JP2532651B2 - Manufacturing method of antinuclear antibody measuring instrument - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention provides a method for producing an instrument for measuring antinuclear antibody, and an instrument produced thereby. According to the measuring instrument of the present invention, antinuclear antibodies in blood or body fluid can be easily measured with high accuracy, and thus can be used for diagnosing autoimmune diseases, and in particular, can be widely used for primary screening. Further, since the present invention can provide a low-cost assay method, the anti-nuclear antibody, which has not been assayed unless it is a special case until now, is generally used in combination with the convenience of its operation. A wide range of medical examinations can be made, and it becomes possible to diagnose and treat autoimmune diseases at a relatively early stage.

[Prior Art] Systemic lupus erythematosus (SLE), systemic sclerosos (PSS), rheumatoid arthritis (RA), Sjoegren syndrome (systemic lupus erythematosus; SLE) known as autoimmune diseases SjS), dermatomyositis (DM), polymyositis (PM), mixed connective tissue disease (mixed)
In patients with connective tissue disease (MCTD), antinuclear antibodies are often detected in blood at high concentrations. Antinuclear antibodies include anti-ds- or ss-DNA antibodies and anti-histone antibodies,
Anti-non-histone nucleoprotein antibody, anti-centromere antibody, anti-U1
-Over 20 types of antibodies against various antigens existing in the cell nucleus such as RNP antibody and anti-Sm antibody are known. For example, as the presence of anti-ds- or ss-DNA antibodies in blood is used as the basis for diagnosing SLE, each antinuclear antibody is known to be associated with various autoimmune diseases, and The detection of these antibodies in the is useful for diagnosing autoimmune diseases.

To date, the most commonly used standard assay method for detecting antinuclear antibodies is the fluorescent antibody method (review;
"Clinical examination", vol.30, No.7, 1986, p684-728), etc.
Double immunodiffusion, RIA, ELISA, hemagglutination, etc. are available.
The fluorescent antibody method uses a tissue section or a cell line fixed on a glass slide as an antigen material, and the presence or absence of antinuclear antibody in a specimen such as blood and the fluorescent staining pattern of cells are observed under a fluorescent microscope. , Determine the type of antinuclear antibody. The relationship between the fluorescence staining pattern and the antinuclear antibody has been well investigated in this assay, and its effectiveness is widely recognized in the diagnosis of autoimmune diseases. However, in this fluorescence method, accurate determination is possible only with the naked eye of an expert,
It has drawbacks such that the fluorescent dye is relatively unstable and difficult to handle, and the sensitivity of the fluorescence microscope and the microscopic conditions influence the determination.

For this reason, ELISA (enzyme linked imm
The unosorbent assay) method has been developed. The ELISA method for antinuclear antibody is roughly divided into two types. One is an ELISA method in which purified antigens such as various DNAs, RNAs, histone proteins, and non-histone proteins separated and purified from cell nuclei are carried on a plastic support, and it is possible to identify antinuclear antibodies. Since it is cumbersome to perform an ELISA for each nuclear antigen, it is mainly used in a secondary screening together with a fluorescence method or separately.

On the other hand, extractable nuclear an
An ELISA method in which tigen; ENA) is supported on a plastic support has been developed mainly for the purpose of primary screening to rapidly detect the presence of many types of antinuclear antibodies (R. Warlow et al., “Diagnostic Immunology. , vol.2, p154−
160, 1984). This method is used to solubilize or acid extract the nuclear antigens of tissues such as liver and cell lines, but among nuclear antigens, antinuclear antibodies against insoluble antigens and antigens that cannot be extracted with acid are found. There was a defect that it was dropped, and it did not always match the determination by the standard fluorescent method.

Furthermore, in 1987, the patent application of "Method and apparatus for detecting antinuclear antibody" by VL Lip et al.
32363). In this measuring method, the nucleus is supported on the solid support by the dry method, and the insoluble nuclear antigen can also be detected.

However, this method does not involve the nucleus alone, but the nuclear antigen (NS) obtained by sonication after detergent treatment and ENA are also prepared, and the three are combined and supported on a solid support. This is a low-sensitivity method that requires manipulation and uses undiluted serum as a sample. Further, since the undiluted serum is directly used, a background reaction due to a large amount of nonspecific antibody contaminating the sample is easily detected. Furthermore, the above-mentioned patent application publication specification lacks an example in which a sample such as blood of an actual autoimmune disease patient is measured, and further improvement is considered necessary for its practical application.

