WO1992014148A1

WO1992014148A1 - Kit for detection of antinuclear antibodies and method for detecting such antibodies

Info

Publication number: WO1992014148A1
Application number: PCT/BE1992/000002
Authority: WO
Inventors: Roger Arsène Joseph Ghislain SMET
Original assignee: Bio-Line S.A.
Priority date: 1991-02-08
Filing date: 1992-02-07
Publication date: 1992-08-20
Also published as: BE1004457A3; EP0579609A1

Abstract

Kit for the detection of antinuclear antibodies in serums, comprising reagents appropriate for performing an ELISA test, and a support having a group of wells per serum to be analyzed, each group of wells having a plurality of detection wells each being provided to perform an ELISA test, the coating of the bottom of each detection well of a group containing one or a plurality of nuclear antigens different from that or from those contained in the coating of other detection wells of said group, and optionally a control well whose bottom is deprived of coating, for a blank test.

Description

Antinuclear antibody screening kit and method for screening for such antibodies

The present invention relates to a kit for screening for antinuclear antibodies in sera, comprising a support in which are arranged several wells having a bottom covered with a coating containing at least one nuclear antigen, reagents suitable for carrying out a test. ELISA as well as possibly a positive control serum and a negative control serum. It also relates to a method of screening for anti-nuclear antibodies in sera.

Antinuclear antibodies are, under certain circumstances, developed by the human body against nuclear antigens. We can distinguish several nuclear antigens:

- chromosomal nuclear antigens: native or double-stranded deoxyribonucleic acid (dsDNA), denatured or single-stranded deoxyribonucleic acid (ssDNA), various histones (Hl, H2a, H2b, H3, H);

- extractable nuclear antigens (ENA), among which there may be mentioned the Sm, RNP (ribonucleoproteins), 5SA, SSB, Sci-70, 30-1, RANA, PCNA, etc. antigens 0 Antinuclear antibodies (ANA ) reacting to dsDNA are present in 60 to 70% of patients with systemic lupus erythematosus (SLE). These antibodies are less frequently detected (<5%) in patients showing other types of autoimmune pathologies, in particular juvenile arthritis -5 or liver disorders. This specificity of ANA is almost always negative in the case of drug-induced lupus. Given their very high degree of specificity (95%), ANA reacting to dsDNA appear among the essential criteria for the diagnosis of LED. The presence of these antibodies can be used either to confirm the diagnosis of SLE in patients where this disease is clinically suspected, or to detect asymptomatic patients. High titers of anti-dsDNA ANA are observed in 60 to 70 ° ό of active LEDs, especially with renal complications. On the contrary, low headings indicate rather inactive LEDs or other disorders. Antinuclear antibodies reacting to AD ss are relatively unspecific; they are detected in chronic progressive polyarthritis (8%), Sjôgren's primary syndrome (13%) and scleroderma (14%). The highest frequencies of positive test results are seen in LED (70%), polydermatomyositis (43%) and drug-induced lupus erythematosus (60%).

Antibodies to ssDNA are sometimes the only positive ANA in photosensitive cutaneous lupus.

Antinuclear antibodies reacting to histones are more frequent in LED (> 90 96) than in other types of connectivities (<20%) where they are present at low levels. Induced lupus erythematosus and idiopathic lupus have a high frequency of ANA reacting to histones but with a different specificity for the different classes of histone. The antibodies present in the LED react both to the free subgroup (Hl) of histones and to the complexed subgroups (H2a / H2b, H3 / H). In contrast, antibodies in lupus-induced procai ^'na- mide are mainly directed against the H2a / H2b complex. In lupus induced by hydralazine, the antibodies show specificity only for the H3 / H complex. ANA to ENA is found in a large number of rheumatic disorders. Some of them are considered as diagnostic markers (5m, S5A, Jo-1, Scl-70).

As is clear from the above, it is often important, in order to make a diagnosis, to add to clinical observations the results of a screening test making it possible to determine the exact specificity of the antinuclear antibodies present in the serum of the patient examined. .

Several methods are currently applied to detect the presence of antinuclear antibodies in sera. Mention may be made, inter alia, of tests by indirect immunofluorescence or immunoprecipitation methods such as double immunodiffusion according to Ouchterlony (see BT NGUYEN and A. MARKOVITS, ANA Atlas, Brussels, 1990, p. 14-30). We also know the technology of electrophoretic transfer of proteins, called Western

Blot. Finally, for certain nuclear antigens, such as DNA, it is known to practice immunoenzymatic techniques, such as the enzyme-linked immunosorbent test (ELISA test). Some of these methods are very expensive, some also not very sensitive. Others are time consuming.

