells. Store uncoated boolate at 2-26°C .

(g> Usage 15.2.2 The secondary reagent, which is required but not provided, is the first reagent described below. It should be adjusted according to the instructions in Section 5.2.5. The names of the following reagents are: List of ingredients needed to prepare the reagent. The famous reagent is distilled deionized water It should be manufactured in

(a) Buffer-coated PBS: Commercially available isotonic phosphate buffer (PBS) [ Dulbecco's PB without calcium or magnesium S is recommended) or NaCΩ, KCΩ, KH2PO4, N’a2 HPO4-7 H20; Thimerosal (trade name: thimerosal).

(b) Washing buffer: Buffer Co1-PBS (reagent a) plus polyethylene sorbitol Tammonourate (Tween 20).

(c) Blocking buffer; wash buffer (reagent b) plus bovine serum albumin (neutral pH 1 fatty acid release preparation).

(d) Substrate buffer: citric acid-hydrate, NaHPO4.7H20, thimerosal , 30% hydrogen peroxide.

(e) Stop solution: Concentrated sulfuric acid.

land Precision pipettes with disposable tips: 50 and 100ul micropipettes .

50-200m! ill adjustable multiwell (mu!tiwell) pipettor .

Disposable reagent throughs for multiwell pipetters A flask and a cylinder are needed for the preparation of reagents.

Microtiter plate cleaning/aspiration equipment.

Cold air incubator, 37±2°C.

Disposable pipette for preparing OPP (o-phenylenediamine) substrate solution.

Microtiter plate reader for measurements at 490 nm.

15.2.3, Preparation of reagents We suggest the following precautionary measures: Do not mix reagents from different kit lots.

Do not use reagents past the expiry date on the label.

Use disposable and pet tips and/or pipetters to prevent reagent contamination. Things.

Sodium azide inactivates HRP. Sodium azide-containing solutions are Should not be used for testing.

Do not expose OPD reagent to strong light during storage or incubation.

Avoid contact of OPD and stop solution to skin and mucous membranes.

If these reagents come into contact with your skin, wash thoroughly with water.

Avoid contact of OPD tablets, OPD substrate solution and stop solution to any metal. Things. Push disposable glassware or test tubes against OPD substrate solution. messenger If you use glassware that cannot be disposed of, pickle it and clean it thoroughly with distilled, deionized water. Should be rinsed.

15.2.4 Kuchi 1 niece, Rokuni Venous blood is collected under sterile conditions. Serum or EDTA plasma is used in this assay. suitable for Remove serum from red blood cells as soon as possible after clotting or separation. or separate plasma.

Samples containing visible sediment should be cleared before use in the assay. ＆ Hemolysis also occurs. should not use lipemic (IipeniC) specimens.

Samples may be stored at 2-8°C for up to 7 days. The time between sample collection and analysis exceeds 7 days. interleukin-2 receptor, it is preferable to store the sample frozen. Freeze-thaw cycles should be avoided as they change the properties of a single molecule.

Prior to assay, the frozen serum or plasma should be gradually brought to room temperature and then slowly cooled. should be mixed by hand. Thawing the sample in a vacuum at 37°C, vortexing the sample or Sudden stirring should be avoided.

Sodium azide inactivates J(RP. Therefore, sodium azide-containing Specimens should not be used in this assay.

15.2.5 i. I ○ Plate coating the following specimens except for PD substrate solution, Section 15.2.6 The following should be prepared prior to: Sectional fluid above PB below; Commercially available isotonic phosphate buffer [Dulbecco] of PBS, without calcium or magnesium]. In some cases, the following method A laboratory PBS can be prepared representing: 8. In about 800 ml of distilled, deionized water. NaCQ of OOg, 0.20 g of KH2PO4, 0, 20g of KCΩ and 2.16g of Na2HPO4. Dissolve 7H20. Add distilled, deionized water to a final volume of 1.00 liters. Ru. Make sure the buffer is pH 7.4±0.2. Mix well.

O,Log CH for either commercially available or laboratory-prepared PBS. Add Lomesal as a preservative. Mix well. 2-26℃ for up to 6 months Save with .

Washiru Sato Zutan: Add 0.40 ml of polyoxyethylene sorbitol to 800 ml of buffer-coated PBS. Add tan monolaurate (Tween 80). Mix well. Until the 30th Store at 24±2°C. Wash M Street is stable for 30 days. Add the washing M solution to the first It can be prepared as required according to Table X.

Table 1X PBS containing M serum Tween 20 '(ml>-b and m- 〇～32 exams 400 0.2 33-64 test 600 0.365-96 test 800 0.4 Add 1.0 g bovine serum albumin to 100 ml wash buffer. mix well . Store at 2-8℃ for up to 30 days before use. 6g citric acid monohydrate, 1.74g N a2 HP 04/7H Dissolve the thimerosal of 2O and O,Olg. Steam to final volume of 100ml Add distilled deionized water. Mix well.

Confirm that the buffer solution has a pH of 6.3 and 0.2. At 2-8℃ for up to 60 days save.

The stop is 2H8O Carefully add <5.8 ml of concentrated sulfuric acid to approximately 800 ml of distilled, deionized water. must be added to the Add distilled deionized water to a final volume of 100 ml. . Mix well.

Store at 2-26°C for up to 6 months.

The next sample should be prepared approximately 30 minutes prior to the conclusion of the incubation with conjugation. (Section 15.2.7, below).

OPD substrate solution: (a> For each small piece of 8 used, add 1 ml of base in a clean glass test tube. Pipette the substrate buffer; (b) OPD tablet for every 1 ml of substrate buffer in the test tube. Add 1 tablet.

OPD tablets should be transferred using non-metallic tweezers or equivalent. be. Cover the test tube tightly and swirl frequently until all OPD tablets are dissolved. 30 Use within minutes.

