KR960005733B1 - Monocyte/macrophage for detecting anti-nuclear antibody - Google Patents

Abstract

This invention is a detection method of ANA using monocyte/macrophage. The cell body used in this method is IT-1(KFCC-10772), monocyte/macrophage cell body derived from marrow. Because the cell body has better adherence to slide than established cell bodies, slides can be prepared even to low cell concentration. And the cell body grows well under various culturing conditions. The detecting steps of anti-nuclear antiboby are (a) to add the cell body to a slide; (b) to react with several drops of body fluids.

Description

[Name of invention]

Cell line for antinuclear antibody detection and antinuclear antibody detection method using the same

Detailed description of the invention

The present invention relates to a mononuclear cell / macrophage cell line for detecting an anti-nuclear antibody (hereinafter abbreviated as "ANA") and a method for detecting ANA using the same.

ANA detection to test for the presence of autoantibodies in the serum includes systemic lupus erythematosus (weak SLE), autoimmune diseases, Sjoegren's syndrome, scleroderma, mixed collagen rheumatoid arthritis, childhood chronic multiple It is applied to systemic rheumatic diseases such as juvenile chronic polyarthritis and other autoimmune diseases. After Hargraves discovered LE cells (or LE factors) used to diagnose systemic lupus erythematosus in 1948, in the 1980s, techniques such as fluorescent antibody staining patterns and double immuno-diffusion methods were used. As nuclear antigens against ANA, DNA, histones, extractive nuclear antigens (ENA), nonhistones such as Sm (Smith), nuclear ribonucleoprotein (nRNP), Ss-A (Sjoegren associated antigen) ), ss-B, scl-70 (scleroderma) and the like have been found. Patients who are primarily diagnosed with autoimmune diseases by detection of ANA should undertake specific antibody assays for each antigen to determine disease name.

In the ANA detection method, an antibody present in the blood of a patient is bound to an antigen present in a fixed substrate (normal mammalian tissue or cell), and then the bound antibody is observed using fluorescence. Therefore, its detection goes through the following steps.

1) Attachment of Substrate: Attaching the substrate to the slide for microscopic observation. After freezing the rat's liver tissue, the frozen tissue section is transferred directly to the slide, or HEp-2 (human epithelial cell), BHK (young hamster kidney) When cell lines such as cells) and KB (human KB cells) are used, slides cultured aseptically on the slides are used. In the latter case, you can use commercialized slides.

2) fixation of the attached substrate; In order to prevent the substrate from falling off during the reaction after the reaction with blood, it is treated to exhibit the effect of attaching to the slide and making the lipid membrane of the stromal cells so as to react well.

3) Reaction with Patient Serum: The serum of patients diluted 2-fold from 1:20 to 1: 1280 is reacted with immobilized substrate and a control experiment is performed on ANA standard serum.

4) After reacting at room temperature for 30 minutes at high humidity, it is washed with phosphate buffer and dried.

5) Administration of anti-immunoglobulin to the primary antibody: In the ANA assay, immunofluorescent method is mainly used, and in the present invention, Dako Co., Ltd., which is a rabbit antibody against human gamma globulin, is labeled with FITC. It was.

6) Wash with phosphate buffer for 10 minutes, stain with Evans blue, and wash again with phosphate buffer.

7) Observed by fluorescence microscope to read. In the general test method, immunofluorescence method using fluorescent material and immunoenzyme method using enzyme reaction are applied, but the former method is applied to ANA test.

In ANA testing, to improve accuracy, consideration should be given to 1) test methods and quantification, 2) selection of substrates and fixatives, 3) establishment of normal ranges and ability to read test results. The development of standard serum in AF-CDC (Sera Standardization Meeting for Antinuclear Antibodies; Arthritis Rheum. 25, 1003, 1982) in 1982 standardized the specificity and titer of the antibody, which is a problem in paragraph 1), and at the same time grade of fluorescence By using the determined slides, inconsistencies and qualitative differences between fluorescence microscopes, the concentration of antiserum bound with FITC, and the mixing ratio of fluorescent substances and proteins were improved.

