KR950011547B1 - Slide and its manufacturing method on anti-nuclear antibody diagnosis - Google Patents

Abstract

The slide for detecting antinuclear antibodies (ANA) contains a macrophage cell line immobilised in wells. The method may be used for diagnosis of autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis and rhematoid arthritis. The specified cell line, namely IT-1 (KCCM 10038), has better glass adhesion that HEp-2 (a tumour cell line), is derived from normal bone marrow, and does not give the false negative associated with HEp-2 ('ANA negative lupus')

Description

[Name of invention]

Anti-nuclear antibody diagnostic slide and its manufacturing method

Detailed description of the invention

The present invention is directed to the development of diagnostic IT-1 cell lines for anti-nuclear antibodies (hereinafter, weakly referred to as ANA), ANA diagnostic slides, and a method for producing the same.

The ANA diagnosis, which examines the presence of autoantibodies in serum, includes systemic lupus erythematusus (weak SLE), Sjoegren's syndrome, scleroderma, mixed collagen, rheumatoid arthritis, and children It is adapted to systemic rheumatoid diseases such as juvenile chronic polyarthritis and other autoimmune diseases. After Hargraves discovered LE cells in 1948, in the 1980s, techniques such as staining patterns and double immuno-diffusion methods by fluorescent antibody methods were introduced, and DNA, histone, Extractive nuclear antigens (ENA), for example Sm (Smith), nRNP (nuclearribonucleoprotein), as-A (Sjoegren associated antigen), ss-B, scl-70 (scleroderma) and the like has been found. Patients who are primarily diagnosed with autoimmune disease by ANA diagnosis should undertake specific antibody assays for each antigen to determine disease name.

The ANA diagnostic method is generally described by binding an antibody present in the patient's blood to an antigen present in a fixed substrate (normal mammalian tissue or cell), and then observing the bound antibody using fluorescence. Therefore, the diagnosis 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 using cell lines such as cells) or KB (human KB kidney), slides cultured aseptically on the slides are used. In the latter case, you can use commercialized slides.

2) Fixation of the attached substrate: After washing with the blood, the substrate is treated to prevent the substrate from falling off.

3) Reaction with Patient Serum: Serum from 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: The immunofluorescence method is mainly used in the ANA assay, and in the present invention, a Dako Corporation product, which is a rabbit antibody to human gamma globulin, is labeled with FITC.

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

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

To improve accuracy in ANA testing, consideration should be given to 1) test methods and quantification, 2) selection of substrates and fixatives, 3) establishment of normal ranges and the ability to read test results. By developing standard sera in AF-CDC (Sera Standardization Meeting for Antinuclear Antibodies; Arthritis Rheum. 25, 1003, 1982) in 1982, the characteristics and titers of the antibody, which is a problem in paragraph 1), were standardized 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 histoblast's tissue blast MF, monkey kidney cells VMK, human cells HA, KB, HEp-2 (deposited as ATCC CLL 23), etc. Compared to frozen tissue sections using organs such as and kidneys has the following advantages. Cultured cells generally have larger nuclei, higher levels of specific nuclear antigens, and more diverse than frozen tissue sections, making them easier to read and less prone to false negatives. Another advantage is that if the cell line is cultured on a slide, the schizophrenic cells can be observed, and fluorescence patterns that are difficult to distinguish from the diagnosis of anti-entromere antibodies and proliferating cell nuclear antigens (PCNA) only observed in proliferating cells are used. Can be objectively confirmed. Frozen tissue slices show VIII-VII specificity or organ specificity. In view of the above, it is advantageous to use a slide using culture cells as a substrate, and Hep-2 cells are cultured and attached by companies such as Kallestad, Behring Diagnostic, and MBL of Japan. Sell slides. In view of the above, the inventors 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 culture cell slides is expensive, and the cultured cells, HEp-2 cells, are human-derived cells but are isolated from laryngeal cancer and are not satisfactory in adherence in slide culture. In order to develop an adherent cell derived from a normal disease for the disease, the present invention was completed because it was stable even in the culture period of 3 years to obtain a cell line with excellent adhesion. Excellent adhesion, low failure rate during the slide preparation, it was possible to produce a slide at a lower cell concentration. The results of the ANA diagnosis compared to the HEp-2 cell line were present within the ANA diagnosis tolerance range. In particular, the developed cell lines showed good growth under various culture conditions. 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

