JPH09166593A - Immunological measuring method and measuring kit for fas anitigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply measure a large amount of specimens without any influence of emzymically cut Fas anitigen and with good sensitivity. SOLUTION: As a standard substance, Fas antigen originating in WR19L-12a cell is used, an anti-Fas antibody specifically coupled to a cell inner area of Fas anitigen is coupled to a microplate to be taken as a solid phase antiboty, and an anti-Fas antiboty specifically coupled to a cell outer area of Fas anitigen is subjected to labeling to be taken as a labeling antibody. A specimen containing Fas anitigen and a solid phase antiboty are made react with each other to form a solid phase antibody-Fas anitigen complex. After that, the complex is processed by a labeling antibody to label the complex trapped to the microplate with POD. The color forming amount of coloring substrate by POD is quantitatively determined to quantatively determine Fas anitigen.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art In multicellular organisms, many cells are eliminated by cell death in the process of 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. Wyll
ie) observed the morphology of dying cells with an electron microscope and classified the cell death into two types morphologically (ie, necrosis and apoptosis) (Wyllie AH 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 after forming a bubble-like structure on the cell surface, it may be divided 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.

[0005] A protein existing in the cell surface layer of the living body, which is thought to be deeply involved in the control of cell death called apoptosis or programmed cell death, is called Fas antigen and is a hydrophobic protein that penetrates the lipid bilayer of the cell membrane once. It is a transmembrane protein that has a peptide sequence and is composed of three main regions, an extracellular region, a transmembrane region, and an intracellular region as a whole. The amino acid sequences of mouse and human Fas antigens, and the cDNA sequences encoding the amino acid sequences have already been clarified (Cell, 66, 233-243, 19).
1991, Ito Naoto et al., J. Immunol., Vol. 148, Vol.
Pp. 1274-1279, 1992, Watanabe (Fukunaga) Rie et al., J. Biol. Chem., Vol. 267, No. 15, 10709-107.
Page 15, 1992, Alexander Oehm et al., J. Exp.
Med., 169, pp. 1747-1756, 1989, Yoneharasin et al., Proc. Natl. Acad. Sci. USA, 87, 9620-9624.
Page, 1990, Kobayashi Nobuyuki et al., Science, Volume 245,
301-305, 1989, Traus (Bernhard C. Trauth)
Et al., J. Immunol., Vol. 149, No. 10, pp. 3166-3173, 1.
992, Jeas Dein et al., The Lancet. No. 335
Volume, 497-500, 1990, Devatin (Klaus-Michael
Debatin) et al., J. Immunol., Vol. 149, No. 11, 3753-3.
758, 1992, Miyawaki Toshio et al., Genomics, 14th.
Volume, Pages 179-180, 1992, Peter Lichter
Et al.).

Yonehara et al. Prepared a monoclonal antibody against the 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 (J. Exp. Med., Volume 169, 1747-17
56, 1989). Moreover, since the cell death showed the characteristic of apoptosis by observation with an electron microscope, it was concluded that Fas antigen mediates cell death by apoptosis. Apart from this, Trauth et al.
We obtained APO-1 antibody, a monoclonal antibody that has properties similar to those of antibodies and exhibits cell-killing activity (SCIENCE, No. 2).
45, pp. 301-305, 1989). The two antibodies are not only common in that they induce cell death, but also very similar in terms of antigen distribution and molecular weight, and it has been clarified 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 very similar to that of the TNF / NGF receptor family and that it is a new type of receptor that causes cell death by receiving an external signal, and is considered to be one of the receptors that form the TNF / NGF receptor family. There is.

Recently, an abnormality in the Fas gene was observed in mice exhibiting autoimmune symptoms (MRL / lpr mice), and a high level of apoptosis was occurring in lymphocytes in HIV-infected individuals.
It has been suggested that the Fas antigen is related, and the relationship between the expression of the Fas antigen and a disease such as an autoimmune disease has been attracting attention.

