JPH0331299A - Cell surface protein of 94 kda in in-vitro examination field of especially systemic lupus erythematodes(sle),its isolation and use - Google Patents

Abstract

NEW MATERIAL: Protein or its fragment forming an immunological composite along with a monoclomal antibody PME 77 being cell membrane protein of 94kDa or optitional other antibody capable of recognizing the same epitope as one recognized by the monoclonal body when this antibody is bonded to a composite consisting of a nucleosome, DNA and histone and not recognized by the monoclonal antibody PME 77 in the absence of the nucleosome or the composite of DNA and histone.

USE: An invitro diagnostic agent of systemic lumpus erythematosus(SLE).

PROCESS: For example, a purified membrane of a Raji human lymphoblast cell is treated with a surfactant to extract protein and this extract is brought into contact with a composite consisting of purified nucleosome or purified nucleic acid and histone and, after the nucleosome or the composite is fixed to a support, protein is eluted from a support by an eluent.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for the implementation of a method for detecting antibodies that are specifically directed against a 94 kDa cell surface protein and that are present in the serum of patients suffering from systemic lupus erythematosus (SLE). , 94 kDa cell surface protein and uses thereof.

SLE is an autoimmune disease characterized by a disturbance in the immune system that causes the production of anti-double-stranded DNA antibodies that are responsible for tissue lesions.

Therefore, most of the tissue lesions observed in human SLE cases are likely due to the formation of DNA-anti-DNA immune complexes and their deposition within the kidney and other tissues (IIE I KK I LA.

R1 et al. (1987) Nature (London)
328, 445-449; HAR[EL-BIE
LLAN A, et al. (1988) J, IMMUNOL.
, 140.

2431-2435).

I: Until now, it has been considered to be a characteristic marker of SLE.

Autoimmune mice, in which the disease can develop in the same way as humans, represent a good model for the study of the immune mechanisms putatively responsible for SLE. In particular, B/W MRL/
1 Autoimmune mice have SLE identical to human SLE. The serum of patients suffering from SLE contains a mixture of antibodies against different nucleic acids. Therefore, the use of hybridoma methods was justified to better characterize the specificity of anti-DNA antibodies.

As a result, - antibodies against double-stranded DNA and double-stranded DNA
An attempt was made to differentiate it from antibodies directed against.

Although this distinction has had some success due to the clinical correlations attributed to it, it has been rather unsatisfactory due to the technical difficulties of characterizing DNA in terms of single-stranded or double-stranded DNA. It quickly became clear that it was something.

Furthermore, it has been observed that the serum of patients suffering from SLE contains not only DNA but also a mixture of RNA (ribonucleic acid) and antibodies capable of reacting with nuclear proteins. It was difficult to assess the individual significance of the antibodies generated.

Obstacles to the detailed analysis of anti-DNA antibodies spontaneously produced in SLE mice have recently arisen from anti-single-stranded DNA monoclonal antibodies (J, 1mmuno 1. (1980)).

124, 1499-1502) and antibodies against double-stranded DNA (J. Ismunol, (1982),
128, 895-89g) by the production of hybridomas.

The experiment was performed on Llle Allerican Rhe
umatism＾5soclaLIon Tan rE
Not only did it show that these titers could not be correlated with the modified criteria for assessment of clinical progression of the disease reported by M Cohen As, Frles J, F, et al. (19g2). Modified criteria for the classification of systemic lupus erythematosus; they showed that certain patients with clinical signs of SLE are not carriers of anti-DNA antibodies.

For all of them, pre-considered DNA-anti-DNA
The involvement of DNA in the formation of immune complexes has already been discussed.

Therefore, double-stranded DNA (hereinafter, for example, M A b
A monoclonal antibody (abbreviated as MAb) that is specific for PME 77 and obtained from autoimmune B/W mice is a monoclonal antibody (abbreviated as MAb) that is directed against Raj I human B lymphoblastoid cells. The binding to the surface and the fact that this binding was caused by DNA5a.I (2
00tt g/ml) (an enzyme that cleaves DNA) but not by treatment with protease K (10% g/ml) (an enzyme that cleaves DNA).
It has already been recognized that the antigen recognized on the cell surface is a protein in nature (PNAS).

81.3B43-3B45 (1984); Eu
r, J, I+nll1uno1. , 14°283~28
B, (19H)). Monoclonal anti-DNA antibodies against some antigens are phospholipids (LAPEI?).

E, M, et al. (1981) J, EB, Mcd, 15
3, 897-909) or proteoglycans (
PAABER, P. et al. (1986) J, ClIn, 1n.
Vest, 77, 1824-1827) suggests that target antigens other than DNA itself may be involved in SLE.

Furthermore, analysis of the cell supernatants obtained after the above-mentioned limiting treatments shows that the latter proteoglycans, in a mixture with a number of other proteins, can be recognized by MAb PME77 after gel electrophoresis and immunotransfer. It was found to contain five polypeptides. Their molecular weights are, in order, 34,000.33□000.1, respectively.
7,000.18.000 and 14,000 (JACO
B, L, et al. (1984) Proc, Natl, Aca.
d, Scj,, (USA) 81, 3843-3845
, JACOB, L et al. (198B) Proe, Nat.
l.

Acad, Sc1. , (USA) 83.6970~6
974). In view of the tactics used, it was suggested that these polypeptides were distributed on the cell surface.

(JACOB L, et al. (198[i) supra).

It was considered to be the best study performed on the cell membrane of leukocytes or mononuclear cells (REKVIC, O, P, et al. (1980) +J
, Exp.

Mcd,, 152, 1720-1733;
ll0LER8, V. M. et al. (1985).

J, Cl1n, 1nvest, 78. 991
~998; l3ENNETT, R,M.

(1987), J, Exp,) 4ad, 16
8, 850-863).

Recently, an 8'OkDa cell surface protein was detected by purification on a cellulose column coupled to oligo(dT) (an oligonucleotide consisting of deoxythymidine). This cell surface protein may play a role in the transport of oligonucleotides from outside to inside the cell, but the internalization suggested in this model is a long process (approximately 50 hours) and therefore has not been formally proven. There was no (LOKIE, S. L. et al. (1989),
Proc, Natl, Acad, Scl.

(USA), 86.3474-347g).

However, various recent studies have not been able to infer the pathological mechanism responsible for the development of SLE.

The present invention is precisely the result of cell membranes implicated in the development of SLE.

More particularly, the present invention is the result of the discovery of biological agents, in particular the in vitro expression of such agents in the blood of four individuals, which can be correlated with SLE.

The present invention particularly relates to a novel method for in vitro diagnosis of SLE.

With the in vitro diagnostic method of the present invention, SLE can be effectively monitored within an individual. This makes it possible in particular to reveal the onset of SLE in an individual, or even to monitor the progression of SLE from the acute phase towards the remission phase in this individual.

The subject of the invention is capable of forming an immunological complex with the monoclonal antibody P M E 77, or that said antibody consists of nucleosomes (polynucleosomes, oligonucleosomes and mononucleosomes) or of DNA and histones. be able to form epidemiological complexes when binding to complexes - not recognized by the purified monoclonal antibody PME77 in the absence of nucleotides or in the absence of complexes consisting of DNA and histones. or any fragment of the protein capable of retaining the above-mentioned immunological properties.

