JPH0875745A - Immunoassay of soluble fas antigen and measuring kit therefor - Google Patents

Abstract

PURPOSE: To easily measure the concn. of a soluble Fas antigen and to foreknow and diagnose an autoimmune disease by combining a solid phase anti-Fas monoclonal antibody with a labelled second monoclonal antibody to constitute a sandwich enzyme immunoassay system. CONSTITUTION: A standard soluble Fas antigen is reacted with a solid phase anti-Fas monoclonal antibody wherein a anti-monoclonal antibody specifically bondable to a soluble Fas antigen is bonded to an insoluble support. A labelled second anti-Fas monoclonal antibody specifically bondable to the soluble Fas antigen is reacted with the soluble Fas antigen and the label quantity of the reaction product is measured to obtain a calibration curve. After a specimen is reacted with the solid phase anti-Fas monoclonal antibody, the labelled second anti-Fas monoclonal antibody specically bondable to the soluble Fas antigen is reacted with the soluble Fas antigen and the label quantity of the reaction product is measured to easily determine the conc. of the soluble Fas antigen in the specimen using the calibration curve.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological assay method for soluble Fas antigen and a kit for the assay.

[0002]

BACKGROUND OF THE INVENTION In multicellular organisms, many cells are eliminated by cell death during ontogeny. It is speculated that this cell death is caused by a predetermined program. In addition, it is considered that in the adult, when the number of cells increases due to cell division on the one hand, cell death occurs on the other hand and the overall cell number balance is maintained. Wyllie et al. Observed the morphology of dying cells by electron microscopy and categorized cell death into two types morphologically (ie necrosis and apoptosis) (Wyllie, A.
H. et al .: Int. Rev. Cytol., 68: 251-306, 1980).

In the case of necrosis, the permeability of the cell membrane is increased at an early stage, swelling of intracellular organelles such as the nucleus and mitochondria, and the destruction of lysosomes eventually occurs.
The cells are destroyed by the released protease. On the other hand, in the case of apoptosis, no significant changes were observed in the structures of mitochondria and lysosomes, and chromosomes were condensed in the nucleus in the early stage and the cytoplasm was also contracted. At the same time, the nucleus may be fragmented into several parts, or a bubble-like structure may occur on the cell surface, followed by division into minicells called apoptotic bodies.

The so-called passive death caused by external causes is mainly caused by necrosis, whereas
The pre-programmed cell death seen during development, differentiation or tissue turnover is thought to occur by apoptosis.

Fas is a transmembrane protein existing on the surface layer of biological constituent cells, which is considered to be closely related to the control of cell death called apoptosis or programmed cell death.
It is called s antigen, and the amino acid sequences of mouse and human Fas antigens and the cDNA sequences encoding the amino acid sequences have already been clarified (Cell, Vol. 66, pp. 233-243, 19).
91, Ito Naoto et al., J. Immunol., Vol. 148, No. 4, pp.
1274-1279, 1992, Watanabe (Fukunaga) Rie et al., J.
Biol. Chem., Vol. 267, No. 15, pp. 10709-10715, 1992, Alexander Oehm et al., J. Exp. Med., Vol. 1
69, pp.1747-1756,1989 Yoneharacin et al., Proc. Natl.
Acad. Sci. USA, Vol. 87, pp. 9620-9624, 1990, Kobayashi Nobuyuki et al., Science, Vol. 245, pp. 301-305, 1989, Bernhard C. Trauth et al., J. Immunol., Vol. 14
9, No.10, pp.3166-3173,1992, Jens Dhein
Et al., The Lancet, Vol. 335, pp497-500, 1990, Klaus-Michael Debatin et al., J. Immunol., Vo.
l.149, No.11, pp.3753-3758,1992, Miyawaki Toshio et al.,
Genomics, Vol. 14, pp. 179-180, 1992, Peter Lichter et al.).

Yonehara et al. Prepared a monoclonal antibody against a cell surface antigen of human FS-7 cells and obtained an anti-Fas antibody. This antibody showed an activity of killing cells expressing the Fas antigen (Yonehara). , S. Et al .: J. Exp. Med., 1
69: 1747-1756, 1989). In addition, the cell death was characterized by apoptosis as observed by electron microscopy.
It was concluded that the as antigen mediates cell death by apoptosis. Apart from this, Trauth et al.
APO-1 antibody, which is a monoclonal antibody having properties similar to that of the as antibody and showing cell-killing activity, was obtained (Trauth, B.
C. et al .: Science, 245: 301-305, 1989). The two antibodies are not only common in that they induce cell death,
The distribution and molecular weight of the antigens are very similar, and it is revealed that they are the same.

As a result of structural analysis of Fas antigen, tumor necrosis factor (TNF) receptor and nerve growth factor (NGF)
It has been clarified that it has a structure similar to that of the above, and it is a new type of receptor that causes cell death by receiving an external signal, and is regarded as one of the receptors that form the TNF / NGF receptor family. There is.

Recently, abnormal Fas gene was observed in mice exhibiting autoimmune symptoms (MRL / lpr mouse), and high-level apoptosis of lymphocytes in HIV-infected persons and Fa.
It came to be suggested that the s antigen was related, and F
The relationship between the expression of as antigens and diseases such as autoimmune diseases has come to the fore.

Cheng. J. et al .: Science, 263:
1759-1762, 1994), in order to investigate the relationship between abnormal transcription of these Fas antigens and autoimmune diseases, intracellular cDNA was extracted from autoimmune patients and healthy individuals, and examined. A normal one that encodes the full length
In addition to 167 bp cDNA, small c of 1104 bp
It was discovered that DNA was found. Furthermore, this 1104
1167 bp cDNA for small bp cDNA
As a result of a comparative examination with a hydrophobic transmembrane site (TM, Tr
Due to the lack of ansmembrane domain, this small cDNA (called FasΔTM) is a Fas antigen (hereinafter referred to as “Fas antigen”) that is soluble in a sample (for example, body fluid such as blood, serum, plasma, urine, saliva, bone marrow fluid). It was revealed that it encodes a soluble Fas antigen ").

[0010] Cheng et al.
CD4 and CD in thymocytes by treatment with s antigen
8 single positive cells and double negative cells increased,
Double positive cells decrease, total number of spleen cells increase, and in SLE patients, the amount of soluble Fas antigen in serum is about doubled as compared with healthy subjects. Shows that overproduction of soluble Fas antigen inhibits apoptosis in thymus and peripheral tissues,
The etiology was developed that the onset of autoimmune disease is triggered by the lack of proper elimination of autoreactive lymphocyte clones.

From this fact, the soluble Fa in the sample is
Measuring the amount of s-antigen is useful for predicting autoimmune diseases, establishing diagnosis and treatment policy, and determining the effect of treatment.

[0012]

However, a simple method for accurately measuring the soluble Fas antigen in a sample with sufficient sensitivity has not been known. The object of the present invention is to analyze soluble F in a sample that could not be measured conventionally.
It is intended to provide a method for measuring a soluble Fas antigen for measuring an as antigen and a kit for the measurement.

[0013]

Means and Actions for Solving the Problems The inventors of the present application have begun to develop a method for detecting soluble Fas antigen in a sample, which includes Western blotting (WB method) and radioimmunoassay ( RIA method), enzyme immunoassay method (EIA method), etc. were considered. However, W. B. Although the method had high sensitivity and high specificity, it lacked in quantification and was not suitable for processing a large amount of samples at one time. Further, in the RIA method, since radioactive substances are used as labeled substances, they can only be handled by qualified facilities in controlled facilities. Compared with this, EIA method (or ELISA method)
Has no problems as described above, can be easily and sensitively measured, and succeeded in establishing a method for detecting soluble Fas antigen using this method, and completed the present invention.