[Problems to be solved by the invention]

An object of the present invention is to provide a method by which a device for ELISA for measuring antinuclear antibody can be manufactured by a simple operation. Furthermore, it is to provide a measuring instrument capable of accurately screening an antinuclear antibody.

[Structure of Invention]

According to the present invention, a suspension of cell nuclei is brought into direct contact with a support to adsorb the cell nuclei to the support, and after being separated from the suspension, the cell nuclei are supported by treating with a 50 to 90% acetone solution. The cell nuclei are supported on the support by being fixed to the body.

Here, the cell nucleus means a nucleus in a state in which a normal structure is retained in the cell (intact) or a fragment (tragment) due to mechanical destruction thereof.

In the present invention, cell nuclei are derived from cell lines of higher animals, especially humans. HEp is a particularly preferred cell line.
-2, Wil-2, A-549, PC-13, ZR-751 and the like can be mentioned. The suspension of cell nuclei can be prepared as follows.

The cells are cultured and collected by a conventional method, then washed with an isotonic buffer solution, and then suspended in a hypotonic buffer solution having a reduced salt concentration to swell the cells to an extent that they are not broken. The swollen cells are loosened by a simple homogenization procedure, so that the cell nuclei are obtained relatively intact.

Examples of the isotonic buffer used here include Tris buffer and phosphate buffer. The hypotonic buffer solution is preferably one having a salt concentration in the range of 1/2 to 1/15 of that of the isotonic solution. It is desirable to destroy cells by 90% or more by homogenization operation. In addition to the homogenization operation, it is also possible to destroy the cells by ultrasonic treatment.

A nuclear fraction is collected by centrifuging the cell lysate thus obtained. The collected nuclear fraction is resuspended in the above hypotonic solution to prepare a suspension of the nuclear fraction.
At this time, the concentration of the nuclear fraction is preferably 2 × 10 ⁵ / ml or more in terms of cell nuclei.

This nuclear fraction suspension is directly contacted with a support, preferably a plastic support to adsorb the nuclear fraction onto the support. Polystyrene as the plastic support,
Surface-treated polystyrene, polyvinyl or the like is used. A polystyrene ELISA plate having 96 wells is commercially available, and it is convenient to use this plate as it is. However, the shape thereof can be any shape such as an examiner's card or a plate. Contact the nuclear fraction suspension with the plastic support at room temperature for 30-
Allow it to stand for 120 minutes. Temperature and time can be varied within a suitable range. When using an ELISA plate with wells, the nuclear fraction suspension may be dispensed and allowed to stand as it is for the required time.

Then separate the plastic support and the nuclear fraction suspension,
The nuclear fraction is fixed on a plastic support by treatment with 50-90% acetone solution. When using an ELISA plate with wells, remove the nuclear fraction suspension by an appropriate means such as suction, and then dispense a 50-90% acetone solution equivalent to the nuclear fraction suspension. Usually, if the cells are left to stand at room temperature for 10 minutes or longer, the cell nuclei are fixed on the plastic carrier and cannot be peeled off even by washing.
After removing the nuclear fraction suspension from the wells, washing is performed to wash away the cell nuclei. However, washing can be performed to control the number of cell nuclei carried on the support. It is desirable to use an 85% aqueous solution as the acetone solution. It is not preferable to use an acetone solution having a concentration higher than 90% because the plastic support is attacked by the acetone. Acetone is preferably an aqueous solution, but a part of water may be replaced with a polar solvent such as ethyl alcohol. The treatment temperature and time can be changed within an appropriate range. In addition to the effect of fixing the cell nucleus to the support, the acetone treatment of the present invention dissolves the phospholipid membrane of the cell nucleus and makes it easier for the antinuclear antibody to approach the nuclear antigen inside the nucleus, which improves the measurement accuracy. Greatly contributes to

By these treatments, in the cell-nucleus-bearing plastic ELISA plate prepared in the present invention, antinuclear anti-nuclear that can be sufficiently measured with 100-fold diluted serum, has high sensitivity, and has low background due to nonspecific antibody coexisting in the sample. It becomes possible to measure the antibody. That is, when this plate is used, the nuclear antigen (N
Even if S) or ENA is not applied, patients with autoimmune diseases can be accurately screened for the presence of antinuclear antibodies. However, ENA or the like can be further loaded on this plate to further facilitate detection. For this, the plate obtained by this method is contacted with the ENA to adsorb the ENA to the plate.