Research into substrates using cryostat tissue sections or cultured cells is still in development. Cultured mammalian cells such as histoblasts of MF (white tissue tissue), VMK (monkey monkey kidney cells), human cells HA, KB, HEp-2 (deposited as ATCC CCL 23), etc. Compared to frozen tissue sections using the organs such as and kidneys has the following advantages. Cultured cells are generally larger in size than frozen tissue sections, have a high content and variety of specific nuclear antigens, and are easy to read in fluorescence and have a much lower chance of false negatives. Another advantage is that if cell lines are cultured on slides, dividing cells can be observed, and fluorescence patterns that are difficult to distinguish from diagnosis such as anti-centromere antibodies and proliferating cell nuclear antgen (PCNA) only observed in proliferating cells can be observed. Can be confirmed more objectively. Frozen tissue slices have been shown to have specific behavior and organ specificity. In view of the above, it is advantageous to use a slide using cultured cells as a substrate, and HEp-2 cells are cultured and attached by companies such as Callestad, Behring Diagnostic, and MLB in Japan. Sell slides. In view of the above, the inventor of the Korean Society of Internal Medicine, Vol. 35, No. 3 (1988), p. 315, entitled "A Study on Antinuclear Antibody Test Using HEp-2 Cell Culture".

However, importing and using cultured cell slides is expensive, and HEp-2 cells, which are cultured cells, are human-derived cells but are isolated from laryngeal cancer tumor cells. In order to improve the development of the adherent cells derived from normal people without disease, while continuing to study, the cell line was stable even in the culture period of three years, and thus the present invention completed the cell line excellent in adhesion.

The cell line according to the present invention is a mononuclear cell / macrophage cell line derived from bone marrow, and when adhered to the slide for the production of the slide for detection of ANA after incubation, the adhesion is excellent and the defect rate is low when preparing the slide. Slides could also be produced at lower cell concentrations. When compared with the HEp-2 cell line, the results of ANA detection were within the ANA detection tolerance. In particular, the developed cell lines showed good growth under various culture conditions.

According to the method of the present invention, detecting the presence of an antinuclear antibody using the mononuclear cell / macrophage cell line described above can be performed according to a conventional method. That is, it is necessary to observe whether the antigen antibody reaction occurs by dropping the body fluid to the target to check the presence of the antinuclear antibody on the slide to which the cell line is attached and fixed. The presence or absence of an antigen-antibody reaction can be confirmed by a conventional method such as fluorescence immunoassay or radioimmunoassay.

Hereinafter, the present invention will be described in detail, but the characteristics of the new cell line are not limited to the examples.

Example 1

Isolation of Cell Lines

Normal bone marrow was aseptically punctured to collect bone marrow cell solution, transferred to a centrifuge plate containing Sistoma's Histopaque 1077, and centrifuged at 400 ° C. at 20 ° C. for 30 minutes to obtain mononuclear cells. Inoculate the isolated mononuclear cells into a 75 square centimeter culture flask containing 15 milliliters of RPMI-1640 (10% calf serum), incubated in a 37 ° C incubator controlled with 5% carbon dioxide. Mononuclear cells, such as lymphocytes, that were not attached, were removed by washing with a culture solution. Cells attached to the incubator were continued to be weighted with RPMI-1640 added 20% calf serum and treated with trypsin after 7 days. The culture was multiplied again in two incubators, and after 5 days of trypsin treatment, the cells were divided into four incubators and cultured. The cultures were exchanged every 3 days and after 28 days the cultures were incubated with a 10% calf serum. This resulted in monocyte / macrophage cell line IT-1.

The cell line is growing well from June 1989 to the present, and was deposited on July 15, 1992 with the deposit number of KFCC-10772 to KFCC. It grew well in either Ham's F12K medium or Dulbecco's Modified Eagle's Medium (weakly referred to as "DEME") and cultured in RPMI-1640 medium added with 5% calf serum. In particular, the additives such as vitamins are not required separately, and in the RPMI-1640 to which 10% calf serum was added, 1 × 10 ⁵ / ml of 0.1 milliliters was inoculated densely after 5 days so that the diet was excellent.

Example 2

Preparation of the slide

After culturing the cultured IT-1 cell solution with RPMI-1640 medium to which 10% calf serum was added to 2 × 10 ⁴ cell / ml, sterilized IF slides with 12 holes and 30 microliters of dilutions in each hole. Inoculation. It was incubated for 24 hours in a 37 ℃ incubator controlled with 5% gas carbonate. Cultures were removed from each slide, washed with phosphate buffer, and soaked in 95% methanol for 10 minutes to fix cells. The slides thus prepared were stored at 4 ° C. for about a year.

Comparative Example

Preparation of Slides Using HEp-2 Cells

Since commercially available IF slides use HEp-2 cells derived from laryngeal cancer tumor cells, a method of culturing this cell line to prepare slides was selected as a comparative example.