That is, the first aspect of the present invention is a method for immunologically measuring a soluble Fas antigen, which comprises an anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen bound to an insoluble support. After reacting the standardized soluble Fas antigen with the phased anti-Fas monoclonal antibody, a labeled second antibody capable of specifically binding to this soluble Fas antigen is added.
Of preparing a calibration curve by reacting the anti-Fas monoclonal antibody of 1. and measuring the labeling amount of the reaction product, and anti-Fas capable of specifically binding to the soluble Fas antigen.
After reacting a sample with a solid-phased anti-Fas monoclonal antibody obtained by binding a monoclonal antibody to an insoluble support, a second labeled anti-Fas monoclonal antibody capable of specifically binding to this soluble Fas antigen is reacted. ,
A step of measuring the labeled amount of the reaction product and quantifying the soluble Fas antigen contained in the sample from the calibration curve.

Here, the standard soluble Fas antigen is a chimera consisting of the extracellular region of human Fas antigen or mouse Fas antigen and the extracellular region of mouse IL-3 receptor β subunit AIC2A or the constant region of immunoglobulin heavy chain. It may be a soluble Fas antigen. In addition, the solid-phased anti-Fas monoclonal antibody bound to the insoluble support is hybridoma clone VB3, C
BE, WB3, ZB4, UB2, AX6, JAE and C
It may be a monoclonal antibody produced from one hybridoma clone selected from the group consisting of H11 (these eight hybridoma clones are commercially available from the Institute for Medical Biology). Furthermore, the labeled second anti-Fas monoclonal antibody is the hybridoma clones VB3, CBE, WB3, ZB4, U.
It may be a monoclonal antibody produced from one hybridoma clone selected from the group consisting of B2, AX6, JAE and CH11. Furthermore, the labeled second anti-Fas monoclonal antibody is a monoclonal antibody labeled with a labeling substance (preferably peroxidase) selected from the group consisting of peroxidase, β-D-galactosidase, microperoxidase, alkaline phosphatase and biotin. May be

In this measuring method, the soluble Fas antigen is measured by appropriately combining a solid-phased anti-Fas monoclonal antibody and a labeled second anti-Fas monoclonal antibody to form a sandwich ELISA system. Here, the reason why the sandwich ELISA method is adopted is as follows. That is, EIA method or ELI
Although there are several SA methods, typically, the sandwich method or the competitive method is used. Since it is necessary to obtain a large amount of purified antigen in the case of measuring by the competitive method, it is preferable to use the sandwich method in consideration of the time and effort required for preparation.

A second aspect of the present invention is a measurement kit for immunologically measuring soluble Fas antigen, which comprises binding an anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen to an insoluble support. Solid phase anti-Fa
s monoclonal antibody and soluble Fa as standard substance
s antigen, and the assay kit may further include a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen.

Here, soluble Fa as the standard substance is used.
The s antigen is the extracellular region of human Fas antigen or mouse Fas antigen and mouse IL-3 receptor β subunit A.
It may be a chimeric soluble Fas antigen consisting of the extracellular region of IC2A or the constant region of immunoglobulin heavy chain. The solid phase anti-Fas monoclonal antibody bound to the insoluble support is hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6,
It may be a monoclonal antibody produced from one hybridoma clone selected from the group consisting of JAE and CH11. Further, the labeled second anti-F
As monoclonal antibody is hybridoma clone V
B3, CBE, WB3, ZB4, UB2, AX6, JA
It may be a monoclonal antibody produced from one hybridoma clone selected from the group consisting of E and CH11. Furthermore, the labeled second anti-F
as monoclonal antibody is peroxidase, β-D
It may be a monoclonal antibody labeled with a labeling substance (preferably peroxidase) selected from the group consisting of galactosidase, microperoxidase, alkaline phosphatase and biotin.

Solid-phased anti-F according to the first and second aspects of the present invention
The as monoclonal antibody has an action of specifically binding to the soluble Fas antigen to trap the soluble Fas antigen on the insolubilized support to form a complex. At this time, the immobilized anti-Fas monoclonal antibody is considered to specifically bind to the antigenic determinant (epitope) of soluble Fas. In addition, the labeled second anti-Fas monoclonal antibody in the first and second aspects of the present invention specifically binds to the soluble Fas antigen trapped by the immobilized anti-Fas monoclonal antibody to form the complex. It has the effect of labeling. At this time, the labeled second anti-Fas monoclonal antibody is the immobilized anti-Fs of soluble Fas antigen.
It is considered that the as monoclonal antibody specifically binds to an antigenic determinant at a position different from that of the soluble Fas antigen.

The immobilized anti-Fas monoclonal antibody and the labeled second anti-Fas monoclonal antibody are usually
Those that recognize different antigenic determinants are used. However, when the same antigenic determinant is present at two positions of the soluble Fas antigen, the same antigenic determinant may be recognized. In this case, one of them binds to the immobilized anti-Fas monoclonal antibody and the other binds to the labeled second
Anti-Fas monoclonal antibody.

The present invention will be described in more detail below. The Fas antigen referred to in the present invention means a mammalian Fas antigen such as human, mouse, rat, dog or monkey, preferably human, mouse or rat Fas antigen, particularly preferably human Fas antigen. To do.

Soluble Fas antigen as a standard substance (standard) used in the detection of soluble Fas antigen, which is the subject of the present invention, can be prepared by using a genetic engineering technique.
soluble region of as antigen and mouse IL-3 receptor AI
Chimeric protein with soluble region of C2A, or Fa
It can be produced and obtained as a chimeric protein consisting of a soluble region of s antigen and a constant region (Fc) of immunoglobulin heavy chain. Specifically, it can be manufactured as follows.

Total RNA from cells capable of producing Fas antigen
Is prepared and a cDNA library is prepared using these as templates. The cells used here can be arbitrarily selected. For example, human T tumor cell line KT3 can be used if it produces a human Fas antigen, and mouse macrophage can be used if it produces a mouse Fas antigen. -BAM3 can be used.

Next, from the cDNA library, Fas
A clone containing a cDNA encoding the antigen is selected, and a full-length cDNA encoding the Fas antigen is obtained from the clone. A cDNA fragment coding for an amino acid sequence that substantially constitutes the extracellular region of Fas antigen is excised from the full-length cDNA and introduced into an appropriate plasmid.

On the other hand, in the same manner as described above, the mouse interface
A cDNA fragment encoding an amino acid sequence that substantially constitutes the extracellular region of leukin-3 receptor-β subunit AIC2A or a cDNA fragment encoding an amino acid sequence that substantially constitutes the constant region Fc of immunoglobulin H chain. To get.

Mouse interleukin-3 receptor-β subunit AIC2 downstream of a cDNA fragment coding for an amino acid sequence which substantially constitutes the extracellular region of Fas antigen, with or without an inserted DNA sequence.
The amino acid sequence that substantially constitutes the extracellular region of A is
CDNA fragment to be introduced or a cDNA fragment encoding an amino acid sequence that substantially constitutes the constant region Fc of immunoglobulin H chain, so as to substantially constitute the extracellular region of Fas antigen opened with a restriction enzyme. The plasmid into which the cDNA fragment encoding the amino acid sequence has been introduced, and the amino acid sequence that substantially constitutes the extracellular region of the mouse interleukin-3 receptor-β subunit AIC2A are encoded c
A DNA fragment or a cDNA fragment encoding an amino acid sequence that substantially constitutes the constant region Fc of the immunoglobulin H chain, and, in the case of an insertion DNA sequence, the insertion DNA sequence is modified so that the DNA ends can be ligated to each other. After that, each DNA fragment is ligated by a conventional method using a DNA ligation kit to obtain an expression vector in which a chimeric protein of Fas antigen and AIC2A is controlled to be produced.

