FR2675805A1 - Antigens specific for rheumatoid arthritis, their constituent proteins, their preparation and their diagnostic application - Google Patents

Abstract

The subject of the present invention is antigens extracted from mammalian malpighian epithelium, especially rat oesophageal epithelium or human epidermis, which are specifically recognised by the auto-antibodies present in patients suffering from rheumatoid arthritis, as well as the constituent antigenic proteins of the said antigens. The invention relates to the use of these antigens and proteins for the preparation of antigenic compositions and their application for the diagnosis of rheumatoid arthritis.

Description

The present invention relates to anti
ozenes extracted from squamous epithelium from mammals and recognized specifically by the autoantibodies present - in patients suffering from rheumatoid arthritis:? R).

 The invention relates more precisely to one part of an antigen extracted from human epidermis and to one part to an antigen extracted from rat oesophageal epithelia, as well as to the antigenic proteins constituting them.

 The invention also relates to the antibodies recognizing these antigens, as well as the antigenic compositions for diagnosing the presence of autoantibodies and the i = tunogenic compositions for the repair of vaccine, comprising said antigens or proteins.

 The invention also relates to a method for diagnosing rheumatoid arthritis using said antigens or proteins.

 Rheumatoid arthritis (RA) is the most common chronic inflammatory rheumatism. It reaches around 2% of the population of developed countries.

 RA is progressive, debilitating and poses difficult diagnostic problems in its forms of Dut Dut, even though it calls for specific treatments. Apart from clinical and radiological signs, the rheumatologist currently only has biological tests which are not very specific for his diagnosis. The most used of them are the Waaler-Rose tests, which detect the rheumatoid factor in 7 PR out of 10. However, the specificity of these tests is very poor since these factors are detected in most other autoimmune pathologies and even in healthy individuals.

 The presence of autoantibodies directed against cellular components is the general characteristic of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma (SCL), polymyositis or connective tissue disease ( MTCD). Among the many types of autoantibodies identified in these diseases, those specifically present in RA patients and reacting with an esophageal epithelial antigen have been described for the first time by B.J.J. Young et al.

in Br.Med.J. 2: 97-99, 1979. These autoantibodies have been incorrectly called anti-keratin antibodies.

These RA-specific autoantibodies have so far been detected and titrated by indirect immunofluorescence on transverse cryoputs of rat esophagus. -.Serre et al. in Rev.Rhum. 53 (11): 604-614 (1986) have shown that the antigen defined by these autoantibodies is squamous and cornified and present only in the
Stratum corneum; they also showed that the antigen recognized by the sera of RA patients is also present in the human epidermis (G. Ser et al.

Revue du Rhumatisme, 1986, 53 (11), 607-614).

 The work carried out by the Applicant in the field of epidermal and squamous differentiation and the study of monoclonal antibodies for the definition of new differentiation antigens but also of human autoantibodies against these same antigens, enabled it to show that the anti- Rat esophageal stratum corneum are dauchentic autoantibodies, which do not recognize human or murine keratins, but other antigens expressed in these tissues.

Further work has enabled the
Applicant to extract, purify and biochemically characterize these antigens as well as the proteins constituting them, specifically recognized by the serum autoantibodies present in RA patients.

The invention therefore relates to a terminal differentiation antigen present in the squamous epithelia of mammals, specifically recognized by the autoantibodies present in a patient suffering from rheumatoid arthritis, characterized in that it has the following properties - it is of a protein nature, - nuclease treatment does not change its
immunoreactivity, - treatment with ethanol does not change its
immunoreactivity, - treatment with trichloroacetic acid does not modify
not its immunoreactivity, - it is water-soluble.

 Among the squamous mammalian epithelia, mention may be made of the epithelia that line the oral cavity, such as the jugal, gingival, lingual or palatal mucous membranes; as well as the upper genital, anal and aerodigestive mucosa.

 The work which led to the present invention more particularly concerned the rat esophageal epithelium and the human epidermis.

A first antigen specifically recognized by the autoantibodies present in patients with
PR was extracted from the human epidermis.

 This antigen has the following properties - after electrophoresis under native conditions, it dissociates into several antigenic proteins whose corresponding molecular weight is between approximately 80 and 400 kD, the most immunoreactive proteins having an apparent molecular weight between 80 and 120 kD , - after isoelectrofocusing, it dissociates into several antigenic proteins whose pHi vary from 5.8 to 7.4.

This antigen is characterized by the following properties - after two-dimensional electrophoresis of the type
isoelectric focusing, followed by gel migration
polyacrylamide-SDS, it appears as a
antigenic protein of apparent molecular weight
average of around 40 kD whose pHi range from 5.8 to
7.4.

This antigen is further characterized by the following property - it has immunological properties in common
with human filaggrin or a proteolysis fragment
of the precursor of filaggrin.

 In addition, this antigen extracted from epiaerm .-. Amain has immunological properties in common with human filaggrin or a fragment of proteolysis precursor of filaggrin.

 The Applicant has also extracted rat esophageal epithelia a second antigen specifically recognized by the autoantibodies present in RA patients.

This antigen has the following properties - after polyacrylamide-SDS gel electrophoresis, it
separates into the following three antigenic proteins a first protein of average molecular weight
about 210 kD, * a second protein of average molecular weight
between approximately 90 and 130 kD, * a third protein of average molecular weight
between approximately 67 and 90 kD, - after polyacrylamide gel electrophoresis in
native condition it separates into the three proteins
following antigens * a first protein with an average molecular weight
corresponding apparent is approximately 440 kD, * a second protein with an average molecular weight
apparent corresponding is approximately 220 kD, * a third protein whose average molecular weight
apparent corresponding is about 120 kD.

This antigen is further characterized by the following properties - after two-dimensional electrophoresis of the type
isoelectric focusing, followed by gel migration
of polyacrylamide-SDS, it dissociates into the three pro
antigenic tines following a first protein of average molecular weight of
approximately 210 kD, the pHi of which vary from approximately 5.85 to 6.85, * a second protein of molecular weight included in
be around 90 and 130 kD whose pHi vary from around
5.85 to 7.35, * a third protein whose molecular weight is reduced
naked from around 120 to 67 kD while its pHi varies
from about 5 to 7.5.

- after gel-filtration chromatography and interaction
hydrophobic then gel electrophoresis
polyacrylamide-SDS, this antigen separates into the two
following antigenic protein fractions * a very hydrophobic first fraction containing the pro
tine of average molecular weight of about 210 kD and the
average molecular weight protein between about
ron 90 and 130 kD, * a second fraction less hydrophobic than the first
containing medium molecular weight protein com
taken between around 120 and 67 kD.

 The proteins that make up these antigens have been purified and tested with the autoantibodies present in RA patients. it appears that these proteins have immunological properties at least equivalent to those of the antigens from which they are derived.

 The invention therefore also relates to the three proteins demonstrated after two-dimensional electrophoresis of the electrofocusing type, followed by migration in polyacrylamide-SDS gel, of the antigen extracted from the rat esophageal epithelium.

 These three proteins have also been demonstrated in the protein fractions obtained after sequential gel-filtration chromatography and hydrophobic interaction of the antigen extracted from rat esophageal epithelium, then electrophoresis under denaturing conditions.

 The first of these three proteins has an average molecular weight of approximately 210 kD and pHi ranging from approximately 5.85 to 7.35; the second has an average molecular weight between 90 and 130 kD and pHi varying from about 5.85 to 7.35; the third has an average molecular weight between 67 and 120 kD and pHi varying from approximately 5.8 to 7.4.

 The invention also relates to the protein demonstrated after two-dimensional electrophoresis of the electrofocusing type followed by migration in polyacrylamide-SDS gel, of the antigen extracted from human epidermis. This protein has a molecular weight of about 40 kD and pHi ranging from about 5.8 to 7.4.

 The antigens and proteins previously described are particularly useful for the preparation of antigenic compositions for the diagnosis of the presence of autoantibodies in a biological sample from a patient suffering from rheumatoid arthritis.

 These compositions allow the formation of an antigen-antibody complex between the proteins or antigens of the invention and the autoantibodies present in a biological sample, such as serum, from a subject suffering from RA; these compositions and the antigens which they contain are therefore particularly advantageous for the in vitro diagnosis of RA.

 An immunoblot test for the detection of anti-Stratum corneum class G autoantibodies, specific for rheumatoid arthritis, has been developed.

 These autoantibodies were previously detected by IIF on a rat esophagus cryocut.