15.2.6 Proposal plate coat preparation form. All 96 μl microtiter plates should be coated at the same time): (a) clean Measure 11.9 ml of buffer-coated PBS in a test tube or flask.

Add 0.1 ml of antibody coated anti-I L2R. Mix well.

(b) Dispense 100 uI of solution into each well for a complete microtiter plate. do.

(C) Cover the plate with plastic encapsulant and leave it in a humid environment for 16-72 hours. , incubate in room m (24±2°C).

(d) Discard the coating solution from all wells.

(e) Add 300u, Q of blocking buffer to complete microtiter plate.

(f> Replace the plastic sealant and ink the plate for 2 hours at 37±2℃. cuveate.

(g> If the sample is to be manipulated immediately, continue with the assay form. If to be manipulated later, contain blocking buffer covered at 2-8 °C until use. Save the antibody coated wells. Wells are stable up to 45 days after coating.

1-5.2.7 Draft protocol 4 This assay should only be performed after completing plate coating, Section 15', Section 2.6 above. can be done.

(a> Mix all reagents thoroughly without stirring before use.

(b) 10 wells and 10 wells needed to run the desired number of patient samples plus blanks. Determine the number of strips needed to test the samples and standards (8 wells per strip). Ho Remove any unnecessary pieces from the molder, cover with plastic sealant, and store at 2-8°C.

(c) Discard blocking buffer from coat well.

(d> Wash the wells three times with wash buffer. Discard the wash buffer after each wash.

(e) Remove any remaining cleaning residue by draining the inverted plate onto clean absorbent paper. Remove the buffer.

(f> Leave the blank well empty and add 50L to both the primary and secondary antibody coated wells. 1. Pipette the Q standard or sample.

(g> Leave the blank well empty and add 1001J, Q samples to all other wells. Add diluent. Care should be taken to avoid cross-contamination of samples.

(h) Covered with plastic encapsulation in constant temperature air incubator for 2 hours at 37 ± 2°C. Incubate with

(i) Remove and discard the sealant. Aspirate solution from all wells. Between washes Aspirate well and wash wells three times with 350 uj of wash buffer.

(j) Leave the blank well empty and add 100+l HRP to all other wells. - Add conjugated anti-IL2R.

(k > 37 ± 2°C in a constant temperature air incubator covered with new plastic encapsulation. Incubate with Approximately 30 minutes before incubation with conjugation, 15. Prepare the OPD substrate solution derived in 2.5.

(Ω) Remove and discard the seal. Aspirate solution from all wells. Between washes Aspirate well and wash three times with 3501J and Q wash buffers.

(m>100 IJJ) of OPDM street solution was added to all wells including blank wells. Ru. Incubate uncovered for 30 minutes at room temperature (24±2°C).

(n) Pipette 50 ml of stop solution into all wells, including blank wells. Ru.

(0) Read the absorbance of the wells at 490 nm against the substrate blank. The absorbance is The assay should be read as soon as possible after completion, but the wells should be kept at room temperature and protected from light. It can be read for up to 2 hours after the addition of the stop solution if maintained at a constant temperature.

See Section 15.2.10 above.

15.2.8 Construction of standard curve (a) Read the absorbance at 490 nm for each standard well.

(b) Average the two values and record the average.

(c) IL2R temperature (water Plot absorbance (vertical axis) against absorbance (flat axis).

(d) Draw the most appropriate curve.

15.2.9 Patient samples (a> Read the absorbance at 490 nm for each patient sample well.

(b) Average the two values and record the average.

(e) Place the average absorbance corresponding to each sample on the vertical axis and horizontally intersect the standard curve. Install the line. At the intersection, read the IL2R concentration (units/m1) from the vertical axis. nothing.

15.2.10 Because test conditions may change from test to test, the standard curve is should be determined. Use disposable pipettes, as mixed contamination between reagents will invalidate the test. A cut tip should be used. Clean reusable glassware before use. All cleaning agents should be rinsed off. Disposable flasks or glassware are preferred. stomach. Thorough washing between incubations is required: (a) new Aspirate well contents completely before applying wash solution.

(b) Fill the wells to the top with wash solution for each wash cycle (approximately 350 uJl) Every time).

(C) Wells should not be subjected to extended periods between incubations.

Only samples with absorbance values that fall within the range of the standard curve will yield an IL2R concentration from the curve. It should be established. Sample samples with absorbance exceeding the highest standard It should be diluted with diluent and retested. The dilution to be applied is 1/10 (50L IN Sample Plus 4501Jj sample diluent). The smaller the percentage of the sample, the larger the Dilution may be required. The exact dilution of IL2R is in units/ml of diluted sample (standard). Multiply the IL2R levels in (from the quasi-curve) by the dilution used to test. obtained by

Using the Cellfree™ Interleukin-2 Receptor Test Kit, The value of soluble or free IL2R is determined by adding up to 2υg/ml of recombinant into the test sample. It is not affected by the addition of IL-2.

15.3 Self-free (standard) test 1 15.3.1 Monoclonal antibodies A mouse monoclonal antibody was prepared using the described method [Lupin.

L, E et al., 1985, Hybridoma 4: pp. 91-102; Ra, G. and Milstein, S., 1975.

Nature 256: pp. 495-497 (Rubin, L, A, etc. at, 1985. Hybridoma 4:9t-102;Kohle r, G, and) lilstein.

C,, 1975, Nature 256: 495-497)] caused it to occur.

The two selected monoclonal antibodies (2R12, 7G7) have different IL2R directed against epitopes. Both antibodies were identified with anti-Tac mAb. It was shown to precipitate 55 kilodaltons (kd) of cell surface proteins.

IgG antibodies were added according to the manufacturer's instructions [Affi-Gel Protein A Mabus II Ki]. Kit (Affi-gel protein A HAPS II kit) instruction map New, Biorat Laboratories.