Specifically, the above-mentioned histones are H and H2A.

■ It is of type HB, H3 or H4. In vivo, histones and DNA combine to form mononucleosomes (unit particles of chromatin).

The 94 kDa protein of the present invention is the antibody P M E 77
D of histones and any kind of DNA, especially phage λ
When binding to the complex formed with NA, the antibody P
It is possible to form immunological complexes with M E 77.

As already pointed out above, the 94 kDa protein of the invention is also compatible with antibody P M E 77 when it binds to nucleosomes, particularly nucleosomes obtained from L 1210 murine leukemia cells. It is also possible to form chemical complexes.

More specifically, the 94 kDa protein of the invention is: -Ra
ji human cells, monkey fibroblasts (CVI cells) RI
Nm is able to be isolated from the surface of cells such as rat pancreatic tumor cells, - is hydrophobic, and contains detergents, especially TrlL.
The solution of onX-114 or X-Ion type does not contain insoluble cell membrane constituents;
It is characterized by being found in tonX-114 and being removed from the cell surface by the action of trypsin.

The present invention is also characterized in that it is capable of forming an immunological complex with the serum of a patient suffering from SLE.
It relates to the above 94 kDa protein or any fragment of this protein.

Another subject of the invention is the treatment of purified animal or human cell membranes suspected of containing the described polypeptides with a Trlton Extraction of said protein by collecting said protein-containing phase, one purified nucleosomes, or
contacting with a complex formed from a nucleic acid and purified histone, provided that these nucleosomes or complexes are immobilized on a carrier (in particular an affinity chromatography carrier or an ion exchange column); A method for isolating and recovering a 94 kDa protein, comprising recovering the protein retained on the carrier by contacting the carrier with a solution that leads to the separation of the 94 kDa protein. Specifically, this solution is: 2M
NacΩ, O, 1M carbonate-bicarbonate buffer, pH
9.3M urea.

The isolation method defined above is advantageously carried out using specific polyclonal or monoclonal antibodies against the 94 kDa protein of the invention or against any of the above-mentioned fragments of this protein.

In this case, the nucleosomes or the complexes consisting of nucleic acids and histones used in the above-mentioned affinity chromatography carrier are treated with an antibody, in particular a monoclonal antibody specific for the protein of the present invention, by 916° with the above-mentioned LOKE S, L, ( It is also possible to carry out the protein isolation method of the present invention using an affinity chromatography carrier enclosing oligo(dT) and gel filtration according to the method described in the paper of 1989).

The invention also relates to the antibodies themselves raised against the 94 kDa protein of the invention or against any of the protein fragments described above.

It will be clear that this production is not limited to polyclonal antibodies.

Such monoclonal antibodies may be produced by hybridoma methods, the general principles of which are described below.

When an animal is first immunized with the above-mentioned 94 kDa protein or a fragment of this protein, the animal's B lymphocytes eventually produce antibodies against this protein. These antibody-producing lymphocytes are then fused with "permanently proliferating" myeloma cells to generate hybridomas. Starting from the heterogeneous mixture of cells obtained in this way, a selection is then carried out of cells that produce specific antibodies and are capable of proliferating indefinitely. Each hybridoma can be propagated in clonal form, each produced a monoclonal antibody, and its recognition properties tested for the protein of the invention, for example by immunotransfer in one or two dimensions and by immunofluorescence.

It will be clear that the invention also relates to polypeptide fractions with a molecular weight lower than the 94 kDa protein, provided that the polypeptide fractions have antigenic sites that can be recognized by the same monoclonal antibody. .

If monoclonal antibodies of the type considered above are available, the same antigen can be isolated from the antigen by known methods of cleavage of polypeptides, e.g. by enzymes capable of cleaving larger polypeptides at specific sites. It will be clear to those skilled in the art that one can seek to isolate shorter peptide sequences containing the site. A specific example of such a protein is the enzyme 5taphyloco
ccus aurcus V8. aChymotrypsin.

Mouse submandibular gland protease commercially available from Bochringcr) f, the peptides Gly-Pro and c
Vlbrl that specifically recognizes ryARa etc.

alginolyLIcus chemovar Io
An example of this is collagenase from PHAUS.

Moreover, after peptide sequencing of the proteins of the invention, the corresponding nucleotide sequences can be reconstructed; these corresponding nucleotide sequences can be synthesized and cloned in sequence.

The present invention further relates to a method for in vitro detection of complexes consisting of antibodies bound to nucleosomes, which may be correlated with systemic lupus erythematosus (SLE), in tissues or biological fluids suspected of containing them. ,
This procedure involves subjecting the tissue or biological fluid to the 94 of the present invention under conditions that cause an in vitro immune reaction between the complex and the protein possibly present in the tissue or biological fluid. kDa protein, or any fragment of this protein, and in vitro detection of the immunological complex that is likely to form between said protein on the one hand and an antibody bound to a nucleosome on the other hand. .

Preferably, the biological medium is constituted by human serum or by renal glomeruli of SLE patients.

Any standard method may be used to perform such detection.

immunization (RIA) method, or equivalent method.

The invention therefore also relates to the 94 kDa protein or any fragment of this protein as defined above, labeled with a suitable marker of the type enzyme, fluorescent, radioactive, etc.

Such a method may include, for example, the following steps: depositing a limited amount of the protein of the invention in the wells of a microtiter plate, and depositing the serum required for diagnosis in increasing dilutions into the wells. - incubating the microplate; repeatedly washing the microplate; - introducing labeled antibodies against blood immunoglobulins into the microplate wells, where the substrate is hydrolyzed; Therefore, by carrying out the labeling of these antibodies with enzymes selected from those capable of changing the energy absorption of this substrate at least at specific wavelengths, and by comparison with a control, the amount of hydrolyzed substrate is determined. and detecting the amount.

The present invention also relates to a tnvttro diagnostic kit for SLE, which kit comprises: (1) a composition containing the 94 kDa protein of the present invention (labeled or unlabeled) or any fragment of this protein as described above; reagents for constituting a medium suitable for carrying out the reaction; and (1) for constituting the detection of a complex produced by an immunological reaction formed between the protein and the antibody-nucleosome complex. a reagent which may in turn be recognized by a labeled reagent, provided that such reagent may contain a marker or, in the more specific case where said protein is not labeled, a reagent which can in turn be recognized by said protein; Vs lacking recognized antibodies! and a target sample containing the eupome complex.

Another object of the present invention is to contact the complex formed between one specific nucleic acid and the other histone with a cell capable of expressing the protein of the present invention on the cell surface, so that the specific nucleic acid, A method of cell transfection comprising constituting internalization within said cell of a complete body consisting of histones and said polypeptide.

According to the advantageous feature, the above-mentioned LOKB S, L, et al.
The above transfection method can cause internalization of the nucleic acid within a few minutes, whereas the procedure described in the article by 989) takes several hours.

The invention more particularly provides that said specific nucleic acid comprises a nucleic acid sequence encoding a specific polypeptide and, if necessary,
It relates to a transfection method as described above, containing regulatory elements for directing the expression of this nucleic acid fragment in a cellular host, in particular a promoter recognized by the polymerase of said cellular host.

placing transformed host cells in culture in a suitable culture medium;
Isolation of the specific polypeptide is then performed by recovering the specific polypeptide from these cells or from the culture medium.