 An immunoblotting protocol, carried out using a murine esophageal epithelial antigenic extract, was optimized to obtain a better definition of the immunoreactive bands and better sensitivity, then three different configurations, either by the method of separation of antigenic proteins ( electrophoresis in native or denaturing conditions), either by diluting the sera analyzed, were validated on the diagnostic level with a large series of sera representative of rheumatological pathology.

 It appeared that in these three conditions, the diagnostic sensitivity of the test hovered around 50% for a specificity close to 95%, when only the presence or absence of immunoreactivity was taken into account.

 On the other hand, the 120 kD antigenic protein of the antigen separated under native conditions, recognized much more specifically by the sera of patients suffering from RA, made it possible to detect the sera with a sensitivity of 68% for a specificity of 99%, performance far superior to that of IIF which has a sensitivity of 43% for a specificity of 99%.

 The parallel approach conducted with the antigen extracted from human epidermis made it possible to show that the 40 kD protein, the only immunoreactive form in this tissue, made it possible to obtain diagnostic performances equivalent to those of the murine antigenic protein of 120 kD. .

 The invention also relates to antibodies formed against the antigens and proteins of the invention, antibodies which can be polyclonal or monoclonal and then produced by any hybridoma prepared according to conventional methods of cell fusion between spleen cells activated in vitro by antigen or from an animal immunized against one of the antigenic proteins or glycoproteins of the invention and cells of a myeloma cell line.

The invention also relates to a method for purifying the autoantibodies specifically present in patients suffering from rheumatoid arthritis, characterized in that it comprises at least the following steps - bringing a patient's biological fluid into contact
rheumatoid arthritis with at least one
antigen or at least one protein, as defined
in the foregoing, under conditions allowing the
formation of immunological complexes with said
antibodies, - the isolation of said complexes from the reaction medium, - the separation of autoantibodies from immuno complexes
logical.

The invention finally relates to a method of in vitro diagnosis of polyarthritis r; aumatolde in a biological fluid, comprising at least the following steps - bringing this biological fluid into contact with at
minus an antigen or a protein according to the invention,
optionally labeled, or a conjugate of this antigen
or protein with a carrier molecule, under conditions
tions allowing the formation of an immunol complex
gic with the autoantibodies possibly present - the detection in the biological fluid of the presence
of the immunological complex, by physical methods
or chemical.

The above diagnostic method can be implemented using a kit or kit comprising - at least one antigen or a protein according to the invention,
optionally labeled, or a conjugate of this antigen
with a carrier molecule - reagents for the constitution of an environment favorable to
the immunological reaction with the autoantibodies even
commonly present in a biological sample - one or more reagents for the detection of the complex
immunological trained - if applicable, a reference sample, such as
negative serum.

 Other characteristics and advantages of the invention will appear on reading the examples which follow and which are illustrated by the appended figures, it being understood that these examples should not be interpreted as tending to reduce the scope of the Claims.

EXAMPLE 1 - CHARACTERIZATION OF THE EXTRACT ANTIGEN
RAT ESOPHAGIC EPITHELIUMS
I - MATERIAL AND METHOD
A - EPITHRLMES PROTEIN EXTRACTIONS
1) Preparation of the esophageal epithelium by chorioetithelial clivaae
The rat esophagus is composed of three main coats which are, from outside to inside: a connective layer (the adventitia), a muscular layer (the muscularis), a layer composed of a chorion covered by an epithelium Malpighian cor nifies.

After elimination, by dissection, of the adventitia and the muscularis, the epithelium is separated mechanically from the chorion (chorioepithelial cleavage) after an athermic shock in liquid medium (KH2 / K2H-PO4 8.5 mM; NaCl 150 mM ; EDTA 5 mM; PMSF 0.4 mM; pH 7.2). The cleaved epithelia are kept hydrated with a minimum volume of PBS (KH2- / K2H-P04 8.5 mM; 150 mM NaCl; pH 7.2) before undergoing a histological control making it possible to verify the absence of contaminating chorion

2) Extraction of epithelial proteins
a) Extraction buffers and sequences
The antigen of the invention being exclusively epithelial, it is from the single epithelium that the extractions were carried out.

 From cleaved epithelia, the extraction of the epithelial proteins is carried out successively by grinding with a potter then stirring at 4 Celsius in buffered medium. After centrifugation, the supernatant is recovered and the pellet is subjected to the extraction steps in order to improve the yields.

Classic protocols (WW Franke et al.,
J. Mol. Biol., 153: 933-959, 1981 - Sun and Green,
J. Biol. Chem. 253 (6): 2053-2060, 1978) adapted to the sequential extraction of proteins from epithelial tissues and in particular from the epidermis, were first used. Then one-step extractions were then tested. Different extraction media corresponding to Tris-HCl buffers with or without added salts (KCl, NaCl), detergents (Triton, NP40), denaturing agents (urea, SDS) and reducing agents (beta-mercaptoethanol) have thus been tested.

 During fractional extractions, the antigen was found exclusively in the fraction corresponding either to a Tris buffer of low ionic strength, or to a TBS-EDTA-Triton buffer. This made it possible to confirm that the antigen is not a cytokeratin, to show that it is extractable in the absence of urea and reducing agents and that it is water-soluble.

 However, when comparing the various extraction techniques, the intensity of immunoreactivity appeared to be slightly higher in the presence of NP40.

 An extraction medium consisting of TBS + inhibitors + NP40 0.5 * therefore appears to be the most effective.

 The high water solubility of the antigen was confirmed when the PBS intended to prevent desiccation of the epithelia during chorioepithelial cleavages and therefore inhibiting them in a very small volume, was analyzed. It was found that this PBS buffer was rich in protein and contained detectable traces of antigens. The antigen was only partially extracted in PBS since it was found in significant quantity after grinding of the esophaguses in TBS-NPQO buffer. This grinding step has therefore not been eliminated.

 During the purification of the antigen, the intense absorption of NP40 at 280 nm interfering with that of proteins, in particular in gelfiltration chromatography, led to comparing the immunoreactivity of extractions carried out with and without NP40. The remaining pellets were in both cases re-extracted in a buffer supplemented with NP40. Very little antigen was detectable in these pellets, which were extracted initially with or without detergent. In addition, the profiles in PAGE-SDS gel were identical and the quantity of proteins extracted was very similar (5.62 mg / ml without NP40 versus 5.76 mg / ml with NP40). This last buffer (TBS + inhibitor without toP40) is therefore the one which must be adopted for the extraction of the antigen before all the purification and characterization works.

b) Ostlmlsatlon of thvsiaues conditions
extraction
In order to optimize the extraction procedure, certain physical parameters have been systematically explored. These are temperature, time, type of grinding, type of agitation and the action of enzyme inhibitors.

- Grinding :
It was carried out either with an electric potter at high speed, or more gently with a manual potter. The extraction efficiency in both cases was roughly equivalent. The electric potter is however preferred because it allows the grinding of a greater number of esophagus.

- Sonification
Optimization of the extraction by sonifications of variable duration, after potterization was tested. It turned out to be very weak and sonification is therefore not necessary.

- Agitation:
Soft agitation and rapid agitation were compared. Rapid agitation in the presence of a detergent leading to the formation of foam and causing over-oxygenation of the medium is indeed likely to alter the antigen. In fact, the quality of the extraction was identical in both cases. Gentle agitation was however used to preserve a maximum of 1 antigen.

- Territory
Ambient temperature and 4-Celsius temperature were compared. Better results have been obtained with extractions conducted at 4-Celsius.

- Duration:
15 minute, 2 hour and 17 hour stirred extractions were compared. Although increasing the extraction time slightly increases the amount of antigen extracted, a period of 15 minutes has been used to minimize the risk of alteration of the antigen.

- Inhibitors
The protective effect for antigen of the following inhibitors has been tested - Inhibitor of bacterial proliferation
Sodium azide, - Inhibitor of lytic enzymes
proteases (aprotinin, PMSF), phosphatases (NaF, PCMB),
glycosidases (deoxymannojirimycin, deoxyJirimycin).

 Extractions without inhibitors were carried out in particular when the extract was intended for the biochemical characterization of the antigen by the action of different enzymes; no modification of the antigen was noted in the absence of an inhibitor.

- Optimized extraction protocol
The study of the previous parameters made it possible to determine the optimized extraction protocol below - Substrate: epithelium of esophagus ae rat obtained by
chorioepithelial cleavage X - Buffer: Tris HC1 40 mM
150 mM NaCl
EDTA 10 mM
pH 7.4 - Inhibitors: Sodium azide 0.1 8
PMSF 1 mM
Aprotinin 2 mg / ml - Substrate / buffer ratio
500 tjl of buffer per esophageal epithelium - Conditions
Grinding: electric potter 15 minutes
Grinding temperature: 4-Celsius
Agitation after grinding: 15 minutes at 4-Celsius - Total protein concentration of the extract
5 to 8 mg / ml - Storage of the raw extract
-20 or -40'C
- PreDaratlon from mucosa
total esoshaaian
In order to simplify the extraction procedure, the extraction of the antigen from the total esophageal mucosa has been studied. It is then a dissection of the mucosa but without chorioepithelial cleavage. The protein contamination thus induced was studied.