Bone marrow was obtained by aseptic puncture of normal human bone marrow, and then transferred to a centrifuge tube containing Sistoma's Histopaque 1077, followed by centrifugation at 400 ° C. for 30 minutes at 20 degrees to obtain only mononuclear cells. RPMI-1640 (10% calf serum) (Gibco Co., Ltd.) 15 days inoculated with the isolated mononuclear cells in a 75 square center culture flask containing 3 ml in a 37 ° C incubator controlled with 5% carbonated gas Cultured and unattached mononuclear cells such as lymphocytes were removed by washing with culture. Cells attached to the incubator continued to proliferate with RPMI-1640 added with 20% calf serum, and after 5 days of trypsin treatment, branched to four incubators. The cultures were exchanged every 3 days and after 28 days the cultures were incubated with a 10% calf serum. This resulted in the mononuclear cell / macrophage cell line IT-1. The cell line is growing well from June 1989 to the present and was deposited on July 15, 1992 as KFCC-10772. It grew well in (Ham's) F12K medium or Dolbecco's medium (weakly referred to as DMEM) and cultured in RPMI-1640 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 and showed excellent growth ability.

Example 2

Preparation of the slide

After diluting the cultured IT-1 cell solution with RPMI-1640 medium to which 10% calf serum was added to 2 × 10 ⁴ cells / ml, sterilized IF slides with 12 holes and 3 microliters of dilutions in each hole. Inoculation. It was incubated for 24 hours in a 37 ° C incubator controlled with 5% carbon dioxide. 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 could be stored at 4 degrees for about a year.

Comparative Example

Preparation of Slides Using HEp-2 Cells

Since commercially available IF slides use HEp-2 cells, a method of preparing the slides by culturing this cell line was selected as a comparative example. Hep-2 cell cells were lined with DMEM medium with 10% calf serum added to 1 × 10 ⁵ cells / ml, followed by inoculation of 30 microliters on the IF slide. When the cells changed and the cells adhered, the inoculum was removed, and 30 microliters of the medium was further branched 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 diagnosis using cell line

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 sold, and reacted at room temperature for 30 minutes at high humidity, followed by phosphate buffer solution. Into this filled Cran bottle, shaken with a shaker at 200rpm per minute and dried. AF-CDC standard serum # 1, # 2, # 3, # 6 was used as a control. 30 microliters of a rabbit antibody (manufactured by Dako) against human gamma globulin labeled with FITC diluted 1:40 was applied to each slide and allowed to react for 30 minutes at room temperature with high humidity. Washed with phosphate buffer for 10 minutes, counter-stained with a Vans Blue solution (preserved at 4 ° C.) at 0.2%, 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 pattern under the microscope is a speckled form in which both resting cells and dividing cells are stained as a whole or only around the cells for standard serum # 1, and not partially stained for standard serum # 2 and # 3. For standard serum # 6, it was stained with nucleolar. 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 an almost discernible amount. HEp-2 cells showed sufficient fluorescence at dilutions of standard serum # 1, # 2 and # 6 at 1: 320, and # 3 at 1: 640. 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; # 2 HEp-2 1: 1280, IT-1 1: 640; For # 3 HEp-2 is also possible at dilutions above 1: 1280, for 2 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 1

Titer comparison of AF-CDC standard serum

When the cells were attached to all 12 holes of the slide during the microscopic observation, it was easy to read, but when examining the defective rate in which the cells were detached and detached, it was 9/600 in the IT-1 cell and HEp-2. 95/600 in the cells. This shows that the adhesion is excellent.

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 result in daily experiment

The positive rate for the test was 96% in SLE and 40% in transdermal 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, the cell lines showed 100% and 95% superiority. 100% accuracy was also found in mixed collagen disease.

In view of the above, the adherent mononuclear cell (monocyte / macrophage) -derived cell line derived from a normal human without disease was stable even for 3 years of culture. When the same results were obtained in ANA diagnosis as compared to HEp-2 cells derived from laryngeal cancer, the concentration and medium of the inoculated cells could be decribed to replace expensive HEp-2 culture cell slides.

Claims (4)

An antinuclear antibody diagnostic slide formed by fixing an antinuclear antibody diagnostic mononuclear cell (monocyte / macrophage) IT-1 cell line (KFCC 1-10772) derived from bone marrow of a normal person in an -IF slide hole. A method for producing an anti-nuclear antibody diagnostic slide comprising inoculating a cell line IT-1 (KFCC-10772) for diagnosis of adherent antinuclear antibody derived from bone marrow of a normal person, culturing for 24 hours, washing with a buffer, and fixing the immobilized antinuclear antibody. The method according to claim 2, wherein the cell line having a concentration of 10 ⁴ cells / ml per slide hole is inoculated at 30 microliters. The method for producing a slide for diagnosing antinuclear antibody according to claim 2, wherein the cell is fixed with methanol.