Chen, J. et al., Science,
263, pp. 1759-1762, 1994), in order to investigate the relationship between abnormal transcription of these Fas antigens and autoimmune diseases,
Autoimmune diseases (Rheumatoid Arthritis; RA),
Intracellular cDNA was extracted from SLE (Systemic Lupus erythematosus)) patients and healthy individuals, and examined. As a result, normal 1167 encoding the full-length Fas antigen was observed in both cases.
1104 bp small cDNA in addition to bp cDNA
I have found that. Furthermore, when this small cDNA of 1104 bp was compared with the cDNA of 1167 bp, the hydrophobic transmembrane site (TM, transmembran
e small domain, so this small cDNA
(Called FasΔTM) is blood, serum, plasma,
It has been clarified that it encodes a Fas antigen soluble in body fluids such as urine, saliva and bone marrow (hereinafter referred to as "soluble Fas antigen").

[0010] Chent 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.

Therefore, measuring the amount of soluble Fas antigen in body fluids such as serum predicts, diagnoses and treats various diseases that are considered to be related to the dynamics of Fas antigen such as autoimmune diseases. It is useful for the establishment of the above-mentioned method and the determination of the therapeutic effect, and the establishment of the assay method and the assay reagent (kit) is eagerly awaited.

As a method for detecting and measuring soluble Fas antigen in body fluids such as serum, Western blot method (Western Blot technique, WB method), radioimmunoassay (Radioimmunoassay, RIA method), enzyme immunoassay. Method (Enzyme Immunoassay, EIA method or Enzyme-link
ed Immunosorbent Assay, ELISA method) and the like can be considered. However, W. B. Although the method has high sensitivity and high specificity, it lacks quantitativeness and is unsuitable for processing a large amount of samples at one time. Moreover, since the RIA method uses a radioactive substance as a labeling substance, it has a drawback that it can be handled only by qualified persons in a controlled facility. Generally, the EIA method or the ELISA method does not have the above-mentioned problems and can be easily measured with high sensitivity, but up to now, high accuracy of soluble Fas antigen in a body fluid such as serum using such a method has been obtained. Moreover, a simple measuring method having sufficient sensitivity is not known.

Further, among Fas antigens, complete Fas antigen has a hydrophilic intracellular region and an extracellular region across a hydrophobic transmembrane region, and soluble Fas antigen lacks the transmembrane region. However, as Fas antigen secreted extracellularly, there may be a molecule that is enzymatically cleaved and has only an extracellular region. It is desirable that this enzymatically cleaved molecule does not affect the quantification of Fas antigen.

Further, in order to measure (quantify) soluble Fas antigen contained in body fluid such as serum, the sensitivity or accuracy of the assay method is confirmed, and the soluble Fas antigen is indirectly determined from the measured value.
In order to prepare a calibration curve for determining the content of as antigen, it is necessary to obtain purified soluble Fas antigen as a standard substance. However, a technique for mass-producing purified soluble Fas antigen has not been established so far.

The present invention has been made in view of the above problems, and provides an immunoassay method for Fas antigen, which is highly sensitive and can easily measure a large amount of sample without being affected by enzymatically cleaved Fas antigen, and The purpose is to provide a measurement kit.

[0016]

Means for Solving the Problems, Embodiments of the Invention, and Effects of the Invention The first aspect of the present invention is a method for measuring Fas antigen, which is specific to the intracellular region (or extracellular region) of Fas antigen. -Extracted Fa as a standard substance on a solid-phased antibody obtained by binding an anti-Fas antibody capable of chemically binding to an insoluble support
After reacting the s antigen, a labeled antibody labeled with an anti-Fas antibody capable of specifically binding to the extracellular region (or intracellular region) of Fas antigen is reacted to measure the labeled amount of the reaction product. Step of producing a calibration curve by, and after reacting the sample with the immobilized antibody, the labeled antibody is reacted, the labeled amount of the reaction product is measured, contained in the sample from the calibration curve And a step of quantifying the Fas antigen.