HEp-2 cells were diluted with DMEM medium supplemented with 10% calf serum to 1 × 10 ⁵ cells / ml and then inoculated with 30 microliters of IF slides. When the cells subsided and the cells adhered, the inoculum was removed, and 30 microliters of the medium was dispensed into each hole, and cultured in the same manner as in Example 1. After removing the medium, it was washed with phosphate buffer, soaked in 95% methanol for 10 minutes and fixed.

Example 3

ANA testing with cell lines

Example 1, Comparative Example and 30 microliters of serum samples of rheumatoid patients diluted 2-fold from 1:20 to 1: 1280 were dropped on the slides of the product to be sold, and reacted at room temperature for 30 minutes at high humidity. Into this filled Copran bottle was shaken and shaken at 200 rpm per minute with a shaker and dried. AF-CDC standard serum # 1, # 2, # 3, # 6 was used as a control. Thirty microliters of rabbit antibody (manufactured by Dako) against human gamma globulin labeled with FITC (Fluorescein isothiocyanate) diluted 1:40 was applied to the slides for 30 minutes at room temperature at high humidity. . Washed with phosphate buffer for 10 minutes, counterstained with 0.2% Evans Blue solution (stored at 4 ° C. with preservatives), and washed with phosphate buffer. The remaining phosphate buffer was absorbed using filter paper and observed at 400-fold by fluorescence microscope using glycerol.

The fluorescence image under the microscope was speckled form of whole or undivided resting cells or dividing cells for standard serum # 1 and nucleoar staining for standard serum # 6. The results of standard serum # 2, # 3 and # 6 were observed only in resting cells. This is consistent with the fluorescence pattern presented by AF-CDC.

In addition, the intensity of fluorescence is divided into 4+ for strong, 3+ for sufficient, and 1+ for almost discernible, and the titer according to the dilution factor is shown in the table. # 1, # 2 and # 6 showed sufficient fluorescence at the dilution ratio of 1: 320 and # 3 to 1; 640, and the same pattern was observed for IT-1 cells. AF-CDC recommends a dilution ratio of patient blood from 1: 160 to 1: 640 for standard serum # 1 and # 3, and 1:80 to 1: 320 for # 2 and # 6. It was in range.

However, when looking at the dilution rate that fluorescence is only detected, HEp-2 is 1: 640 and IT-1 is 1: 1280 for standard serum # 1; For HE2, HEp-2 is 1: 1280 and IT-1 is 1: 640; For # 3 HEp-2 is also possible at dilution of 1: 1280 or higher, IT-1 is 1: 1280; For # 6, HEp-2 was 1: 1280 and IT-1 was 1: 1280. But I do not see much meaning.

[table]

Titer comparison of AF-CDC standard serum

If the cells were attached to all 12 holes of the slide during microscopic observation, it was easy to read, but when examining the defective rate where the cells were detached and detached, it was 9/600 in IT-1 cells and HEp-2. 95/600 in the cells. As a result, the cell line according to the present invention was found to have superior adhesion compared to known HEp-2 cells.

In addition, the number of cells inoculated on the slide was reduced by 1/5 and the culture medium was also saved by half.

Example 4

Application Results to Clinical Trials

The positive rate for the test was 96% in SEL and 40% in scleroderma when using frozen whole tissue of white kidney, and 99% and 88% when using HEp-2, a human-derived cell. In the case of using 1, 100% and 95% showed the superiority of the cell line. 100% accuracy was also found in mixed collagen disease.

In addition, adherent monocyte / macrophage-derived cell lines derived from healthy individuals without disease were stable even for 3 years of culture. Compared with HEp-2 cells derived from laryngeal cancer, which was used for the detection of ANA, the same results can be obtained in the ANA test, thus conserving the concentration of the inoculated cells and the medium, thereby replacing expensive HEp-2 culture cell slides. It became possible.

Claims (5)

Adherent, mononuclear / macrophage cell line for detection of antinuclear antibody derived from bone marrow of normal humans. The cell line according to claim 1, wherein the monocyte / macrophage cell line is IT-1 (KFCC-10772). Collecting body fluids from a subject to check whether or not they have an antinucleus antibody; Attaching the cell line of claim 1 to a slide to produce a slide for detecting an antinuclear antibody; The mononuclear cell / macrophage system of claim 1, comprising the step of dropping the body fluid collected in the step on the slide and reacting the cell line on the slide with the antinuclear antibody in the body fluid. A method of detecting the presence of an antinuclear antibody using a cell line. 4. The method of claim 3, wherein the cell line is obtained by culturing without the addition of a special additive. The method according to claim 3 or 4, wherein the monocyte / macrophage cell line is IT-1 (KFCC-10772).