 Parallel extractions, either from mucous membranes, or from cleaved epithelia.

 The SDS-PAGE analysis of the proteins extracted ("PBS" fraction and extraction by grinding according to the Sun and Green method) shows that the presence of the chorion does not lead to protein contamination in the molar mass zone of the antigen . After immunoblotting of the epitheliochorial fraction extracted by grinding, the antigen is perfectly detectable.

c) Concentration of the crude extract
As the antigen is poorly represented in the crude extract (approximately less than 1% of total proteins), a step of concentrating the extracts is necessary, especially when these have to be analyzed by electrophoresis on an FPLC system (deposits of l 'microliter order).

 Among the various concentration techniques tested, that of concentrating the extracts by precipitation with 10% TCA was chosen. This easy-to-use technique produces no alteration of the antigen since the immunological response is perfectly preserved in immunoblotting.

 Similarly, precipitation with methanol or ammonium sulphate as well as controlled lyophilization do not induce any significant alteration of the antigen and are therefore perfectly usable.

B - ELECTROPHORESES
For all the extraction media studied below, the soluble proteins are separated by electrophoresis in polyacrylamide-SDS gel (PAGE-SDS) and then electro-transferred to nitrocellulose.

1) Isoelectrofocallsatlon tar separation (IEF)
horizontal. Gel: 9 X 15 cm, 4% acrylamide containing ampholines
forming a pH gradient from 3.5 to 9.5.

Sample deposited: raw extract prepared according to the pro
previous toole, concentrated by acid purification
trifluoroacetic (TCA) at 10% and taken up in water.

2) Monodimensional electrothoreses - Classic vertical electrophoresis according to the method
described by Laemmli (Nature, 227: 680-685, 1970). Gel: 10 X 10 cm, 7% acrylamide. Migration buffer: Tris 25 mM, glycine 192 mM,
Sodium Dodecyl Sulfate (SDS), 1%, pH 8.3.

sample deposited: crude lyophilized extract and taken up
in 10 mM Tris-HCl pH 7. ~ 4, SDS 2%, 2-Mercaptoethanol
(2ME) 1%, bromophenol blue 0.1%, glycerol 10%.

- Miniaturized horizontal electrophoresis "under conditions
denaturing.

Gel: 4 X 4 cm, 7.5% acrylamide
200 mM buffer, 200 mM Tricin, 0.55% SDS, pH 8.1.

Sample deposited: crude extract concentrated by preci
pitation with 10% TCA and taken up in 10 mM Tris-HCl,
pH 7.4, SDS 2%, 2-ME 1%, Bromophenol blue 0.1%.

"Miniaturized" horizontal electrophoresis under conditions
natives.

Gel: 4 X 4 cm, gradient from 8 to 25% in acrylamide Migration buffer: Tris 250 mM, L-Alanine 880 mM,
pH 8.8.

Sample deposited: crude extract concentrated by preci
pitation with TCA 10% and taken up either in water or in
200 mM Tris-HCl buffer at different pH.

 3 IEF / PAGE-SDS two-dimensional electroohoreses first dimension: horizontal IEF.

Gel: 4 X 4 cm, 4% acrylamide, pH gradient 3.5 to 9.5 Sample deposited: crude extract concentrated by preci
pitation with ethanol (4 volumes) and taken up in water.

second dimension: denaturing electrophoresis as
previously described.

. Sample deposited: IEF gel track incubated in Tris
112 mM, acetic acid 112 mM, SDS 2.5%, 2-ME 1%.

C - IMMMNODETECTION
Immunodetection of the antigen is carried out using human sera from RA patients in which the rat esophageal anti-Stratum corneum autoantibodies were previously titrated by semiguantitative indirect immunofluorescence and assigned a value- titer from 0 to 8: GIFOR parameter (Rat G-Immunofluorescence-Esophagus) The threshold value, GIFOR = 2, which corresponds to a diagnostic specificity for RA greater than 99%, allows the patient's sera to be classified into two groups: GIFOR + and GIFOR- (C. Vincent et al., Ann. Rheum. Dis. 48: 712-722, 1989).

In the selected sera, the following immunological parameter was also taken into account: the titer of epidermal class G anticytokeratin autoantibodies determined in ELISA (GELISA) (G.Serre et al,
J. Invest. Dermatol. 88: 21-27, 1987). These autoantibodies react in immunotransfer with contaminating human cytokeratins possibly present in the extract and due to the natural scaling of the manipulators. The sera used were chosen to also form two groups, GELISA + and GELISA-, depending on the high (OD> 0.6) or low (OD <0.2) titer of these natural autoantibodies. - the following groups of PR sera were used
- GIFOR + / GELISAs
- GIFOR + / GELISA
- GIFOR- / GELISA +
- GIFOR- / GELISA
Finally, control sera from normal individuals or from patients suffering from rheumatoid rheumatic infections as well as murine monoclonal antibodies specific for human cytokeratins were included in the tests (G.Serre et al., Colloquium
INSERM, Vol. 171: 524, 1988).

Under these experimental conditions, the antigen extracted from rat esophageal epithelia is detectable in immunoblotting by a specific immunoreactivity of PR GIFOR + sera corresponding to a broad band located between 90 and 120 Kd. Natural anti-keratin autoantibodies result in the presence of immunoreactive bands located around 65-67 Kd, present in GELISA + sera alone, more or less intense depending on their titer and completely independent of the value of GIFOR
D - CHROMATOGRAPHY
The antigenic molecules were separated into different fractions after sequential gel-filtration chromatography / hydrophobic interaction. Then these fractions were analyzed by PAGE-SDS and PAGE under native conditions.

E - ENZYMATIQUR DIGESTION
1) Proteinase K
Two extracts of rat esophageal epithelium in TBS / NP40 buffer are precipitated with 10% TCA and the proteins taken up in a 50 mM Tris-HCl buffer pH 7.5 10 mM CaCl 2. One of the extracts is supplemented with proteinase
K (preincubated for 1 hour at 37 ° C. to eliminate other hydrolases possibly present in the commercial preparation) at the final concentration of 50 Rg / ml. The two samples are then incubated for 1 hour at 37 ° C. and then analyzed comparatively in immunoblot after migration to PAGE under native conditions or to SDS-PAGE.

2) Nuclease
Two extracts of rat esophageal epithelium in TBS / NP40 buffer are precipitated with 10% TCA. One of them is hydrolyzed with 2 U / L of micrococcal nuclease (hydrolysis of DNA and RNA) in 50 mM Tris-HCl, pH 7.5, 5 mM CaCl 2. The second sample, untreated, serves as a control. The two preparations are incubated for 1 hour at 37 ° C. and then analyzed comparatively in immunoblot after migration to PAGE under native conditions or to PAGE-SDS
F - DEGLYCOSYLATION
1) OxYdatlon ~ with rerlodloue acid
WOODWARD and COLL protocol. (J. IMM.

 METH. 78: 143-153, 1985) was used; this protocol allows periodic oxidation of a sample previously transferred to a nitrocellulose membrane, 1 periodic oxidation can be followed by immunodetection.

 An extract of rat esophageal epithelium is transferred after migration in SDS-PAGE. The nitrocellulose membrane is cut into several tracks which are subjected to periodic oxidation (incubation in 50 mM sodium acetate buffer pH 4.4, periodate 20 mM sodium, for 1 hour in the dark and at room temperature) followed by reduction with sodium borohydride (50 mM NaBH4 in PBS pH 7.4 for 30 min at room temperature). Control samples undergoing no chemical treatment or undergoing only reduction were included. All of the samples are then immunodetected.

2) Acid tar treatment
trifluoromethanesulfoniaue (TFMS)
The use of TFMS, according to the method of
EDGE and COLL. (ANAL. BIOCHEM. 118: 131-137, 1981), allows almost total deglycosylation of the glycoproteins while retaining their peptide axis. This method was applied to a control glycoprotein, bovine fetuin and to extracts of rat esophageal epithelium containing the antigen. Extracts are dialyzed against water and then lyophilized; Their treatment is carried out by adding a mixture (66/33-V / V) of TFMS and amisol (agent protecting the peptide axis) and incubation with shaking after nitrogen saturation of the atmosphere of the tubes. reference protocol was optimized by varying different parameters - proportion of TFMS / Anisol mixture compared to the
mass of proteins treated: 33 to 266 Rl of mixture per
mg of total protein from the esophageal extract.

