FR2682113A1 - Peptides derived from the protein A of the ribonucleoprotein snRNP-U1 and their use for the diagnosis of mixed connective tissue disease - Google Patents

Abstract

The invention relates to peptides derived from the protein A of snRNP-U1 which are capable of forming an immunological complex with autoantibodies present in a biological fluid from patients suffering from mixed connective tissue disease; the said peptides consist of or comprise all or part of the sequence of the following formula: SQFGQILDILVSRSLKMRGQAFVI

Description

 Peptides derived from the snRNP-U1 ribonucleoprotein protein A and their use for the diagnosis of mixed connective tissue disease.

 The invention relates to peptides having the property of being specifically recognized by the antibodies present in patients with mixed connective tissue disease.

 The invention also relates to the applications of these peptides and compositions containing them for the in vitro diagnosis in humans of mixed connective tissue disease, as well as their use for the constitution of diagnostic kits for this disease.

 The invention further relates to the antibodies raised against these peptides and the application of these antibodies and compositions containing them for the in vitro diagnosis in humans of mixed connective tissue disease, as well as to the production of drugs against this disease.

 The invention finally relates to the applications of these peptides to the production of immunogenic compositions and vaccinating compositions against this disease.

 Patients with mixed connective tissue disease produce antibodies that react with a variety of cellular components such as DNA, RNA, proteins and RNA-protein complexes (Smeenk et al., 1985, Morrow and Isenberg, 1987; 1989). Some of these antibodies are markers specific to a clinical syndrome; for example, anti-DNA antibodies and antibodies to Sm antigen are markers of systemic lupus erythematosus, anti-centromere antibodies are markers of CREST syndrome and anti-topoisomerase I antibodies are markers of scleroderma .

 Among the many types of autoantibodies identified in the preceding diseases, those reactive with Sm antigen are present in 20 to 30 subjects with systemic lupus erythematosus and very rarely in subjects with other systemic autoimmune diseases of connective tissue.

The Sm antigen is associated with a particular class of ribonucleoproteins called snRNP; these ribonucleoproteins contain five species of uridine-rich RNA called U1, U2, U4, U5 and U6 (Lerner and Steitz 1979, Brunel et al. Seven proteins, named for their electrophoretic mobility on polyacrylamide gels, band B4 (29 kd), band B (28 Kd), band D (16 Kd), band D '(15, were identified in each ribonucleoprotein snRNP. 5
Kd), E band (12 Kd), F band (11 Kd) and G band (9 Kd).

In addition to these proteins that make up the common core, snRNP ribonucleoproteins include other proteins; ribonucleoprotein snRNP-U1 comprises three proteins of 70 Kd, 34 Kd and 22 Kd, the protein of 34
Kd is identified in the literature by the letter A and called protein or polypeptide A or U1A. European Patent Application 0 313 156 in the name of AKZO NV

describes the amino acid sequence of protein A shown in FIG.

 Among the antibodies reactive with snRNP ribonucleoproteins which can be detected in mmunoblot, it is possible to distinguish the anti-snRNP-U1 antibodies reacting with at least one of the three proteins of snRNP-U1, the anti-Sm antibodies reacting with the B proteins. ', B and D which are common to snRPs U1, U2, U4, U5 and U6, and the anti-antibodies (snRNP U1, U2) reactive with the proteins A'or B "of the ribonucleoprotein snRNP-U2.

 Cloning of the snRNP antigens made it possible to demonstrate sequence homologies between these proteins. SnRNP-U2 protein B "has similarities to snRNP-U1 protein A, these proteins will be referred to in the following as U2B- and UlA; in particular, the aminoterminal part of U1A between the amino acid residues 7 and 101 has about 77% homology with the segment of the protein U2B "between the amino acid residues 4 and 98, and the part The carboxy-terminal U1A between amino acid residues 206 and 283 has about 86% similarity to the 147-225 segment of U2B-.

The existence of common sequences between the two proteins explains the cross-reactions observed between the anti-U2B- and anti-UlA antibodies.

 The research that led to the peptides of the invention specifically aimed to determine in the amino acid sequence of the protein UlA segments with little or no homology with the sequences of other antigens associated with autoimmune diseases.

 The developments of the work of the inventors made it possible to isolate thirteen segments from the U1A sequence, of which three not yet described are recognized by the sera of patients suffering from autoimmune diseases; among these, the inventors have demonstrated an amino acid sequence specifically recognized by the autoantibodies present in patients with mixed connective tissue disease.

The subject of the present invention is therefore a peptide comprising at least a part of the amino acid sequence of the protein A of snRNP-1 L, characterized in that it consists of or comprises all or part of the following sequence of formula
SQFGQILDILVSRSLKMRGQAFVI (I)
The meaning of the symbols representing the amino acids is recalled in the appendix.