The invention also provides, together with physiologically acceptable excipients,
The present invention relates to pharmaceutical compositions containing specific therapeutically active nucleic acids bound to histones or nucleotides.

refers to any nucleotide (particularly an oligonucleotide) that is active.

"Selective blockade" refers to the blockade of sequences associated with the initiation, propagation, or termination of nucleic acid replication during transcription of one or more genes and/or of the RNA messengers corresponding to those genes. Translation means both.

It is also the use of these pharmaceutical compositions in the treatment of diseases of therapeutic or tumor origin that prevents the "blocked" sequences from acquiring proteins, such as enzymes or chemicals.

The present invention also relates to anti-ideotype monoclonal antibodies against the above-mentioned monoclonal antibodies of the present invention.

The invention further relates to pharmaceutical compositions comprising an anti-ideotionic antibody as described above and a physiologically acceptable excipient, and the use of these compositions, particularly in the treatment of SLE.

The present invention also relates to compositions constituted by human or animal pancreatic cells covalently linked to a limited amount of the 94 kDa protein or 1,000 unique fragments of this protein of the invention, according to techniques known to those skilled in the art. .

After introduction into a host (human or animal) using these compositions with physiologically acceptable excipients, 94%
It is possible to induce suppressor T cells specific for kDa proteins.

The amount of 94 kDa protein bound to pancreatic cells is determined as a function of the desired degree of immunosuppression.

According to a particularly interesting embodiment, the present invention provides for the production of antibodies associated with SLE, possibly associated with nucleosomes, in biological tissues or body fluids likely to contain them.
The method involves: contacting the biological tissue or body fluid with the complex formed between the 94 kDa protein and the nucleosome; or contacting the biological tissue or body fluid with the complex formed between the 94 kDa protein and the nucleosome; Contacting the complex formed between the nucleosome and any fragment of this 14 kDa protein, if bound, if necessary, by ~IAb PME77 and/or or having one or several sites recognizable by an antibody present in the blood of an SLE patient, one between said antibody and said complex possibly present in a biological tissue or body fluid. carrying out the contact under conditions that allow an immunological reaction to occur in vitro, and detecting in vitro' the immunological complex formed between said complex and said antibody. It is characterized by

The invention more particularly relates to the above-mentioned method, which method comprises: - preparing said 94 kDa protein or a protein fragment thereof as above on a solid support, in particular in a microtitration for use in ELISA (enzyme-linked immunosorbent assay); binding to the wells of a plate, such as a plate; - removing unbound 94 kDa protein or fragment thereof bound to said solid support;
4 kDa protein or a fragment thereof is incubated with the nucleome to form a complex between the 94 kDa protein or fragment thereof and the nucleosome (hereinafter these complexes are referred to as 94 kDa-nucleoworm 1 summer coalescence). removing nucleosomes that are not bound to the 94 kDa protein bound to a solid support or fragments of this protein; - incubating with a body fluid; - removing components of the biological tissue or body fluid that are not bound to the 94 kDa-nucleosome complex bound to the solid support; - when present in the biological tissue or body fluid; This includes detecting the immune complex, if present, formed between the antibody that presumably binds to the suspected nucleosome and the 94 kDa-nucleosome complex.

The invention also relates to the above method, which method comprises: binding the solid-94 kDa-nucleosome complex to a columnar support, in particular to the wells of a plate such as a microtiter plate used in ELISA (enzyme-linked immunosorbent assay); provided that these 94 kDa-nucleosome complexes are preformed by binding said 94 kDa protein or its protein fragment as described above to nucleosomes.

94 above, which is not bound to a solid support;
removing the 94 kDa-nucleosome complex bound to a solid support, incubating the 94 kDa-nucleosome complex bound to a solid support with said biological tissue or body fluid; removing the constituents of said biological tissue or body fluid, the immunological formation likely to form between the antibody and the 94 kDa-nucleosome complex that presumably binds to the nucleosomes believed to be present in the biological tissue or body fluid; including detecting the target complex.

According to a preferred method of carrying out the above method, antibodies specific for SLE are prepared using anti-immunoglobulins, preferably human, which are likely to be immunologically reactive with antibodies possibly present in biological tissues or body fluids. Detection phase is carried out. These anti-immunoglobulins are especially labeled using enzymes such as alkaline phosphatase.

The expression "nucleosome" refers to polynucleosomes (containing several hundred side nucleosomes), oligonucleosomes (containing approximately 10-20 nucleosomes), mononucleosomes, chromatin, or complexes formed by DNA and histones. includes.

Preferably, the 94 kDa protein is composed of a DNA fragment of 150 to 200 base pairs and histones, especially of the HA, HB, H3 or 2 H4 type, in particular a leukemic mouse L.
Binds to mononucleosomes derived from 1210.

The subject of the present invention is the application of the method described above for the in vitro diagnosis of SLE, more particularly for monitoring the progression of SLE from the acute phase to the remission phase.

Preferably, the biological medium is constituted by human serum or by renal glomeruli of SLE patients.

The present invention also relates to a biological complex formed between the 94 kDa protein described above and a nucleosome.

A preferred biological complex of the present invention is such that the 94 kDa protein is obtained from leukemic mouse L 1210,
It is characterized by binding to mononucleosomes composed of 200 base pair DNA fragments.

In the complex between the 94 kDa protein and the nucleosomes, it is to be understood that all types of bonds (covalent or non-covalent) are likely to occur between these nucleosomes and said protein.

The present invention also provides that these polypeptide fractions have binding sites that are likely to be recognized by nucleosomes and that, if necessary, the binding sites can be recognized by the same monoclonal antibody that recognizes the 94 kDa protein. If possible, it relates to a complex formed by binding a nucleosome to a polypeptide fraction with a lower molecular weight than the 94 kDa protein.

As used herein, the expression "r94kDa-nucleosome complex" refers to the complex formed between a nucleosome and a 94kDa cell surface protein, as well as the complex formed between a nucleosome and a fragment of the 94kDa protein mentioned above. A name is given to the complex formed.

Another subject of the invention is biological reagents (or laboratory reagents) comprising biological complexes as defined above and the use of these reagents, in particular for carrying out the methods described herein. It is.

The present invention further provides antibodies that bind to SLE in vltr.
For the diagnostic kit for detection at
a protein or any fragment of this protein and - for constituting a medium suitable for the formation of a complex between a nucleosome and said 94 kDa protein or a complex between a nucleosome and said fragment of this protein. a reagent and a nucleosome - an antibody possibly present in a biological tissue or body fluid and the 94 kDa-nucleosome complex;
- only reagents possibly present in biological tissues or body fluids, and one for configuring the detection of the complex formed during the immunological reaction between said antibody and the 94 kDa-nucleosome complex. and reagents.

A particularly useful diagnostic kit of the invention for the in vitro detection of antibodies bound to SLE is the biological 94 kDa antibody described above, optionally attached to a solid support.
- a suitable medium for carrying out an immunological reaction between the 94 kDa nucleosome complex and an antibody possibly present in a biological tissue or body fluid. and reagents for detecting a complex formed during an immunological reaction between said antibody and a 94 kDa-nucleosome complex.