Finally it is mandatory to apply several different methods to be carried out in succession. We generally have to count on a 72-hour delay. for precise determination of the nuclear antigens concerned. In general, in order for the exams to be profitable, it is expected that there are approximately 5 patients to be tested.

It can sometimes take a very long time to obtain 5 patients for whom it is necessary, for example, to determine which extractable nuclear antigens are present in their organism.

The object of the present invention is to avoid the abovementioned drawbacks and to develop a screening kit which is easy to implement and which makes it possible to rapidly obtain selective detection of the antinuclear antibodies sought. Preferably, this kit will be of low cost and it can be implemented in a cost-effective manner even if there is only one patient serum to be tested. This kit will advantageously allow greater sensitivity of the test, in particular with the possibility of detecting antibodies from the start of the disease.

These problems are solved, according to the invention, by a kit as described at the beginning, in which the support comprises, per serum to be examined, a group of wells, each group of wells comprising several screening wells each provided for carrying out a test ELISA, the coating of the bottom of each screening well of a group of wells containing one or more nuclear antigens different from that or those contained in the coating of the other screening wells of this group, and optionally a control well, the bottom of which is uncoated, for a blank test.

According to one embodiment of the invention, the support comprises at least one bar in each of which the wells of a group of wells are arranged one behind the other.

Bars are known which are stored on a support and in which are arranged one behind the other wells provided for performing ELISA tests. Such bars are for example placed on the market by the firm Nunc A / S, Roskilde, Denmark, under the name of Nunc-Immuno-Module. In known screening kits, the bottoms of the wells of a strip are all covered with a coating containing one or more identical antigens. Each well of a strip is therefore intended to receive the serum of a different patient and, for reasons of profitability, we must then wait until there are enough patients to be examined, five for example, to start the test. . According to the present invention, on the other hand, the bottoms of each of the screening wells of a strip are covered with a coating containing one or more different antigens and all the screening wells of a strip are intended to receive the serum from the same patient. This results in the possibility of immediately starting the examination, which can be completed 2 hours later.

According to an advantageous embodiment of the invention, each group of wells comprises at least three screening wells, the coating of a first screening well containing at least one deoxyribonucleic acid (DNA), the coating of a second well of screening containing at least one extractable nuclear antigen and the coating of a third screening well containing at least one histone. The coating of the first well may advantageously contain dsDNA and the coating of an additional screening well may then contain sDNA.

According to another embodiment of the invention, each group of wells comprises several screening wells having a bottom covered with a coating containing at least one histone and the bottom coating of each of these contains one or more different histones of that or those contained in the others screening wells containing at least one histone. For example, the coating of a well will contain at least one histone H l rich in lysine while the coating of another well will contain a mixture of histones H2a, H2b, H3 and H4. According to a particular embodiment of the invention, each group of wells comprises several screening wells having a bottom covered with a coating containing at least one extractable nuclear antigen and the coating of the bottom of each of these contains one or more extractable nuclear antigens different from that or those contained in the other screening wells containing at least one extractable nuclear antigen.

According to a preferred embodiment of the invention, the kit also comprises a control well for the positive control serum, this control well having a bottom covered with a coating containing a mixture of the antigens contained in the coatings of at least two separate screening wells from a group of wells, and the positive control serum is a mixture of sera positive for the antigens of this mixture of antigens. Advantageously, the kit also comprises a control well for the negative control serum, this control well having a bottom covered with a coating containing said mixture of antigens and the negative control seruτι is a serum or a mixture of sera which is negative vis- against all the antigens of said mixture. The kit may optionally contain a control well for the serum to be examined, this control well having a bottom covered with a coating containing the said mixture.