A second aspect of the present invention is an immunoassay kit for Fas antigen, which comprises an insoluble support containing an anti-Fas antibody capable of specifically binding to the intracellular region (or extracellular region) of Fas antigen. A solid-phased antibody to be bound, a labeled antibody labeled with an anti-Fas antibody capable of specifically binding to the extracellular region (or intracellular region) of Fas antigen, and cell-extracted Fas antigen as a standard substance It is characterized by including.

When an anti-Fas antibody capable of specifically binding to the intracellular region of Fas antigen is used as the immobilized antibody, it can specifically bind to the extracellular region of Fas antigen as the labeled antibody. An anti-Fas antibody is used. On the contrary, when an anti-Fas antibody that can specifically bind to the extracellular region of Fas antigen is used as the immobilized antibody, F is used as the labeled antibody.
An anti-Fas antibody that can specifically bind to the intracellular region of the as antigen is used.

Examples of the insoluble support used for the immobilized antibody include plastics such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, and the like.
There is no particular limitation as long as it is a water-insoluble substance such as glass. The loading on the insoluble support may be mere physical adsorption or chemical bonding.

In the present invention, the anti-Fas antibody capable of specifically binding to the intracellular region of Fas antigen is an anti-Fa antibody which specifically reacts with the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
s antibody is preferred. Further, the anti-Fas antibody capable of specifically binding to the extracellular region of Fas antigen is preferably an anti-Fas antibody that specifically reacts with the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. The cell-extracted Fas antigen as the standard substance is preferably a Fas antigen derived from WR19L-12a cells in mass production.
The method for producing WR19L-12a cells has been reported by, for example, Ito et al. (Cell, 66, 233-243, 1991). As the labeled antibody, an anti-Fas antibody labeled with a radioactive element as a labeling substance may be used, but in this case, it is necessary to handle it in a facility where qualified personnel are controlled, so consideration of ease of handling is required. Then, it is preferable that the anti-Fas antibody is labeled with a labeling substance selected from the group consisting of peroxidase, β-D-galactosidase, microperoxidase, alkaline phosphatase and biotin.

In the immunological assay method and assay kit of the present invention, one of the immobilized antibody and the labeled antibody is prepared by using an anti-Fas antibody that specifically binds to the intracellular region. The other is prepared using an anti-Fas antibody that specifically binds to the extracellular region. Therefore, according to the present invention, a complete Fas antigen (ie, a Fas antigen having a hydrophilic intracellular region and an extracellular region with a hydrophobic transmembrane site) and a soluble Fas antigen (ie, complete Fas antigen). Fas antigen lacking the transmembrane region) can be measured. It should be noted that since the Fas antigen enzymatically cleaved into the extracellular region alone is not detected by the assay system of the present invention, the quantitative value is not affected by the extracellular region alone. .

According to the immunological assay method and assay kit for Fas antigen of the present invention described above, enzymatically cleaved Fa is used.
Since the standard substance can be mass-produced with high sensitivity without being affected by the s-antigen, it is possible to easily measure a large amount of sample. In addition, by quantifying Fas antigen in a sample, prediction of autoimmune diseases, establishment of diagnosis and treatment policy,
This is useful because the effect of treatment can be determined.

[0023]

EXAMPLE The structure of a complete Fas antigen is schematically shown in FIG. Here, the peptide numbers 17 to 33, 54 to 72, and 321 to 33 counted from the N-terminal of the amino acid
5 is a hydrophilic region from the result of amino acid analysis, in which an antigenic determinant may exist.