Reagents for effectively constituting the detection of the complex formed between the antibody and the 94 kDa-nucleosome complex may carry a marker or be recognized in turn by a labeling reagent.

The present invention also provides nucleosomes with associated 94 kD
a complex constituted by the protein or by any fragment of this protein as described above associated with a nucleosome. (23) Claim 1
Item 7 or regarding monoclonal antibodies.

These antibodies capable of immunologically reacting with the 94 kDa-nucleosome complexes defined above are more commonly designated by the expression "anti-94 kDa-nucleosome antibodies".

Such monoclonal antibodies may be produced by hybridoma methods, the general principles of which are described below.

An animal is first immunized with the 94 kDa-nucleosome complexes described above, and over time the animal's B lymphocytes produce antibodies against these complexes.

These antibody-producing lymphocytes are then fused with "permanently proliferating" myeloma cells to generate hybridomas.

Starting from the heterogeneous mixture of cells thus obtained 1
．． Next, cells that can produce a specific antibody and can proliferate indefinitely are selected. According to a particularly advantageous feature of the invention, each hybridoma is propagated into clonal form and each is led to the production of monoclonal antibodies, the recognition properties of which are determined with respect to the conjugate of the invention, e.g.
The above-mentioned anti-94 kDa-nucleosome antibody is of great interest in carrying out in vitro quantitative analysis (or administration, or titration) methods of antibodies bound to SLE that may be contained in biological samples. be.

More specifically, the present invention aims to determine the amount of antibodies correlated with SLE that are likely to be contained in biological tissues or body fluids.
For in vitro measurements, the method involves contacting a 94 kDa-nucleosome complex with a defined amount of an anti-94 kDa-nucleosome antibody in such a way that all of the complex is engaged in immunological binding with the antibody. an antibody possibly present in a biological tissue or body fluid;
Anti-9 against the above 94 kDa-nucleosome complex
Under conditions to constitute the performance of a competitive reaction between the 4 kDa-nucleosome antibody, the product of the previous immunological reaction, i.e. (94 kDa-nucleosome) (anti-94 kD
a- nucleosome) complexes with a biological tissue or body fluid, - an amount of anti-94 kDa-nucleosome antibody that is not bound to the 94 kDa-nucleosome complexes or is still bound to these complexes; Detecting any of the amounts of said antibodies in 1n vltro, provided that the amount of antibodies detected is S contained in the biological tissue or body fluid.
It is characterized by including a correlation with the amount of antibody bound to LE.

The above in vitro administration method includes the following steps:
-94 kDa-nucleosome complex on a solid support,
binding to the wells of a plate, such as a microtiter plate used in particular for ELISA; removing the 94 kDa-nucleosome complex that is not bound to the solid support; Incubating the bound complex with a determined amount of anti-94 kDa-nucleosome antibody in excess of the complex, and incubating the bound complex with a defined amount of anti-94 kDa-nucleosome antibody in excess of the complex, The complex formed during the incubation step (94 kDa-nucleosome)-(anti-94 kDa-
removing the anti-94 kDa-nucleosome antibody that is no longer bound to the carrier-bound 94 kDa-nucleosome complex, and if necessary removing the anti-94 kDa-nucleosome antibody that is no longer bound to the carrier-bound 94 kDa-nucleosome complex. in a washing solution; and collecting either the amount of anti-94 kDa-nucleosome antibody that is not bound to -94 kDa-nucleosome complexes or the amount of said antibody that is still bound to these complexes in It is effective to perform the step of detecting by vltro, provided that the amount of antibody detected is correlated with the amount of antibody bound to SLE contained in biological tissues or body fluids.

According to an effective method of carrying out the in vHro administration described above, the anti-94 kDa-nucleosomal antibody is specifically labeled using an enzyme. Determination of the amount of SLE-specific antibodies possibly present in biological tissues or body fluids: - by direct detection of the amount of labeled 94 kDa-nucleosomal antibodies recovered after the final washing step of the above method;
The amount of LE-specific antibody is determined by the amount of labeled anti-94k recovered in the washing solution.
directly corresponds to the amount of Da-nucleosome antibody - or by indirectly detecting the amount of labeled anti-94 kDa-nucleosome antibody bound to the 94 kDa-nucleosome complex; in this case, the amount of SLE-specific antibody , on the one hand, the amount of labeled anti-94 kDa-nucleosome antibody bound to the 94 kDa-nucleosome complex before the incubation step with the biological tissue or body fluid, and on the other hand, the incubation with the biological tissue or body fluid and the ln above. Corresponding to the difference between the amount of anti-94 kDa-nucleosome antibody bound to the 94 kDa-nucleosome complex after the final step of washing according to the in vitro administration method.

Another method of carrying out the ln vitro administration method described above is to leave the anti-94 kDa-nucleosome antibody unlabeled and to determine whether the amount of anti-94 kDa-nucleosome antibody recovered in the wash solution after the final wash step or the biological After the final step of incubation with biological tissue or body fluids and washing, the 94 kDa-nucleoworms remained 1M.
. The amount of these antibodies bound to the complex is detected using stray anti-immunopropurine, which is expected to be immunologically reactive with these antibodies.

The present invention also provides 1nVItrO of the above-mentioned SLE-specific antibody.
A diagnostic kit for the in vitro detection and administration of antibodies specific for SLE, characterized in that it contains all the components of the detection kit V, and if necessary, especially with the aid of enzyme or radioactive markers, the labeled J' J Concerning the defined amount of anti-94 kDa-nucleosome antibody identified.

The method for detecting 94 kDa-nucleosomes of the present invention will be explained below using specific examples.

Nucleosomes were detected by immunoblotting at 94
TritonX - directly binds to kDa protein
After extraction with 5DS-polyacrylamide (7,
5%) and then transferred onto a nitrocellulose sheet. Nucleus, t-some (1
μg) was incubated on the sheet for 1 hour at room temperature, and then the sheet was incubated with buffer T BST (
Tris loOmM, N a C1) 150mM
.

7ween 0.05%, pH 8) for 10 minutes
Wash twice.

Next, this sheet was coated with refined TAMAb PME77 (1
0 μg).

After washing, the sheet is incubated with a 1/1000 dilution of mouse anti-immunoglobulin labeled with alkaline phosphatase. After washing, the anti-immunoglobulin bound to the sheet was washed with Tris loOmM
, N a CΩ 100mM and MgCΩ 25IfiM
V! with a pH of 9.5 consisting of Visualize using a buffer solution.

Stop the reaction using EDTA 10IIIM.

The hybridoma secreting MAb PME77 was deposited with accession number 1-555 at the Collection Nation, Institut Pasteur, Paris (France).
ale des cultures de micro-
It has been deposited with the National Institute of Organs and Organs (CNCM).

The nucleosomes bound to the 94 kDa protein or the above-mentioned protein fragments in the biological complexes of the invention, and more particularly mononucleosomes, are among those that appear to be bound to the 94 kDa protein. Generally selected from

In particular, Luttcr C, (1978) J, Mo1. B
Nucleosomes are obtained from any biological tissue or body fluid likely to contain nucleosomes by classical techniques used to obtain nucleosomes from cell nuclei, according to the method described in J.L.

An aliquot of the nucleosome preparation obtained as above can be used to test the binding of the 94 kDa protein to nucleosomes in order to establish whether these nucleosomes can be used in the complexes of the invention. .