Preferably, said mixture of antigens contains all the antigens present in the screening wells, preferably in equal parts. It is advantageous to provide in the kit according to the invention, as a reagent, an anti-human IgG immunoglobulin of the type (H + L). This anti-human immunoglobulin makes it possible to detect the presence in the serum of both immunoglobulin IgM and IgG, that is to say that a very early detection of the appearance of anti-nuclear antibodies becomes possible. This is not possible, according to the state of the art. The screening kit according to the invention can further include an interpretation table of the optical densities obtained in each well of a group at the end of the ELISA test, this interpretation table presenting maximum values of negative serum and minimum values of positive serum for each of the wells of screening, the values having been established on the basis of the results obtained on a large series of positive and negative sera, as well as possibly the values of positive control and of negative control established as a function of the positive and negative sera contained in the kit used. The subject of the invention is also a method of screening for antinuclear antibodies in sera, which allows in a single operation the selective detection of these antibodies and this in a minimal time and with an advanced security of the result. To solve this problem, a method of screening for antinuclear antibodies has been provided according to the invention, comprising, for each serum, several screenings by ELISA test, carried out simultaneously, each of the screenings making it possible to selectively detect the presence in the serum examined of one or more antinuclear antibodies which are different from that or those detectable in the other simultaneous screenings, possibly a blank test, a simultaneous reading of the optical densities of the screenings and the blank test and, on basis of results of said reading, a determination of the absence of anti-nuclear antibodies or of the presence of one or more of the anti-nuclear antibodies sought. The invention therefore offers the advantage of carrying out an ELISA test simultaneously on several samples of the serum of the same patient. This examination may advantageously be preceded by routine detection of anti-nuclear antibodies by indirect immunofluorescence, which makes it possible to already eliminate certain sera, for example those containing anti-centromeres.

The invention will be described in more detail below, using a non-limiting embodiment.

A screening kit is provided comprising a frame supporting several strips, for example of the type placed on the market under the name of Nunc-Immuno-Module. Each strip includes S wells: five screening wells, two control wells and a control well.

The screening wells are developed as follows. First, five solutions of anti-nuclear antigens are prepared:

Solution 1: histones other than Hl (Sigma N 7755). As a stock solution, 10 mg of Sigma N 7755 are stored in 10 ml of carbonate buffer (3.18 g of Na_C, 5.86 g of NaHCO, 0.20 g of NaN, and HO distilled in sufficient quantity for 1 liter) , at a temperature of -20 ° C. For use, dilute 1/10 in carbonate buffer.

Solution 2: histones H1 (Sigma H 5880). As a stock solution, 2 mg of Sigma H 5880 are stored at -20 ° C. in 1 ml of carbonate buffer. For use, dilute 1/10 in the same buffer.

Solution 3: DNAs (Sigma D 3636 - sodium salt of type II calf thymus, highly polymerized).

As a stock solution, 10 mg of this product are stored in 1 ml of carbonate buffer. For use, we dilute. to ι / ιo in this same buffer.

Solution 4: ssDNA.

1 ml of the stock solution is heated

3 at 100 ° C in a water bath, for 10 minutes, and cooled in an ice bath. This solution then contains denatured single-stranded DNA. It is diluted 1/20 in carbonate buffer, for use.

Solution 5: Extractable nuclear antigens. Two extracts containing such anti¬ genes are mixed: calf thymus extract (CTE) and primate spleen extract (PSE), and diluted 50 times in carbonate buffer, after reconstitution of the extracts freeze-dried according to the manufacturer's recommendations (e.g. Mardx Diagnostics Inc., Scotch Plains, New Jersey, United States of America).

These solutions are used for raising the awareness of the screening wells of the strips of a kit according to the invention. 100 μl of the different solutions 1 to 5 are distributed in their wells correspondents and we cover these. It is then left to incubate for 3 hours at a temperature between 0 ° C and 40 ° C, preferably between room temperature and 40 ° C, and advantageously at 37 ° C. The solution residues are removed from the wells, for example by aspiration or centrifugation.

Then distributed in the wells 125 μl of carbonate buffer supplemented with 1% bovine albumin, and the wells are covered. It is again incubated for 1 hour at a temperature between 0 ° C and 40 ° C, preferably 37 ° C. Wash four times with 0.05% PBS Tween buffer (76.5 g NaCl, 9.025 g Na-HPO ^. 2H-.0, 2.1 g KH-PO ^, HO distilled in quantity sufficient to obtain 10 1, 0.05% of Tween 20 FLU A), which is added with 1% of bovine albumin.

Then distributed in the wells 125 μl of a fixing reagent based on PBS Tween 0.05% buffer, supplemented with 10% glycerol. Wait approximately 15 minutes and remove the liquid residues, for example by aspiration or centrifugation.