 In formula (I), the N-terminal amino-serine amino acid and the C-terminal isoleucine amino-acid correspond respectively to residues 35 and 58 of the U1A sequence shown in FIG.

A preferred peptide comprising a part of the sequence of formula (I) is constituted by or comprises the following sequence of formula
GQILDI LV SRSLK (II)
Preferred peptides comprising the entire sequence of formula (I) are constituted by or comprise the sequence of formula (I) extended at its N-terminus by a peptide group of 1 to 10 amino acids corresponding to residues 25 to 34 of U1A represented by in FIG. 1, and / or extended at its C-terminal end by a peptide group of 1 to 10 amino acids corresponding to residues 59 to 68 of U1A shown in FIG.

 The subject of the invention is also the peptides constituted by or comprising a fragment and / or a derivative and / or a variant of the sequences of formulas (I) and (II), provided that they are specifically recognized by the autoantibodies present in patients with mixed connective tissue disease.

"Variations" means the preceding sequences modified by insertion and / or deletion and / or substitution of one or more amino acids, provided that the antigenic properties of said sequences are not modified; variants are also understood to mean sequences in which the peptide bond (-CO-NH-) is replaced by the structures
CO-N (CH3) -, -CH2-CH2-, -CO-CH2-, or else the sequences in which the peptide backbone has one or more intercalated groups such as the -CH2-, -NH-, -O- groups. . The invention also contemplates peptides in which the amino acids having asymmetric carbon are in D or L form.

The invention also relates to the conjugates obtained by coupling the peptides of the invention with carrier molecules that are optionally physiologically acceptable and non-toxic; as carrier molecules, mention may be made of natural proteins such as tetanus toxoid, albumin or albumin-containing serums or carriers such as liposomes (Frisch et al., J. Immunol., 1991, 21: 185). 193)
The invention also relates to the antibodies formed against the peptides of the invention. Antibodies which may be polyclonal or monoclonal and then produced by any hybridoma prepared by conventional cell fusion methods between spleen cells activated in vitro by the antigen or from either an animal immunized against one of the peptides of the invention or an unimmunized autoimmune animal, and cells of a myelomaous line.

 Because of the specificity of the peptides of the invention with respect to autoantibodies present in patients with mixed connective tissue disease, the antibodies prepared from the peptides constitute immunological probes that are also very specific to mixed connective tissue disease.

 Moreover, the antibodies formed against the peptides of the invention and the autoantibodies of the patients reacting with said peptides and obtained after affinity chromatography can be used to prepare anti-idiotypic antibodies, which anti-idiotype antibodies constitute in part an exact copy. of the original antigenic peptide and thus able to bind to the autoantibodies observed in patients with mixed connective tissue disease. The present invention thus relates to these anti-idiotypic antibodies and compositions containing them and their application for the in vitro diagnosis in humans of the presence of autoantibodies observed in the case of mixed connective tissue disease.

 The peptides of the invention have antigenic properties and can therefore be used in diagnostic methods for the determination and monitoring of patients with autoimmune diseases and more particularly for the diagnosis of mixed connective tissue disease. The subject of the invention is therefore also compositions containing at least one of said peptides or peptides conjugated to a carrier molecule, and capable of being recognized by autoantibodies present in the serum or any other biological fluid of patients with mixed connective tissue disease. . The detection of the peptide-antibody complex in vitro is carried out by immunoenzymatic tests of the ELISA, immunofluorescence, radioimmunoassay or radioimmunoprecipitation type, or immunotransfer immunoblots (immunoblot or dot blot).

The invention therefore also relates to a method for in vitro diagnosis of connective tissue disease, characterized in that it comprises the following steps
contacting a biological fluid capable of containing autoantibodies with at least one peptide according to the invention or that peptide conjugated to a carrier molecule, under conditions allowing the formation of an immunological peptide complex with autoantibody,
the detection of the immunological complex formed by physical or chemical methods.

 For the implementation of these tests, the invention relates to cold peptides unmarked or labeled with a suitable marker which may be biotin or its derivatives, an enzyme such as peroxidase, a fluorescent marker such as fluorescein , a radioactive marker, etc.

Such tests include for example the following steps
depositing a determined quantity of a composition containing at least one peptide of the invention or a conjugate of the peptide or an antiidiotype antibody according to the invention, in the wells of a microtiter plate or on another support such as that beads or nitrocellulose membrane,
depositing in the wells of the biological liquid to be tested or incubating it with the beads or the membrane, in the presence of saturating agents or after prior saturation of the activated supports,
after incubation and rinsing of the microplates or beads, deposition in the wells or incubation with the bilids of a system for revealing the peptide-autoantibody complex that may be formed.