- temperature: O'C, 4-C or room temperature.

- Duration of incubation: 1, 3 or 24 hours.

 In all cases, after incubation, the reaction is stopped by adding 2 volumes of glacial ether and 3 volumes of a pyidine / water mixture (50/50-V / V). The precipitate of pyridinium trifluoromethanesulfonate, soluble in the ethereal phase, was removed with it. After a further extraction with glacial ether, the aqueous phase was dialyzed against water.

The samples were then lyophilized, separated by
PAGE, transferred, then immunodetected.

3) Traltement ar tettide N-alvcosldase F (PNGase F)
PNGase, also marketed under the name of Glycopeptidase F or N-Glycanase, hydrolyzes the amide bond existing between the asparaginyl residue of a glycoprotein and the N, N 'diacetyl-chitobiose of the structure common to all N-linked oligosaccharides .

 Fetuin contains three N-linked oligosaccharide chains, the elimination of which by PNGase F results in a reduction in molar mass that can easily be viewed in SDS-PAGE (S. HIRANI et al., ANAL. BIOCHEM.

162: 485-492, 1987).

Bovine fetuin (2 mg / ml) and a lyophilized extract of rat esophageal epithelium (8 mg / ml of protein) are denatured for 5 min at 90 ° C. with SDS at 28 and 2ME 0.05 M. Then the proteins are placed in a reaction medium of the following composition - Nonidet P40 (NP40) 1.25% (protection of the enzyme against
vis the denaturing action of SDS), - potassium phosphate 166 mM pH 8 (conservation of pH
optimal enzyme), - 1,10-orthophenentroLine 10 mM (protease inhibitor)
solubilized in methanol at the final concentration
10%, - PNGase F 0.3 U / Al.

 In this reaction medium, the respective final concentrations of SDS and 2ME are 0.166% and 16 mM. The incubation is 24 hours at 37 ° C.

"Fetuin" and "antigenic extracts" controls incubated without enzymes are included in the proto zone. After hydrolysis and concentration by precipitation with TCA, the samples are separated by PAGE, transferred
es and immunodetected.

G - REACTIVITY WITH DIFFERENT LECTINS
The inventors sought to confirm the glycoprotein nature of the antigen by specifically detecting certain sugars by lectins coupled to peroxidase. Two plant lectins: wheat germ lectins (WGA) and concanavalin A (ConA) were chosen for their different specificity tI.J. GOLSTEIN ez RD PORETZ, The Lectins: properties, function and application in biology and medicine) - the WGA recognizes residues of ssD-N acetylglycosamine
with the following growing affinities
(1) GlcNAc <<GlcNAcpl - 4GlcNAc <GlcNAcBl-4 GlcNAc51-4GlcNAc and also binds with less affinity to sialic acid residues.

- Con A associates with the following sugars classified by affi
growing nity
(2) aGlcNAc <aGlc <oMan <Manal-2Man <Manal-ZManal-2Man the proteins of two antigenic extracts, crude or pu rified, are transferred to nitrocellulose after migration in one-dimensional PAGE or IEF / PAGE-SDS. The nitrocellulose bands were incubated with Con A and WGA coupled with peroxidase at the minimum concentrations of 20 and 15 μg / rtLi respectively (JCS CLEGG,
ANAL. BIOCHEM 127: 389-394, 1982 and WF GLASS et al.,
ANAL. BIOCHEM. 115: 219-224, 1981). The incubation is carried out in a 50 mM Tris-HCl buffer, pH 7.4, 200 mM NaCl, 1 mM CaCl 2, 1 mM MnC 12 and 0.18 Tween for 1 hour at 37 ° C. and 1 night at 4 ° C. is preceded and followed by washing steps in the same buffer. Various additional saturation agents such as gelatin or bovine serum albumin oxidized by a treatment with periodic acid do not allow to amplify the response obtained when using the Tween alone.

After the last wash, the lectin-antigen association is revealed by demonstrating the peroxidase activity with H2O2 and 4-chloro-1-naphthol as during immunodetections.

II - PYSICOCHEMICAL CHARACTERIZATION OF ~ 'ANTLGENPI
The physicochemical characterization of the antigen was carried out using crude extracts of rat esophagus epithelium, then from purified antigenic fractions In both cases, after electrophoretic separation, the proteins were transferred and then immunodetected by sera from RA patients.

A - MOLAR MASS
Immunoblot analysis of an extract of rat esophageal epithelium separated by PAGE-SDS and immunodetected with PR GIFOR + and GIFOR sera shows that the antigen is in the form of a large immunoreactive zone and spread corresponding to a protein of average molar mass between 90 and 130 kD, a narrow immunoreactive band corresponding to a protein of average molar mass of approximately 210 kD and an immunoreactive zone corresponding to a protein of average molar mass between 67 and 90 kD.

B - ISOELECTRIC POINTS
The crude extract is concentrated five times by TCA 10% precipitation, taken up in calf, separated by IEF (pHi 3.5 to 9.5), transferred then immunodetected with sera from patients with GIFOR + and GIFOR - PR. The antigenic molecules have a very heterogeneous distribution, the proteins of the crude extract recognized specifically by most GIFOR + sera are distributed in a bundle of fine bands whose isoelectric points vary from 5.2 to 7.5.

C - CHARACTERTS OF MIGRATION IN CONDITIONS
NATIVES
FIG. 1 represents the immunoblot of an extract of rat esophageal epithelia taken up in an acid medium and separated by PAGE under native conditions. The crude extract concentrated ten times by precipitation with TCA, is taken up in water (pH less than 4), separated by PAGE under native conditions (8-25%), transferred then immunodetected with PR GIFOR + sera (tracks 74, 52, 5, 11) diluted 1/100; track Tg represents a negative control. In these conditions of recovery at acidic pH, the immunoblotting carried out with four sera from RA patients reveals three immunoreactive zones corresponding to proteins of apparent average molecular weight of approximately 440 kD, 220 kD and 120 kD, which are recognized with a variable affinity according to the sera.

D - TWO-DIMENSIONAL ELECTROPHORESIS IHP '/ PAGE-SDS
This technique which combines IEF in the first dimension and PAGE-SDS in the second makes it possible to simultaneously determine the isoelectric points and the molar masses of the various antigenic molecules, to confirm their great heterogeneity of charge and to show their great heterogeneity of mass.

 FIG. 2 represents the immunoblot of an extract of rat esophageal epithelia separated by two-dimensional electrophoresis (EF / PAGE-SDS). The crude extract concentrated 20 times by precipitation with ethanol then taken up in water, is separated by IEF (pHi 5 to 8) then by SDS-PAGE (7.5 *), transferred then immunodetected with two patient sera with PR GIFOR +, diluted 1/100. The immunoreactive zone of 90 to 130 kD has pH i varying from 5.85 to 7.35, those of the 210 kD band vary from 5.85 to 6.85. A third immunoreactive population, in the form of a "comma", migrated from 120 to 67 kD while these pHi are distributed continuously between 5 and 7.5. The antigenic molecules with a molar mass of less than 90 kD, clearly detected after two-dimensional electrophoresis only appear in one-dimensional electrohores with high titer sera, in the form of an extension of immunoreactivity towards areas of low molecular weight. The greater quantity of antigen deposited in IEF / PAGE-SDS, allowing better immunodetection sensitivity, explains this apparent discrepancy.

E - CHARACTERISTICS OF SEPARATION C} IROMATOGRAPHIQW3
FIG. 3 represents the separation in hydrophobic interaction chromatography of a prepurified antigen solution by gel-filtration (elution profile followed by measurement of the OD at 280 nit). Immunoreactive fractions purified by gelfiltration chromatography (Superose 12) from a crude extract are separated on Phenyl-Superose (50 mM phosphate equilibrium buffer, 1.7 M NH 2 SO 4, pH 7; 50 mM phosphate elution buffer , pH 7; volume injected: 30 mm; Flow rate 0.5 ml / min; DO 280 nm full scale: 0.05; Collection 1 ml / tube).

 The elution profiles and the immunoblot study of the different antigenic fractions proved to be very reproducible during multiple purifications. The distribution of antigenic molecules in the different fractions could therefore be modeled.