California (BioRad Laboratories, California) romatograph on immobilized protein A (manufactured by Biorat) according to It was purified to greater than 95% homogeneity using a high-speed filtration method. Comprehensive description of enzyme-antibody complexes [Wilson, M., B. and Nakane, B., Gay, 1978, Munofluorescence and Related Steining Techniques, Ka Natsub, Niichi Daburi, Kei, Holver and Gee.

Edited by Uzigzis, Elezavich/Norton-Holland Biomedical Pre Wilson, p. 215 (Wilson) 1. B. and Nakane, P. , 1978. Immunofluorescence and Re1at ed 5taeds, eds, , Elsevich/Norton-Hol land Biomedical Press.

P, 215)] by labeling IgG with horseradish peroxidase. Prepare.

15.3.2.　′ I L2R, standard phytohemageltin (PHA) stimulated peripheral blood mononuclear cells (PB MC) was prepared from the supernatant of the culture fluid.

Leukocytes were collected using Ficoll-paque [Pharmacia] according to the manufacturer's instructions. c ia)] was collected after treatment. 10B cells/ml to 1000/ln l penicillin, 100 g/ml streptomycin and 15% Koji serum and suspended in RPM II 640 supplemented with PHA [Welcome Diag] Made by Nostakes (Wellcome Diagnostics)] 2. Q Added to a concentration of uCl/ml. Cultures were kept at 37°C for 3-6 days.

The supernatant was collected by shaking at 200×g for 8 minutes and incubated for 10 minutes. At OOOXg It was purified by high speed rotation.

The IL2R level was determined in relation to the supernatant given a value of 1000 units/ml. Units per m1 (IJ/m l) Given the value [Lupin, L., A. et al., 1985, G.I., Imuts. Rubin, 135: pp. 3172-3177 (Rubin, L, A, etc. al,, t985. J, Immun.

1, 135: No. 3172-3177)].

15.3.3 Disease Normal serum was collected from healthy blood hosts and kept at 4°C for up to 7 days, and for longer periods of time. Stored at -70°C.

Adult T-cell leukemia patient serum was obtained from Dr. N. Yasud (or N. Yasud). o) Provided by (Kyoto University, Japan). Abnormal clinical serum should be taken to a local hospital or and clinics.

15.3.4 Purification I L2R Purified IL2R onto anti-IL2R immunoadsorbent Affinity chromatography of supernatant from PHA-stimulated P B M C culture of Prepared by Raffy. Affinity power ram to 1m according to manufacturer's instructions l Reactigel (Pierce) 1■ Prepared by linking anti-IL2R antibody (2R12>.100d PH A supernatant culture was applied to a 1+nl column. 100 ml of phosphate buffered saline (P After washing unbound material from the gel with BS), IL2R was washed with 5 ml of 0.2 M Elute in solution, pH 2,8; dialyzed in water; lyophilized and 0.5 ml PB resuspended in S.

15, 3.　5　I≦IjiOtsu: 12 and 1! En2=2? - commercially available The recombinant IL-2 produced by Dr. James Kernick, James Kun ick) [Massachusetts General Hospital, Boston, Mass. Massachusetts General Ho5pital, Boston, HA)] was a generous gift.

15.4 Cell y! J-(*li! > Cellfree (trademark) interleukin- A two-receptor kit was used for whole serum assays as follows; Wells were incubated with anti-IL2R mice in PBS at 100 u per well at room temperature overnight. Coat the antibody by incubation with a 1,5 Uj7/ml solution of SmAb. I started. Discard the coating solution and add 300 UJl of 1.0% bovine serum albumin to the wells. Blocked with Min and 0.05% Tween 20 in PBS for 2 hours at 37°C. . The wells were then washed three times with washing buffer (0.05% Tween 20 in PBS). did. Pre-coated microspheres can be used in a two-step assay or a modified shorter one-step assay. used in any of the methods. For a two-step assay, 50 u1 of sample, standard or A control was placed in each well.

Add 100 uJ of sample diluent consisting of animal serum proteins and supernatant to each well. added. Wells were incubated for 2 hours at 37°C and then washed three times with wash buffer. Purified. 100 uJ directed against a second epitope on the IL2R receptor. Add HRP-conjugated anti-IL and 2R antibodies to each well and incubate at 37°C for 2 hours. I bet. After washing three times with washing buffer, 100 uJ of substrate solution (0.2% 0- Phenyldiamine [0PD3.0.0225%H2O2,63n+H diphosphoric acid Sodium hydrogen, 17ml citric acid) was added to each well. Wells at room temperature Incubated for 30 minutes. Add 50U, l! of reaction to each well. 2 regulations H2SO The absorbance of the wells at 490 nm was stopped by the addition of Guinatec (D ynatech) MR6000 microtiter plate reader.

All assay buffers and diluents contain 0.1% thimerosal as a preservative. Ta. The unknown sample contained 0°100.400 and 1600 units/ml of IL2R. The values were determined based on the standards drawn using the standards that had been established.

If necessary, use a faster one-step method as follows: 50 UN standard, sample or control was added to each well followed by 100ufl of HRP-conjugated antibody.

Incubate the wells at 150-180 rpm for 2 hours at room temperature on a shaking table at °C. I did it. After washing the wells three times with wash buffer, 1100 u of substrate solution was added. . Wells were incubated for 20 minutes at room temperature. Add 100uJ of reaction to each well The reaction was stopped by adding 2N sulfuric acid. Read the absorbance and analyze as described above. did.

Yotai-A Niecy, Tsuji-'! L-adic A typical standard curve is shown in FIG. The sensitivity of the assay exceeds the zero reference absorbance. Calculated as the amount of I L2H that gives two standard deviations, approximately 50 units/ml It is. Samples with IL2R activity up to 1600 units/ml can be processed using this kit. Exceeding levels can be quantified if the sample is diluted prior to assay. can be done.