As a specific example, this test involves contacting these nucleosomes with a 94 kDa protein bound to a solid support, followed by washing to remove nucleosomes that are not bound to the 94 kDa protein, followed by 94 kDa protein binding to the 94 kDa protein.
Any labeled reagent that seems to specifically recognize nucleosomes bound to Da protein, especially MA
b using PME77 and anti-immunoglobulin labeled with alkaline phosphatase according to the method described above.
4 kDa'ffi involves detecting nucleosomes bound to white matter.

Further features of the present invention will become clear in the course of describing the method and conditions for obtaining the protein according to the present invention.

■-Materials and Methods - RI Na-based cell system of rat pancreatic islet of Langerhans tumor cells (
GAZDARA, F. et al. (1980) Proc, Na
tl, Acad, Scl.

(USA) 77, 3519-3523), the Rajl human lymphoblastoid cell line and monkey fibroblasts (CVI cells), TR0N F, et al. (1984) IEur, J.
I+n+uuno1. , 14.283-28G.

- Mouse Monoclonal Antibody (MCA) Hybridoma PME 77 secreting DNA antibodies was co-incubated with a non-secreting myeloma line (P 3 X 133A g 8.05) and (N
It was isolated after fusion with Z B selection of hybrids, cloning and sack rolling of this cell line,
Purification of antibodies, characterization of antibody classes and subclasses,
As well as the specificity of the antibody, 1"l?ON F, et al. (198
0) J, 1mn+uno1. ．． 125, 2805-2
809; and JACOBl7. (1982) J, I
mmunol, +128, 895-89g. MCA PME77 is an IgG
JACOB L, et al. (1999), which is described as a 2b antibody and specific for double-stranded (+15 DNA) (above).
g2)).

- Preparation of purified membrane fractions GASTINEL L, N. (1982) Blo
ch! m, Blophys, Acta.

Both purified membranes were prepared according to the method described in 684.117-126. Ten million live cells were washed three times with phosphate buffered saline (PBS). This cell'1
゜mM Tris-HCΩ, lomM CaCΩ2
and suspended in 7 ml of buffer A containing 0.25 M sucrose and adjusted to pH 7.4. Homogenization was performed in a Dounce homogenizer in the presence of protease inhibitors. This homogenate is 1000×
The mixture was centrifuged for 20 minutes. Next, this supernatant was added to 2 (1,
(corresponds to a preparation of 10 (IX g).

- Extraction of purified membrane fraction with TrltonX-114 (T
114 (Fluka) (BORDIIERJ, (1
981) J, Blol, Chant, 25G,
11304-1807) at 4°C,
% sucrose solution at 100.000 x g for 1 hour at 4°C, then transferred to a 37°C tube and incubated for 3 minutes. After centrifugation at 3.00 rpm for 3 minutes at room temperature, the pellet and supernatant were saved and aliquoted. Hydrophobic membrane proteins (pellet) were collected in the surfactant phase and soluble proteins (supernatant) were collected in water tn.

! - Ribsin-treated RI Nm live cells were incubated at 0°C5. tO and 20 minutes 1147
．． Treated with 50 Mg/ml trypsin. Cell viability was assessed by trypan blue exclusion test: the viability was 98% in all preparations and remained unchanged at the end of the trypsin treatment.

Cell extracts were extracted using T X-114 as described above. The surfactant phase obtained by trypsin treatment was analyzed by immunotransfer method.

The supernatant of monoclonal antibody P M E 77 was loaded onto a DNA cellulose (Sigma) column. After washing with PBS, MAb PME77 was diluted with 2MNa (1!
pl+8).

It a n k containing MgCj72 (20Oug)
DN A sc I (Worth
lngton) at 4°C for 1 hour.

- Immunotransfer analysis JACOB L, et al. (1984) Proc, Natl.
, Acad, Sci, (IISA).

MAbP M eluted from this analytical DNA-cellulose column according to the method described in J. H., 3843-8845.
E77 was passed through a column consisting of histones covalently bound to activated Tris-acrylic beads (IBF) in PBS buffer. The eluate was purified M used in these experiments.
Corresponds to Ab PME77.

-DNA and histone λ phage (Boebringer Mannheim)
) DNA was treated with protease K (Boehrlng
er Mannhelm; 100μ for 1 hour at 4°C
g/ml) followed by phenol extraction for purification. Histones were fixed with lOμH cells in a freshly prepared solution of paraformaldehyde and MAb PM
Incubation with E77 and fluorescein (BIosys, CoLIlplcgnc-France)
This was revealed using mouse anti-IgG labeled with .

Preparation of chromatin and nucleosomes LUTTERC
, (1978) J, Mo1. Biol,, 12
4. Chromatin and nucleosomes were prepared from the cough of leukemia cells L 1210, which were cultured and proliferated by the method described in 391-420. Electrophoresis on a 0.896 agarose gel and a 3.6% Na DodS O4/polyacrylamide gel in ethidium bromide was performed to analyze the purity of the preparation. One band related to mononucleosomes was visualized. Their purity is at least 9596.

KO'r'ZIN 13. L. et al. (1984) J.I.
mIIIunol, 133.

According to the method described in the paper by 2554-2557,
This method was implemented.

(2) -Results In order to analyze the mechanism by which anti-DNA antibodies can recognize cell surface proteins, the partial sequence of the polypeptide released by the action of elastase and recognized by the monoclonal antibody during immunotransfer was measured.

These polypeptides have sequences identical to intact histones, and cell culture supernatants of MAb PME77 also react with histones purified by immunotransfer. These results suggest that the binding of monoclonal antibody P M E 77 at the cell surface may imply the formation of DNA-histone complexes.

Because MAb PME77 may have a high affinity for DNA-histone complexes, free antibody cannot be obtained by conventional purification methods.

As a result, we subsequently selected a purification method for MAb P-IE77. This antibody was first bound to a DNA-cellulose column. The monoclonal antibody eluted with 2M NaCg contains heavy and light chains, but no histones, on electrophoresis on a NaDodSO4/polyacrylamide gel. The eluted antibodies were then loaded onto a column of histones covalently bound to activated Trisacrylic to remove any contaminating DNA.

The obtained eluate was then purified with MAb PME77 (Pm
Ab). PmAb was dsDNA (first
) but not with histones purified by immunotransfer (see FIG. 4A, line C) and by ELISA (FIG. 1). PmAb
+DNA reacts with histones purified by immunotransfer (see Figure 4A, line d) and ELISA, which contrasts with what occurs in the reaction with PmAb in the absence of DNA (see Figure 4A, line d). (See line C)
. Therefore, PmAb can form complexes with DNA and histones. PmAb showed higher binding of purified histones than DNA alone in ELISA.
Reacts better with DNA (Figure 1).

Because PmAb reacts with DNA-histone complexes,
The possibility that PmAb also reacts with chromatin and nucleosomes was tested. As shown in Figure 2, EL
In ISA, PmAb binds to chromatin, polynucleosomes, oligonucleosomes and mononucleosomes. These results suggest that PmAb has a higher affinity for DNA-histone complexes, particularly for mononucleosomes and oligonucleosomes, than for DNA alone.