 FIG. 4 represents the modeling of the immunoreactivity profiles of the purified antigenic fractions by sequential gelfiltration / hydrophobic interaction chromatography, obtained after PAGE-SDS (1) or PAGE under native conditions (2), The differences in intensity of the bands or immunoreactive zones reflect, for the same band or zone, the relative richness in antigenic proteins of each fraction.

 In denaturing electrophoresis, the band at 210 kD (fractions F, G and H), the broad band ill-defined from 90 to 130 kD (D, E, F, G and H) and a band from 67 to 90 kD (C , D and E) have been visualized.

 In native electrophoresis, the fine band at 440 kD (F, G and H), the band at 232 kD (C, D, E, F, G and H) and the wide band at 120 kD (B, C, D and E) have been found.

The comparison of the antigenic molecules analyzed in PAGE-SDS in the various purified fractions and of the molecules identified after two-dimensional electrophoresis (IEF / PAGE-SDS) in a crude extract, as indicated in FIG. 7, provides information on the extent of respective hydrophobicity.
- The 210 kD band, the pHi of which varies from 5.85 to 6.85, is found in the fractions obtained at the end of elution (F, G and H), it is therefore very hydrophobic.

 - The diffuse zone from 90 to 130 k, whose pHi are between 5.85 and 7.35, has the same elution profile as the band at 210 kD (fraction E, F, G and H) and therefore the same hydrophobicity.

- On the other hand, the population in the form of a comma which has pHi more spread out from 5 to 7.5, is found mainly at the start of elution (fractions B,
C, D and E) and therefore has a less hydrophobic character than the two previous molecules.

III - BIOCHEMICAL CHARACTERIZATION OF THE ANTIGEN
A - ANTIGENIC MOLECCLES ARE NATURAL
PROTEIN
FIG. 5 represents the immunoblot analysis of the proteins of a digested antigenic extract (B) or not (A) by proteinase K, separated by PAGE under native conditions (8-25z), transferred then immunodetected with three patient sera with RA
GIFOR + (tracks I to 3) and a patient serum
PR GIFOR - (track 4); track Tg represents negative controls.

 The red culvert stains of the nitrocellulose bands corresponding to the treated samples show the total digestion of the proteins of the extract.

Immunodetection after PAGE in non-native conditions shows that the specific immunoreactivity of sera
GIFOR + disappears When the sample is previously digested with proteinase K. Immunodetection after transfer of the separated samples to PAGE-SDS confirmed this result.

 The protein nature of the molecules carrying the antigenicity being affirmed, the inventors determined whether they were homo- or heteroproteins, which associations and / or substitutions of the antigen were capable of accounting for the diversity. isoelectric weight and apparent molar mass and what was the biochemical nature of the recognized epitope (s).

B - ANTIGENIC PROTEINS ARE NOT
ASSOCIATED WITH NUrtwIQZES ACIDS
The possible association of the antigen with nucleic acids has been studied. An extract of rat esophageal epithelia was treated or not with micrococcal nuclease and then analyzed by electrophoresis in 1% agarose gel, followed by staining with ethidium bromide.

 After separation of the samples by electrophoresis in 1% agarose gel, the absence of nucleic acids in the treated sample proves the efficiency of the enzymatic digestion.

 FIG. 6 represents the analysis by immunotransfer after PAGE under native conditions (8-25%) of an extract of epithelium of rat esophagus treated (b) or not (a) with micrococcal nuclease. The analysis was carried out with three sera from patients with PR GIFOR + (lanes 1 to 3) and one serum from PR GIFOR - (lane 4); track Tg represents negative controls.

 Immunodetection after PAGE under native conditions shows the insensitivity of the antigenic proteins to nuclease treatment both from the point of view of their immunoreactivity with sera from RA patients and from that of their apparent molar mass, proving that they are not not associated with nucleic acids.

C - ANTIGENIC PROTEINS ARE RT.'T.KS DES
GLYCOPROTEINS?
The glycoproteins often exhibit a high heterogeneity of isoelectric points and an inconstant rate of SDS binding which makes their resolution in PAGE-SDS poor. Antigenic proteins also having these characteristics, the inventors sought their possible substitution with carbohydrate residues. To this end, techniques aimed either at altering the sugar molecules possibly carried by the peptide axis, or at ridding the protein part of its glycan residues, were used.

1) Destruction of possible etitotes
alucidioues because 1 acid teriodiatie
FIG. 7 shows the immunodetection of an extract of rat esophageal epithelia which, after separation by PAGE-SDS (7.5%) and transfer, was - incubated for 1 h 30 min in TBS buffer (controls
"incubation": track a), - incubated for 1 hour in sodium acetate buffer, then re
duit for 30 min with sodium borohydride (controls
"reduction": lane b), - oxidized by periodic acid in acetate buffer of so-
dium for 1 hour then reduced for 30 minutes with boro
sodium hydride ("test": track c),
three PR GIFOR + sera (tracks 1 to 3), two sera
from PR GIFOR - (tracks 4 and 5) and two sera from
control (tracks 6 and 7) were used; the Tg track
represents negative controls.

Treatment with periodic acid did not change the proteins specifically recognized by the sera of RA patients. Three interpretations of this result are possible - the antigenic proteins are glycoproteins but
sugars sensitive to periodic oxidation
that they wear are not involved in the
epitopes mainly recognized by autoantibodies
specific for rheumatoid arthritis.

- the antigenic proteins are glycoproteins but
the sugars which they carry by their nature and / or
their type of chain are insensitive to an oxida
periodic (periodic acid cleaves the bond
between two vicinal carbons initially bearing either
each an alcohol function, or 1 a function
alcohol and the other an acid or ketone or amide function
primary or secondary).

- antigenic proteins are not glyco
proteins.

In order to decide between these three possibilities, the inventors used a method of total deglycosylation and then sought modifications of the mass or
The immunoreactivity of antigenic proteins.

2) Déalocosvlatlon
Two main types of link allow the association of an oligosaccharide with a peptide axis the O-glycoside link which allows the association of an oligosaccharide with a Serine or Threonine (and rarely hydroxylysine) residue via a galactosamine. N-acetylated and the N-glycosidic bond which attaches an oligosaccharide to an asparagine residue via an N-acetylated glucosamine.

To deglycosylate the glycoproteins, two types of treatment can be applied - specific treatments allowing to eliminate all
types of carbohydrate radicals, whether related to
the protein by an N- bond or a bond
O-glycosidic. This is the case, for example, with treatment
chemical with trifluoromethanesulfonic acid (TFMS); - specific treatments for one or the other of these
types of binding. This is the case of ss-elimination by
soda for O-glycosia bonds and some
more or less specific number of enzymatic hydrolysis
of the chemical nature of N-linked sugar for bonds
N-glycosides.

a) Tralement assécifioue tar TFMS
Analysis by immunoblot after SDS-PAGE (7.5%) of an antigenic extract treated with TFMS and of an extract incubated in water shows that the antigenic proteins show a slight decrease in apparent molar mass compared to the control sample.

Although small, this difference of approximately 10 to 20 kD is observed for all the treatment conditions and found in two separate experiments. Immunoblot analysis after IEF of the proteins of an extract of rat esophageal epithelia treated with TFMS shows that the specific immunoreactivity of the sera of patients with -PR on the control samples spreads over an area of pHi from about 5.2 to 7.3. After treatment for TFMS, the proteins immunodetected by the sera of patients with RA presented more basic pHi between 6 and 8.

 This change in pHi and apparent molar mass of the antigenic proteins is in favor of a glycan substitution of the molecules. However, the persistence of a poor definition in PAGE-SDS and a wide range of pHi in IEF suggests that anti geniou proteins could also be substituted by other non-carbohydrate radicals and / or that the observed effects could be due to cleavage of a particularly fragile peptide bond.

 However, the conservation of the immunoreactivity after treatment with TFMS is a strong argument in favor of the non-carbohydrate nature of the epitope (s) recognized by the specific autoantibodies of RA.

b) Specific treatment for PNGase F
In order to confirm the results obtained with TFMS, the inventors undertook deglycosylation experiments with the PNGase F enzyme which specifically eliminates N-linked sugars.

Deglycosylation with PGNase F was carried out on a crude antigenic extract after dialysis against water and lyophilization. Analysis of this extract by immunodetection in PAGE-SDS and transfer does not show any difference between the control antigen and the antigen treated with PNGase F. Two interpretations of this result are possible - the antigen is not substituted by
N-oligosaccharides, - oligosaccharides connected to asparagine residues
of protein have an original chemical structure which
prevents them from being cleaved by PNGase F.

 However, a single one-dimensional analysis does not make it possible to clearly state the insensitivity to PNGase F of the antigenic proteins since these are partially superimposed, as shown by the analysis in two-dimensional electrophoresis.