! 5.4.2 Office The internal (1ntra) and intermediate ((inter) test accuracies are shown in Table X. The assay changes to testing 20 pairs of samples from each pool in one assay run. Therefore, it was determined for three serum pools with different IL2R levels. of change The percentage (CD) ranged from 3.2 to 3.9%. The cross-validation change is 20 was determined from the average of paired samples for each individual run. The cross-validation CV consists of three It ranged from 10-12% relative to the test serum pool.

Table X I L2R-'s PP information Internal verification accuracy a: Average Standard deviation Rate of change <n (Ll/ml> (Ll/ml) (%) Serum pool A 386 14 3.7 Serum pool B 636 25 3.9 Serum pool C1,029333,2 Mutual validation accuracy b: Average Standard deviation Rate of change -Trial 1-1- (U/lnri Artificer Lice and (%) Serum Pool A 414 50 1 2 Serum pool B 688 73 11 Serum pool C1, 10910710 a. Internal assay precision was determined from the average of 20 assays per sample.

b. Cross-validation precision determined from mean-pair sample averages for 20 different runs. It was.

15.4. 3 PP Affinity-purified IL2R purified endogenous IL2R by passage over an anti-IL2R affinity gel. I took it out. I spiked samples with different levels of added IL2. Tested in assay against L2R. The recovery rate of TL2R was 95 to the expected result. It was 103% (Table XI).

Table XI Upper prefecture λ and a Expected value Recovery value Recovery rate -Ih illusion- (U/m1) -Wisdom 0- 0 Fixed samples contain varying amounts of purified IL2R and endogenous IL2R from isolated blood. Prepared by adding to the fresh water pool - Ichikami Kunin 11111 ----------------------- --m---15,4,4 assay specificity and interleuin-2 origin Both antibodies used in the immunoassay are specific to human IL2R. Anti-Tac antibodies have been shown to recognize molecules similar to anti-Tac [our Jama, T. et al., 1981, Jay, Imtsuru, 126: No. 1393-1 397 pages (Uchiyama, T., et at., 1981. J. Im. munol, 126:1393-1397) 1. Using these antibodies, Immunoprecipitation of 1251-labeled surface proteins from tohemagellin-stimulated lymphocytes A diffuse 55 kd band was revealed, and sequential immunoprecipitations revealed that this molecule was anti-T. showing that it is exactly the same as that precipitated by ac [Lupin, L., E. et al., 1985, Hybridoma 4: pp. 91-102 (Rubin, L. A, et at, 1985゜Hybridoma 4:9l-102) ], therefore one of the antibodies (2R12>) is suitable for cell fluorescence analysis of activated lymphocytes. This shows that the anti-Tac compound competitively binds to anti-Tac. Enzyme immunization using these antibodies The assay was carried out in the medium supernatant of TL2R surface-positive cells [Lupin, L., A., et al. 1985, Jay, Imzuru, 135; pp. 3172-3177 (Rubin , L.A., et al., 1985. J, 1mmu mouth of, 13 5:3172-3177)] and the flattening of the IL2R gene rather than its normal duplication. In the medium supernatant of mouse cells transformed with the head morphology [Triger, B., F. et al., 1986, J.A.

Imtsur, 136: pp. 4099-4105 (Treiger, B, F, et al.

1986, J, Tmmunol, 136:4099-4105) I Detecting the free form of L2R.

It was decided to perform the IL2R assay kit in the presence of added IL-2. 51 (1/ml The serum concentration of added recombinant IL-2 (rIL-2) up to (Figure 14). 5000 (1/ml With added rIL-2, IL2R levels were only 40% of expected values. . Concentrations of IL-2 proposed for therapeutic trials are in the 1000/ml range, and showed a half-life of less than 10 minutes in the serum after the first injection [Rossocher, M., T. -etc.

1985, Zei, Imzuru, 135; pp. 1865-2875 (LOtZe .

H, T, et at, 1985. J, Itnmunol 135: 2865-2857)].

Therefore, therapeutic levels of IL-2 are significant when measuring IL2H using this assay. Not expected to cause disturbances.

IL2R expected values of greater than 80% were obtained at these concentrations of rIL-2. addition However, no detectable levels of IL-2 in normal serum were observed.

15゜4.5 I = L2R level lt in human serum - IL in normal human serum The distribution of 2R levels is shown in FIG. Average I L2R rate of 174 healthy blood hosts Bell was 273 U/ml. The upper limit of normal for this group is 47 70/m! (mean + 2 standard deviations).

Serum levels for adult T. 22 in 23 patients with alveolitic disease were below normal levels. It rises above the bell and rises 7': (Fig. 15).

The majority of these patients showed markedly elevated IL2R levels (1600 Place/　m! excess).

Current tests have determined that detectable levels of free rL2H present in normal human serum and Patients with human subcellular leukemia present with elevated serum levels [Lupin, L., E. et al., 1 986L r FIDENTIFICATION AND CHARACTERIZATION On a released form on the interleukin-2 receptor ”, Tsuno, Leuco Sites and Host Defense, Oppenheim, J.D. Ijay, and Dee, M., Jacobs, eds., Alan Earl, Rhys, Parakeet. -Holated, New York.

pp. 95-102 (Rubin, L.A., et at., 1986. ”Identification and Characterization of a Re1eased Form of the Interleuki n-2Receptor, in Leukocytes and Ho5t D efense, Oppenheim, J, J, and D, H, Jaco bs, eds,, ^tan R, l.

iss, Inc., New York, Dp, 95-102) l.

As an alternative to cell surface measurements, for the detection of free or soluble IL2H. The utility of commercial enzyme immunoassays to evaluate altered immune status in novel methods make it possible to Therefore, avoid invasive sampling methods to obtain classified cells. . Additionally, serum IL2R assays provide insight into immune regulation.

The next section detects soluble CD8 in patients. Antibodies and assays that can be used to do so are described.

