IE41739B1 - Reagent for the detection of antinuclear factors and process for stabilizing it - Google Patents

Abstract

Reagents suitable for use in clinical diagnosis to detect antinuclear factors are distinguished by containing tissue cells, in particular tissue culture cells such as the BHK-21 cell, which are immobilised on slides enclosed in a container with an atmosphere with a low water vapour content. The reagents are prepared by a tissue culture preparation being immobilised on slides under mild conditions, dried and enclosed together with a desiccant in an air-tight container. To maintain an atmosphere with a low water vapour content, a desiccant with a water partial pressure at 25 DEG C of less than 2 x 10<-1> Torr is used.

Description

The present invention relates to a reagent for the detection of antinuclear factors and to a process for conserving the antigenic properties of cell nucleus constituents in tissue culture cells which are to be used for the detection of antinuclear factors.

Antibodies against the nuclear substances termed nuclear factors are found in various auto-immune diseases of connective tissue, especially in collagen diseases, for example, polyarthritis rheumatioa, sclerodermia, lupus erythematodes visceralis (LE), dermatomyositis and periarteriitis nodosa. These antibodies can be directed against various of, the nuclear factor antigens, for example, against deoxyribonucleic acid (DNA) , nucleoproteins, histones (often considered as nucleoproteins), the ribo15 nucleic acid (ENA) in the nucleolus, or against antigens that can be extracted from cell nuclei by means of a physiologically tolerable sodium chloride solution (the Smantigen).

For the detection of these antibodies, different 2o methods may be used; the antiglobulin-consumption test, the complement fixation reaction, the precipitation reaction, the passive haemagglutination reaction,, the latex test, and immunohistological and immunocytological methods.

Of the methods for the detection of antibodies against nuclear substances, the immunohistological and the immunocytological tests, especially those in which fluores- - 3 cent dyestuffs are used for the visualization of the positive reaction, have proved to be especially advantageous for the following reasons:a) they are distinguished by their high sensitivity and specifity. b) they detect antibodies against every nuclear factor antigen mentioned above and thereby permit a differentiation according to the pattern and the degree of the fluorescence. c) the test can be carried out relatively easily.

In the known processes, the following antigens may be used:1) fresh cryostat slices of an organ of a test animal or of human tissue, 2) fresh human cells, for example, buccal cells or leucocytes. 3) tissue culture cells of human or animal origin. 4) stabilized calf thymocytes.

To avoid the extraction of the water-soluble nucleoproteins (Sm-proteins) from the cells or organ slices used, the cells or slices are generally fixed on slides according to a suitable method, for example, with the aid of acetone. A serum to be tested for antibodies to nuclear factor antigens is applied to slides in different dilutions, and the slides are incubated and washed.

An antiserum having antibodies against human immunoglobulins and which is labelled with a fluorescent dyestuff, for example, fluorescein-isothiocyanate-(TITC-) , or peroxidase is then applied to the slides, which are then incubated again and washed. The specifically bound 4173® - 4 antibodies are detected by microscopical examination for fluorescence or by histochemical staining of the slides.

Freshly prepared antigens have the basic drawback of poor stability at room temperature or at refrigerator temperatures, so, it is necessary to produce fresh material for each test or to store the material at temperatures ranging from -20 to -30°C or below. This drawback gives rise to particular problems in the commercial preparation of suitable reagents, because they must be dispatched at temperatures below freezing point in order not to adversely affect the antigenicity of the reagent.

The use of tissue culture cells has certain advantages over other antigenic material because the uniformity of this antigen material is readily ensured. The tissue culture cells can be prepared in any desired amount, and, furthermore, the culture can be carried out directly on slides. In this case too, though, the drawback is that the cells have to be stored at temperatures below 0°C. In some cases, furthermore, storage in glycerol is recommended. The glycerol, however, must be washed off before the slides are used as reagents, whereupon it appears that the Smantigen, which is necessary for the detection of the socalled spotted fluorescence, is lost.

The present invention is based on the observation that the stability of the antigenic material in tissue culture cells can be significantly prolonged when the tissue culture preparation is stored together with a drying agent in an airtight vessel, for example, a box.

This invention accordingly provides a reagent for the detection of antinuclear factors whioh comprises tissue culture cells fixed on a slide, which is or has been maintained in an airtight vessel in the presence of a desiccant.

This invention also provides a process for conserving the antigenic properties of cell nucleus constituents in tissue culture cells for use in the detection of antinuclear factors, which process comprises fixing tissue - 5 culture cells on a slide, drying the slide and then maintaining it in an airtight vessel in the presence of a desiccant.

The fixation may be carried out with one of the volatile organic solvents generally used for this purpose, for example, acetone. Drying is then carried out advantageously at room temperature or an elevated temperature up to about 50°C at atmospheric or under reduced pressure. Suitable desiccants are those substances used in chemistry for binding water vapour, especially those having a water partial pressure over the fresh drying agent of 0.2 torr or less at 25°C, for example, CaCl2, CaO, Al2O3, anhydrite or P20g. Direct contact of the preparations with the desiccant should be avoided.

