The present invention relates to an antinuclear antibody assay utilizing stress-treated cells or cell extracts. More precisely, the invention relates to an antinuclear antibody assay for screening patient sera, wherein stress-treated cells derived from humans, or protein extracts from said cells, are used. Optionally patient antibodies against nuclear stress-specific proteins are determined as well.

Background A stress situation for a human cell can be defined as exposure to a hostile environment threatening the proper functioning or the survival of the cell. In response to severe forms of stress, the cell reacts in a specific manner involving the coordinated activation of a number of genes presumed to bring protection to the environmental threat.

Different types of stress can be identified depending on the initiating factor (such as W radiation, DNA-damaging drugs, heat or oxygen radicals), each associated with induction of a set of genes, including some overlap in these responses. The function is known only for a minority of these stress-induced genes (Holbrook N J and Fornace Jr A J. 1991. Response to adversity: molecular control of gene activation following genotoxic stress. New Biologist, 3, 825-833).

Antinuclear antibodies (ANA) are a heterogenous group of autoantibodies that react with various structures of the cell nucleus. They are associated with various autoimmune disorders, and therefore their determination in patient blood is of diagnostic interest.

Antinuclear antibody (ANA)-screening tests are the most common immunological examination methods for analysis of patient sera to determine if the patient possibly suffers from a disease which induces cells under stress to produce an abnormality related to nuclear proteins not present in the normal cell, especially an autoimmune disease.

The ANA screening tests used at present are performed by examination of animal tissue sections or cultivated human cells by fluorescent microscopy, adding patient serum sample and allowing patient antibodies directed against cell nuclear proteins to form complexes with said proteins, adding fluorescent antibodies which are directed against the protein-complexed patient antibodies. In a small number of patients it is possible to detect with an ANA test, antibodies not only against cell nuclear proteins but also against proteins of the surrounding cytoplasma. These antibodies against cytoplasma proteins may also indicate disease.

The presence in human cells of a large number of proteins, in detectable quantities, is dependent on the exposure of said cells to a stress situation. Such stress-specific proteins can be found both in the cell nucleus and the surrounding cytoplasma.

The ANA screening tests presently used all utilize non-stress-treated cells and they lack the sensitivity to detect antibodies directed against stress-specific proteins.

It would be desirable to have an ANA assay which would detect not only antibodies directed against cell nuclear proteins but also antibodies directed against cell nuclear stress- specific proteins.

Description of the invention The present invention makes use of stress-treated cells derived from humans, or protein extracts from such cells.

The preparation of a cell extract containing stress-induced proteins has been previously disclosed in the literature. (Schreiber E., Matthias P., Muller M. M., Schaffner W.

1989. Rapid detection of octamer binding proteins with"mini-extracts", prepared from a small number of cells. Nucleic Acids Research 17: 6419; Lavasani S., Henriksson G., Brant M., Henriksson A., Radulic M., Manthorpe R., and Bredberg A. 1998. Abnormal DNA Damage-inducible Protein in Cells from Sjogren's Syndrome Patients. Journal of Autoimmunity 11: 363-369.) The present invention provides an antinuclear antibody assay for screening patient sera, wherein stress-treated and subsequently cultured cells derived from humans, or protein extracts from said cells, are used as a protein source for immunological determination of the presence of patient antibodies against nuclear proteins in screening samples of patient sera.

Compared to a common ANA screening assay which utilizes non-stress-treated cells, the assay of the invention is more sensitive to nuclear stress-specific proteins and it can be performed with different types of immunological assays, not only immunofluorescence.

Additionally, a distinction between patient antibodies against nuclear proteins and stress- specific nuclear proteins is possible with the assay of the invention.

Thus, in an embodiment of the invention the immunological determination is performed utilizing anti-human antibodies labeled with a marker selected with regard to the immunological assay used for the determination.

In a preferred embodiment of the invention the immunological assay used for the determination is selected from the group consisting of immunofluorescence assay, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blot and dot blot,

and the anti-human antibodies are labeled with a fluorescent marker, enzyme marker, radioactive marker or no marker, depending on the assay used.

The assay according to the invention may be performed so that additionally patient antibodies against nuclear stress-specific proteins are determined by comparison of the assay result with the result from a parallel run performed with non-stress-treated (mock-treated) cells or protein extracts from said cells.

Another aspect of the invention is directed to a diagnostic kit for the determination of patient antibodies against nuclear proteins in samples of patient sera, which comprises stress- treated and subsequently cultured cells derived from humans, or protein extracts from said cells.

In an embodiment of the diagnostic kit according to the invention the kit will additionally comprise, separately, non-stress-treated (mock-treated) cells or protein extracts from said cells, for the additional determination of patient antibodies against nuclear stress-specific proteins.

In a preferred embodiment of the diagnostic kit according to the invention the kit will additionally comprise, separately, an optionally labeled secondary (anti-human) antibody, a buffer solution, a positive standard serum sample, and a negative standard serum sample.