16.1 Monochrome power holding mushroom Anti-CD8 monoclonal antibody 5F4 was developed by Peinherz et al. [1 979, Bros, Natl, Akad, Sai, Ni, Nis, Ei, 76th; 406th 1-4065 pages (1979, Proc, Natl, Acad, Sci, U,S,A,76:4061-4065)] Cut (Jurkat) '1'''C' To immunize BALB/C mice Therefore, it occurred. Monoclonal antibody 5F4 was recognized by mAb 0KT8. Recognizes an epitope on a CDS that is different from the epitope on the CDS.

Yotachi Nisu Goshu and Emperor's remains Soluble CD8 was detected in the samples using the method summarized below: (a) Polystyrene microtiter well [Flow Lab oratory) 1 to 100 uJ) mAb OKT [orthodiagmeste sisis. Raritan, New Chassis (Ortho Oiagnostics) , Raritan, N1. Moon's anti-CD8 mouse monoclonal antibody (2,5 The plate was coated with 0 (1/ml) PBS solution at 4°C overnight.

(b) Discard the coating solution and coat the well with 300 uj! 0.05% Tween 20 and Tris containing sodium chloride (NaCQ) 251IIH Tris pH 7.4 Blocked with 1% BSA in buffered saline for 1-2 hours at room temperature.

(c) Wells were filled with 1011IH Tris 0.05% Tween 20. pH8.0 (tri Washed three times with water (cleaning solution).

(d) Add 10 uJ sample per well, followed by Tris-buffered saline and 0.1 μJ sample per well. 4%NP-40 [fern? (sinma)] diluted with 50% FC8 in 90uj) Added the agent. Wells were incubated for 2 hours at 37°C.

(e) Wash wells three times with Tris wash solution.

(f) 100uJ) of 50% FC3, 24mHl Tris pH 7°4.0.15M Horseradish peroxidase conjugated anti-CD8 in NaCC and Np-40 Mouse monoclonal antibody (0.21 g/ml m, Ab 5F4) was added to each well. and incubated at 37°C for 2 hours.

(g) Wash wells four times with Tris wash solution.

(h) 0°2% 0-phenylene in citrate-phosphate buffer solution (100 uJ) diamine (OPD) and 0.015% H2O2 were added per well. pre The plates were incubated for 30 minutes at room temperature.

(i) Add 50 μl of 2N H2SO to each well to minimize the absorbance of each well. Titer was measured at 490 nm in a plate reader.

16.3 CD8 control standard The supernatant of the tissue culture system [Jurkatt] was used as a standard. . The measured value was determined to be a relative number of about 2000/ml. The culture supernatant is 10 RPM-16 containing %FC3 and 100 units of penicillin-streptomycin The outfit consists of 40 16.4 The amount of cell-released TM3 cell CDS-like molecules... Monoclonal antibodies directed against distinct epitopes on the CD8 antigen, human Using suppressor/sociotoxic T cell markers, we demonstrated that human T cell C D8-like antigen. Elevated levels of this antigen are associated with certain leukemias, allograft transplants and and some diseases including rheumatoid arthritis and autoimmune diseases such as lupus or observed in the state.

In longitudinal patient studies, increased serum levels of cellular iICD8 were associated with soluble Different time frames compared to expression of interleukin-2 receptor (IL2R) It is found in In certain instances, an increase in cell migration %fCDS-like molecules is After several days with an increase in serum IL2R levels. This cellular NCD5-like molecule Increased levels are recognized from immune activation as measured by elevation of cell-free IL2R. A significant number of suppressor/cytotoxins are associated with specific clinicopathological events can be shown. The presence of cell-bound CD8 and IL2R molecules can be determined by cell surface staining. Measured superficially. These cell release assays provide a more complete evaluation of certain immunological disorders. can provide understanding.

The assay used for CD8 is the Sandwich test described in Section 16.2 above. It is an enzyme immunospecies. Anti-CD8 mAb (mAb 1>, for example, in a microtiter wafer) coated onto solid substrates such as silica. CD8 in the sample binds to the antibody-coated well. Combine; wash out unreacted sample components. Recognized an epitope different from that of mAb 1. The enzyme-conjugated second anti-CD8 mAb (mAb2) that recognizes the It binds to the loaded CD8 antigen and completes the sandwich. Wash away unbound mAb2 Don't leave. Substrate solution is added to the well and colored product is added to the well in proportion to the amount of CD8 present in the sample. It was formed by The reaction was stopped with a stop solution and the absorbance was measured. Standard curve C D8 standard (see Section 17.3 below). Determine the unknown value from the standard curve. Established.

16.4.1 Evaluation of anti-CD8 monoclonal nine bodies as capture antibodies Antibody that detects various monoclonal antibodies (mAb) induced against CD8 antigen In enzyme immunoassay using anti-CD8 mAb 5F4 as (rnAb 2) were tested for their suitability as capture antibodies.

Plate [Flora Microstrips, 70 U Laboratory, Pine Clean , Virginia (Flow m1crostrips, Flow Labor atory, McClean, VA)] to 2. 5ug/ml capture antibody I coated it all day and night. The non-reactive site was 0.15MN a CΩ, 0.025M Lis-CQ (pH 7,4>.

A solution containing 0.01% methylosal, 0.15% and 1% BSA in Tween 20. It was blocked with solution the next day. Composed of CD8-containing Jurkat cells, 25% calf serum in squirrel buffered saline, 0.25% Nonidet P-40 [Non 10ufJ sample diluted 1:4 in 1det P-40 (NP-40)] added. After incubation for 90 minutes at 37°C, transfer the plate to 110ff1 plate. Washed with squirrel, 0605% Twee 720, pH 8.0. 100u, Q Tris 25% calf serum in buffered saline, NP-40 (1:5 of 1ml strain) ．． Add horseradish peroxidase-conjugated mAb5F4 at OOO dilution. Ta. Plates were incubated for 90 minutes at 37°C and washed as above. 10 0uj of 0.2% 0-)1-2-diamine was added in 0.015% H202 and After incubating at room temperature for 30 minutes, absorbance was read at 45 onm.