Then, an appropriate host cell is transformed with this expression vector, the transformed cell is cultured in a medium, and soluble Fas antigen as a chimeric protein is collected from the culture supernatant. As the expression vector, a plasmid vector can be used, and it can be selected and used according to the cells to be transformed and, depending on the purpose, from plasmids usually used in test research or industrial fields. For example, pME18S, pCEV4, pE
F-BOS and pHβAPr-1 can be used.

The expression promoter is a promoter usually used in test research or industrial fields, depending on the host cell to be transformed and depending on the purpose.
Can be selected and used. For example, SV40 promoter, LTR promoter, SRα promoter, EF-1α promoter, β-actin promoter.
And an immunoglobulin promoter etc. can be used.

The host cell is not limited to a natural cell or an artificially established recombinant cell, which is suitable for the expression vector and is usually used in the test research or the industrial field depending on the purpose, and is not limited to animals. It can be selected from cells and used. For example, mouse-derived cells (CO
P, L, C127, Sp2 / 0, L5178Y and NS
-1, etc.), rat-derived cells, hamster-derived cells (BH
K and CHO), monkey-derived cells (COS1, COS)
3, COS7, CV1 and Velo, etc.) and human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells, Namalwa, etc.) and the like can be used.
Of these, preferred are COS1 cells, COS3 cells, and C
OS7 cells, cells derived from human diploid fibroblasts, myeloma cells, and the like.

Selection of transformed cells can be carried out by a conventional cell selection method by providing transformed cells with one or more properties such as drug resistance, temperature sensitivity, auxotrophy and radiation mutability. . The selection marker DNA used in the present invention may be selected and used from the selection marker DNAs which are usually used in test research or industrial fields depending on the purpose. For example, ampicillin (Amp) resistant DNA, tetracycline (Tc) resistant DNA, thymidine kinase (Tk) resistant DNA, 6-thioguanine (HGPRT)
Drug resistant DNA such as resistant DNA, neomycin (Neo) resistant DNA and hygromycin resistant DNA can be used.

As a method for purifying and isolating the desired soluble Fas antigen from the culture supernatant of the transformed cells, the obtained culture is filtered or centrifuged to obtain a culture filtrate (supernatant), and the culture is carried out. The filtrate can be purified and isolated according to a conventional method. That is, for example, a method utilizing solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, sodium dodecyl sulfate-
Polyacrylamide gel electrophoresis (SDS-PAGE)
Such as a method utilizing a molecular weight, a method utilizing an electric charge such as ion exchange chromatography or hydroxylapatite chromatography, a method utilizing a specific affinity such as affinity chromatography, a hydrophobic phase such as reverse phase high performance liquid chromatography. How to take advantage of the difference,
A method utilizing an isoelectric point such as isoelectric focusing can be used.

The monoclonal antibody used as the solid-phased anti-Fas monoclonal antibody and the labeled second anti-Fas monoclonal antibody can be produced by a general method for producing a known monoclonal antibody.
For example, it can be produced from a hybridoma (fused cell) produced by so-called cell fusion. That is, a fused hybridoma is formed from an antibody-producing cell and a myeloma cell, and the hybridoma is cloned. For example, a Fas antigen-expressing natural cell or an artificially produced transformed cell, or as described above. It is produced by using a purified and isolated soluble Fas antigen as an antigen and selecting a clone producing an antibody showing a specific affinity thereto. For the operation, conventionally known means can be used except that the Fas antigen-expressing cells or Fas antigen described above is used as an immune antigen.

The animals to be immunized include mammals such as mice, rats, guinea pigs, hamsters and rabbits, preferably mice, rats or hamsters. Immunization is performed by injecting the immunogen subcutaneously, intramuscularly, intravenously, in the hood pad, or intraperitoneally in these mammals once or several times or by transplantation.

Usually, about 1 to 2 weeks after the first immunization, 1 to
Immunization is performed 4 times, and final immunization is performed about 1 to 4 weeks later, and antibody-producing cells are collected from the immunized animal about 3 to 5 days after the final immunization. Hybridomas that secrete monoclonal antibodies are prepared by the method of Kohler and Milstein (Nature, Vol. 256, pp. 495-497,
1975) and modification methods similar thereto. That is, the monoclonal antibody of the present invention, the spleen obtained from the animal immunized as described above, lymph nodes, bone marrow or tonsils, preferably antibody-producing cells contained in the spleen, preferably the same species of mouse, rat. , Mammals such as guinea pigs, hamsters, rabbits or humans,
More preferably, it is prepared by culturing fused cells (hybridomas) obtained by fusing with mouse, rat or human myeloma cells (myeloma). Culture is in vitro, or in mouse, rat, guinea pig,
It can be carried out in vivo in a mammal such as hamster or rabbit, preferably mouse or rat, more preferably ascites fluid of mouse, and the antibody can be obtained from the culture supernatant or the ascites fluid of the mammal, respectively.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8, P3 / NS1 / 1
-Ag4-1 (abbreviated as NS-1), P3 / X63-Ag8.U1, SP2 / 0-Ag14,
F0 or BW5147, rat-derived myeloma 21ORCY3-Ag1.
2.3, human-derived myeloma U-266AR1, GM1500-6TG-A1-2,
UC729-6, CEM-AGR, DIR11 or CEM-T15 can be mentioned.

The screening of fused cell clones producing a monoclonal antibody is carried out by culturing the fused cells in, for example, a microtiter plate, and measuring the reactivity of the culture supernatant of the wells in which the proliferation is observed, for example, RIA or E.
It can be performed by measuring with an enzyme antibody method such as LISA.

Purification and isolation of the monoclonal antibody is carried out by subjecting the serum or ascites containing the monoclonal antibody of the present invention obtained by the above-mentioned method to ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein. It can be carried out by subjecting it to affinity column chromatography such as A column.

The anti-Fas monoclonal antibody used in the present invention is IgG, IgM, IgA, IgD or I.
It is preferable to use a monoclonal antibody belonging to any immunoglobulin class of gE. Particularly preferred among these is IgG or IgM.

In the method for measuring soluble Fas antigen of the present invention, at least two kinds of monoclonal antibodies are appropriately selected and used from the anti-Fas monoclonal antibodies prepared as described above, depending on the purpose or conditions of the measurement. However, it is usually desirable to use a monoclonal antibody that binds to the insoluble support and another monoclonal antibody that is labeled, which recognize different antigenic determinants. However, when the same antigenic determinant is present at two positions of the soluble Fas antigen, the same antigenic determinant may be recognized.

Specifically, for example, International Immunology Vol. 6,
Yonehara et al., No. 12, pp. 1849-1856 (1994), Journal of Experimental Medicine
Report in Vol. 169, pp. 1747-1756 (1989), Cell Vol. 66, 233-243.
Clone ZB4 (IgG), UB2 (I), which are anti-human Fas monoclonal antibody-producing hybridomas described in the report on page (1991) and in the Examples described later.
gG), WB3 (IgG), VB3 (IgG), CBE
(IgG), JAE (IgM), AX6 (IgM), CH11 (IgM), etc. can be selected and used from the monoclonal antibodies produced.