 Advantageously, this test is carried out with a mixture of peptides, on the one hand at least one peptide of the invention reacting specifically with the antibodies present in the serum of patients with mixed connectivitis and secondly at least one peptide reacting with the antibodies present in the sera of patients suffering from another autoimmune disease.

Kits for carrying out the diagnostic method of the invention include
at least one peptide according to the invention, or this peptide conjugated to a carrier molecule,
reagents for the constitution of a medium which is suitable for carrying out an immunological reaction between the peptide (s) and the autoantibodies possibly present in a biological sample,
one or more optionally labeled reagents capable of reacting with said peptide (s) for the detection of the peptide-autoantibody complexes formed,
where appropriate, at least one reference biological medium, such as a serum devoid of autoantibodies.

 Finally, the invention relates to immunogenic compositions for the production of vaccines whose active principle is constituted by at least one peptide or an anti-idiotype antibody according to the invention, optionally conjugated to a carrier molecule, inducing the production of antibodies against said antibodies. peptides and which are capable of interfering with the pathologies and / or clinical manifestations of connective tissue disease. These pharmaceutical compositions according to the invention, which can be used as vaccines, consist of solutions or suspensions injectable or administrable by other routes, which can be given at doses of between 10 Fg / kg and 100 mg / kg of peptide.

 Other features 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 can not be interpreted as tending to reduce the scope of the claims.

EXAMPLE 1 PREPARATION OF PEPTIDES
Fourteen peptides corresponding to segments 1-11, 7-37, 35-58, 38-50, 56-77, 74-96, 103-135, 131153, 149-166, 163-184, 180-205, 203-225 , 224-251 and 257282 of the snRNP-U1 protein A, the sequence of which is shown in FIG. 1, were prepared according to standard solid phase peptide synthesis techniques (Briand et al., 1990), or by successive condensation. amino acid residues in the required order, either by condensation of the amino acid residues on a previously formed fragment already containing several amino acids in the appropriate order or by condensation of several previously prepared fragments, taking care to to protect beforehand all the reactive functions carried by the amino acid residues or the fragments, except the functions engaged in the peptide bond formed during the condensation.

 According to an example of preparation, the amine function of the added amino acid is protected by a terbutyloxycarbonyl (Boc) group, the side chains of the trifunctional amino acids are protected for example by the following groups: cyclohexyl for glutamic acid and aspartic acid, benzyl for threonine and serine, 2-chlorobenzyloxycarbonyl for lysine, 2,6-dichlorobenzyl for tyrosine, p-toluenesulfonyl for arginine and histidine; methionine is introduced as terbutyloxycarbonyl (O) sulfoxide methionine which is reduced to methionine upon final cleavage of the peptide from the resin by the action of anhydrous hydrofluoric acid. The amino acid Bocs are coupled in the form of benzotriazole ester except Boc histidine which is introduced as a symmetrical anhydride.

 The total coupling time is approximately 45 minutes; a ninhydrin test is used to verify that the coupling has been successfully completed, which, if necessary, can be doubled.

 At the end of the synthesis and after the last deprotection step, the resin is washed and dried.

Depending on the presence or absence of (O) methionine in the sequence a strong or weak treatment with hydrofluoric acid is used for the deprotection and cleavage of the peptide of the resin.

 After lyophilization, the crude product is dissolved in 10% acetic acid and purified by medium pressure chromatography (Barakat et al., 1990).

The final purity of each peptide is monitored and their characterization is carried out by high pressure liquid chromatography as well as by the analysis of their amino acid composition by mass spectrometry.

 In order to be used in ELISA, peptides 1-11 and 38-50 are conjugated to bovine serum albumin (BSA) via the SH function of a cysteine introduced into the peptides during synthesis, using coupling agent the m-Maleimido benzoyl-N-hydroxysuccinimide ester (Muller, 1988). Peptides 35-58, 56-77, 131-153, 149-166, 163184, 180-205, 203-225 and 224-251 are conjugated to bovine serum albumin (BSA) using 1% or 0.5% of glutaraldehyde according to the composition of the lysine sequence (Muller 1988). Peptides 7-37, 74-96, 103-135 and 257-282 were not conjugated to any carrier protein.

The peptides synthesized comprise between 11 and 33 amino acid residues and represent 96% of the 282 amino acid residues that comprise the protein A of the ribonucleoprotein snRNP-U1, hereinafter referred to as
U1A.

Peptides 149-166, 163-184 and 180-205 correspond to the domain of snRNP-U1 protein A not showing homology with snRNP protein B ".
U2. Peptides 7-37, 35-58, 56-77, 74-96, 203-225, 224-251 and 257-282 show strong homology with the corresponding portion of snRNP-U2 protein B ".