In the diagnostic kit according to the invention the cells or protein extracts are provided in/on slides, wells of ELISA plates/strips, gels for electrophoresis, or membranes, including cellulosic membranes. All of these support systems are well known in the art of immunoassays. The diagnostic kit will also be accompanied by instructions for use.

Yet another aspect of the invention is directed to the use of stress-treated and subsequently cultured cells derived from humans, or protein extracts from said cells, in diagnostic assays.

In a preferred embodiment of the use according to the invention the diagnostic assay is an antinuclear antibody assay for screening patient sera.

The invention will now be illustrated by description of specific examples, which, however, should not be construed as limitations to the scope of the invention.

General description of ELISA analysis of patient serum antinuclear antibodies to stress- specific proteins in human cell extracts.

Cell culture Human B-lymphoblastoid cell lines were established from peripheral blood by Epstein-Barr virus transformation and cultured at 37°C and 5% C02 in RPMI 1640 (Gibco,

UK) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 25 mM Hepes and 12 ug/ml gentamicin.

Stress treatment of cells with short-wave UV radiation Cells kept in 0.5 ml of PBS (pH 7.4) in a 4 cm diameter Petri dish were exposed to 6 J x m 2 ouf short wavelength ultraviolet radiation (WC) from an ordinary germicidal lamp emitting 1 W x m-2 at 254 nm as measured by a UV-X Radiometer (Ultraviolet Products), and then immediately suspended in culture medium.

Extraction of cellular protein A cell nucleus preparation was collected 24-48 hours after exposure of the cells to UV radiation by lysis of pelleted cells in a neutral pH buffer containing 0.5% NP-40,10 mM KC1,0.2 mM EDTA and a mixture of proteinase inhibitors ("Cocktail", Boehringer- Mannheim, Germany). Pelleted cell nuclei were then lysed on ice in a hyperosmolar neutral pH solution with 0.40 M NaCl, 2 mM EDTA and proteinase inhibitors. Total protein concentration was determined spectrophotometrically in 96 well microtiter plates using copper sulphate (Pierce, Ill.). A typical yield from 25 X 106 cells was for the nuclear preparation 400-1100 ig protein/ml, total volume 40 1. All extracts were stored at-70°C.

Detection of antinuclear antibodies to stress-specific proteins by ELISA.

Polystyrene microwell plates (F96 Maxisorp, Nunc-Immuno Module, Roskilde, Denmark) were coated overnight at 4°C with 2 ug per well of cell nuclear extract protein dissolved in 100 ul 0.1 M NaHC03 buffer (pH 9.4). Neighboring wells were coated with extract from stress-treated and mock-treated cells, respectively, permitting an evaluation of the presence of stress-specific antibodies. The plates were then washed four times with washing buffer (phosphate-buffered saline [PBS] containing 0.05% Tween 20, pH 7.4) and incubated for 1 h at room temperature with 200 ul of blocking buffer (PBS containing 1.5% ovalbumin and 0.05% Tween 20, pH 7.2). After renewed washing, 100 gl per well of patient sera diluted 1: 500 in blocking buffer was added. A positive control and a negative control (sera from patients determined by a conventional Western immunoblotting procedure to be positive and negative, respectively) diluted in blocking buffer was included on each plate. The sera and controls were allowed to react at room temperature for 1 h. The plates were then washed four times with washing buffer and incubated once more for 1 h at room temperature with 100 gl per well of peroxidase-conjugated rabbit anti-human IgG (DAKO A/S, Glostrup, Denmark) diluted 1: 6000 in blocking buffer. Finally, the plates were washed again and 100 p1 of 1 mM tetramethylbenzidine in citrate buffer (0.1 M, pH 4.25) with 2 mM H202 at a 1: 20 ratio was added as substrate. The enzymatic reaction was stopped 5 min later by the addition

of 100 p1 of 1 M H2SO4 and the optical density (OD) at 450 nm was determined by a spectrophotometer (Multiskan Plus, Labsystems, Finland).

All sera and controls are analyzed in triplicate, of which two were run in wells coated with cell extract protein and one in an uncoated well on the same plate. The result may be expressed as the optical density (OD) difference between the two coated wells (mean value) and the uncoated well. In this way, any false positive reactions due to nonspecific binding of patient IgG to factors employed in the assay other than the cell extract protein were excluded for.

Example 1 Comparison between the prior art ANA test and the ANA assay of the invention by use of enzyme-linked immunosorbent analysis (ELISA) A commercial antinuclear antibody test kit (Immunoconcept, Sacramento, Calif.) for the golden standard HEp-2 immunofluorescence method was used to screen a consecutive series of 200 sera submitted by physicians to a hospital laboratory requesting ANA analysis.

The commercial kit uses human cells which are not stress-treated. The instructions of the kit supplier were used The same sera were tested by the ANA assay of the invention utilizing stressed HEp-2 cells cultivated and exposed to a stress situation according to the General description of ELISA analysis, Cell culture, Stress treatment of cells.

The test results obtained with the commercial kit were compared with the results obtained from parallel testing with the above described ELISA-analysis according to the invention. The results are presented in Table 1, and are expressed as positive/negative.