- Does not bind to the binding of PmAb-DNA-histone complexes to the cell surface (Figure 3C). However, if dsDNA bound to purified histones is added to PmAb, the complex formed reacts strongly and uniformly at the cell surface (Figure 3a). No significant response is observed when DNA or histones are missing. These results demonstrate that PmAb binds to the cell surface through the mediation of DNA-histone complexes.

The 94 kDa protein has properties that correspond to cell surface proteins.

This 94 kDaffl white matter requires the use of detergents to be solubilized and detergent DNA
- Binding of PmAb in the presence or absence of histone complexes was tested in the detergent T X -114 enriched phase obtained from the membrane fraction of purified RI Nm cells. PmA
When applying the b-DNA-histone complex to a nitrocellulose strip, immunotransfer experiments
kDa protein was detected (Figure 4B, line C).

If DNA was missing, no polypeptide was detected (Figure 4B, line a). PmAb-DN
In the presence of A complex, a weak reaction was observed (4th B
Figure, line b).

(Figure 5A, line b). Furthermore, it has been shown that viable cells can be easily isolated with trypsin, which does not alter cell viability (Figure 5B). Immunotransfer analysis of the supernatant obtained after trypsin treatment did not find any tryptic fragments, suggesting that the binding site of the nucleosome to the 94 kDa protein is highly sensitive to trypsin. are doing. The 94 kDa protein is expressed in different cell types: cells of the human B lymphoblastoid cell line of Rajl, CVI monkey cells, and RINI cells.
11 was found on the surface of the pancreatic islets of Langerhans of the rat S tumor cell line.

PME7F-nucleosome complex and 94 kD
The same results obtained previously were observed by transfer of purified chromatin, polynucleosomes, oligonucleosomes, and mononucleosomes bound to a protein. However, it has been demonstrated that mononucleosome-PmAb complexes react with the cell surface by immunotransfer and with a 94 kDa protein on the cell surface from immunotransfer.

- Active phase SLE mediated by DNA-histone complexes
Sera from patients suffering from the disease were passed through a DNA-cellulose column. Antibodies were eluted with 2M NaCΩ. When DNA-histone complexes are added, the purified antibody is 94k
It causes an immunological reaction with Da protein (Figure 6, line C). Eluates from three acute-phase patient sera that appear to contain antibodies composed of DNA and histones react with the 94-kDa protein, whereas inactive-phase (
Or, the eluate obtained from two types of serum (in remission phase) was 94 k
Does not react with Da protein.

Below is an explanation of the drawings. Figure 1: PmAb in ELISA and ds
Reaction with DNA, histones, and dsDNA+histones.

The composition is: DNA (1u g/ml) + histones (2,
5μg/ml) ・The symbol indicates: DNA (1ug/ml), and the circle indicates histone (2.5μg/ml).

- Box 2: PmAb in ELISA (15u g
/ml), dsDNA (Δ), and ri07tin (・)
, polynucleosome (mu), oligonucleosome (
0), and binding of PmAb with mononucleome (■) to the cell surface.

(a) PmAb (20ug) 10 DNA (lμg)
+ histones (5 μg).

(b) PmAb (20ug) + mononucleosomes (8μg).

(c) PmAb (20ug).

- Figure 4 Figure 4A: Reaction of PmAb with Rajl cell extract and purified histones by immunotransfer in the presence or absence of DNA.

Line (or column or vertical row): a, PmAb (20ug) and Raj! Reaction with cell extract; b, PmAb (20ug) + DNA (lμg)
And 1? Reaction with ajl cell extract; c, PmAb (20ug) and purified histones (1μg)
Reaction with; d, PmAb (20ug) + DNA (1,cz
Reaction of r) with purified histones (1 μg).

- Figure 4B; PmAb and 94 in the presence or absence of DNA-histone complexes by immunotransfer
Binding with kDa protein. Extraction of both purified membranes obtained from RI Nm cells was performed using T X-114.

A phase rich in the surfactant T X-114 was used as the antigen in these experiments.

Linear a, PmAb (20 μg); b, P
mAb (20 μg) + DNA (t μg)
;c, PmAb (20μg) + DNA (lμg)
+ histones (1 μg).

LMW (low molecular weight marker) is the low molecular weight marker used.

Detection of 94 kDa protein by Spha. PmAb (20 μg) before trypsin treatment in immunotransfer
) + DNA (1 μg) + histone (1 μg) binding.

Line (vertical): a, TX-114 supernatant before phase separation; b, phase rich in surfactant TX-114: c, TX-114
Water phase (LMW has the same meaning as given above with respect to Figure 4).

- Figure 5B: 5.10 at 0°C by immunotransfer
and PmAb (20, ur) + DNA (after trypsinization at 50 μz/ml concentration for 20 min)
1 μg) + histone (1 μg) binding.

Vertical line: a, trypsin-free surfactant T X-1
14-enriched phase; b, surfactant T X after trypsin (50 μg/ml for 5 min) treatment; c,) surfactant T -114-rich phase: d, trypsin (5
Surfactant T after treatment (0 μz/ml for 20 min)
Phase enriched in
Binding with proteins.

A phase rich in the surfactant T X-114 obtained from membrane fractions purified from RINOI cells was used as antigen.

Serum from individuals suffering from SLE was first bound to a DNA-cellulose column. The eluted polyclonal antibody was then passed through a column consisting of histones co-conjugated to activated Trisacrylic. The resulting eluate was purified anti-DN
Corresponds to A polyclonal antibody.

Line (vertical); a, PmAb (20 μg) + DNA (l μg) + histone (1 μg); b, PmAb (20 μg); C1 purified polyclonal anti-DNA antibody (20 μg) + D
NA (1 μg) + histone (1 μg); d, purified polyclonal anti-DNA antibody (20 μg)

[Brief explanation of drawings]

The present invention will be explained in more detail with the help of the following figures: Figure 1 shows that purified monoclonal antibody (MAb) PME77 was purified by ELISA (enzyme-linked immunosorbent assay)
Figure 2 shows the reaction of MAbP M with double-stranded DNA (dsDNA) and with dsDNA bound to histones.
E77, dsDNA, ri07tin, polynucleosomes, oligonucleosomes and mononucleosomes
A and histones in the presence (mBa diagram) or in the presence of mononucleosomes (Figure 3b) or alone (Figure 3C),
Figure 4A shows studies binding purified MAb PME77 to CVI cells;
Figure 4B shows immunotransfer studies of the reaction of Ab PME77 with Raji cells and purified histones.
Figure 2 shows the binding of the reaction with ME77 to the immunotransfer surface protein. Study on the presence of a 94 kDa protein in both membranes , and Figure 6 shows:
As a result of immunotransfer experiments, polyclonal anti-DNA antibodies purified from the serum of individuals suffering from SLE in the presence or absence of DNA-histone complexes and engravings of 94 kDa cell diagrams (no changes in content) . igure igure pg/ml (anhi1body) ug/ml (antigen Engraving of the drawings (no changes to the contents) Engraving of the drawings (no changes to the contents) 1079- Dou Lucierche Medical France, 75654 Paris Sedex 13, Tolbiatsk, 01 Dou Dou Ioso- Case Indication Procedure Amendment (Method) % Formula % 7. Contents of amendment (1) In the description , page 83, line 2, line 5 to line 6 of the same page, line 10 of the page, and line 13 of the same page, the words ``indicate research,'' were replaced with ``research,'' respectively. In addition, in the first line of page 84, the text ``Shows the combination.'' is corrected to ``Photograph showing the combination.'' (2) Clearly drawn in black. Appropriate drawings should be supplemented as shown in the attached sheet. (No change in content) Name of the invention, especially systemic lupus erythematosus (SLE) o wi
94 kDt cell surface protein in the field of LRO diagnosis, its isolation method, and its relationship with the case of the person who corrects its use