Peptide 38-50, although corresponding to the high homology domain between U1A and U2B-, has only four amino acid residues common at positions 38, 42, 43 and 50.

EXAMPLE II: SEROLOGICAL STUDY
1) Preparation of antisera in rabbits
Antisera against peptides 1-11, 7-37, 35-58, 56-77, 74-96, 103-135, 131-153, 149166, 163-184, 180-205, 203-225, 224- Unconjugated 251 and 257-282 were obtained in rabbits. The reactivity of the antisera with their corresponding peptide was measured in ELISA using microplates activated by the different peptides. A strong response was obtained with each of the thirteen previous peptides, making it possible to establish that the thirteen peptides were satisfactorily bound to the surface of the plates and could therefore be used as an antigen in an ELISA test with patient sera. studied.

2) Materials and methods
The ELISA procedure used to measure the immunological reaction between the preceding peptides and the antibodies present in the human sera tested was as follows
0.25 to 2 pLM of each peptide diluted in a 0.05 M solution of carbonate buffer are deposited in the wells of microtiter plates at a temperature of 370 Celsius; the plates are then incubated for 1 hour at 37 ° C. with a 10 mg / ml solution of bovine serum albumin (BSA) in PBS buffer at pH 7.4 and containing 0.05% of Tween 20 (PBS-T-BSA). ).

 After three washes of the microtiter plates with PBS-T buffer, patient serum diluted 1/1000 in PBS-T-BSA buffer is added and incubated for 1 hour at 37 ° C.

After several washes with PBS-T, the reaction is detected by incubation for 30 minutes at 37.degree.
OC with anti-human IgG conjugate of goat peroxidase. The reaction is then visualized by addition of a peroxidase substrate such as 3,3 ', 5,5'-tetramethylbenzidine in the presence of H 2 O 2 for 15 minutes at 37 ° C. The reaction is stopped by the addition of HCl (0.25 M final concentration) and the optical density (OD) is read at 450 nm.

 In order to determine the threshold of positivity of the test, 60 sera of healthy individuals diluted 1/1000 were tested according to the previous protocol with 0.25% of peptide 257-282 and 2 FM of the thirteen other peptides.

The average absorption is 0.16 with a standard deviation of 0.070 for peptide 257-282 and less than or equal to 0.12 with a standard deviation of 0.075 for the other peptides. A serum was thus considered positive when the value of its OD is greater than the mean value of OD found for the peptide 257-282 supplemented with twice the standard deviation, ie 0.3 units of OD. Using this threshold value, respectively, 5, 1.7 and 5% of the sera of normal individuals were found positive with peptides 1-11 (OD between 0.03 and 0.46), 38-50 (OD between 0.03 and 0.39) and 257-282 (OD between 0.07 and 0.40); whereas no positive reaction was observed with the other peptides.

2) Study of sera from patients with autoimmune diseases
The following sera were tested for the presence of antibodies capable of reacting with
ELISA with synthesized peptides
- 18 sera of patients with mixed connective tissue disease (MCTD),
- 145 sera from patients with systemic lupus erythematosus (SLE),
- 120 sera from patients with other rheumatoid diseases: 40 sera from patients with Sjögren's primary syndrome (SS), 41 sera from rheumatoid arthritis (RA) patients and 39 sera from juvenile chronic arthritis patients (JCA) )
- 60 sera of normal subjects were used as controls (NHS).

 Table I below reports the results observed with the peptides 1-11, 35-58 and 257282 using the threshold values of 0.3 and 0.4. The protocol implemented corresponds to that described above. The sera of patients diluted 1/1000 are brought into contact with 0.25 μM of the peptide 1-11 and 2 μM of the peptides 35-58 and 257-282 absorbed on the microtitration plate, then measurement of the OD after incubation of 15 minutes of the anti-human IgG peroxidase conjugate with the substrate. The results are expressed as a percentage of positive sera relative to the total number of sera tested. A serum is considered positive when the OD value is greater than or equal to 0.3 (which corresponds to the average value of the OD for the 60 sera of normal subjects added by 4 times the standard deviation).