As an example of a specific combination, a monoclonal antibody obtained from clone CBE is used as a monoclonal antibody for preparing a solid-phased anti-Fas monoclonal antibody, and a labeled second anti-Fas monoclonal antibody is prepared. The monoclonal antibody obtained from clone VB3 can be used as the monoclonal antibody of.

Regarding the solid-phased anti-Fas monoclonal antibody, examples of the insoluble support to which the monoclonal antibody is bound include plastics such as polystyrene resin, polycarbonate resin, silicone resin and nylon resin, and
Water-insoluble substances such as glass are applicable. The loading on the insoluble support may be mere physical adsorption or chemical bonding.

Regarding the labeled second anti-Fas monoclonal antibody, the labeling substance is peroxidase, β-
In addition to various enzymes such as D-galactosidase, microperoxidase, alkaline phosphatase, and isotopes, a method of reacting a biotinylated antibody and then reacting with avidin peroxidase can be used as a method for obtaining higher sensitivity. If a radioactive element is used as the labeling substance, the concentration of soluble Fas antigen can be measured by the RIA method. In this case, it is necessary to be carried out by a licensed facility at a managed facility.

By thus using the immobilized anti-Fas monoclonal antibody and the labeled second anti-Fas monoclonal antibody, the concentration of the soluble Fas antigen in the sample can be easily and specifically achieved in the clinical setting. Can be measured.

[0045]

The present invention will be described in more detail with reference to its preferred embodiments. [Example 1] Preparation of standard substance soluble Fas antigen Soluble Fas antigen used as a standard substance (standard) was prepared as follows. That is, Reference Example 1 described later
Transformed cells prepared according to JM109 (deposition number F
ERM BP-4436), plasmid pME18
Take out S / hFas EXT-AIC2A EXT and use Gene Pulser (Kit of Bio-Rad Co., Ltd.)
L5178Y cells were co-transformed with pMAMneo (manufactured by Clontech) by electroporation. The cells were cultured in a G418-containing medium, and a G418-resistant clone was selected to obtain a soluble human Fas antigen-expressing clone. The selected clones were cultivated in a large amount using 10% horse serum-containing Fischer medium, the culture supernatant was collected and concentrated, and the resulting clone was subjected to column chromatography using Q Sepharose (Pharmacia (manufactured)) to prepare a standard substance. Chimeric soluble Fas antigen was obtained. [Example 2] Production of anti-human Fas monoclonal antibody (2-1) Preparation of antibody-producing cells Human diploid fibroblast F expressing human Fas antigen
Balb / c female mice were immunized with the S-7 cell line. The sensitized mouse was laparotomized and the spleen was excised.
After mashing on a stainless mesh in MI1640 culture solution, the spleen cell solution was centrifuged (7 at 1500 rpm).
Minutes). Centrifuge residue is collected and serum-free RPMI164
It was suspended in culture medium 0. Furthermore, serum-free RPMI164
The cells were washed twice with 0 culture medium to obtain antibody-producing mouse spleen cells. (2-2) Preparation of mouse myeloma cells Mouse myeloma cell NS-1 (ATCC TIB18)
ASF-104 medium (manufactured by Ajinomoto Co., Inc.) containing 10% FCS and 50 U / ml of kanamycin at 5 ° C., 5% CO _2.
Cultured in (2-3) Preparation of Monoclonal Antibody-Producing Hybridoma Serum-free RPMI1640 solution obtained by mixing mouse myeloma cells NS-1 cells washed with serum-free RPMI1640 culture medium and mouse spleen cells prepared as described above is centrifuged. Separation (1000 rpm for 10 minutes), collection of the centrifugal residue, and the method of Herzenberg (LA) et al. (Selected Methods in Cellular Immunology)
Cell fusion was performed according to page 351 (1980) to obtain a plurality of hybridoma colonies.

To screen the hybridoma producing the anti-human Fas monoclonal antibody, Fa was screened.
The reactivity of the culture supernatant of each hybrid-macronii to human thymocytes expressing s antigen was measured by flow cytometry.
Of the hybridoma clone CH11 (Ig
M) was obtained. (2-4) Acquisition of anti-human Fas monoclonal antibody Hybridoma clone CH11 (2 × 10 ⁸ ) was cultured in serum-free ASF104 medium (Ajinomoto Co., Inc.) at 37 ° C. for 5 days, and the obtained culture supernatant was obtained. Was subjected to ultracentrifugation with an Omega cell (Filtron (manufactured)). The centrifugation supernatant was collected and applied to a hydroxylapatite column (Asahi Optical Co., Ltd.). Using an FPLC system (Pharmacia Fine Chemical Co., Ltd.), sodium phosphate (pH 7.
4, 10 to 400 mM).

The eluted fractions were collected, subjected to SDS-PAGE, and purified with a purity of 95% or more (J. Exp. Med., Vol. 169, 1747-). See page 1756 (1989) report). Further, another anti-human Fas monoclonal antibody was prepared as follows. (2-5) Preparation of immunizing antigen Plasmid pF58 containing human Fas antigen cDNA
Was digested with XhoI to obtain a fragment containing human Fas antigen cDNA, which was used as an expression vector pEF-BOS.
(Nucl. Acid Res.
), See report on page 18, 5322),
It was introduced using a BstXI adapter to obtain the expression plasmid pEFF58.

25 μg / ml pE digested with VspI
2.5μ digested with FF58 fragment and EcoRI
1 × 10 ⁷ (0.8 ml) mouse T lymphoma WR19L cells (ATCC TIB52) using g / ml of pMAMneo (Clontech) fragment.
Was co-transformed by electroporation. The cells were cultured in a medium containing G418 to select G418 resistant clones. The G418-resistant clones thus obtained were analyzed by a flow cytometer and a limiting dilution method to give human Fas antigen-overexpressing transformed cells WR19L1.
2a was cloned (cell volume 66, 23)
See the report on pages 3-243 (1991)).

Homogenize WR19L12a cells,
The plasma membrane fraction was obtained by centrifugation and used as an immunogen. (2-6) Preparation of antibody-producing cells Human Fas antigen-overexpressing transformed cells WR19L12a
Balb / C mice were immunized with the plasma membrane fraction of and the antibody-producing mouse spleen cells were obtained in the same manner as described above. (2-7) Preparation of Monoclonal Antibody-Producing Hybridoma Using mouse myeloma NS-1 cells and the obtained mouse spleen cells, a plurality of hybridoma clones were obtained in the same manner as described above, and seven anti-human Fas antibody-producing hybridoma clones were obtained. ZB4 (IgG), UB2 (IgG), V
B3 (IgG), CBE (IgG), WB3 (Ig
G), AX6 (IgM) and JAE (IgM) were obtained (these hybridoma clones are commercially available from Institute of Medical Biology). (2-8) Acquisition of anti-human Fas monoclonal antibody Seven kinds of anti-human Fas monoclonal antibodies were purified and acquired from each of the obtained hybridoma clones in the same manner as described above (International Immunology No. 6). Volume, No.12, 18th
49-1856 (1994)). [Example 3] Measurement of soluble Fas antigen-Part 1 (3-1) Immobilization on insoluble support (preparation of microplate type reagent) The monoclonal antibody IgG fraction of clone CBE was adjusted to 0.
It was dissolved in 1M phosphate buffer (pH 7.4, 0.1% sodium azide added) and the concentration was adjusted to 0.005 mg / ml. A 96-well microplate (Nunc Co., Ltd.)
Add 100 μl to each well of Maxisorp)
Monoclonal antibody was allowed to bind by leaving still at 18 ° C. for about 18 hours.