The inventors have found that complete Fas antigen (including extracellular domain, transmembrane domain, intracellular domain) and soluble Fas
In order to specifically measure s antigen (complete Fas antigen lacking the transmembrane region), antibodies against the intracellular region and extracellular region of Fas antigen are separately prepared, and these antibodies are used to generate so-called It was thought that it should be measured by the sandwich ELISA method. [Example 1] Preparation of antibody against intracellular region of Fas antigen [1-1] Preparation of synthetic peptide Antibodies against intracellular region of Fas antigen include anti-Fa
It was decided to use s polyclonal antibody. That is, the inventors of the present invention (Cell, Volume 66, 233-243) by Ito, Yonehara et al.
Page, 1991), the Fas antigen was subjected to hydrophobic mapping based on the amino acid sequence of human Fas antigen, and the hydrophilic region of amino acids 321 to 335 at the C-terminal in the intracellular region (SEQ ID NO: in the sequence listing). 1) was selected to prepare a synthetic peptide. The synthetic peptide was prepared by a solid phase method using the Fmoc method using a fully automatic synthesizer (for details of the synthetic method, see Shujunsha, Cell Engineering, separate volume, "Anti-Peptide Antibody Experimental Protocol"). To the actual synthetic peptide, one cysteine residue unrelated to human Fas was added to the N-terminal side, which is advantageous when the synthetic peptide was bound to a polymer carrier (see the following formula).

[0025]

Embedded image

[1-2] Bonding of Synthetic Peptide to Polymer Carrier The synthetic peptide prepared in Example 1-1 (see the above formula) is relatively small with 16 amino acids, and such a low molecular compound (hapten and In the case of (i), since the antigenicity is weak and antibodies cannot be easily formed as it is, it is necessary to bind to a polymer carrier in order to enhance the antigenicity during immunization. As such a polymer carrier, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) and the like are used, but KLH was used here.

As the binding method with KLH, generally, the glutaraldehyde method, the carbodiimide method, the diazo condensation method, the maleimidobenzoyloxysuccinimide (MB
S) method and the like, but synthetic peptide may be chemically modified because of non-specific binding except MBS method. The MBS method is a method in which KLH and a cysteine residue of a synthetic peptide are cross-linked by MBS and bonded, and this method avoids chemical modification of the synthetic peptide and produces a high-quality peptide antibody. Is possible. Therefore, the MBS method is used here for the first embodiment.
The synthetic peptide prepared in -1 was bound to a polymer carrier (for the specific method of binding, see the above-mentioned "anti-peptide antibody experiment protocol", pages 48 to 52). [1-3] Immunization Using the conjugate (conjugate of synthetic peptide and KLH) obtained in Example 1-2, rabbit (Japanese white female 3.5k).
g), subcutaneously immunized about 10 times once a week,
After immunization 5 times, a small amount of blood was collected from the parotid vein, the serum was separated, and the antibody titer was checked by the ELISA method. That is,
K in 1 / 100M physiological phosphorylation buffered saline (PBS)
0.1 mg / by dissolving synthetic peptide that does not bind to LH
A 100 ml solution was prepared, 100 μl of this solution was added to a Nunc 96-well microplate “Maxisorp”, and the solution was left standing at room temperature (20 to 25 ° C.) for 3 hours. 30 μl of PBS containing serum albumin was added and allowed to stand at 4 ° C. for about 18 hours to block the unreacted part of the cup. Excluding the blocking solution, PBS 300μl
After washing 3 times with PBS, a series of antisera diluted with PBS was prepared, and 100 μl of this solution was added to each cup at room temperature (2
After standing at 0-25 ℃ for 1 hour, the reaction solution is removed and PBS3
Wash 4 times with 0 μl. Next, diluted peroxidase-labeled anti-rabbit IgG (Medical and Biological Research Institute, Inc.)
After 100 μl was added, the mixture was allowed to react at room temperature (20 to 25 ° C.) for 1 hour, then washed again in the same manner, and 3,3 ′, 5,5′-
100 μl of tetramethylbenzidine and hydrogen peroxide solution
Was added as a chromogenic substrate and reacted for a certain time, then 1.5M
The reaction was stopped by adding phosphoric acid, and the absorbance at a wavelength of 450 nm was measured. The measurement results are shown in the graph of FIG.
As is clear from this FIG. 2, the obtained antiserum shows a sufficient antibody titer. With respect to the rabbit showing a sufficient antibody titer, 70 ml of blood was collected from the parotid vein to obtain about 30 ml of antiserum. [1-4] Preparation of Specific Antibody Against Synthetic Peptide The antiserum obtained in Example 1-3 contains serum components other than the desired antibody and other antibodies. In order to increase the sensitivity of the assay system, it is preferable to bind only the antibody that reacts with the synthetic peptide of Example 1-1 to the insoluble support, and thus the specific antibody was collected by affinity chromatography. That is, using a magnetic stirrer, ammonium sulfate powder was gradually added to rabbit serum to 50% saturation, and the precipitated fraction was separated from the supernatant by centrifugation and dissolved in the minimum amount of PBS. It dialyzed using PBS of and the ammonium sulfate was removed.
This was applied to Sepharose 4B gel to which a synthetic peptide was bound, washed with a sufficient amount of PBS, and then the immunoglobulin bound to the gel was eluted with 0.17 M glycine-HCl buffer pH 2.3. Elute the synthetic peptide-specific antibody promptly to avoid a decrease in antibody activity.
/ 10 volume of 1 M Tris-HCl buffer pH 9.0 was added for neutralization. By the above method, 0.5 mg of synthetic peptide-specific antibody (ie, Fas
Antibodies against the intracellular region of the antigen) were obtained.