TABLE I

<tb><SEP> Peptides <SEP> Peptides
<tb><SEP> Number <SEP> of <SEP> (Threshold <SEP> 0.30) <SEP> (Threshold <SEP> 0.40)
<tb><SEP> Serums <SEP> patients
<tb><SEP> 1-11 <SEP> 35-58 <SEP> 257-282 <SEP> 1-11 <SEP> 35-58 <SEP> 257-282
<tb> MTCD <SEP> 18 <SEP> 50.0 <SEP> 94.4 <SEP> 94.4 <SEP> 44.4 <SEP> 77.8 <SEP> 94.4
<tb> LED <SEP> 145 <SEP> 33.8 <SEP> 19.3 <SE> 64.8 <SE> 17.2 <SE> 13.8 <SE> 53.8
<tb> SS <SEP> 40 <SEP> 10.0 <SEP> 0 <SEP> 25.0 <SEP> 10.0 <SEP> 0 <SEP> 17.5
<tb> PR <SEP> 41 <SEP> 4.9 <SEP> 0 <SEP> 14.6 <SEP> 2.4 <SEP> 0 <SEP> 0
<tb> JCA <SEP> 39 <SEP> 2.6 <SEP> 0 <SEP> 10.2 <SEP> 2.6 <SEP> 0 <SEP> 2.6
<tb> NHS <SEP> 60 <SEP> 5 <SEP> 0 <SEP> 5.0 <SEP> 1.7 <SEP> 0 <SEP> 1.7
<Tb>
Using a threshold value of 0.30, 39 of the 145 sera of LED patients (ie 33.8%) react with the peptide 1-11, 28 (ie 19.3%) react with the peptide 35-58 and 94. (ie 64.8%) react with peptide 257-282. The OD values measured for each of the peptides are as follows
peptide 1-11: OD of between 0.06 and 1.11; mean value of OD 0.27 with a standard deviation of 0.19.

 peptide 35-58: OD between 0.01 and 1.45; mean value of OD 0.21 with a standard deviation of 0.19.

 peptide 257-282: OD of between 0.02 and greater than or equal to 2; mean value of the OD 0.74 with a standard deviation of 0.69.

 These results are also reported in Figure 2 in the appendix.

 Table II below shows that among the 145 sera of LED patients all those with antibodies reactive with peptide 1-11 and / or peptide 35-58 also react with peptide 257-282.

TABLE II

<tb> Reactivity <SEP> with <SEP> Reactivity <SEP> with
<tb><SEP> the <SEP> peptide <SEP><SEP> peptides <SEP> Number <SEP> of
<tb><SEP> 257-282 <SEP> 1-11 <SEP> and / or <SEP> 35-58 <SEP> sera
<tb><SEP> + <SEP> + <SEP> 47 <SEP> 32.4
<tb><SEP> + <SEP> ~ <SEP><SEP> 47 <SEP> 32.4
<tb><SEP> + <SEP> 5 <SEP> 3,4
<tb><SEP> 46 <SEP> 31.7
<Tb>
None of the other ten peptides 7-37, 56-77, 74-96, 103-135, 131-153, 149-166, 163-184, 180-205, 203225 and 224-251 were recognized by the one of the sera.

 The thirteen peptides were also tested with sera from patients with other rheumatoid diseases. It is noted in Table I that only a few of them from region 1-250 react with less than 5% of these sera, except for sera from SS patients that react with peptide 1- 11 (OD between 0.10 and 1.21, average OD of 0.21 with a standard deviation of 0.22). Moreover, it is found that the antibodies binding peptide 257-282 are present in 25% of the sera. of patients with SS (OD between 0.07 and 1.89, mean OD of 0.29 with a standard deviation of 0.31), 15% of sera from patients with RA (OD between 0.08 and 0.38, mean OD of 0.18 with a standard deviation of 0.09) and 10% of patients with JCA (OD between 0.05 and 0.41, mean OD of 0.33 with deviation standard of 0.04).

These results show a high frequency of antibodies reacting with peptide 257-282 both in the case of patients with
MCTD (94%) and LED (65%), and a lower frequency but still significant of these antibodies in the case of patients with other autoimmune diseases studied. In contrast, the peptide 35-58 corresponding to the peptide of the invention of formula I, allows a good discrimination between the sera of patients suffering from
MCTD (94%) and those with LEDs (19%).

3) ELISA reactivity of sera from patients with autoimmune diseases with peptide 38-50
Peptide 35-58 of snRNP U1 protein A has about 15 amino acid residues common to the 32-55 sequence of snRNP-U2 protein B. As a result, a portion of the LED reactive serum sera with peptide 35-58 could correspond to sera containing antibodies reactive with the snRNP-U2 protein In order to have a more specific marker of the connective tissue disease, the inventors prepared the peptide 38-50 corresponding to the peptides of the invention of formula II.

This peptide has only four amino acid residues common with the 35-47 sequence of U2B-, it is the glycine residue at position 38, the aspartic acid residue at position 42, the isoleucine residue at position 43 and the residue lysine in position 50, in addition, it does not contain the RNP1 site corresponding to the RNA binding domain, and having the formula: RGQAFVIF, which site is located in U1A at position 52-59, in U2B "in position 49-56 and in the 70 Kd protein of snRNP-U1 at position 320-327 in the sequence determined by Spritz et al (1987).