After antibody binding, the monoclonal solution in the well was removed, 300 μl of phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 5% sucrose was added, and the mixture was allowed to stand at 4 ° C. for about 24 hours. The reaction was allowed to occur and the unreacted portion of the wells of the microplate was blocked. This blocking is to prevent the labeled antibody and the like from being adsorbed on the wall surface of the microplate so as not to impair the quantitativeness of the measurement system. After blocking, the blocking solution in the well was removed, and the plate was completely air-dried at room temperature to obtain a microplate type reagent (immobilized anti-Fas monoclonal antibody). (3-2) Preparation of labeled monoclonal antibody 5 g of monoclonal antibody IgG fraction of clone VB3
This IgG fraction was used to collect Journal of Histochemistry and Site Chemistry (J.Histoche
m. Cytochem) Vol. 22, p. 1084 (1974)
POD [Horse radish per
oxidase: manufactured by Sigma (manufactured by the company)] and bound to a peroxidase-labeled monoclonal antibody (labeled second anti-F
as monoclonal antibody) was obtained. (3-3) Measurement of Soluble Fas Antigen A measurement method using each reagent prepared in (3-1) and (3-2) above will be described. (3-3-1) Preparation of sample and standard soluble Fas antigen Serum was diluted 50 times with PBS to prepare a sample. At the same time, the standard soluble Fas antigen prepared and prepared in Example 1 was similarly diluted to prepare a standard solution. When the amount of soluble Fas antigen in the sample is large and exceeds the detection limit, the sample is diluted with PBS so that it falls within the measurement range, and the quantitative value of the diluted sample is multiplied by the dilution factor. The concentration of soluble Fas antigen in the medium. (3-3-2) Antigen-antibody reaction between microplate-type reagent and soluble Fas antigen in sample Specimen prepared in (3-3-1) and microplate-type reagent prepared in (3-1) above, and 100 μl of the standard solution was dispensed into each well. At the same time only PBS 100μ
It was dispensed in an amount of 1 and was blinded. The above reaction system was allowed to stand at room temperature for 1 hour. After the reaction was completed, the plate was washed 3 times with 5% PBS, and then the buffer solution was thoroughly removed. (3-3-3) Peroxidase-labeled anti-F to a conjugate of a microplate type reagent and soluble Fas antigen in a sample
Binding of as monoclonal antibody Peroxidase-labeled anti-Fa prepared in the above (3-2)
s monoclonal antibody 0.1% BSA, 0.15M
Dilute 250-1000 times with 10 mM sodium phosphate buffer (pH 8.0) containing NaCl, and add (3
-3-2) 100 μl of each well was dispensed to the microplate reacted with the antigen and antibody, and allowed to stand at room temperature for 1 hour.
After washing 3 times with BS, the same buffer was removed. (3-3-4) Coloring reaction Tetramethylbenzidine dihydrochloride (manufactured by Sigma) was dissolved in 10 mM citrate buffer (pH 6.8), and 1.
A 6 mM solution was made. 10 parts of this solution and hydrogen peroxide
10 mM diluted with mM citrate buffer (pH 6.8)
An equal amount of the above solution was mixed to prepare an enzyme substrate solution.

This enzyme substrate solution was applied to a microplate treated with a peroxidase-labeled anti-Fas monoclonal antibody in (3-3-3), 100 μ per well.
Dispense 1 by 1 and leave at room temperature for 10 to 20 minutes, then 1.5
Dispense 100 μl of N-phosphoric acid per well and adjust the pH.
The enzyme activity of peroxidase was stopped by lowering.

The degree of color development thus generated in each well of the microplate was measured by an absorbance meter (MPR-manufactured by Tosoh Corp.).
It was detected by measuring the absorbance at a wavelength of 450 nm in A4). From this absorbance, the concentration of soluble Fas antigen in the sample can be known. To determine the concentration from the absorbance, a calibration curve was prepared as shown below. (3-3-5) Preparation of calibration curve The soluble Fas antigen prepared in (3-3-1) was detected using the above microplate type reagent, and the absorbance was measured. The results are shown in Table 1 and FIG.

[0053]

[Table 1]

(3-3-6) Quantification of sample Using the above reagents and measuring method, 50 SLE patients, R
Serum collected from 50 patients A and 200 normal persons by a conventional method is used as a sample, and (3-3-1) to (3-3-5)
Table 2 shows the results of the measurement according to.

[0055]

[Table 2]

As can be seen from Table 2, it can be seen that the measured value shows a remarkably high concentration in SLE patients having a large content of soluble Fas antigen. Therefore, if the assay method of this example is performed using the reagent of this example, the concentration of soluble Fas antigen can be easily measured even in a clinical setting, and SLE
Detection of patients with autoimmune diseases such as

If a radioactive element is used as a labeled substance,
The concentration of soluble Fas antigen can be measured by the RIA method. In this case, it is necessary to be carried out by a licensed facility at a managed facility. Example 4 Measurement of Soluble Fas Antigen—Part 2 The standard substance soluble Fas antigen was prepared in the same manner as in Example 1, and the anti-human Fas monoclonal antibody was prepared in the same manner as in Example 2. (4-1) Immobilization on insoluble support (preparation of microplate type reagent) The monoclonal antibody IgG fraction of clone CBE was adjusted to 0.
It was dissolved in 1M phosphate buffer (pH 7.4, 0.1% sodium azide added) and the concentration was adjusted to 0.015 mg / ml. A 96-well microplate (Nunc Co., Ltd.)
Add 100 μl to each well of Maxisorp)
Monoclonal antibody was allowed to bind by leaving still at 18 ° C. for about 18 hours.

After binding the antibody, the monoclonal solution in the well was removed, and 300 μl of phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 10% sucrose.
Was added and allowed to react at 37 ° C. for 2 hours to block the unreacted portion of the well of the microplate. This blocking is to prevent the labeled antibody and the like from being adsorbed on the wall surface of the microplate so as not to impair the quantitativeness of the measurement system.

After blocking, the blocking solution in the well was removed, and the well was completely air-dried at room temperature to obtain a microplate type reagent (immobilized anti-Fas monoclonal antibody). (4-2) Preparation of labeled monoclonal antibody The monoclonal antibody IgG fraction of clone VB3 was adjusted to 0.
Dialyze against 1M acetate buffer (pH 4.2) and adjust the protein concentration to 5
After adjusting to mg / ml, pepsin (3% of total protein amount) was added, and the reaction was carried out at 37 ° C. with stirring overnight. After stopping the reaction by adding an appropriate amount of 2M Tris-HCl buffer (pH 8.0), Ultrogel AcA44 (LKB company) equilibrated with 0.1M phosphate buffer (pH 6.5) containing 0.2M sodium chloride. (Manufactured)) column (2.
Gel filtration was performed at 0 × 60 cm) to obtain F (ab ′) 2 fraction 2.5 mg.

Succimidyl 4- (N-maleimidomethyl)-
Cyclohexane-1-carboxylate [Succimidyl 4- (N
-maleimi-domethyl) -cyclohexan-1-carboxylate: Zieben Chemical Co., Ltd.) [Hinge method (J. Immunoassay, Vol. 4, Vol. 209 (19
1983)), POD [Horseradish peroxidase:
Sigma] was bound to IgG / Fab ′ of the mouse anti-Fas monoclonal antibody obtained above through the SH group to obtain a peroxidase-labeled monoclonal antibody (second anti-Fas monoclonal antibody labeled). (4-3) Measurement of Soluble Fas Antigen Specimen in the same manner as in (3-3-1) of Example 3 above
A standard soluble Fas antigen was prepared, and the soluble F in the microplate type reagent and the sample was prepared in substantially the same manner as in (3-3-2).
An antigen-antibody reaction with an as antigen is performed, and a peroxidase-labeled anti-Fas is bound to the bound product of the microplate-type reagent and the soluble Fas antigen in the sample in substantially the same manner as (3-3-3).
Monoclonal antibody was bound.