There may be used any type of tissue culture cell that a person skilled in the art would consider to be suitable as a reagent for the detection of the antinuclear factors. For the diagnosis of lupus-erythematodes, for example, the cells that are especially suitable are kidney cells of baby hamsters, so-called BHK-cells, rabbit kidney cells, human amnion cells, and HeLa-cells. Preferred are BHK-21 cells that have been described in more detail by N.Stoker and I.Macpherson in Nature 203, 1355-1357 (1964).

The reagent of the invention can be stored at refrigerator temperatures. At 4°C, over a period of about 12 months, no decrease in the quality of the reagent was observed, whereas tissue culture cells made stable in a way different from that of the invention, after only a few days at the same storage temperature showed only a slight fluorescence with sera containing specific antibodies or even no fluorescence at all.

This invention also provides a method of determining nuclear factor antigens, which comprises applying 43.739 - 6 a serum or other test fluid to a reagent of the invention, Incubating and then washing It, applying to It antibodies against human immunoglobulins, which antibodies are labelled with a fluorescent dyestuff or enzyme for example, fluorescein isothiocyanate or peroxidase, incubating the reagent and washing it, and detecting any specifically bound antibodies by microscopal examination for fluorescence or by histochemical staining.

The following Example illustrates the Invention. l0 EXAMPLE BHK-cells were cultured according to the method of X. Macpherson and M. Stoker, Virology IS, 147-151 (1962) on slides having labelled reaction fields. After 24 hours the slides, loaded with the cells, were removed from the nutrient medium and rinsed with physiologically tolerable sodium chloride solution, put for 10 seconds into physiologically tolerable Sodium chloride solution and were then rinsed first With 50% acetone (diluted with physiologically tolerable NaCl solution) and then 2q with undiluted acetone. The slides were dipped twice for 10 seconds each time in fresh acetone and then fixed for 10 minutes in fresh acetone. After having been dried for a short time in the open air, the slides were wrapped in silk paper and put into a vessel having 2g dimensions of 18 x 8 x 10 cm, the bottom of which was covered with a layer of phosphorus pentoxide 2.5 cm deep, the layer of phosphorus pentoxide being covered with cellulose gauze to avoid direct contact with the slides. The vessel was then closed, by means of a screw top.

Claims (15)

1. CLAIMS:1. A reagent for the detection of antinuclear factors comprising tissue cells fixed on a slide, which is or has been maintained in an airtight vessel in the presence of a desiccant.

2. A reagent as claimed in olaim 1, wherein the tissue cells are cells grown in a tissue culture.

3. A reagent as claimed in claim 2, wherein the cells are grown in a tissue culture on the slide.

4. A reagent as claimed in any one of claims 1 to 3, for the detection of a collagen disease. 5. 20. 22. A method of determining nuclear factor antigens, which comprises applying a serum or other test fluid to a reagent as claimed in any one of claims 1 to 10 or claim 21, incubating the reagent, washing it, applying to it

5. A reagent as claimed in claim 4, for the detection of lupus erythematodes visceralis.

6. A reagent as claimed in claim 4, for the detection of polyarthritis rheumatioa, sclerodermia, dermatomyositis or periarteriitis nodosa.

7. A reagent as claimed in any one of claims 1 to 5, wherein the tissue cells are kidney cells of baby hamsters, BHK-cells, rabbit kidney cells, human amnion cells or HeLa cells.

8. A reagent as claimed in claim 7, wherein the cells are BHK-21 cells.

9. A reagent as claimed in any one of claims 1 to 8, wherein the desiccant has a water partial pressure at 25°C of 0.2 torr or less. 10. Antibodies against human immunoglobulins, which antibodies are labelled with a fluorescent dyestuff or enzyme, incubating the reagent, washing it and detecting any specifically bound antibodies by microscopal examination for fluorescence or by histochemical staining. 10 desiccant Is calcium chloride, calcium oxide, aluminium oxide, anhydrite or phosphorus pentoxide.

10. A reagent as claimed in claim 1, and which is substantially as described in the Example herein.

11. a process for the conservation of the antigenic properties of cell nucleus constituents in tissue culture cells, which comprises fixing the cells on a slide, drying the slide, and maintaining it in an airtight vessel 5 in the presence of a desiccant.

12. A process as claimed in claim 11, wherein the desiccant has a water partial pressure at 25°C of 0.2 torr or less.

13. A process as claimed in claim 12, wherein the

14. A process as claimed in claim 11 or claim 12, wherein the cells are cultured on the slide. 15. A process as claimed in claim 11 or claim 12, 15 wherein the cells are suitable for the detection of a collagen disease. 16. A process as claimed in claim 15, wherein the cells are suitable for detecting lupus erythematodes visceralls. 20 17. A process as claimed in claim 15, wherein the cells are suitable for detecting polyarthritis rheumatica, sclerodermia, dermatomyositis, or periarteriitis nodosa. 18. A process as claimed in claim 16, wherein the cells are kidney cells of baby hamsters, BHK-cells, rabbit 25 kidney cells, human amnion cells, or HeLa cells. 19. A process as claimed in claim 18, wherein the cells are BHK-21 cells. 417 - 9 20. A process as claimed in claim 11, conducted substantially as described in the Example herein. 21. A reagent as claimed in claim 1, whenever prepared by a process as claimed in any one of claims 11 to

15. 23. A method as claimed in claim 22, wherein the anti bodies are labelled with peroxidase or fluorescein-isothiocyanate.