 Peptide 38-50 was tested in ELISA with 12 sera from MCTD patients, 145 sera from LED patients and 60 sera from normal subjects as a control.

 A preliminary experiment using anti-38-50 peptide antiserum prepared in the rabbit demonstrated that the 38-50 peptide was not adsorbed on the solid phase. The plates were therefore activated with a conjugate of peptide and bovine serum albumin, conjugated with which the antiserum reacted.

The 38-50 conjugated peptide was recognized by 29% of MCTD patient sera (OD between 0.07 and 0.82, mean value of OD 0.29 with a standard deviation of 0.19), only 4% of the sera of patients with SLE (OD between 0.07 and 0.63, mean value of OD 0.17 with a standard deviation of 0.09) and by less than 4% of patients with SLE other rheumatoid diseases or normal subjects (1.7%)
The standard AF / CDC 4 standard serum provided by CDC as an anti-UlRNP serum, calibrated for fluorescence use, reacted by ELISA with peptide 38-50 (title 80), as with peptides 1-11 (titre 160) and 257-282 (Title 40).

EXAMPLE 3 COMPARISON BETWEEN ELISA AND IMMUNOBLOT
1) Immunoblot
Immunoblotting experiments were performed according to the protocol described by Abuaf et al. (1990) using as antigen a rabbit and non-calf thymus extract which does not contain the 70 Kd protein specific for U1-SnRNP. The proteins are then separated in a 13% SDS-polyacrylamide gel and transferred to nitrocellulose membranes as described by Towbin et al. (1979).

Immunoreactivity is detected on stains with sera of patients diluted 1/100 and conjugate of anti-human IgG and horseradish peroxidase.

The ANA titre is measured by immunofluorescence (IF) on cell monolayers
HEp-2 and cryocoupes of rat liver.

 Antibodies raised against the extracted nuclear antigen (ENA) are detected by immunodiffusion (ID).

The immunoblot is a particularly suitable method for detecting anti-RNP antibodies (Pettersson et al., 1986, Hoch 1989, Combe et al.
Figure 3 in the appendix illustrates the immunoblot analysis of sera from patients detected by immunodiffusion of anti-Sm and / or anti-snRNP antibodies.
U1. The extracted nuclear antigen (ENA) is separated into
SDS-PAGE 13% and transferred to nitrocellulose band.

Each band is then incubated with patient sera diluted 1/100, and contacted with a goat-peroxidase anti-human IgG conjugate and then with a chloronaphthol substrate. Strips A, B, C were incubated with a pateint serum containing anti-Sm antibodies, the J band was incubated with a patient serum containing anti-snRNP-U1 antibodies and the K band with a subject serum. normal; groups
D, E, F, G, H and I were respectively incubated with the sera of pateints 11, 17, 14, 15, 3 and 4 of Table III below.

2) Results
Nineteen sera of male (M) or female (F) patients diagnosed with MCTD or SLE are tested in parallel with ELISA peptides 1-11, 35-58, 257282 and immunoblotted with a thymus nuclear extract as antigen. .

Table III below reports the results of this comparative study between immunoblot and
ELISA.