The results are shown in Table XII.

Table XII CD8 monoclonal iFjmAb 5F4 to CD8 as a supplement Supplementary -jl-] and purchase 9th 4502B98.1.1 Partial 0.02 8 B116.1.1 Yes 0.027 B99.1.1 None 0.039 0KT8 None 0.413 No 4C9 1.106 5F4 Yes 0.042 62EC3 (control) with HRP-conjugated mAb as detection antibody 0.0421 3 Keyhole Limpet Hemosyanin (Keyhole 1impe hemocyanin) [Pacific Biolabs, California (Pacific Biotabs, CA) -ship The results of the binding competition assay to mAb 5F4 are also shown in Table XII. These tests are 50 uJl competing antibody (log/ mriplus 50u1HPR-conjugated mAb 5F4 (1mg/ml of 1:250 was carried out using a similar recipe as above, except that it was a mixture of (at 0 dilution) Ta.

The seven mAbs tested as capture antibodies were directed to the CD8 antigen as shown in Table XII. Three groups were selected based on their ability to competitively inhibit the binding of mAb 5F4. It can be classified into groups. 5F4m as both capture and detection antibody as expected Use of Ab does not work (capture antibody 5F4 outcompetes detection antibody 5F4 binding) (complete out). Surprisingly, 5F4 binding to CD8 4C9 gives the best assay results among the group of mAbs that do not competitively inhibit worked best, followed by 0KT8, whereas B99°1.1 was negative. subordinate Therefore, even on the basis of competitive ability, anti-CD8 monoclonal antibodies It is not obvious that one would expect it to work in the Hitch test.

In addition, it was measured using mAb OKT8 against mAb 4C9 as a capture antibody. A comparison of serum CD8 levels was also made. Using mAb 5F4 as a detection antibody did. The results shown in Table XII show that in most cases the capture antibody Figure 4 shows large detection levels using 4C9.

Table XIII Two different anti-CD8 capture antibodies Use CD8 level 1 Patient group normal 3B 270 Table XIII (continued) Two different anti-CD8 capture antibodies Use CD8 level kidney transplant patient 1 Use anti-CD8 mAb 5F4 as a detection antibody. (Some diseases and disorders are caused by the patient's serum CD8 level being low) It can be staged or diagnosed by measuring levels. Example below: Section.

17.1 Rheumatoid Arthritis 9 Problems 1 Seasonal Pictures Above CD8 Sandwich Enzyme Immunization The assay can be used to distinguish rheumatoid arthritis from other combined diseases (Figure 16) . The results shown in Figure 16 are based on mAb 4C9 as the capture antibody and detection antibody as Based on the level of serum or synovial fluid CD8 concentration measured using mAb 5F4. Ku.

17.2 Kidney Six Types Subplant Blood It Bell Patient A received a non-synonymous kidney from a deceased person. The patient received an allogeneic graft. This patient was transplanted on May 23rd, and Cyclos It was maintained with porin A (CsA). From May 23rd to May 31st, this patient , experienced CsA toxicity and some early rejection reactions. June 28th, rejection reaction Pisode was diagnosed on the basis of elevated cretinine levels, and patients were given 10 days until July 7. 0KT3 rnAb was administered intravenously 4 times a day in 5 doses of indigo. July 8th, The reatinine level decreased and the patient entered a phase of recovery. July 18th, Pred There was another increase in creatinine levels that was treated with administration of Nizon, but another dose Creatinine levels decreased until August 26th when prednisone was administered. caused.

IL2R and CD8 in patient serum were measured at appropriate intervals from May 23rd (transplant date). Keep measuring and episodes of comb CsA poisoning after rejection episodes Figure 17 shows the peak in serum CD8 levels. this is , demonstrating the role of measuring serum CD8 levels in the examination and specific diagnosis of kidney transplantation. suggest

17.3 2CD8 levels in children with non-Hodgkin lymphoma - CD8 levels were used to study IL2R serum levels as described in Section 9 above. Non-Hodgkin lymphoma (NHL) or B-cell acute lymphoblastic leukemia (A LL) was measured in the serum of children with LL.

The enzyme immunoassay described above was performed using anti-CD8 mAb 4C as the capture and detection antibody, respectively. Used with 9 and 5F4.

The results shown in FIG. present a likely association with severe disease and poor outcomes.

17.4 CD8 level in infectious diseases The enzyme sandwich immunoassay described in Section 16.2 should be used in patients with infectious diseases. It was used to measure CD8 levels in the serum of individuals. Anti-CD8 mAb 4C9 and 5F4 were used as capture and detection antibodies, respectively. Results shown in Figure 19 are associated with elevated levels of the CD8 antigen, especially hepatitis B, Epstein-Barr virus, and AI. Elevated levels of CD8 antigen in patients with DS-related complexes and AIDS disorders shows the file.

Top 8. - Jurkat cell supernatant and cell lysis The molecular properties of CD8 in its soluble or cell-associated molecules are recognized by antibodies. For research, immunoprecipitate with anti-CD8 mAb.

18.1 2 Falcon T 257 Lasco 5X106 Jacket Thin Cells were pulsed with 2mC135S-methionine in met-RPM1640 medium. (pulse-1aheled).