The color development reaction was performed as follows. 1 mg / m of ortho-phenylenediamine (manufactured by Sigma Co.) in 0.1 M sodium citrate buffer (pH 5.1)
Dissolve at a concentration of 1 and dispense 100 μl per well into a microplate treated with peroxidase-labeled anti-Fas monoclonal antibody, leave at room temperature for 10 to 20 minutes, and dispense 2 N sulfuric acid 100 μl per well. Then, the enzymatic activity of peroxidase was stopped by lowering the pH.

The degree of color development thus generated in each well of the microplate was measured by an absorbance meter (MPR-manufactured by Tosoh Corp.).
It was detected by measuring the absorbance at a wavelength of 492 nm in A4). From this absorbance, the concentration of soluble Fas antigen in the sample can be known. The preparation of the calibration curve and the quantification of the sample were carried out by substantially the same procedure as in (3-3-5) and (3-3-6) of Example 3 above. The data of the calibration curve are shown in Table 3 and FIG. 2, and the results of actually measuring the sera of normal persons and SLE patients by the above reagents and measuring method are shown in Table 4.

[0063]

[Table 3]

[0064]

[Table 4]

As can be seen from Table 4, it can be seen that the measured values show significantly high concentrations in SLE patients. Therefore, if the assay method of this example is performed using the reagent of this example, the concentration of soluble Fas antigen can be easily measured even in a clinical setting, and a patient with an autoimmune disease such as SLE can be quickly detected.

If a radioactive element is used as a labeled substance,
The concentration of soluble Fas antigen can be measured by the RIA method. In this case, it is necessary to be carried out by a licensed facility at a managed facility. [Example 5] (Preparation of biotin-labeled monoclonal antibody) (3-2) of the above Example 3 and (4-2) of the above Example 4
Instead, a biotin-labeled monoclonal antibody can be prepared, and the soluble Fas antigen can be measured in substantially the same manner as in Examples 3 and 4 above.

Monoclonal antibody Ig of clone VB3
The G fraction was dissolved in 0.1 M carbonate buffer (pH 8.5) to prepare an IgG concentration of 5 mg / ml. This I
NHS-LC-BI, which was also dissolved in 0.1 M carbonate buffer (pH 8.5) in the gG solution to prepare 25 mg / ml.
OTIN (PIERCE (manufactured)) IgG10
891 μl per 0 mg was added, and the mixture was stirred with a stirrer at room temperature for 4 hours to prepare a biotin-labeled monoclonal antibody.

When the biotin-labeled monoclonal antibody thus obtained is used, the enzyme (peroxidase, galactosidase, etc.) labeled with avidin is further reacted to form a solid phase anti-monoclonal antibody-soluble Fas antigen. The concentration of soluble Fas antigen in the specimen was determined by forming a complex of biotin-labeled antibody-avidin-labeled enzyme and measuring the color development of the chromogenic substrate in the same manner as in Examples 3 and 4 above. When the combination of the biotin-labeled antibody and the avidin-labeled enzyme is used, the soluble Fas antigen is measured with higher sensitivity than when the usual enzyme-labeled antibody is used as in Examples 3 and 4 above. I was able to. [Reference Example 1] E. Production of E. coli cell line JM109 (deposition number FERM BP-4436) Hereinafter, hFas means human Fas antigen and EXT means extracellular region (soluble region). (Reference 1-1) Acquisition of cDNA encoding full-length hFas (Reference 1-1-A) Preparation of cell line The cell line used in this Reference Example was prepared in advance. Human T tumor cell line KT3 (sold by Dr. Shimizu, Kanazawa Medical University): RPMI1640 medium containing 10% fetal calf serum (FCS) and 5 ng / ml human recombinant IL-6 (manufactured by Ajinomoto Co., Inc.) Cultured in・ Monkey cell line COS7 (ATCC CRL 1651): 10% FCS
It culture | cultivated in the DMEM culture medium containing. (Reference 1-1-B) Preparation of antibody Mouse anti-hFas antibody CH used in this reference example
Yonehara et al. (J. Exp. Med., Vol.169, p.
p.1747-1756, 1989) and prepared in the same manner as in Example 2. (Reference 1-1-C) Construction of plasmid The foreign cDNA cloning vector (plasmid) used in this reference example was constructed in advance. -Plasmid pCEV4 (Science, Vol.247, pp.324-32
7, 1990, Ito et al.): Plasmid pcDSRα (Mol.
CEll Biol., Vol.2, pp.161, 1982, Okayama et al., Mol.
CEll Biol., Vol.3, pp.280, 1983, Okayama et al. And Mo
l. CEll Biol., Vol.8, pp.466-472, 1988, Takebe et al.) as a basic skeleton,
a virus) and a replication origin DNA (Pyori) extracted as a BglI-BclI cleavage fragment and a plasmid CDM8 (Proc. Natl. Acad. sci. USA, Vol.
84, pp.3365-3369, 1987, Brian Sade
eed et al., Nature, Vol. 329, pp. 840-842, 1987, Brian
Seed et al.), A stuffer having two BstXI cleavage sites extracted as XbaI cleavage fragments.
(stuffer) DNA was inserted into the NdeI and PstI sites of pcDSRα,
The NotI cleavage site in (XbaI cleavage fragment) was removed by filling in, and the NotI linker was inserted into the ClaI cleavage site of pcDSRα to construct plasmid pCEV4. -Plasmid pME18S (contributed by Dr. Kazuo Maruyama of the University of Tokyo): a cDNA cloning vector suitable for expression in animal cells constructed with pCEV4 as the basic skeleton.
And the origin of replication sequence of pUC, 16S intron sequence of SV40, polyA signal sequence and polyA.
It has translation termination of each frame immediately before the signal sequence (Experimental Medicine "Genetic Engineering Handbook" (separate volume), p.
p.101-107,1992, Kazuo Maruyama et al.).

Restriction enzyme maps of plasmids pCEV4 and pME18S are shown in FIGS. 3 and 4, respectively. (Reference 1-1-D) Preparation of cDNA library derived from human T tumor cell KT3 Guanidine isothiocyanate / acid phenol method (Anal.
Biochem., Vol. 162, pp.156-159, 1987, Chomczynski and Sacchi et al.) To prepare total RNA from KT3,
Poly (A) RNA was selected by oligo (dT) cellulose column chromatography. Random hexamer-oligonucleotide (pdN6) (manufactured by BRL) and oligo (dT) (manufactured by BRL) were used as primers for M-MLV RNAaseH ^- reverse transcriptase, and double-stranded cDNA was prepared from each RNA. Was synthesized
(See Cell, Vol.61, pp.341-350, 1990, Fukunaga et al.).
Pre-synthesized BstXI non-palindromic adapter was ligated, and cDNAs longer than 2 kb were separated by agarose gel electrophoresis, and they were digested with BstXI-digested pCEV4.
Vector-ligated into fragment. Electroporation method (Nucl. Acids Res., Vol. 16, pp. 6127-6145, 1988,
(See Dower et al.) Coli WM1
Human T tumor cells KT3 transformed into 100
A derived cDNA library was prepared. (Reference 1-1-E) Acquisition of hFas cDNA fragment Method by Brian Seed et al. (Proc. Natl. Acad. Sci. US
A, Vol.84, pp.3365-3369, 1987) and the following operations were performed.