TABLE III

Patients <SEP> Gender <SEP> Diagnosis <SEP> ANA <SEP> IF <SEP> Anti <SEP> ENA <SEP> ID <SEP> IMMUNOBLOT <SEP> ELISA
<tb><SEP> Title <SEP> 70Kd <SEP> A <SEP> BB '<SEP> C <SEP> D <SEP> 1-11 <SEP> 35-58 <SEP> 257-282
<tb><SEP> 1 <SEP> M <SEP> MTCD <SEP> 1000 <SEP> RNP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> + <SEP> ++
<tb><SEP> 2 <SEP> F <SEP> MTCD <SEP>> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> - <SEP> - <SEP> + <SEP > + <SEP> ++
<tb><SEP> 3 <SEP> M <SEP> MTCD <SEP>> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> + <SEP> - <SEP> + <SEP > + <SEP> ++
<tb><SEP> 4 <SEP> F <SEP> MTCD <SEP>> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> + <SEP> - <SEP> + <SEP > + <SEP> ++
<tb><SEP> 5 <SEP> F <SEP> MTCD <SEP> 1000 <SEP> RNP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++ <SEP> ++
<tb><SEP> 6 <SEP> F <SEP> MTCD <SEP> 500 <SEP> RNP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 7 <SEP> F <SEP> MTCD <SEP> 1000 <SEP> RNP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 8 <SEP> F <SEP> MTCD <SEP> 500 <SEP> RNP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 9 <SEP> F <SEP> MTCD <SEP> 500 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 10 <SEP> F <SEP> MTCD <SEP> 200 <SEP> RNP <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> + <SEP> + <SEP> ++
<tb><SEP> 11 <SEP> F <SEP> MTCD <SEP>> 1000 <SEP> RNP <SEP> - <SEP> + <SEP> + <SEP> + <SEP> - <SEP> - <SEP > + <SEP> ++
<tb><SEP> 12 <SEP> F <SEP> MTCD <SEP> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 13 <SEP> F <SEP> MTCD <SEP> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 14 <SEP> F <SEP> LED <SEP> 1000 <SE> RNP + Sm <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> ++
<tb><SEP> 15 <SEP> F <SEP> LED <SEP> 500 <SE> RNP <SEP> + <SEP> + <SEP> + <SEP> - <SEP> + <SEP> + <SEP> + <SEP> ++
<tb><SEP> 16 <SEP> F <SEP> LED <SEP> 1000 <SEP> RNP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> ++
<tb><SEP> 17 <SEP> M <SEP> LED <SEP>> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP > + <SEP> ++
<tb><SEP> 18 <SEP> F <SEP> LED <SEP>> 1000 <SEP> RNP <SEP> + <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP > + <SEP> ++
<tb><SEP> 19 <SEP> F <SEP> LED <SEP> 500 <SE> RNP <SEP> + <SEP> + <SEP> + <SEP> - <SEP> - <SEP> + <SEP> + <SEP> ++
<Tb>
As shown in Table IV below, 4 of 19 sera reacted with peptide 1-11 in ELISA and immunoblot with polypeptide A, while 4 sera were negative in both tests; 2 ELISA positive sera with peptide 1-11 are immunoblot negative whereas 4 negative sera are negative in
ELISA and positive with the immunoblot polypeptide A. Interestingly, all sera reacting with immunoblot band A are ELISA positive with both peptides 35-58 and 257-282; in addition, 6 sera (1
LED and MCTD) reacted with both peptides but will not bind the A polypeptide in immunoblot.

TABLE IV

<tb><SEP> Reactivity <SEP> in <SEP> ELISA
<tb> Reactivity <SEP> with
<tb><SEP> the <SEP> band <SEP> A <SEP> in <SEP> Peptide <SEP> 1-11 <SEP> Peptides
<tb><SEP> real estate <SEP> 35 <SEP> - <SEP> 48 <SEP> and <SEP> 257 <SEP> - <SEP> 282
<tb><SEP> Positive <SEP> Negative <SEP> Positive <SEP> Negative
<tb><SEP> Positive <SEP> 8 <SEP> 5 <SEP> 13 <SEP> 0
<tb><SEP> Negative <SEP> 2 <SEP> 4 <SEP> 6 <SEP> 0
<Tb>
EXAMPLE IV: PREDICTION OF SITES
ANTIGENIOUES
The previous study allowed the inventors to identify several regions of the protein A of snRNP-U1 recognized by autoantibodies present in the sera of patients with various autoimmune diseases. Protein A interacts directly with snRNA-U1 via a RRM recognition pattern located in the N-terminal region of the protein at position 12-84. This region contains the RNP1 consensus sequence at position 52-59, which is also present in the 70Kd and B "proteins and in the La 60 Kd antigen. A second RRM motif located in the C-terminal region of the protein at position 210 -277 is not necessary for the specific binding between RNA and U1A (Scherly et al., 1990). However, the U1A and U2B "proteins have significant sequence homology, including about 77% homology at 7-101 of U1A and 4-98 of U2B ", the region defined by Scherly et al. (1990) as a major determinant of the specific binding with RNA is located very close to the RNP consensus sequence in one the most variable regions of the preserved motif.

This seems to explain the difference between U1A and U2B- at the level of binding properties between RNA and protein.

 Three of the peptides studied previously are recognized by the antibodies present in the sera of patients tested. Peptide 257-282 and at the opposite end peptide 1-11 are recognized by sera from patients with SLE and MCTD, as well as antibodies present in the sera of patients with other rheumatoid diseases. The 35-58 peptide is recognized by 94% of the MCTD patient sera and only 19% of the LED sera.

 The methods for prediction of antigenic sites use algorithms based on certain parameters such as hydrophobicity or hydrophilicity or the mobility of short segments of the protein structure.

Figure 4 in the appendix shows the antigenic prediction profiles of the U1A protein constructed according to the following scales
- in A: according to the hydrophilicity scale of
Parker et al. (1986)
- in B: according to the mobility scale of
Karplus and Schultz (1985).

