Chemistry: analytical and immunological testing – For preexisting immune complex or auto-immune disease – Antinuclear
Patent
1994-03-17
1996-12-10
Housel, James C.
Chemistry: analytical and immunological testing
For preexisting immune complex or auto-immune disease
Antinuclear
422 57, 435 724, 4352402, 435970, G01N 33564
Patent
active
055830536
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention, related to the macrophage cell line, IT-1, is a method for detecting an antinuclear antibody (abbreviated as ANA, hereinafter) by employing the said macrophage cell line and a slide preparation.
BACKGROUND OF THE INVENTION
The ANA test for detecting the presence of an autoantibody in the serum or any other body fluid is applied to autoimmune diseases such as systemic lupus erythematosus (hereinafter, abbreviated as SLE), Sjoegren's syndrome, systemic sclerosis, mixed connective tissue disease, rheumatoid arthritis, juvenile chronic polyarthritis and other autoimmune diseases.
When a patient is initially thought to have a systemic rheumatic disease by the ANA test, the precise disease is diagnosed through a definite antibody identifying test against antigens such as DNA, histone, extractable nuclear antigens (ENA) such as Sm (Smith), nRNP (nuclear ribonucleoprotein), SS-A (Sjoegren's syndrome antigen A), SS-B, Scl-70, Jo-1, rRNP, etc.
The characteristics and titer of the antibody were standardized by using the standard serum developed from AF-CDC (Arthritis Foundation-Centers for Disease Control) in 1982 (Arthritis Rheum. 25, 1003, 1982) and concurrently, in an examination utilizing an immunofluorescence assay. The inconsistency or qualitical differences between the fluorescent microscopes employed, the concentration of FITC (fluorescein isothiocyanate) conjugated antisera and fluorescent label/protein (f/p) ratio were improved by employing an optical slide with graded degrees of fluorescence (Am. J. Clin. Path., 82, 57, 1984).
The most important factor in FANA (fluorescent antinuclear antibody), a test which is essential to the diagnosis of an autoimmune disease, is the selection of a substrate. In earlier ANA tests, cryostat tissue sections of organs such as the livers or kidneys of experimental animals had been used as the substrate. Recently, cultured human cells have mainly been used. An example of cultured human cells is the HEp-2 cell (ATCC CCL-23). By employing said cultured human cells, the species-genus specificity or the organ specificity could be overcome.
Since cultured human cells generally possess a larger nucleus and contain higher contents and a greater variety of specific nuclear antigens in comparison with the cryostat tissue section, an interpretation of the fluorescent pattern is easier and the possibility of false-negative results can be significantly decreased.
When the cell lines mentioned above were cultured on a slide, mitotic cells could be observed, and using these mitotic cells, it became possible to detect the anti-centromere antibody, anti-PCNA (proliferating cell nuclear antigen), an antibody which is found only in proliferating cells, etc. and to interpret the indistinguishable fluorescent patterns more precisely and objectively.
Although the HEp-2 cell was derived from a human, it is a tumor cell isolated from the laryngeal carcinoma, and when the cell was cultured on a slide without any pre-treatment, its glass adherence did not reach a satisfactory level. Moreover, another defect was that ANA is not detected in some patients, which is termed "ANA negative lupus".
To improve the defects mentioned above, the inventor tried to develop a new substrate, and as a result, discovered that the macrophage cell line (IT-1, KCCM-10038) derived from normal bone marrow does not have the above defects and can be employed as a useful substrate for the ANA test.
The theoretical background for research of macrophage as the substrate for the ANA test is as follows:
First, since the macrophage serves as an antigen presenting cell in an immune response, it is highly presumable for the cell to contain autoimmune antigens evenly and abundantly for the purpose of distinguishing self from nonself;
Second, since the glass adherence of the macrophage is excellent, the preparation of a slide for substrate is easy and economical; and
Third, since the IT-1 cell is one of nontumor origin, the possibility of false-negative results in the ANA test can be lowered
REFERENCES:
patent: 4487830 (1984-12-01), Coates et al.
Sundstrom, C. and K. Nilsson. "Establishment and characterization of a human histiocytic lymphoma cell line (U-937)." International Journal of Cancer, vol. 17 (1976), pp. 565-577.
Wing, E. J. and J. S. Remington. "Delayed hypersensitivity and macrophage functions." in: Basic and Clinical Immunology, 3rd edition, (Lange Medical Publications, 1980), p. 134.
Housel James C.
Murthy Prasad
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