Table 1 Commercial ANA IF kit (HEp-2 immunofluorescence method with no stress treatment) + Present invention (ELISA with + 5 10 stress treatment) -26 159 Table 1 shows that 31 patient sera were analyzed to contain antinuclear antibodies by the commercial method, whereas 15 of the 200 sera samples analyzed by the assay of the invention were positive. However, 10 out of the 15 were found positive only in the ELISA

test of the invention, which indicates that this test more specifically detects antinuclear antibodies to stress-specific proteins.

The results from the commercial ANA IF-kit are based on assessment by ocular inspection and are thus to some extent subjective, whereas the results from the ELISA are based on measured values of optical density. Therefore, the results from the two methods are not directly comparable, but it is evident from Table 1 that the ELISA test of the invention is able to detect stress-specific antibodies not found with the commercial ANA IF method. This is probably due to the higher concentration of nuclear stress-specific proteins as antigens in the test of the invention.

Example 2 Comparison between ANA ELISA assay using protein antigens from non-stress treated cells and ANA ELISA of the invention using protein antigens from stress treated cells.

A consecutive series of 200 sera submitted by physicians to a hospital laboratory requesting ANA analysis were subjected to screening by ELISA assays performed as disclosed in the General description of ELISA analysis. Two parallel concepts were used, one commercial and one according to the invention. The antigens used in the ELISA of the invention were from a culture of HEp-2 cells stress treated as in Example 1.

The test results obtained were compared, and the results are presented in Table 2, and are expressed as positive/negative.

Table 2 Results from ANA ELISA (stress antigen) positive negative Results from ANA ELISA positive 18 25 (no stress antigen is used) negative 6 151 Table 2 shows that 43 patient sera were analyzed to contain antinuclear antibodies by the ANA ELISA with no stress antigens, whereas 24 of the 200 sera samples analyzed by the ANA ELISA with stress antigens were positive. However, 6 out of the 24 were found positive only when stress antigens were used in the ELISA test, which indicates that this test more specifically detects antinuclear antibodies to stress-specific proteins.

The reason why the ANA ELISA with no stress antigen detected 43 sera containing antinuclear antibodies, whereas the ANA ELISA with stress antigen only detected 24, is

probably due to the fact that the latter analysis has not yet been optimized, i e there were not enough of no stress antigens present in the ELISA. However, this example clearly shows that the ANA ELISA of the invention can detect sera containing antibodies directed to stress- specific nuclear antigens not detected with the ANA ELISA utilizing no stress antigens.

Example 3 Comparison between the prior art ANA test and the ANA assay of the invention bv use of immunofluorescence microscopy The same commercial ANA test kit as in Example 1 was used for screening a consecutive series of 60 sera submitted by physicians to a hospital laboratory requesting ANA analysis. The same sera were tested by an ANA assay of the invention utilizing stressed HEp- 2 cells cultivated and exposed to a stress situation according to the General description of ELISA analysis, Cell culture, Stress treatment of cells, except for that Eaglens MEM medium was used instead of RPMI 1640. The results were in both cases determined by immunofluorescence microscopy.

Table 3. Immunofluorescence-based analysis of antinuclear antibodies (ANA) in patient sera, using stressed HEp-2 cells (the invention) compared to the results using a commercial ANA kit ANA IF assay of the invention Commercial ANA IF kit +-Total + 25 1 26 4 56 60 The results show that 25 patient sera were analyzed to contain antinuclear antibodies by both methods, whereas the commercial kit gave additionally one positive sample. Out of the 60 negative sera analyzed by the commercial kit, 4 sera were found positive by the assay of the invention. These preliminary results indicate that the assay of the invention is more sensitive than the commercial ANA test kit.

Example 4 Comparison between the prior art ANA test using fluorescence microscopy and the ANA assay of the invention using Western blot.

The same commercial ANA test kit as in Example 1 and 3 was used for testing patient sera. An ANA assay of the invention was used to assay the same sera. Western blot and protein extract from stressed human cells prepared according to the General description of ELISA, Cell culture, Stress treatment of cells, was used in the ANA assay of the invention.

The patient sera came from patients with Sjögren's syndrome, which syndrome induces the production of antinuclear antibodies in a large fraction of the patients.

Table 4. Commercial ANA IF test kit results compared with Western analysis of antinuclear antibodies (ANA) in patient sera, using protein extracts from stressed human cells according to the invention.

ANA Western blot of the invention Commercial ANA IF kit +-Total + 6 0 6 -2 2 4 Table 4 shows that the ANA Western blot assay of the invention gave 2 positive results in addition to the 6 positive results also analyzed with the commercial ANA IF kit.

These preliminary test results indicate that the ANA Western blot analysis of the invention is more sensitive than the commercial ANA IF test.

The increased sensitivity of the ANA assays of the invention compared to the commercial ANA IF test is probably due to the fact that the prior art assay is incapable of detecting patient antibodies directed against stress-specific cellular proteins.