After 6 hours, remove the medium, wash the cells with complete RPM11640 medium, and They were incubated in complete RPM 11640 medium for 16 hours before harvest. Centrifuge cells It was made into pellets. 20. Dispense the supernatant sample for 30 minutes. On cell culture with OOOXg The clear liquid was centrifuged, and 0 and 22 um Millipore (HilliporeTH) ) Prepared by filtering the supernatant through a filter. Dissolve 1ml of cell lysate. Prepared by resuspending 5 x 10G cells in lysis buffer: i.e. lysis slow The solution was 0.15M NaCD, 25ml Tris-CΩ (pH 8,0> , 1mM MgCN2.1% Nonidet P-40 (NP-40, polyoxyethylene) Tyrene (9>p-1ert-octylphenol), 1mM phenylmethyl Sulfonyl fluoride (PMSF>, 0.2μ/ml alpha-2-mac Loglupurin, 10mM iodoacetamide, 0.1■/ml Ovomucoi It is a dotrypsin inhibitor.

The cell plus lysed M solution was left at 4°C for 0.5 hours, then 20 minutes for 30 minutes. At OOOXg Centrifugation and supernatant <m cell lysate sample) were removed for immunoprecipitation.

Culture supernatants and cell lysates were treated with anti-CD8 mAbs 5F4.4C9 and 0K. Purified by immunoprecipitation with T8. Immunoprecipitation is achieved in two steps: i.e. a non-specific adsorption step and a specific binding step.

Locate lysate or cell supernatant samples at room temperature to nonspecifically remove bound material. Pac-Sepha linked to 5% volume of mouse immunoglobulin G for 3 hours on a Loin (5epharose) [Anoie gel. Violat, Richmond , California (Affigel, Biorad, Ricbmond, CA)]. After removing Sepharose by rotation, supernatant liquid (1 ml culture Cultured supernatant sample or 0. ．． 2 ml lysate sample) to 10 u1 mAb Linked to either 0KT8 or 4C9 or 5F4 or control mAb 0KT3. The cells were incubated with back-bound Sepharose. Stir the sample for 2 hours at room temperature. Stir and then wash 10 times with 1.5 ml phosphate buffered saline. sample 2001 ) of 50mM dimethylamine.

Eluted in 0.15M NaCN, pH 11.0. Electrophoresis of equal volume of eluate Do not dilute with sample buffer. Half of each eluate was dithiothretated to a concentration of 1.0 mM. (DTT) and leave half of each eluate unreduced. Ta. Boil the sample and add 10-20% sodium dodecyl sulfate-polyacrylamide. placed on the gel. The gel was dried and exposed for autoradiography.

18.2 Anti-CD8 mAb is 52 to 55 kg Anti-1U adsorption 1 and 1 moxibustion unreduced cell lysate immunoprecipitates were predominantly 52-55 kd Contains the form of This was previously reported [Fujimoto.

J et al., 1983, J. Exp. Met. 159: No. 752-76. 6 pages (Fujimoto, J, et at, 1983. J, Exp , Hed, 159 Nia 52-766)] 66 kd CD8 antigen cell surface It is somewhat smaller than the surface form. Surprisingly, unreduced cell culture supernatant immunoprecipitates , contains only the dimeric form of the 52-55 kd CD8 antigen. This is Sando 2 under either reducing or non-reducing conditions for soluble antigen in the itch immunoassay. This is compared with the results of Fujimoto et al., which detected only the 7kd monomer form (Fujimoto et al. et al., supra). In the reduced supernatant immunoprecipitates described here, the 27kd CD 8 monomers were observed, with a 52-55 kd soluble form of the same as a homodimer. Check the identity. Therefore, anti-CD8 mAbs are both soluble and cell-associated molecules. recognized the dimeric 52-55 kd form of the CD8 antigen.

Sandwich enzyme immunoassay for CD8 antibodies in supernatant samples with reducing agent, DTT No binding was observed when included in Therefore, the soluble dimer in the monomer Reduction of the CD8 mAb may compromise the ability of the anti-CD8 mAb to bind soluble antigen. confirmed that these anti-CD8 mAbs could only bind to soluble CD dimers. did.

19. Hybridoma 1 The next hybridoma cell line producing the indicated monoclonal antibodies was American Ta ipu culture collection.

Rockville, Maryland (American Type Culture) C.

11ection, Rockville, HD,) and set the deposit number. Being admired: Hybridoma Tennoizu1nigo Jd light body cell-cell system A) 192/2R12 8H92/2R12 (anti-IL2R) cell line 7G7 7G7 (anti-IL2R) H 88784 cell line 4C94C9 (anti-CD8) Cell line 5F4/7812 5F4/7B12 (anti-CD8) Deposited embodiment It is intended as a brief illustration of one embodiment of the invention and that any functionally equivalent cell line To be within the scope of this invention, the invention should be limited in scope by cell line deposits. isn't it. In fact, various modifications of the invention in addition to those shown and described herein may be made from the foregoing. It will become clear to those skilled in the art from the description and accompanying drawings. Such changes are It is intended to be within the scope of the appended claims.

Japanese sex is T#1114 (%) Log@I L2R LogelL2R 'J#, it IL2R (single yen/m1) fJ2f+, l7iI L-21/beginning "Cure Hale" (lit/ml) FIG, 12 FIG. 15 FIG. 16 Yu L2R7BtJ784g/mf (<1oo0)FIG, 18 FIG. 19 international search report 1″1″1lJlxllll″””””””-’ PCT/[JS8710043 0

Claims (40)