6 cm dish for bacteriology (Falcon1007)
10 μg goat anti-mouse IgM antibody (Cappe
l) company (manufactured)) containing 50 mM Tris-HCl (3 m
1; pH 9.5) was incubated at room temperature for 90 minutes to prepare a panning dish. The unreacted antibody was removed by washing the panning dish 3 times with 0.15 M NaCl, and then 0.5 mM EDT was added.
A, P containing 0.02% NaN ₃ and 5% FCS
Blocking was carried out by overnight incubation using BS (phosphate-buffered saline) (3 ml).

108 6 cm dishes (Falcon 1007)
At the stage where COS7 seeded on the cells grew to 50%, spheroplast fusion; Mol. Cell. B
iol., Vol. 1, pp. 743-752, 1981, Sandi-Goldrin et al.
Transformed into S7. 72 hours later, 0.5 mM EDT
PBS containing A and 0.02% NaN ₃ (PBS / ED
TA / NaN ₃ ) was added and the cells were detached from the dish by incubating at 37 ° C. for 30 minutes. Peeled cells were collected, centrifuged and 10 μg / ml mouse anti-hF
as antibody CH11 (IgM) (J. Exp. Med., Vol.169, p.
p.1747-1756, 1989, Yonehara et al.))
Resuspended in BS / EDTA / NaN ₃ (9 ml). After incubation on ice for 60 minutes, PBS /
Cells were diluted 1: 2 with EDTA / NaN ₃ , overlaid with 2% Ficoll 400 and centrifuged at 1000 rpm. 5% of live cells collected in the upper layer of phycol
The aggregate was removed by suspending in FCS-containing PBS / EDTA / NaN ₃ (27 ml) and filtering with a nylon mesh (pore size 100 μm).

The filtered cells were plated on the panning dish (54 sheets) prepared above. The cells were left to stand at room temperature for 2 to 3 hours, allowed to attach to the dish, and then gently washed 3 times with 5% FCS-containing PBS / EDTA / NaN ₃ (2 ml) to remove cells that did not bind to the dish. Har
COS bound to the dish according to the method of Hirt et al.
Plasmid DNA was prepared from the cells. That is, 10 mM EDTA-containing 0.6% SDS solution (0.4 ml) was added to each dish and left at room temperature for 20 minutes. The lysate was collected in a microtube, NaCl was added to a concentration of 1M, and the tube was allowed to stand on ice for 5 hours or more.
Then, after centrifugation at 13000 rpm for 5 minutes, the supernatant was extracted with phenol / chloroform, and the plasmid DNA was recovered by the ethanol precipitation method. The recovered plasmid DNA was used for E. E. coli WM1100,
About 3.2 × 10 ⁵ colonies were obtained, the colonies were analyzed and transformed cells were selected, and then transformed into COS7 by the spheroplast fusion method (48 dishes). The same procedure was repeated two more times to obtain plasmid clones containing 14 hFascDNA fragments (pF1-pF1).
4) was acquired. These plasmid clones were cleaved with the restriction enzyme XhoI, clones containing the same 3.0 kb insertion sequence (pF1, 2, 5 and 11) and the same 1.
A clone containing the 5 kb insert (pF3, 4, 6, 7).
And 9). pF1 and pF3 plasmid clones
After transformation of COS7 into COS7, it was confirmed by flow cytometry analysis using an anti-Fas antibody that these clones were cDNA fragments encoding the Fas antigen. (Reference 1-1-F) Acquisition of full-length hFas cDNA 0. 5'-terminal side of pF3 obtained in (Reference 1-1-E).
Ten cDNA clones were obtained from the cDNA library prepared in (See 1-1-B) by the colony-hybridization method using a 5 kb XhoI-BamHI cleavage fragment as a probe. Restriction enzyme mapping confirmed that these clones contained a 1.8-2.6 kb insert with the same restriction map. The longest clone pF58 by various analyzes consisted of 2534 bp, consisting of a 3'-terminal polyadenylation signal sequence (ATTAAA), a 1005 bp o-position starting from the translation initiation codon located at the 195th to 197th bases. Punreading frame (open reading frame) and 120
It has a translation stop codon (TAG) located at the 0th to 1202nd bases, and clone pF58 has a full-length hFasc.
It was confirmed to have DNA. pF58 black
A 2555 bp DNA fragment containing the full-length Fas antigen cDNA extracted as XhoI digested fragment from
mammalian expression plasmid pC using stXI adapter
The integration plasmid pCEV4 / hFas was constructed in EV4. (See 1-2) Extracellular region of hFas (hFas.EX
Acquisition of cDNA coding for T) X-ray of pCEV4 / hFas constructed in (See 1-1-F)
After digestion with hoI, the full-length Fas cDNA was extracted as an XhoI-cut fragment (2555 bp). Also,
pME18S was digested with XhoI and the stuffer part was removed as an XhoI digested fragment. To remove the phosphate group at the 5'end of the cleavage plane, alkaline phosphatase (BAP) (E.
The pME18S fragment was treated with E. coli C75; Takara Shuzo. Full length hFas cDNA was digested with XhoI fragment (2555 bp) and BAP-treated pM
The E18S fragment was ligated with a DNA ligation kit (Takara Shuzo Co., Ltd.) to prepare plasmid pME18S / hFas.
Was built.

PME18S / hFas was added to BglII and P
Digestion with stI removed the cDNA region corresponding to the transmembrane region of hFas and the subsequent intracellular region. Then, using the DNA blunting kit (Takara Shuzo Co., Ltd.), the cleaved ends were blunted, the pME18S fragment was self-ligated, and the plasmid pME18S / hFas.EXT containing only the cDNA encoding hFas.EXT was introduced.
Was built.

Plasmid pME18S / hFas.EX
The construction process of T is shown in FIG. (Reference 1-3) Acquisition of cDNA encoding mouse IL-3 receptor-β subunit AIC2A (AIC2A) (Reference 1-3-A) Preparation of cell line The cell line used in the present Reference Example was previously prepared. Prepared. -Mouse mast cell line MC / 9 (ATCC CRL 8306): 10
R containing 0 U / ml recombinant mouse IL-3 and 10% FCS
Grow in PMI1640 medium.・ Monkey cell line COS7 (ATCC CRL 1651): 10% FCS
Grow in DMEM medium containing. (Reference 1-3-B) Preparation of antibody Mouse anti-AIC2 antibody (IgM) used in this reference example
Yonehara et al. (Int. Immunol., Vol.2, No.2, pp.143-150, 1
990) and prepared in advance. (See 1-3-C) Construction of plasmid The plasmid pCEV4 constructed in (See 1-1-C) was used. (Reference 1-3-D) Mouse mast cell MC / 9-derived cDNA
Preparation of library In the same manner as described in (Reference 1-1-D), MC /
Double-stranded cDNA from poly (A) RNA selected from 9
A was synthesized, ligated to a BstXI non-palindromic adapter, and cDNAs longer than 1.5 kb were separated by agarose gel electrophoresis and they were digested with BstXI-digested pCE.
V4 vector-ligated into the fragment. The vector was transformed into E. coli by the electroporation method. E. coli DH5α was transformed to prepare a mouse mast cell MC / 9-derived cDNA library (Science, Vol. 247, pp324-327, 1).
990, see Ito et al.). (See 1-3-E) Obtaining AIC2A cDNA fragment In the same manner as described in (See 1-1-E), a goat anti-rat IgM antibody (produced by Cappel Co.) was used as a secondary antibody. -Creating a panning dish and using rat anti-Aic2 antibody (IgM) as the primary antibody, 24 COS7-transformed plasmid clones were obtained, and each clone was cloned. 1.6 kb with the same 13 clones analyzed
The plasmid clone pAIC2-2 was selected for use with the probe used in the next step after confirmation that it had the AIC2A cDNA fragment (insertion sequence) of
cience, Vol.247, pp324-327, 1990, Ito et al.). (Reference 1-3-F) Acquisition of full-length AIC2A cDNA pAI was prepared in the same manner as described in (Reference 1-1-F).
CD prepared in (See 1-3-D) by the colony hybridization method using C2-2 as a probe.
18 cDNA clones were obtained from the NA library, and a cDNA clone pAIC2-26 having the full length AIC2A cDNA was obtained (GenBank deposit number: M2
9855). PAIC2-26 was found to be 3 by various analyzes.
351 bp, having an open reading frame coding for 878 amino acid residues.
The Punreading frame encodes a signal peptide consisting of 22 amino acid residues and a mature protein consisting of 856 amino acid residues.
It was confirmed to be composed of an extracellular region consisting of 17 amino acid residues, a transmembrane region consisting of 26 amino acid residues, and an intracellular region consisting of 413 amino acid residues.