It is then dried for 1 hour at 37 ° C., the wells are covered and they are stored at 4 ° C., or preferably at -20 ° C. The bars are now sensitized and ready to use.

For the preparation of the two control wells, the solutions I to 5, mentioned above, were mixed in equal parts and it is this mixture which is distributed in these two wells at the start of the sensitization of the wells of the strips. The sensitization process is continued in the same way as that described above for the screening wells, and simultaneously with this one. Screening can be done as soon as the serum of a single patient needs to be examined.

The wells are washed 4 times, before use, with a washing solution formed from PBS Tween 0.05% buffer.

The serum to be examined is diluted to 1/100 in this same buffer, added with 1% bovine albumin. 100 μl are distributed in the screening wells and the control well. The procedure is the same with the positive and negative sera in the control wells, but by distributing there the positive serum and the negative serum. We then incubate for

30 minutes at 37 ° C. After washing 4 times with the washing solution, 100 μi of an anti-human antibody, for example human IgG anti-immunoglobulin, preferably of H + L type (for example that produced by the firm SERA-LAB Ltd, Sussex, Great Britain, under the code: SDL-2515), this anti-human antibody being conjugated to an enzyme, in particular alkaline phosphatase (at a dilution of 1/200 in the 0.05% Tween PBS buffer, supplemented with 1% bovine albumin). It is left to incubate, for example for 60 minutes at 37 ° C. After washing 4 times with the washing solution, 100 μl of a substrate for the enzyme are distributed in the wells, for example paranitrophenyl phosphate, which has been diluted in a substrate buffer, for example diethanolamine . The wells are then covered, and incubated in the dark for 30 minutes at 37 ° C.

The reaction is then blocked with 50 μl of 3N NaOH. The absorbance of the samples can then be read on a standard microtiter plate reader at 405 nm.

During this reading, the value of optical density observed in the screening wells must be subtracted from the value observed in the control well. In the kit of this example, the optical density observed for the negative control must advantageously be less than 0.317 and that for the positive control greater than 1.246. As it is complicated to obtain a standard positive serum and a standard negative serum, since these are mixtures of several sera which must be reactive or not with several antigens at the same time, it is preferable that a table of The interpretation of the results as given below by way of example accompanies each kit, with values corresponding to the control sera actually present in the kit. TABLE Result

It should be understood that the present invention is in no way limited to the embodiment described above and that many modifications could be made thereto without departing from its scope.

One could for example provide kits where each strip contains a number of wells greater than 8. This would allow further selective detection, in particular extractable nuclear antigens.

One could also imagine that, in certain cases, the test as described above with 5 screening wells is not sufficient to screen for the antibody present. This is particularly the case for rheumatic disorders induced by certain NES. We can then provide a second kit similar to the first, but in which the five screening wells each contain one or more ENA different from that or those contained in the other wells. After the first screening (duration of about 2 hours), the second of the same duration is immediately carried out and a very sensitive diagnosis can be made in a very short time compared to what exists in practice.

It is obvious that the ELISA tests are known per se and that various reagents equivalent to those described above can be used for this purpose.

One could also plan not to have control wells, or only a control well for positive serum or for negative serum. We could also provide a control well, no longer for a positive or negative serum, but for the serum to be examined. Such a control well covered with a coating advantageously containing all the antigens provided in the screening wells would allow a comparative control. If the test serum is negative, it must also be negative in the control well. If it is positive for one of the screening wells, it must also be positive in the control well. The presence of this concordance makes it possible to verify the validity of the test.

We could also plan to no longer have control wells for a blank test.

It should be noted that the method according to the invention covers not only the use of a screening kit according to the invention, but also the simultaneous application of screenings by ELISA test even if the wells used do not come from the same bar. Indeed, for example in the case of screening for ENA, it turns out that these are quite sensitive and that the manufacture of a strip each of whose wells is coated with a different ENA can be complex. We can therefore provide in the kit a series of parallel bars, for example five in number. Each of these bars contains wells coated in a similar manner, with a determined ENA, but each of the wells of the bar is provided for separable from the others. , for example by cutting. It then suffices to combine 5 separate wells from a different strip and simultaneously apply an ELISA test in these 5 wells on the same serum to be examined in order to fulfill the conditions of the process according to the invention.