3) Detection of lectins tar sugars
The possible reactivity of the antigen with wheat germ lectin (WGA) or concanavalin (conA) was tested by "affinodetection" on antigenic nitrocellulose and compared with the specific immunodetection profiles of RA sera.

 The proteins of the crude antigen extract, recognized after PAGE-SDS or PAGE under native conditions, by a serum of RA patients, were different from the proteins recognized by lectins. However, common zones of reactivity, in particular at the level of the 210 kD protein in SDS-PAGE, suggests that the WGA and the ConA recognize part of the antigen.

Antigenic fractions purified by gel filtration chromatography were analyzed under the same conditions. This experiment confirmed that glycoproteins of the same molecular weight as certain antigenic proteins were recognized by ConA and that the reactivity profile of WGA seemed to approximate that of a serum of RA patients.

 Evidence for an affinity of the lectins for the antigenic proteins was obtained after tEF / PAGE-SDS of the crude antigenic extract.

 FIG. 8 represents the detection of the proteins of a crude antigenic extract, after two-dimensional electrophoresis (IEF pHi 5 to 8 / PAGE-SDS 7.5%) and transfer to nitrocellulose, with - in (2): a serum of patients with of PR GIFOR +, - in (4): ConA, - in (5): WGA, in order to amplify the reactivity, three deposits of antigenic extracts were made on the gel of IEF. A fraction of the antigen (band at 200 kD and immunoreactive zone between 90 and 130 kD) deposited on the anode side precipitated there, producing an artefactual reactivity in the zone of acid pHi.

 This study made it possible to show that the molecules recognized by ConA were different from the antigen, but that the WGA associated specifically with the antigenic proteins of 90-130 kD and 210 kD. The immunoreactive proteins in the form of a "comma" ranging from 120 kD to 67 kD with pHi of 5 to 7.5 do not, on the other hand, show any reactivity towards WGA.

EXAMPLE 2 - CHARACTERIZATION OF THE ANTIGENH EXTIT OF
THE SPECIFIC HUMAN EPIDERMIS OF THE
POLYARTHRITIS RHDDATO IDE
I - MATERIAL AND METHOD
A - PATIENTS AND SERUMS
The present study was carried out on 64 sera from patients suffering from various rheumatic diseases perfectly characterized clinically and biologically. Among these 64 sera, 45 come from patients suffering from rheumatoid arthritis scrolling according to the criteria of the American Rheumatism Association. 32 sera from healthy subjects, men and women, were used as a control.

The immunodetection was carried out using two monoclonal antibodies, one specific for human cytokeratints called F 12-19 (ref. Iblio. Or or ret
Biosoft-Clonatec), the other specific for human filaggrin called AKH-1 (ref. Blblio. Or ref. Biomedical
Technologies Inc.).

B - EXTRACTION EXTRACTION OF EPIDERMAL PROTEINS
Skin fragments collected after breast and abdominal plastic surgery or after circumcision were stored at -80 ° C. The epiaerm was mechanically separated from the dermis, according to the method described by Kassis and Sondergaard (Arch. Dermatol. Res. 273 301-306, 1982), by heat treatment at 57 ° C. in PBS buffer (KH2- / K2H- 8.5 mM PO4; 150 mM NaCl) containing 5 mM EDTA and 0.5 mM Phenylmethylsulfonylfluoride (PMSF) then mechanically milled in 40 mM Tris-HCl buffer pH 7, *; 150 mM NaCl; 10 mM EDTA; NP40 0.5 mM, hereinafter called buffer A.

 After centrifugation at 12,000 xg for 15 minutes, the supernatant was separated from the pellet containing the epidermal cytokeratins.

 In addition, other extractions were carried out according to the same protocol but in a 20 mM Tris-HO1 buffer, pH 7.4; 0.5 mM PMSF, hereinafter referred to as buffer B.

 The different fractions were kept at -20 ° C.

C - ELECTROPHORESES
The proteins of the various fractions were analyzed by isoelectric focusing at equilibrium or before equilibrium, or by acrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (denaturing conditions; PAGE-SDS) or in the absence of sodium dodecyl sulfate (native conditions; PAGE-native).

Two-dimensional electrophoresis was also carried out.

 The proteins were sometimes precipitated by 4 volumes of ethanol or by 15% trichloroacetic acid (TCA) final, in order to increase the concentration.

D - INODETECTION
The proteins were transferred onto a nitrocellulose membrane according to the technique described by TOWBIN et al. (PNAS 76: 4350-4354, 1979). The sera were used diluted 1/10 to 1/100 after incubation of the nitrocellulose strips in PBS, Tween-20 0.05%.

Incubation in the presence of the sera was continued for 1 hour at 37 ° C. and then 12 hours at 4 ° C.

 The monoclonal antibodies AKH-1 diluted 1/100 or F12-19 diluted 1/500 were incubated for 2 hours at 37 C. After washing, the nitrocellulose strips were revealed by secondary antibodies, anti-human IgG or murines marked with peroxydas e.

E - ENZYMATIC TREATMENTS
The concentrated epidermal extract (80 Rg / l) was treated with proteinase K (1 mg / ml, 30 minutes, 37 C in the presence of 1% SDS) or with microccocal nuclease (100 U / ml, 30 minutes, 37 C in a 5 mM Tris-CaC12 buffer, 30 mM Tris-HO1 pH8).

F - IMMUNOPRECIPITATION -
Preadsorbed epidermal extract on Protein
A-Sepharose was incubated at 37 ° C for 2 h in the presence of the antibody AKH-1 or F12-19 and NaCl, 1 M final.

 The antibody-antigen complexes formed were then collected by the protein A-Sepharose and then analyzed by immunoblotting after PAGE-SDS.

G - PURIFICATION OF CYTOKERATINS
The water-soluble cytokeratins were purified from mammary epidermis according to the method described by SUN and GREEN (J. Biol. Chem. 252: 2053-2060, 1978).

H - PURIFICATION OF HUMAN FILAGGRIN
Filaggrin was extracted from breast skin according to the technique described by LIMLEY and DALE (BBA 774: 28-35, 1983) with the following modifications: epidermal extract in 6M urea, prepared in the absence of reducing agent, was purified on FPLC chromatography with an anion exchange resin. The cationic proteins not retained were precipitated with 4 volumes of acetone, redissolved in a small volume of buffer containing 1.5% of SDS and 2.5% of 2-mercaptoethanol and deposited on top of an SDS preparative gel of 10 cm in height. This gel was then transferred to a Immobilan-PVDF type membrane marketed by the Company
MILLIPORE. The area corresponding to filaggrin was identified by immunodetection with the antibody AKE-1 and cut out; the filaggrin was then eluted with 0.3 ml / cm2 of elution buffer (50 mM Tris-HCl pH 8.8 2% SDS
Triton X-100 1t) and concentrated by precipitation.

I - PURIFICATION OF THE ANTIGEN
20 cm2 of mammary epidermis were ground in an electric potter in buffer A. The homogenate was centrifuged for 15 minutes at 10,000 g and the supernatant precipitated by TCA (15% final). The precipitate was resolubilized in a small volume of 30 mM Tris-HCl pH8, 5 mM CaCl 2; the soluble fraction was clarified by centrifugation, added with SDS 1.5 and 2ME 2.5 * and deposited on top of an SDS preparative gel 10 cm in height. The antigen was then purified as described above for filaggrin, the zone corresponding to the antigen being referred by immunodetection with a serum of rheumatoid arthritis.

II - RESULTS
A - PHYSICO-CHEMICAL CHARACTERIZATION AND
BIOCHEMICAL ANTIGEN
In order to characterize the antigen recognized by the rheumatoid arthritis sera, a mammary epidermal extract was prepared by homogenization of the mammary epidermis in buffer A, in the presence of a nonionic detergent (NP40). This soluble extract was analyzed by immunodetection after PAGE and transfer of the proteins to nitrocellulose.

 FIG. 9 represents the immunoblot analysis of the preceding NP40 extract with rheumatoid arthritis sera and control sera.

The NP40 extract (8 mg / ml) was precipitated by TCA, taken up in Tris-CaC12 buffer by concentrating it 7 times then analyzed by immunoblotting after PAGE-SDS (gradient 8-25%).

The sera were diluted 1/10 in 0.05% PBS-Tween 20.

sera 1 and 3 are from patients with myolan malui, serum 2 is from a patient with SHARP disease, serum 4 is from a patient with osteoarthritis, serum 5 is from a patient polymyositis, serum 6 is from a patient with BIAGET disease, sera 7 to 21 are from patients with rheumatoid arthritis and sera 22 to 38 are from normal subjects; t0 is a control incubated only with the labeled anti-human IgG antibody.