The synthetic peptide-bonded Sepharose 4B was prepared in accordance with the operating instructions of Pharmacia. That is, 6 g of CNBr-activated Sepharose 4B purchased from Pharmacia was added to 600 ml of 1 mM HCl, and allowed to stand for 15 minutes to swell the gel. Then, 1 mM HCl was removed using a glass filter, and 1 mM HCl600 was further added.
The gel was washed similarly with ml. 3 gels washed
0.1M NaHCO 3 containing 0 ml 0.5M NaCl
₃ and suspended in 30 ml of pH 8.3 (coupling buffer). On the other hand, 20 μmol of the synthetic peptide was dissolved in 20 ml of coupling buffer, mixed with the above gel suspension, gently stirred at room temperature for 1 hour, and then washed with 200 ml of coupling buffer using a glass filter to remove excess. The peptide was removed. 0.1M with additional 0.5M NaCl to block excess active groups
Acetate buffer pH 4.0 and 0.1 M Tris-HCl buffer pH 8.0 containing 0.5 M NaCl 10 each
Alternately wash 3 times with 0 ml. [Example 2] Preparation of antibody against extracellular region of Fas antigen As an antibody against extracellular region of Fas antigen, clone VB3 (manufactured by Medical Biology Research Institute, Inc.) was used and anti-F was used.
It was decided to prepare an as monoclonal antibody. This clone VB3 is located at the 126th to 135th positions (extracellular region) from the N-terminal of the amino acid on the human Fas molecule, and its amino acid sequence is as shown in SEQ ID NO: 2 in the sequence listing.