[Claims] 1. Elevated levels of soluble interleukin-2 receptor indicate advanced stages of the disease. Soluble interferon in body fluids obtained from patients with malignant lymphoma Staging of malignant lymphoma consisting of measuring the concentration of Ikin-2 receptor How to do it. 2. Elevated levels of soluble interleukin-2 receptor may be associated with a modest response to treatment. Soluble interleukin-2 receptors in body fluids obtained from patients that are indicative of Therapeutic treatment for patients with malignant lymphoma consisting of measuring the concentration of How to test the effectiveness of 3. Elevated levels of soluble interleukin-2 receptors Patients receiving interleukin-2 therapy that demonstrate a positive response to 2 therapy. measuring the concentration of soluble interleukin-2 receptor in body fluids obtained from Effects of interleukin-2 treatment on patients with non-lymphoid malignancy consisting of How to test the fruit. 4. Elevated levels of soluble interleukin-2 receptor are associated with allograft rejection. Soluble interleukins in body fluids obtained from transplant patients who exhibit severe reactions Allogeneic xenograft rejection in transplant patients consisting of measuring the concentration of 2 receptors How to detect. 5. Elevated levels of soluble interleukin-2 receptor indicate active viral infection Soluble interleukin-2 receptor in body fluids obtained from patients exhibiting A method of testing a patient for viral infection that consists of measuring the amount of 6. Claims 1, 2, 3, 4, or 5, wherein the body fluid is serum. The method described in section. 7. Conditions under which soluble interleukin 2 receptor recognizes (a) immunospecific binding (b) Contacting monoclonal antibodies 2R12 and 7G7 with body fluid; Immunospecific binding indicates the presence of soluble interleukin-2 receptor in body fluids The immunospecific binding is similar to that of monoclonal antibodies 2R12 and 7G7. Claims detected by detecting whether components of body fluids that have The method according to item 1, item 2, item 3, item 4, or item 5. 8. 8. The method of claim 7, wherein the body fluid comprises serum. 9. In claim 1 or 2, the malignant lymphoma includes non-Hodgkin's lymphoma. Method described. 10. The person according to claim 1 or 2, wherein the malignant lymphoma is leukemia. Law. 11. 4. The method of claim 3, wherein the non-lymphoid malignancy comprises lung cancer. 12. 5. The method of claim 4, wherein the allograft comprises a liver transplant. 13. 5. The method of claim 4, wherein the allograft comprises a heart transplant. 14. 6. The method of claim 5, wherein the virus consists of HTLVIII. 15. The method of claim 5, wherein the virus comprises herpes simplex virus. . 16. (a) The sample is mixed with the first anti-CD8 antibody under conditions that allow immunospecific binding. contacting the anti-CD8 antibody with a second anti-CD8 antibody that does not compete for the same binding site. and (b) immunization of components of the sample of both the first and second anti-receptor antibodies. specific binding of cell-free soluble CD8 or fragments, derivatives or derivatives in the sample. immunospecific binding is indicative of the presence of the first and second anti-CD8 antibodies. Detecting whether the components in the sample have both A method for detecting cell-free CD8 T cell antigen in a sample comprising: 17. 17. The method of claim 16, wherein the first antibody comprises 4C9. 18. The method according to claim 16, wherein the second antibody comprises 5F4/7B12. . 19. The first antibody consists of 4C9 and the second antibody consists of 5F4/7B12. A method according to claim 16. 20. A first anti-CD8 antibody is immobilized and immunospecific binding is indicated by detection of the immobilized label. 20. The method of claim 19, wherein the second anti-CD8 antibody is labeled for the purpose of labeling the second anti-CD8 antibody. 21. 21. The method of claim 20, wherein the immobilized antibody consists of 4C9. 22. 21. The method of claim 20, wherein the labeled antibody consists of 5F4/7B12. 23. Claim in which the immobilized antibody consists of 4C9 and the labeled antibody consists of 5F4/7B12 The method according to item 20. 24. Claims 16, 19, 20, or 2, wherein the sample is a body fluid. The method described in Section 3. 25. 25. The method of claim 24, wherein the body fluid comprises serum. 26. malignant lymph, where elevated levels of soluble CD8 indicate an advanced stage of the disease; consisting of measuring the concentration of soluble CD8 in body fluids obtained from patients with tumors. How to stage malignant lymphoma. 27. Patients whose elevated levels of soluble CD8 indicate a modest response to treatment malignant lymphoma consisting of measuring the concentration of soluble CD8 in body fluids obtained from A method of testing the effects of therapeutic treatment on patients with . 28. Elevated levels of soluble interleukin-2 receptor are associated with non-specific complications. In contrast, the potential in body fluids obtained from patients who are indicative of a history of rheumatoid arthritis. A method of diagnosing rheumatoid arthritis in a patient consisting of measuring the concentration of soluble CD8 Law. 29. Elevated levels of soluble CD8 are indicative of allograft rejection Transplant patients comprising measuring the amount of soluble CD8 in body fluids obtained from transplant patients. A method for detecting allogeneic rejection. 30. Patients whose elevated levels of soluble CD8 indicate an active viral infection Viral sensitization of patients consisting of measuring the amount of soluble CD8 in body fluids obtained from How to test for staining. 31. Claim 26 or 2, wherein the malignant lymphoma includes non-Hodgkin's lymphoma The method described in Section 7. 32. 29. The method of claim 28, wherein the body fluid comprises synovial fluid. 33. 30. The method of claim 29, wherein the allograft comprises a liver transplant. 34. 31. The method of claim 30, wherein the virus consists of HTLVIII. 35. Claim 30, wherein the virus comprises Epstein Barr virus. the method of. 36. 31. The method of claim 30, wherein the virus comprises hepatitis B virus. 37. Substantially pure cell-free soluble T cell CD8 antigen. Claims consisting of homodimers having a molecular weight of 38.52 to 55 kilodaltons Soluble CD8 antibody according to paragraph 37. 39. Cell-free CD8 antigen identified by monoclonal antibodies 4C9 and 5F4 , fragment, derivative or complex. 40. (a) Immobilized monoclonal antibody 4C9 under conditions that allow immunospecific binding adding a cell-free sample; (b) removing unbound material; (c) Add labeled antibody 5F4 to the reaction mixture of step (a) under conditions that allow immunospecific binding. In addition, (d) remove unbound material; and (e) testing whether immunospecific binding is induced in the reaction mixture of step (c); give out Claim 37 which responds positively to a sandwich immunoassay consisting of the steps cell-free CD8, fragment derivatives or complexes.