Claims (1)

[Scope of Claims] (1) A 94 kDa cell membrane protein which: - is recognized by the monoclonal antibody PME77 when it binds to the purified monoclonal antibody PME77 or when the monoclonal antibody PME77 binds to a nucleosome or a complex consisting of DNA and histones. be able to form an immunological complex with any other antibody capable of recognizing epitopes similar to that of the antibody - in the absence of nucleosomes or in the absence of complexes consisting of DNA and histones Monoclonal antibody P purified with
A protein characterized in that it is not recognized by ME77 or any fragment of said protein capable of retaining the above immunological properties. (2) The protein according to claim 1, which is capable of forming an immunological complex with the serum of a patient suffering from systemic lupus erythematosus (SLE). (3) The protein: has a molecular weight of -94 kDa, - Raji human cells, monkey fibroblasts (CVI cells),
- be able to be isolated from the surface of cells such as RIN_m rat pancreatic tumor cells; - be hydrophobic; - contain cell membrane components that are insoluble in detergent solutions, especially of the Triton X-114 or X-100 type. - The protein according to claim 1 or 2, wherein the protein is removed from the cell surface by the action of trypsin. (4) A monoclonal or polyclonal antibody against the protein according to any one of claims 1 to 3. (5) A method for isolating and recovering a protein according to any one of claims 1 to 3, comprising: -Trito
Extraction of the protein by treating purified membranes of animal or human cells possibly containing the protein on the cell surface with a detergent of the nX-114 type and then recovering the detergent phase containing the protein. - contacting said protein-containing phase with purified nucleosomes or a complex consisting of purified nucleic acid and histones and immobilizing these nucleosomes or complexes on a carrier, and - bringing said carrier into contact with said protein-containing phase. and recovering the protein retained on the carrier by contacting it with a solution that leads to separation of the complex. (6) A complex consisting of a nucleic acid and histone is added to a nucleosome carrier used in an affinity chromatography carrier, and a specific antibody against the protein according to any one of claims 1 to 3, especially the claims Claim 5, characterized in that the monoclonal antibody according to Claim 4 is replaced with the monoclonal antibody according to Claim 4.
The method described in section. (7) A method for detecting in vitro a complex correlating with SLE, consisting of an antibody bound to a nucleosome, in a tissue or biological fluid that is thought to contain the complex, comprising: and said complex possibly present in tissues or biological fluids.
contacting the protein according to any one of claims 1 to 3 with this tissue or biological fluid under conditions conducive to an immunological reaction in vitro; and contacting the protein with the nucleosome. A method comprising the in vitro detection of a complex capable of forming between an antibody bound to an antibody. (8) A diagnostic kit for in vitro detection of an antibody-nucleosome complex characteristic of SLE, comprising: - the protein according to any one of claims 1 to 3; - immunological reagents for constituting a medium suitable for carrying out the reaction; - reagents allowing the detection of the complex formed between said protein and said antibody-nucleosome complex produced by the immunological reaction; - optionally, a tissue or biological fluid lacking an antibody-nucleosome complex recognized by the protein according to any one of claims 1 to 3, - further optionally and a comparative sample containing these antibody-nucleosome complexes. (9) The reagent that enables the detection of the complex formed between the protein and the antibody-nucleosome complex has a marker or can be sequentially recognized by a labeled reagent. The diagnostic kit according to scope item 8. (10) A method for in vitro detection of antibodies capable of correlating with SLE and capable of binding to nucleosomes in biological tissues or body fluids thought to contain them, the method comprising: - said organism; a complex formed between a 94 kDa protein and a nucleosome, which itself probably forms an immunological complex with the purified monoclonal antibody PME77 when said monoclonal antibody binds to a nucleosome, or , a fragment of the 94 kDa protein that is likely to bind to nucleosomes, MAb
Contacting with a complex formed between a nucleosome and any fragment of the 94 kDa protein that is a fragment with one or several sites that can be recognized by PME77 and/or by antibodies present in the serum of SLE patients - carrying out this contact in vitro between said antibody and said complex, possibly present in a biological tissue or body fluid;
and - in vitro detection of the immunological complex formed between said complex and said antibody. (11) The method: - Transfers a 94 kDa protein or the protein fragment according to claim 1 to a solid support, in particular a well of a plate such as a microtiter plate used in ELISA (enzyme-linked immunosorbent assay). - washing said solid support to remove 94 unbound to the support;
removing the 94 kDa protein or fragment thereof, - incubating the 94 kDa protein or fragment thereof bound to the solid support with nucleosomes to remove the 94 kDa protein or fragment thereof;
A complex of protein a or its fragment and nucleosome (
- forming a solid support (hereinafter referred to as a 94 kDa-nucleosome complex); - washing the solid support to remove nucleosomes that are not bound to the 94 kDa protein or fragments of this protein; incubating the carrier-bound 94kDa-nucleosome complex with said biological tissue or body fluid; - washing the solid support to remove components of the biological tissue or body fluid that are not bound to the 94kDa-nucleosome complex; - detecting the immune complexes, if any, formed between the 94 kDa-nucleosome complex and the antibody that is likely present in the biological tissue or body fluid and possibly bound to the nucleosome. 11. The method according to claim 10, characterized in that: (12) The method: - A 94 kDa-nucleosome complex preformed by binding a 94 kDa protein or the protein fragment according to claim 1 with a nucleosome is transferred to a solid support, particularly by ELISA (enzyme-linked immunosorbent absorption). assay)
- washing the solid support to remove the above-mentioned 94 kDa-nucleosome complex that is not bound to the support; - binding to the solid support. incubating the 94 kDa-nucleosome complex with said biological tissue or body fluid; - washing the solid support to remove components of the biological tissue or body fluid that are not bound to the 94 kDa-nucleosome complex; - detecting an immunological complex that may be formed between the antibody, which is likely present in a biological tissue or body fluid and possibly associated with a nucleosome, and a 94 kDa-nucleosome complex. 11. The method according to claim 10. (13) The step of detecting SLE-specific antibodies is preferably performed using anti-immunoglobulin labeled with an enzyme such as alkaline phosphatase, which is immunologically compatible with said antibodies possibly present in biological tissues or body fluids. 13. The method according to any one of claims 10 to 12, characterized in that it is carried out using reactive, preferably human, anti-immunoglobulins. (14) A nucleosome composed of a DNA fragment of 150 to 200 base pairs, especially leukemic mouse L1210.
13. The method according to any one of claims 10 to 12, characterized in that a 94 kDa-nucleosome complex is formed using a 94 kDa protein that binds to a mononucleosome derived from a nucleosome. (15) for in vitro diagnosis of SLE, characterized in that the biological fluid is a human serum sample;
A method according to any one of claims 7 and 10 to 14. (18) The method according to any one of claims 7 and 10 to 14, for monitoring the progression of SLE from the acute phase to the remission phase. (17) The complex is associated with nucleosomes, 94