The anti-Fas monoclonal antibody was obtained as follows. That is, clone VB3 (2 × 10 ⁸ ) was cultured in serum-free ASF104 medium (Ajinomoto Co., Inc.) for 5 days at 37 ° C., and the obtained culture supernatant was used in Omega cell (Filtron Co., Ltd.). Ultracentrifuged. 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 fraction was collected and subjected to SDS-PAGE to obtain a purified anti-Fas monoclonal antibody with a purity of 95% or higher (Journal of Experimental Medicine (J. Exp. Me.
d.) Volume 169, pp. 1747-1756 (1989)
Year))). [Example 3] Preparation of immobilized antibody The synthetic peptide-specific antibody obtained in Example 1-4 was prepared in 0.1 M carbonate buffer pH 9.0 to a concentration of 5 µl / ml, and a Nunc 96-well microplate was prepared. 10 μl was added to each well of “Maxisorp”, and the reaction was allowed to stand at 4 ° C. for 20 hours. After discarding the antibody solution, 200 μl of PBS containing 1% BSA and 5% sucrose was added to each well, and the mixture was allowed to stand at room temperature (20-2
Blocking was carried out by allowing to stand at 5 ° C. for 2 hours. The blocking solution was discarded and the plate was air-dried to obtain the immobilized antibody. This immobilized antibody was sealed and stored with a desiccant. [Example 4] Preparation of labeled antibody Purified IgG of anti-human Fas monoclonal antibody (clone VB3) obtained in Example 2 0.056 U per 1 mg of IgG
Ficin was added and reacted at 37 ° C for 8 hours, then U
gel filtered using ltrogel ACA44, F
(Ab) ' ₂ fractions were obtained. This F (ab) ' ₂ fraction was labeled with peroxidase by the maleimide method to give a peroxidase-labeled antibody. The labeling was carried out in accordance with "Enzyme Immunoassay 3rd Edition" written by Eiji Ishikawa, published by Ikusho Shoin. [Example 5] Preparation of cell-extracted Fas antigen (standard) As the Fas standard, a cell WR19L-12a (Yonehara et al., Cell , First
66, pp. 233-243, 1991).

That is, cDNA encoding human Fas antigen
The fragment containing human Fas antigen cDNA obtained by digesting the plasmid pF58 containing A with XhoI was used as an expression vector pEF-BOS (Nucleic acid resea).
rch, Vol. 18, p. 5322, 1990) using the BstXI adapter to obtain the expression plasmid pEFF58. 25 μg / ml pEFF58 digested with VspI
2.5 μg / ml digested with fragment and EcoRI
1 × 10 ⁷ (0.8 ml) of mouse T lymphoma WR19L cells (ATCC TIB52) were cotransformed with the pMAneo fragment (manufactured by Clontech) of. The cells are treated with antibiotic G418
G418-resistant clones were selected by culturing in a medium containing. The G418-resistant clones thus obtained were analyzed by a flow cytometer and a limiting dilution method to clone human Fas antigen-overexpressing transformant WR19L-12a.

WR19L-12a cells contained 10% FC
S, cultured in DMEM medium containing 400 μg / ml of G418, and 1 ml of R was added to 5 × 10 ⁷ cells collected.
IPA buffer (150 mM NaCl, 1% nonidet P-40, 0.5% deoxycholic acid, 50 mM
Tris-HCl buffer (pH 8.0) was added and the mixture was crushed by an ultrasonic crusher. Ultracentrifugation (4 ℃, × 5
0000 g, 30 minutes) and solubilized F in the supernatant
As was used as a standard. [Example 6] Measurement of Fas antigen in serum The test serum was diluted 5-fold with PBS containing 10% normal rabbit serum, and the diluted 1 was added to a microplate (immobilized antibody of Example 3).
00 μl was added to each. The standard was prepared by diluting the same buffer at a concentration of 2 ng / ml in multiples, and 100 μl was added in the same manner.

After reacting at room temperature (20 to 25 ° C.) for 1 hour with gentle shaking, 0.1% tween was added.
30 μl of PBS containing 20 was added to each well for washing.
Washing was performed 5 times. After removing the washing solution, the peroxidase-labeled antibody obtained in Example 4 was treated with 1% BSA and 0.79%.
NaCl, 0.1% para-hydroxyphenylacetic acid (p-Hy
droxyphenyl acetic acid), diluted to 1 μg / ml with 20 mM HEPES buffer pH 7.2, added 100 μl to each well, reacted at room temperature (20-25 ° C) for 1 hour with gentle shaking, and washed again in the same manner. did. After removing the washing solution, 1.6 mM of 3,3 ', 5,5'-
Tetramethylbenzidine dihydrochloride, 20% N, N-dimethylformamide, 1.25% polyethylene glycol 4000 in 10 mM sodium citrate solution, 1
An equal volume of 10 mM citric acid solution containing 0 mM hydrogen peroxide was mixed, and 100 μl of this solution was added at room temperature (20 to 2
(5 ° C) for 30 minutes with gentle shaking to react,
The reaction was stopped by adding 100 μl of 1.5N phosphoric acid, and the absorbance at a wavelength of 450 nm was measured. Fas in the sample
The concentration was read using a standard curve (see the graph in FIG. 3) prepared from the concentration of standard and the absorbance.