The detection of the antinuclear antibody using the cell nucleus-supporting plastic support prepared in the present invention can be carried out in the same manner as the ELISA method in which the extracted nuclear antigen is supported on the plastic support.

That is, according to the present invention, a test serum is added to and reacted with a plastic well carrying cell nuclei, unreacted serum is removed and washed, and then anti-human immunoglobulin antibody such as rabbit or goat labeled with horseradish peroxidase (HRPO) or the like is used. Alternatively, protein G or the like is reacted and bound to the antinuclear antibody bound to the cell nucleus. After washing, develop HRPO with tetramethylbenzidine (TMB) coloring solution.

According to the present invention, a plastic plate for highly accurate detection of antinuclear antibody can be obtained by a simple operation. If the plastic plate thus obtained is stored at low temperature and low humidity, it can be stored for 1 year or more without lowering its efficacy.

The present invention will be specifically described below with reference to Examples and Test Examples.

Example (cell culture) As a material for preparing the nuclear fraction of eukaryotic cells, ATCC (American
HEp-2 cell line (ATCCCCL23) derived from human laryngeal epithelial cell carcinoma was purchased from Type Culture Collection). The purchased HEp-2 cells are immediately cultured in 10% FBS-RPMI1640 medium and cryopreserved. The actual preparation of cell nuclei is performed by thawing the cryopreserved cells and allowing the cells to grow for about 1 week.
Collect × 10 ⁸ cells. When detaching the cells from the culture flask, 0.02% EDTA-phosphate buffer solution (PBS) (Na ₂ HPO ₃ ·
12H ₂ O; 14.5 g, KH ₂ PO ₄ ; 1.0 g, NaCl; 40.0 g, KCl; 1.0 g was dissolved in distilled water to adjust the pH to 7.2, and the total volume was adjusted to 5 liters. Dissolve 2H ₂ O and sterilize by autoclaving. ) Is used.

(Extraction and fixation of cell nuclei with hypotonic buffer) 1 × 10 ⁸ HEp-2 cells were washed with Tris buffer (TBS) (0.01M Tris-Hcl, 0.15M NaCl, pH7.4) 5
The cells were suspended in 0 ml, and the cells were collected as a precipitate by centrifugation at 500 × g for 5 minutes. This washing operation was repeated once. 20 ml of the hypotonic buffer solution (0.01M Tris-HC
l, 0.01M NaCl, 1mM MgCl ₂ , pH7.4), and suspend on ice.
Let stand for 15 minutes. The cells were disrupted with a Dounce homogenizer, and after confirming that the cell membrane was disrupted by 90% or more under a microscope, the cells were again centrifuged at 2,000 xg for 10 minutes.
The nuclear fraction obtained as a residue was suspended in the above-mentioned hypotonic buffer solution so that the concentration when converted into cell nuclei would be 2 × 10 ⁵ / ml, and a plastic microplate (for example, commercially available from Nunc) 50 μl was added to each of the wells.
The plastic plate was left at room temperature for 30 minutes.
After standing, the supernatant of the added solution was sufficiently removed by suction using an automatic washing machine or the like (for example, Model BEP-II manufactured by Bering Co.). Next, 30% of 85% acetone aqueous solution was added to each well and left at room temperature for 10 minutes. The acetone solution was sucked off and the plastic plate was dried in a dryer. The plastic plate supporting the cell nuclei prepared in this manner is wrapped in Saran wrap etc.
It was possible to store it for more than half a year.

(Extraction and Fixation of Cell Nucleus by Sonication) HEp-2 cells cultured by the method described above were 1 × 10 ⁸
Individually suspended in TBS. The cells were washed 3 times with 50 ml TBS,
The cells were collected as a pellet by centrifugation at 500 xg for 5 minutes. After suspending the cells in 10 ml of TBS, the cells were disrupted by sonication. The ultrasonic treatment was performed by using a commercially available ultrasonic oscillator (Nippon Seiki Seisakusho Co., Ltd.) with an output of 2.0 and a phase of 1.2 for 40 seconds. After ultrasonic treatment, at 2,000 × g
After centrifugation for 10 minutes, a precipitate containing cell nuclei was obtained. This precipitate was resuspended in 80 ml of TBS (2.5 × 10 ⁵ cells / ml), and 50 μm was added to each well of the plastic plate. This was fixed as in the previous example. That is, this plastic plate was left for 30 minutes, and the supernatant of the additive solution was removed by suction. Then, 50% of 85% acetone aqueous solution was added to each well, left standing for 10 minutes at room temperature, the acetone aqueous solution was suctioned off, and then the plastic plate was dried.