 Most of the sera from patients with rheumatoid arthritis (80%) have recognized a protein having an SDS gel (denaturing conditions) - a molecular weight of approximately 40 kD. Its migration was not modified by the omission of the reducing agent (2ME) in the sample buffer, which indicates that this molecule probably does not contain a disulfide bridge. Conversely, 89% of the sera from patients suffering from rheumatoid arthritis other than rheumatoid arthritis and none of the control sera recognized this antigen. A 68 kD protein was detected non-specifically by a large portion of sera belonging to all the groups tested and therefore probably by natural autoantibodies.

FIG. 10 represents the immunoblot analysis of the preceding NP40 antigenic extract, whether or not treated with a nuclease (1) or a protease (2). In (1), the NP40 extract of breast epidermis was precipitated by the
TCA, taken up in Tris-CaC12 buffer then treated with (+) or without (-) microccocal nuclease (100 U / ml) for 30 minutes at 37 ° C. In (2), the NP40 extract of human epidermis identical to the previous one was precipitated by TCA then taken up in SDS 1% and treated without (-) or with (+) 10 mg / ml of proteinase K for 30 minutes at 37'C. In all cases, the proteins were separated into SDS-PAGE (8-25%), transferred then immunodetected with control sera (a) and sera from patients suffering from rheumatoid arthritis (b) diluted to 1, 100 . The disappearance of the immunoreactive region after treatment of the extract by proteinase K confirms the protective nature of the molecule carrying the epitope (s) recognized as autoantibodies, whereas this region has not been modified after digestion of the acids. nucleic acid by Micrococcus nuclease, the molecules specifically recognized by the sera of RA patients after transfer and immunodetection of the epidermal proteins analyzed by PAGE under native conditions, are heterogeneous.

 FIG. 11 represents the analysis of the antigen by this electrophoresis under native conditions this analysis consisted in precipitating the NP40 extract of human epidermis with ethanol, then in carrying out an immunodetection after PAGE under native conditions ( 8-25%) and transfer to nitrocellulose with sera from RA patients (sera 1 to 4 and 8 to 8) and control sera (sera 5 to 7), diluted to 1/100.

 FIG. 12 represents the analysis of the antigen by isoelectric focusing (IEF). The proteins of the NP40 extract of breast epidermis were precipitated by ethanol, separated by IEF (pHi 3 to 9), transferred to nitrocellulose with sera from patients suffering from rheumatoid arthritis (sera S to 12) and sera controls (sera 1 to 4) diluted 1/100, then immunodetected. The antigen also has a very heterogeneous distribution of pHi, these varying from 5.8 to 7.4.

This heterogeneity of pHi was verified by 2D electrophoresis (IEF / PAGE-SDS). The protein recognized specifically by the sera of patients suffering from RA, after transfer of such a 2D geL shows a characteristic image in the form of a comma, the apparent molecular weight decreasing as the pHi becomes more basic.

FIG. 13 represents the analysis of the antigen in two-dimensional gel IEF / PAGE-SDS; the proteins of the NP40 extract of breast epidermis were precipitated with ethanol, separated by IEF (pHi 5 to 8) followed by
SDS-PAGE (12.5%), transferred to nitrocellulose and immunodetected by a serum from a patient suffering from rheumatoid arthritis (RA) and a control serum.

 The marked heterogeneity of the antigen, both in mass and in pHi, probably indicates the presence of modified isoforms; it suggests that it could be glycosylated and / or phosphorylated. However, treatment with N-peptide N-glycosidase-F did not modify its migration or its immunoreactivity after PAGE-SDS.

3 - HROSOLUILITY OF THE ANTIGEN
5 cm 2 of mammary epidermis were mechanically ground in buffer A or in buffer B. The homogenates were then centrifuged for 10 minutes at 10,000 g and an equal quantity of the soluble proteins of the two supernatants was precipitated with ethanol and then separated by PAGE under native conditions (8-25), transferred to nitrocellulose then immunodetected from sera from RA patients and from control sera. This study indiluates that the antigen is extracted not only in the presence of a detergent (buffer A) but also in its absence (buffer B). In addition, the antigen is always found mainly in soluble fractions and present in the pellets in very small quantities. These results indicate that the human antigen, like that of rat esophagus is a highly water-soluble molecule.

C - EXPRESSION OF ANTIGANG IN DIFFERENT
EPIDERMAL ANATOMICAL TERRITORIES
Immunoblot analysis of proteins from an NP40 extract of abdominal epidermis and preputial epidermis indicates that the antigen associated with RA is present in the human epidermis regardless of sex and anatomical territory (breast, abdomen or foreskin ), as well as age (children and adults). The antigen being histologically located in the Stratum corneum, it is legitimate to think that its abundance will depend on
The thickness of it which varies according to the anatomical territory.

D - IDENTIFICATION OF THE ANTIGEN
The antigen recognized by the sera of patients suffering from RA is not a human epidermal cytokeratin.

FIG. 14 represents - in (a): an NP40 extract of immuno-mammary epidermis
detected, after PAGE-SDS (8-25 *) and transfer to nitro
cellulose, with RA patient serum, pre
incubated on the one hand with the extraction buffer (1), with
the extract itself (2) and with an insoluble fraction
enriched with epidermal cytokeratin (3).

- in (b): the immunosoureasible cytokeratins
detected, after PAGE-SDS (8-25%) and transfer, with
monoclonal anti-cytokeratin antibody F12-19 pre
incubated either with the extraction buffer (1) or with
epidermal extract (2), either with an enri fraction
shit in cytokeratin (3), either with cytokeratins
water soluble (4).

- in (c): the proteins of the epidermis extract (5), a
fraction enriched in cytokeratin z) and cyto
pure water-soluble keratins (7) separated by PAGE-SDS
(8-25%), transferred and immunodetected with mAb
anti-cytokeratin F12-19.

The immunoreactivity of sera from RA patients was not inhibited by preincubation of these on epidermal cytokeratins (enriched or purified), which then disappeared in the presence of water-soluble skin extract. Similarly, the monoclonal antibody F12-19 has not recognized in immunoblot, no protein of the antigenic extract
The characteristic comma form of the 2D gel antigen and its apparent gel molecular weight
SDS are similar to those of filaggrin, a protein which appears in the Stratum granulosum during epidermal differentiation (SARRET et al., Path. Biol.

 37: 297-303, 1989). The inventors therefore sought to determine whether the filaggrin and the antigen of the invention were identical and whether the sera of patients suffering from RA contained anti-ilaggrin autoantibodies.

 FIG. 15 represents analysis of an immunoblot of the proteins of an extract of mammary epidermis separated in (A) by PAGE-SDS (12.5%), in (B) by PAGE in native conditions and in (C) by IEF (pHi 3 to 9), then transferred to nitrocellulse and immunodetected by sera from RA patients (sera 1, 3, 6, 7 and 8), the anti-filaggrin AKH-1 monoclonal antibody (2), a control monoclonal antibody (4) and a control serum (5). These proteins were separated into triplica (D) by NEpHGE followed by PAGE-SDS (8-25%) then transferred to nitrocellulose and stained with Ponceau Red (a) or immunodetected by the monoclonal antibody AKH-1 (b), and with a serum from a patient with RA (c). The antigen is recognizable by its characteristic comma shape.

The human antifilaggrin monoclonal antibody
AKH-1 specifically recognizes in immunotransfer a human epidermal protein soluble in the presence of NP40, having the same migration characteristics under denaturing or native electrophoresis conditions as the RA antigen. After IEF and 2D gel, the antigen and protein recognized in immunoblotting by AKH-1 present an identical image.

 The inventors then sought to determine whether the monoclonal antibody AKH-1 could immunoprecipitate the antigen from the epidermal extract in NP40.

FIG. 16 presents the comparative analysis of the proteins of an NP40 epidermal extract, preabsorbed on protein A-Sepharose, separated by PAGE-SDS (12.5%) - before immunoprecipitation with the monoclonal antibody
AKH-1 (total), - after immunoprecipitation with the monoclonal antibody
AKH-1 (IP KAH-1), - immunoprecipitation with the monoclonal antibody of
G36 control (IP G36),
then, after transfer to nitrocellulose, immuno
detected with AKH-1, G36, a patient serum
of PR, a control serum (CO) or with the only
secondary anti-mouse IG antibodies (Tg S) and
anti-human GI (Tg h); the supernatants of the fractions
immunoprecipitated with G36 (Surn G36) and AKH-1
(Surn AKH-1) were analyzed in the same way after
precipitation with TCA.