Table 1 shows the results of measurement of samples using this system.
Shown in

[0034]

[Table 1]

As is clear from Table 1 above, the measured values of Fas antigen in RA patient sera, RA patient ascites, and SLE patient sera are significantly higher than those in healthy subjects. 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.

[0036]

[Sequence list]

SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 1 5 10 15

SEQ ID NO: 2 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Protein

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the structure of Fas antigen.

FIG. 2 is a graph showing the antibody titer of antiserum in [1-3] of Example 1.

FIG. 3 is a graph of a standard for measuring Fas antigen in serum.

Claims (10)

[Claims] 1. An immunological assay method for Fas antigen, which comprises the following steps a) to c) and d) to f). a) Cell-extracted Fas as a standard substance on a solid-phased antibody obtained by binding an anti-Fas antibody capable of specifically binding to the intracellular region (or extracellular region) of Fas antigen to an insoluble support
After reacting the antigen, b) a labeled antibody labeled with an anti-Fas antibody capable of specifically binding to the extracellular region (or intracellular region) of Fas antigen is reacted, and c) the labeled amount of the reaction product And a step of producing a calibration curve by measuring d), after reacting the sample with the immobilized antibody, e) reacting with the labeled antibody, and f) measuring the labeled amount of the reaction product, A step of quantifying the Fas antigen contained in the sample from the calibration curve. 2. The anti-Fas antibody capable of specifically binding to the intracellular region of Fas antigen is an anti-Fas antibody which specifically reacts with the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Immunoassay for Fas Antigens. 3. The anti-Fas antibody capable of specifically binding to the extracellular region of Fas antigen is an anti-Fas antibody that specifically reacts with the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. 2. The immunological assay method for Fas antigen according to 2. 4. The immunoassay method for Fas antigen according to claim 1, wherein the cell-extracted Fas antigen as the standard substance is a Fas antigen derived from WR19L-12a cells. 5. The labeled antibody is peroxidase,
The immunoassay method for Fas antigen according to any one of claims 1 to 4, which is an anti-Fas antibody labeled with a labeling substance selected from the group consisting of β-D-galactosidase, microperoxidase, alkaline phosphatase and biotin. 6. A solid-phased antibody obtained by binding an anti-Fas antibody capable of specifically binding to the intracellular region (or extracellular region) of Fas antigen to an insoluble support, and the extracellular region of Fas antigen (or A kit for immunological measurement of Fas antigen, comprising a labeled antibody obtained by labeling an anti-Fas antibody capable of specifically binding to an intracellular region) and a cell-extracted Fas antigen as a standard substance. 7. The anti-Fas antibody capable of specifically binding to the intracellular region of Fas antigen is an anti-Fas antibody which specifically reacts with the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. A kit for immunological measurement of Fas antigen. 8. The anti-Fas antibody capable of specifically binding to the extracellular region of Fas antigen is an anti-Fas antibody that specifically reacts with the amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing. 7. A kit for immunological measurement of Fas antigen according to 7. 9. The kit for immunological measurement of Fas antigen according to claim 6, wherein the cell-extracted Fas antigen as the standard substance is a Fas antigen derived from WR19L-12a cells. 10. The anti-Fas antibody labeled with a labeling substance selected from the group consisting of peroxidase, β-D-galactosidase, microperoxidase, alkaline phosphatase and biotin, according to any one of claims 6 to 9. A kit for immunological measurement of Fas antigen according to Crab.