A biological complex comprising a kDa protein or the protein fragment according to claim 1. (18) A 94 kDa protein or a fragment thereof was obtained from leukemic mouse L1210 and a 150-200 base pair D
18. The biological complex according to claim 17, which binds to a mononucleosome composed of NA fragments. (19) Claim 17 for carrying out the method described in any one of Claims 10 to 16.
19. A biological reagent comprising a complex according to paragraph 18 or paragraph 18. (20) A diagnostic kit for in vitro detection of antibodies characteristic of SLE, comprising: - a 94 kDa protein or the protein fragment according to claim 1, optionally bound to a solid carrier; - a nucleosome and the nucleosome and the above 94
reagents for forming a suitable medium for the formation of a complex with the kDa protein or a complex of a nucleosome with said protein fragment, - an antibody possibly present in a biological tissue or body fluid, and said 94 kDa protein. - reagents for constituting a medium suitable for carrying out an immunological reaction between the nucleosome complex formed during said immunological reaction between said antibody and the 94 kDa-nucleosome complex; and a reagent that enables detection of the complex. (21) A diagnostic kit for in vitro detection of antibodies characteristic of SLE, comprising: - the biological 94 kDa according to claim 17 or 18, optionally bound to a solid carrier; −
a nucleosome complex, or a reagent according to claim 19, and - suitable for carrying out an immunological reaction between said 94 kDa nucleosome complex and an antibody possibly present in a biological tissue or body fluid. A diagnostic kit comprising reagents for constituting a medium; - reagents allowing the detection of the complex formed during the immunological reaction between the antibody and the 94 kDa-nucleosome complex. (22) A claim characterized in that the reagent that enables the detection of the complex formed between the antibody and the 94 kDa-nucleosome complex carries a marker or can be sequentially recognized by a labeling reagent. Section 20 and Section 2
Diagnostic kit according to item 1. (23) A polyclonal antibody or monoclonal antibody against the complex according to claim 17 or 18. (24) SL, possibly contained in biological tissues or body fluids
A method for in vitro determination of the amount of antibodies correlatable with E.
a-nucleosome complex with a defined amount of the anti-94 kDa-nucleosome antibody according to claim 23;
contacting in such a way that all of said complexes engage in immunological binding with said antibody, - the product of the immunological reaction, i.e. (94kDa-nucleosome) - (anti-94kDa-nucleosome);
a-nucleosome) complex under conditions that allow for the performance of a competitive reaction between antibodies possibly present in biological tissues or body fluids and an anti-94 kDa-nucleosome antibody against said 94 kDa-nucleosome complex. contacting with a biological tissue or body fluid, - the amount of anti-94kDa-nucleosome antibody that is not bound to the 94kDa-nucleosome complexes or the amount of said antibody that is still bound to these complexes;
This is nvitro\detection, and the amount of this antibody detected is
A method characterized in that it can be correlated with the amount of antibodies characteristic of SLE contained in a biological tissue or body fluid. (25) The method includes the following steps: - 94kD according to claim 17 or 18.
The a-nucleosome complex is transferred to a solid support, especially for ELIS
binding to the wells of a plate such as the microtiter plate used in A; - washing the solid support to remove any of the above 9 that is not bound to the support;
removing the 4 kDa-nucleosome complex; - converting said complex bound to the carrier to a determined amount of the anti-9 according to claim 23 in excess of the amount of said complex;
incubation with a 4kDa-nucleosome antibody; - a complex formed during the previous incubation step between a 94kDa-nucleosome and an anti-94kDa-nucleosome antibody (94kDa-nucleosome);
(anti-94kDa-nucleosome) with biological tissue or body fluid; - washing the carrier to remove anti-94kDa-nucleosome antibodies that are no longer bound to the 94kDa-nucleosome complex; and the amount of anti-94kDa-nucleosome antibody that is not bound to -94kDa-nucleosome complexes or the amount of said antibody that is still bound to these complexes.
Claims 1 and 2 include an in vitro detection step in which the amount of antibodies detected can be correlated with the amount of antibodies characteristic of SLE contained in biological tissues or body fluids. The method described in Section 24. (26) characterized in that the antibody according to claim 23 is labeled, in particular with an enzyme, and the determination of the amount of SLE-specific antibodies possibly present in biological tissues or body fluids: - patent directly by detection of the amount of labeled anti-94 kDa-nucleosome antibody recovered after the final washing step of the method of claim 25, provided that in that case S
The amount of LE-specific antibody is determined by the amount of labeled anti-94k recovered in the washing solution.
Corresponds directly to the amount of Da-nucleosome antibody - or indirectly by detecting the amount of labeled anti-94kDa-nucleosome antibody that binds to the 94kDa-nucleosome complex, provided that in that case the amount of SLE-specific antibody is on the one hand, the amount of labeled anti-94kDa-nucleosome antibody bound to the 94kDa-nucleosome complex before the step of incubation with the biological tissue or body fluid, and on the other hand, the incubation with the biological tissue or body fluid and the claimed The anti-94 kDa-nucleosome complex bound to the 94 kDa-nucleosome complex after the final step of washing as described in scope item 25.
26. The method according to claim 25, characterized in that the amount corresponds to the difference between the amount of 4 kDa-nucleosomal antibodies. (27) Not labeling the antibody according to claim 23 and the amount of anti-94kDa-nucleosome antibody recovered in the washing solution after the final washing step or the incubation with biological tissue or body fluid and washing. The amount of these antibodies still bound to the 94 kDa-nucleosome complex after the final step of the process is detected using a labeled anti-immunoglobulin that can immunologically react with these antibodies. The method according to scope item 25. (28) A diagnostic kit comprises the components of the kit according to any one of claims 20 to 22 and a defined amount of the antibody according to claim 23, labeled if necessary. An SLE-specific antibody characterized by comprising ￥i
Diagnostic kit for nvitro detection and administration. (29) It is believed that the complex formed between one specific nucleic acid and the other histone causes the protein according to any one of claims 1 to 3 to appear on the cell surface. A cell transfection method comprising contacting a cell to allow internalization within said cell of a complete body consisting of a specific nucleic acid, histone and said protein. (30) said specific nucleic acid is recognized by a nucleic acid sequence encoding a specific polypeptide and optionally a regulatory element capable of expressing this nucleic acid fragment in a host cell, in particular a polymerase of said cellular host; Claim 29, characterized in that it includes a promoter that
The method described in section. (31) Claim No. 1 applies to the production of specific polypeptides by placing transformed host cells in culture in an appropriate culture medium and the recovery of specific polypeptides from these cells or the culture medium. The method described in Section 30. (32) An anti-idiotype antibody against the monoclonal antibody according to claim 4. (33) A pharmaceutical composition comprising the antibody according to claim 32 together with a physiologically acceptable excipient. (34) Claim 3 particularly in the treatment of SLE
Therapeutic use of the antibody according to paragraph 2. (35) A composition comprising the 94 kDa protein according to claim 1 bound to pancreatic cells of human or animal origin. (36) Claim 3 for immunosuppressive therapeutic purposes
Use of the composition according to item 5.