 These results show that AKE-1 has effectively immunoprecipitated an epidermal protein of the same molecular weight as the RA antigen, this molecule being moreover specifically recognized in immunoblotting by the sera of patients suffering from RA.

This immunoprecipitation was performed in the presence of
1 M NaCl, which makes co-immunoprecipitation unlikely due to an association between the two proteins (filaggrin and antigen). In order to verify that the proteins of the extract recognized by AKH-1 and by the sera of RA patients were the same, the supernatants of an immunoprecipitation by the anti-filaggrin monoclonal antibody were subsequently analyzed by immunoblotting with a serum from RA patients.

FIG. 16 shows that the extract has been exhausted (partially or completely) in antigen and in filaggrin by the monoclonal antibody antifilaggrin. Conversely, sera from RA patients did not immunoprecipitate the antigen. This negative result may be due to a low affinity of the autoantibodies for the antigen, to a too small amount of autoantibodies in the serum or to a non-recognition by them of the antigen in its native form. The latter hypothesis is unlikely since the antigen is immunodetectable on nitrocellulose after native gel electrophoresis.

 In order to confirm that the filaggrin and the RA antigen at least have in common the epitog detected by the monoclonal antibody AKH-1, the antigen was purified by elution from a transfer membrane.

FIG. 17 represents the analysis by PAGE-SDS and immunotransfer of the proteins of the various fractions during the purification. The total proteins of a human epidermis extract (1), the soluble proteins after precipitation of the extract by TCA (2) and those of the final purified antigenic fraction (3) were separated by PAGE-SDS ( 12.5%), transferred to nitrocellulose and stained with amldo-plate (Amido) or immunodetected with a serum from RA patients (PR), a control serum (cl), a monoclonal anti-filaggrin antibody (AKH -1) and a monoclonal control antibody (G36).

 The major proteins, soluble after precipitation with TCA, appear in the form of an apparent molecular weight (PM) doublet of approximately 40 kD. This doublet is the main immunoreactive band recognized by a serum from RA patients. This protein was then purified from a preparative PAGE-SDS gel by transfer then elution. The RA antigen was specifically immunodetected after PAGE-SDS by the monoclonal antibody antifilaggrin AKH-1.

 In order to verify that the sera of patients with RA also recognized human filaggrin and therefore that it was closely related to the antigen, the inventors purified filaggrin according to the method described by LINNEY and DALE (BBA 744: 28 -35, 1983). FIG. 18 represents the analysis of purified human filaggrin, analyzed by PAGE-SDS (12.5%) transferred onto nitrocellulose and immunodetected with sera from patients with RA (sera 1 to 4), a control serum ( serum 5), a control mAb (6) and the AKH-1 mAb (7) or stained with Rouge Ponceau (8). Figure 18 shows that sera from RA patients specifically recognize human filaggrin.

Claims (18)

CLAIMS -  1. Terminal differentiation antigen present in squamous epithelia of mammals, specifically recognized by the autoantibodies present in patients suffering from rheumatoid arthritis, characterized in that it has the following properties - it is of a protein nature, - treatment with a nuclease does not change its  immunoreactivity, - treatment with ethanol does not change its  immunoreactivity, - treatment with trichloroacetic acid does not modify  not its immunoreactivity, - it is water-soluble.  2. Antigen according to Claim 1, characterized in that it is extracted from human epidermis.  3. Antigen according to Claim 2, characterized in that it has the following properties - after electrophoresis under native conditions, it  dissociates into several antigenic proteins including  corresponding molecular weight is between  about 80 and 400 kD, the most l; mmuno- proteins  reagents with an apparent molecular weight included  between around 80 and 120 kD, - after isoelectric focusing, it dissociates further  several antigenic proteins whose pHi vary from  5.8 to 7.4.  4. Antigen according to one of Claims 2 to 3, characterized in that after two-dimensional electrophoresis of the isoelectric focusing type followed by migration into polyacrylamide-SDS gel, it appears as an antigenic protein of average apparent molecular weight of approximately 40 kD whose pHi range from 5.8 to 7.4.  5. Antigen according to one of Claims 2 to 4, characterized in that it has immunological properties in common with human filaggrin or a proteolysis fragment of the precursor of filaggrin.  6. Antigen according to Claim 1, characterized in that it is extracted from rat esophageal epithelia.  7. Antigen according to Claim 6, characterized in that it has the following properties - after electrophoresis in polyacrylamide-SDS gel, it  separates into the three antigenic proteins  following * a first protein of average molecular weight  about 210 kD, * a second protein of average molecular weight  between approximately 90 and 130 kD, * a third protein of average molecular weight  between approximately 67 and 90 kD, - after polyacrylamide gel electrophoresis in condi  native tion, it separates into the three anti proteins  following genes * a first protein with an average molecular weight  corresponding apparent is approximately 440 kD, * a second protein with an average molecular weight  apparent corresponding is approximately 220 kD, * a third protein whose average molecular weight  apparent corresponding is about 120 kD.  8. Antigen according to one of Claims 6 to 7, characterized in that after two-dimensional electrophoresis of the isoelectric focusing type followed by migration in polyacrylamide-SDS gel, it dissociates into the following three antigenic proteins - a first protein average molecular weight of  approximately 210 kD with pHi values varying from approximately 5.85 to  6,85, - a second protein of molecular weight included in  be around 90 and 130 kD whose pHi vary from around  5.85 to 7.35, - a third protein whose molecular weight decreases  naked from around 120 to 67 kD while its pHi varies  from about 5 to 7.5.  9. Antigen according to one of Claims 6 to 8, characterized in that after gel chromatography and hydrophobic interaction followed by electrophoresis on polyacrylamide-SDS gel, it separates into the following two antigenic protein fractions - a first fraction very hydrophobic containing pro  tine of average molecular weight of about 210 kD and the  average molecular weight protein between  about 90 and 130 kD, - a second fraction less hydrophobic than the first  containing medium molecular weight protein  between approximately 120 and 67 kD.  10. Antigen protein extracted from rat esophageal epithelium according to Claim 8, characterized in that it has an average molecular weight of approximately 210 kD and pHi varying from approximately 5.85 to 6, 85.  11. Protein- of the antigen extracted from rat esophageal epithelium according to Claim 8, characterized in that it has an average molecular weight of between 90 and 130 kD and pHi varying from approximately 5.85 at 7.35.  12. Antigen protein extracted from rat esophageal epithelium according to Claim 8, characterized in that it has an average molecular weight of between 67 and 120 kD and pHi varying from approximately 5 to 7.5 .  13.- Protein of the antigen extracted from human epidermis according to Claim 4, characterized in that it has a molecular weight of approximately 40 kD and pHi varying from approximately 5.8 to 7.4.  14. Antigenic composition for the diagnosis of the presence of autoantibodies in a biological sample from a patient suffering from rheumatoid arthritis, characterized in that it = comprises at least one antigen according to one of Claims 1 to 9 or a protein according to one of Claims 10 to 13.  15. Monoclonal antibody characterized in that it recognizes an antigen according to one of Claims 1 to 9 or a protein according to one of Claims 10 to 13.  16. Method for purifying the autoantibodies specifically present in patients suffering from rheumatoid arthritis, characterized in that it comprises at least the following steps - bringing a patient's biological fluid into contact  rheumatoid arthritis with at least one  antigen according to one of Claims 1 to 9 or to  at least one protein according to one of Claims 10 to  13, under conditions allowing the formation of com  immunological plexes with said antibodies, - the isolation of said complexes from the reaction medium, - the separation of the autoantibodies from the complexes  immunological.  17. Method for in vitro diagnosis of rheumatoid arthritis in a biological sample characterized in that it comprises at least the following steps - bringing this biological sample into contact with  at least one antigen according to one of Claims 1 to  9, or at least one protein according to one of the Revendica  tions 10 to 13, possibly marked, or a  conjugate of this antigen or protein with a  carrier molecule, under conditions allowing the  formation of an immunological complex with auto  antibodies possibly present; - the detection in this biological sample of the pre  sence of the immunological complex, by phy methods  sic or chemical.  18. Necessary for the in vitro diagnosis of rheumatoid arthritis in a biological sample characterized in that it comprises - at least one antigen according to one of Claims 1 to  9 or at least one protein according to one of the Revendica  tions 10 to 13, possibly marked, or a conjugate of  this protein with a carrier molecule - reagents for the constitution of an environment favorable to  the immunological reaction with the autoantibodies even  commonly present in said biological sample - one or more reagents for detecting the complex  immunological trained - if applicable, a reference sample, such as  negative serum.