Test example 100 human sera obtained from normal humans (37 cases) or autoimmune disease patients (71 cases) were placed on a plastic plate carrying cell nuclei extracted with hypotonic buffer, respectively.
A 40-fold diluted sample was added to each well. At this time, dilute human serum with casein 0.01 M Tri
s-HCl / 0.15M NaCl buffer (pH 7.4, preservative 0.1% NaN ₃
The diluted solution was prepared by dissolving it until it became saturated. Approximately 1 plate at room temperature after adding diluted serum to the sample
Left for hours. After standing, the unreacted supernatant was removed by suction from each well, and the washing solution (1.3 mg / ml Na ₂ HPO ₄ , 3.3 mg / ml KH ₂ PO ₄ ,
Wash the wells three times with a concentrated washing solution containing 100 mg / ml NaCl and 20 mg / ml polyoxyethylene sorbitan monolaurate diluted 20 times before use. Then in each well HR
A PO-labeled protein G (protein G is a protein that reacts specifically with immunoglobulins and is commercially available from Cosmo Bio Co., Ltd.) is a casein solution (same as the diluent,
However, add 40 µl of the labeling solution prepared by dissolving 0.2% phenol (preservative) at a concentration of 5 mg / ml, and leave at room temperature for about 1 hour. After re-standing, the unreacted labeling solution is removed by suction from each well, and each well is washed again with a washing solution. Next, add 50μ of tetramethylbenzidine (TM) to each well.
B) Color solution (5mg / ml tetramethylbenzidine, 0.2mg / ml phenoxymethylpenicillin potassium, 3.2μ / ml chromogen solution containing hydrochloric acid and 0.27mg / ml urea / hydrogen peroxide, 1
Substrate solution containing 3.1mg / ml sodium hydroxide and 1.64μ / ml acetic acid was mixed in 1:10 for work) and left at room temperature for 30 minutes, then 50μ 0.5N sulfuric acid was added to each well for reaction. Was stopped and the absorbance was measured at 450 nm.

The results are shown in the figure. In the figure, one ○ or ● mark is 1.
Represents one specimen. The ∘ mark indicates the absorbance of each sample in the case of the conventional ELISA method using the solubilized antigen (ENA) performed as a control, and the ● mark indicates the absorbance of each sample when the measuring instrument of the present invention is used. In addition, I is the standard method at present, when the serum of a positive autoimmune patient is measured by the fluorescent antibody method using a glass slide on which HEp-2 cells are immobilized, II is when the serum of a normal person is measured, and III is measured.
Shows the case where the serum of a negative autoimmune patient was measured by the fluorescent antibody method. The circle marked with low absorbance at I indicates that the sensitivity of the conventional measuring instrument is poor.

ELIS with plates loaded with nuclear fractions according to the invention
A correlation rate of 85-95% was found between the positive / negative determination by the A method and the determination by the fluorescent antibody method, whereas the conventional ELISA method using the conventional ENA and the fluorescent antibody method were used. Only a 60-70% correlation was found between the decisions. Also, a high correlation was observed between the measured value by ELISA of the present invention and the fluorescent staining intensity of the fluorescent antibody method.

[Brief description of drawings]

The figure shows the results of measuring the antinuclear antibody in the serum of a sample using the present invention and the conventional measuring instrument.

Claims (3)

(57) [Claims] 1. A suspension of cell nuclei is contacted with a plastic support to adsorb the cell nuclei onto the support, and the plastic support is separated from the suspension, and then the surface thereof is 50 to 90%.
A method for producing an instrument for measuring antinuclear antibodies, which comprises treating cell nuclei on a support by treating with an acetone solution. 2. A suspension of cell nuclei is added to a well of a plastic support and allowed to stand at room temperature for 30 to 120 minutes to adsorb the cell nuclei on the plastic support, and after removing unadsorbed supernatant from the wells. A 50 to 90% acetone solution is added to the wells and allowed to stand at room temperature for 10 minutes or longer to immobilize the cell nuclei on the plastic support. 3. An antinuclear antibody measuring instrument manufactured by the method according to claim 1.