Claims

1. Antinuclear antibody screening kit in sera, comprising a support in which are arranged several wells having a bottom covered with a coating containing at least one nuclear antigen, reagents suitable for performing an ELISA test, as well as possibly a positive control serum and a negative control serum, characterized in that the support comprises a group of wells per serum to be examined, and in that each group of wells comprises several screening wells each provided for carrying out an ELISA test, the coating of the bottom of each screening well of a group of wells containing one or more nuclear antigens different from that or those contained in the coating of the other screening wells of this group, and possibly a control well, the bottom of which is devoid of coating , for a blank test.

2. Screening kit according to claim

1, characterized in that the support comprises at least one bar in each of which the wells of a group of wells are arranged one behind the other.

3. Screening kit according to either of claims 1 and 2, characterized in that each group of wells comprises at least three screening wells, the coating of a first screening well containing at least one deoxy acid ¬ ribonucleic acid (DNA), the coating of a second screening well containing at least one extractable nuclear antigen and the coating of a third screening well containing at least one histone. . Screening kit according to claim

3, characterized in that the coating of the first screening well contains native or double-stranded deoxyribonucleic acid (dsDNA) and in that an additional screening well contains denatured or single-stranded deoxyribonucleic acid (ssDNA).

5. Screening kit according to either of claims 1 to 4, characterized in that each group of wells comprises several screening wells having a bottom covered with a coating containing at least one histone and in that the the bottom coating of each of these contains one or more histones different from that or those contained in the other screening wells containing at least one histone.

6. Screening kit according to either of claims 1 to 5, characterized in that each group of wells comprises several screening wells having a bottom covered with a coating containing at least one extrac¬ nuclear antigen tible and in that the bottom coating of each of these contains one or more extractable nuclear antigens different from that or those contained in the other screening wells containing at least one extractable nuclear antigen.

7. Screening kit according to any one of claims 1 to 6, characterized in that it further comprises a control well for the positive control serum, this control well having a bottom covered with a coating containing a mixture of antigens contained in the coatings of at least two separate screening wells from a group of wells, and in that the positive control serum is a mixture of sera positive for the antigens of this mixture of antigens.

S. Screening kit according to any one of claims 1 to 7, characterized in that it further comprises a control well for the negative control serum, this control well having a bottom covered with a coating containing a mixture of the antigens contained in the coatings of at least two separate wells of a group of wells and in that the negative control serum is a serum or a mixture of sera which is negative with respect to all the antigens of said mixture.

9. Screening kit according to any one of claims 1 to 8, characterized in that it further comprises a control well for the serum to be examined, this control well having a bottom covered with a coating containing a mixture antigens contained in the coatings of at least two separate wells from a group of wells.

10. Screening kit according to either of claims 7 to 9, characterized in that said mixture of antigens contains all the antigens present in the screening wells, preferably in equal parts.

11. Screening kit according to any one of claims 1 to 10, characterized in that it comprises, as reagents, a conjugate of an anti-human IgG immunoglobulin and alkaline phosphatase as well as paranitrophenyl phosphate.

12. Screening kit according to claim 11, characterized in that the conjugation product comprises an anti-human IgG immunoglobulin type (H + L).

13. Screening kit according to any one of claims 1 to 10, characterized in that it further comprises a table for interpretation of the optical densities obtained in each well of a group at the end of the ELISA test, this table of interpretation presenting maximum values of negative serum for each of the screening wells and minimum values of positive serum for each of them, these values having been established according to the results obtained on a large series of positive and negative sera, as well that possibly positive control and negative control values established as a function of the positive and negative sera contained in the kit used.

14. A method of screening for antinuclear antibodies in sera comprising, for each serum, several screenings by ELISA test, carried out simultaneously, each of the screenings simultaneously making it possible to selectively detect the presence in the examined serum of one or more antibodies antinuclear which are different from that or those detectable in the other simultaneous screenings, possibly a blank test, a simultaneous reading of the optical densities of the screening and bianc test and, on the basis of the results of said reading, a determination of the absence of antinuclear antibodies or of the presence of one or more of the antinuclear antibodies sought.

15. Screening method according to claim

14, characterized in that it simultaneously comprises, for each serum, at least one screening for deoxyribonucleic acid, at least one screening for histone and at least one screening for extractable nuclear antigen.

16. Screening method according to either of claims 14 and 15, characterized in that it comprises, before said screenings, detection of antinuclear antibodies by indirect immunofluorescence.