Full-length AIC2AcDN taken out from pAIC2-26 clone as an XhoI digested fragment.
The DNA containing A was integrated into the mammalian expression plasmid pCEV4 using the BstXI adapter to obtain the plasmid pC.
EV4 / AIC2A was constructed. (Reference 1-4) Acquisition of cDNA consisting of cDNA encoding hFas · EXT and cDNA encoding extracellular region of AIC2A (AIC2A · EXT) Plasmid pME18S / hF constructed in (Reference 1-2)
As EXT was digested with NotI, linearized, blunted with a DNA blunting kit, and treated with BAP to remove the phosphate group at the 5'end of the cleaved surface.

Plasmid p constructed in (See 1-3-F)
CEV4 / AIC2A was digested with DraIII to extract the cDNA encoding AIC2A.EXT, and the cut ends of the fragment were blunted using a DNA blunting kit. D using a DNA ligation kit
The RaIII-cleaved AIC2A EXT cDNA fragment was converted into the previously prepared NotI-cleaved pME18S / hFas
-Introduced into the EXT plasmid fragment, plasmid pME18S / hFas.EXT-AIC2A.EXT
Was built.

PME18S / hFas.EXT-AIC
The construction process of 2A EXT is shown in FIG. (See 1-5) J expressing soluble hFas-AIC2A
Obtaining M109 (see 1-4) Plasmid pME18S / hF constructed
as EXT-AIC2A EXT, E. The E. coli cell line JM109 was transformed and the colonies were analyzed to select transformed cells. In addition, this transformed E. co
The li cell line JM109 has the deposit number FERM BP-44
36 has been internationally deposited at the Institute of Biotechnology, Ministry of International Trade and Industry.

[0078]

As described in detail above, the soluble F of the present invention is used.
According to the method for measuring as antigen and the kit for measuring the same, it is possible to easily measure the concentration of soluble Fas antigen in a sample, which has been impossible in the past. It is useful because it can predict the autoimmune disease, establish a diagnosis and treatment policy, and determine the therapeutic effect based on the amount of soluble Fas antigen in the sample measured by this measurement method and the measurement kit. .

[Brief description of drawings]

FIG. 1 shows a graph of a calibration curve for determining the concentration of soluble Fas antigen from the absorbance of Example 3.

FIG. 2 shows a graph of a calibration curve for determining the concentration of soluble Fas antigen from the absorbance of Example 4.

FIG. 3 is a restriction map of plasmid pCEV4.

FIG. 4 is a restriction map of plasmid pME18S.

FIG. 5: Plasmid pME18S / hFas.EXT
FIG.

FIG. 6: Plasmid pME18ShFas.EXT-
It is a construction process drawing of AIC2A / EXT.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takahisa Hachiya 1063-103 Ohara, Ina City, Ina City, Nagano Prefecture

Claims (11)

[Claims] 1. A method for immunologically measuring a soluble Fas antigen, which comprises: a) an anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen bound to an insoluble support. After reacting standard soluble Fas antigen with, b) reacting with a labeled second anti-Fas monoclonal antibody capable of specifically binding to soluble Fas antigen, and c) measuring the labeled amount of the reaction product. Step of preparing a calibration curve, and d) after reacting the sample with a solid-phased anti-Fas monoclonal antibody prepared by binding an anti-Fas monoclonal antibody capable of specifically binding to soluble Fas antigen to an insoluble support, e) A labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen is reacted, and f) the labeled amount of the reaction product is measured. And a step of quantifying the soluble Fas antigen contained in the sample from the calibration curve, the method of immunologically measuring the soluble Fas antigen. 2. The standard soluble Fas antigen is human Fa
Extracellular region of s antigen or mouse Fas antigen and mouse I
The soluble Fas antigen according to claim 1, which is a chimeric soluble Fas antigen comprising the extracellular region of L-3 receptor β subunit AIC2A or the constant region of immunoglobulin heavy chain.
Immunological measurement method of s antigen. 3. The solid phase anti-Fas monoclonal antibody bound to the insoluble support is hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX.
The immunological assay method for soluble Fas antigen according to claim 1 or 2, which is a monoclonal antibody produced from one hybridoma clone selected from the group consisting of 6, JAE and CH11. 4. The labeled second anti-Fas monoclonal antibody is hybridoma clones VB3 and CB.
E, WB3, ZB4, UB2, AX6, JAE and CH
The immunological assay method for soluble Fas antigen according to any one of claims 1 to 3, which is a monoclonal antibody produced from one hybridoma clone selected from the group consisting of 11. 5. The labeled second anti-Fas monoclonal antibody is a monoclonal antibody labeled with a labeling substance selected from the group consisting of peroxidase, β-D-galactosidase, microperoxidase, alkaline phosphatase and biotin. Item 5. A method for immunologically measuring the soluble Fas antigen according to any one of Items 1 to 4. 6. An immobilized anti-Fas monoclonal antibody prepared by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fas antigen to an insoluble support, and a soluble Fas antigen as a standard substance. A kit for immunological measurement of a characteristic soluble Fas antigen. 7. The kit for immunological measurement of soluble Fas antigen according to claim 6, which contains a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen. 8. A chimeric soluble F comprising, as a standard substance, an extracellular region of human Fas antigen or mouse Fas antigen and an extracellular region of mouse IL-3 receptor β subunit AIC2A or a constant region of immunoglobulin heavy chain.
The soluble Fas according to claim 6 or 7, which uses an as antigen.
A kit for immunological measurement of an antigen. 9. The solid-phased anti-Fas monoclonal antibody bound to the insoluble support is hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX.
The kit for immunological measurement of soluble Fas antigen according to any one of claims 6 to 8, which is a monoclonal antibody produced from one hybridoma clone selected from the group consisting of 6, JAE and CH11. 10. The labeled second anti-Fas monoclonal antibody is hybridoma clones VB3 and CB.
E, WB3, ZB4, UB2, AX6, JAE and CH
The immunological assay kit for soluble Fas antigen according to any one of claims 6 to 9, which is a monoclonal antibody produced from one hybridoma clone selected from the group consisting of 11. 11. The labeled second anti-Fas monoclonal antibody is a monoclonal antibody labeled with a labeling substance selected from the group consisting of peroxidase, β-D-galactosidase, microperoxidase, alkaline phosphatase and biotin. Item 6-10
9. A kit for immunological measurement of the soluble Fas antigen according to any one of 1.