The scales were normalized according to the method of Pellequer et al. (1991). Charts are plotted between the fourth residue and the n-3 residue. The localization of epitopes recognized MCTD sera and
LEDs (regions 1-11, 35-58 and 257-282) are shown in thick lines.

Table V below indicates, for the three peptides reactive in ELSA (peptides 1-11, 35-58 and 258-282), the result of the antigenic prediction obtained according to the following four methods.
- mobility scale, Karplus and Schultz (1985); result obtained from the data of Figure 4,
hydrophilicity scale, Parker et al.

(1986); result obtained from the data of Figure 4,
hydrophilicity scale according to the HYDRO 3 program of Hopp (1986); Pollard and Cohen (1990)
- acrophilicity scale according to the program
ACRO de Hopp (1986); Pollard and Cohen (1990).

TABLE V

<tb><SEP> PEPTIDE
<tb><SEP> 1-11 <SEP> 35-58 <SEP> 258-282
<tb> Scale <SEP> of <SEP> mobility <SEP> Karplus <SEP> and <SEP> + <SEP> - <SEP> +
<tb><SEP> Schultz <SEP> (1985)
<tb> Scale <SEP> of Hydrophilicity, <SEP> Parker <SEP> +/- <SEP> - <SEP> +
<tb><SEP> and <SEP> in (1986) <SEP>
<tb> Scale <SEP> of hydrophilicity <SEP> according to
<tb><SEP> the <SEP> program <SEP> HYDRO <SEP> 3 <SEP> of <SEP> Hopp <SEP> - <SEP> + <SEP>
<tb><SEP> (1986)
<tb> Scale <SEP> of acrophilicity <SEP> according to <SEP>
<tb><SEP> program <SEP> ACRO <SEP> from <SEP> Hopp <SEP> (1886) <SEP><SEP> - <SEP> - <SEP> - <SEP>
<Tb>
The HYDRO 3 program (Hopp, 1986) based on hydrophilicity indicates that the 43-56 sequence might contain an antigenic site of the U1A protein (Pollard and Cohen, 1990). This site is however not noticed using the Parker et al. hydrophilicity scale, Karplus and Scultz mobility scale, or Hopp acrolegality scale (ACRO). Regions 1-11 and 258282 are not highlighted with programs
HYDRO 3 and ACRO and 9 regions determined by the programs HYDRO 3 and ACRO as potentially antigenic have not been recognized in the study described previously, these are the regions 15-28, 8396, 99-112, 106-119 , 123-136, 134-147, 153-166, 198-211 and 212-225 described by Pollard and Cohen (1990).

 These results illustrate the limitations of antigenic prediction scales, particularly when applied to RNP autoantigens.

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Symbols of amino acids
Ala alanine
Cysteine Cysteine
D Asp aspartic acid
E Glu glutamic acid
F Phenylalanine
Gly glycine
H his histidine
I Isoleucine Island
K Lily lysine
Leucine leucine
M Met methionine
N Asn asparagine
P Pro proline
Gln glutamine
R Arg arginine
Serin serine
TH threonine
Valine Valley
W Trptophan Trp
Tyr Tyrosine Y

Claims (7)

 1 - Peptide comprising at least a portion of the amino acid sequence of protein A of snRNP-U1, characterized in that it consists of or comprises all or part of the following sequence of formula SQFGQILD ILVSRSLKMRGQAFVI  2 - Peptide according to claim 1, characterized in that it consists of or comprises the following sequence of formula  GQILDILVSRSLK  3 - Antibody characterized in that it is (or are) directed specifically against a peptide according to any one of claims 1 and 2.  4 - antigenic composition characterized in that it contains at least one peptide according to one of claims 1 and 2, or a conjugate of this peptide with a carrier molecule.  5 - Method for in vitro diagnosis of connective tissue disease, characterized in that it comprises the following steps  contacting a biological fluid that may contain autoantibodies with at least one peptide according to one of claims 1 and 2, or this peptide conjugated to a carrier molecule, under conditions allowing the formation of an immmunological complex peptide autoantibodies,  the detection of the immunological complex formed by physical or chemical methods.  6 - Kits for the in vitro diagnosis of connective tissue disease characterized in that it comprises  at least one peptide according to one of claims 1 and 2, or this peptide conjugated to a carrier molecule,  reagents for the constitution of a medium which is suitable for carrying out an immunological reaction between the peptide (s) and the autoantibodies possibly present in a biological sample,  one or more optionally labeled reagents capable of reacting with said peptide (s) for the detection of the formed peptide-autoantibody complex,  where appropriate, at least one reference biological medium, such as a serum devoid of autoantibodies.  7 - immunogenic composition characterized in that it contains at least one peptide according to one of claims 1 and 2, or a conjugate of this peptide with a carrier molecule.