# Patent

## Title
Method for detecting rheumatoid arthritis-specific autoantibodies

## Abstract

A method for detecting anti-filaggrin-autoantibodies specific to rheumatoid arthritis in a biological sample, by providing a filaggrin with no citrulline residues (FNC) having an arginine residue or a filaggrin peptide with no citrulline residues (PFNC) having an arginine residue. Also provided are a citrulline-containing filaggrin (FC) having the peptide sequence of the FNC where some arginine residues have been converted to citrulline residues or a citrulline-containing peptide (PFC) having the peptide sequence of the PFNC, where at least one arginine residue has been converted to a citrulline residue. The sample is contacted with the FNC or PFNC and the FC or PFC, and the resulting immune complexes are detected and quantified, with a value XNC and XC, respectively. Anti-filaggrin-autoantibodies specific to rheumatoid arthritis are determined to be present in the biological sample if the value of XC is greater than the value of XNC.

## Background
Rheumatoid arthritis (RA) is the most common of the chronic inflammatory rheumatisms. It is a systemic autoimmune disease characterized by an inflammation of the joints, which affects more than 500 000 individuals in France and more than 2 million in the United States. The serum of affected patients contains autoantibodies, some of which are specific and may constitute a marker for the disease allowing diagnosis thereof even at an early stage.
Autoantibodies which are specifically present in patients suffering from RA and which react with a rat esophageal epithelial antigen were described for the first time by B. J. J. Young et al., in Br. Med. J. 2:97-99 (1979). At the time, these autoantibodies were named “anti-keratin antibodies” (AKAs), but it has subsequently been shown that these antibodies are in fact antifilaggrin autoantibodies (AFAs). The antigens for these antibodies are the neutral/acidic forms of filaggrins (Simon et al., J. Clin. Invest., 92, 1387, (1993)).
Filaggrin is generated from profilaggrin, which is a phosphorylated polyprotein of epidermal keratohyalin granules. Profilaggrin has a high molecular weight (approximately 400 000 in humans), and it is soluble in the presence of high concentrations of salts or of urea. It has a high content of basic amino acids (arginine and histidine), and also of glycine, serine and glutamic acid. It has a low content of nonpolar amino acids and contains neither methionine, nor cysteine, nor tryptophan. It is highly phosphorylated on serine residues, which confers on it an isoelectric point to close to neutrality.
Profilaggrin is cleaved into filaggrin units in the course of a complex process of maturation involving dephosphorylation followed by cleavage by proteases in the interdomain segments. This cleavage first generates fragments of intermediate size, and then the functional filaggrin molecules.
Filaggrins derived from the dephosphorylation and cleavage of profilaggrin are basic proteins, the amino acid content of which is similar to that of the profilaggrins. They contribute to keratin filament organization and undergo gradual maturation during which the arginine residues, which are basic, are converted to citrulline residues, which are neutral, under the action of peptidylarginine deiminase (Harding C. R. and Scott I. R., J. Mol. Biol. 170, p. 651-673 (1983)). This leads to a decrease in their affinity for the keratins, from which they detach; they are then completely degraded by various proteases.
The properties of filaggrins and of pro-filaggrins have been particularly well studied in rats, in mice and in humans. The size of profilaggrin varies, according to species, from 300 to 400 kD, and that of the filaggrins from 27 to 64 kD.
The gene encoding profilaggrin is made up of repeated subunits, each one of which encodes a filaggrin molecule, separated by portions encoding the interdomain peptide segments. All the repeat units encoding each of the human filaggrins have the same length (972 base pairs in humans); however, in humans, considerable sequence variations (10-15%) are observed from one subunit to the other. While most are conservative, some of these variations induce amino acid changes and, in some cases, changes in the electric charge of the protein. Thus, human filaggrins form, independently of the post-transcriptional modifications, a heterogeneous population of molecules of similar size but of different sequences and charges (pHi equal to 8.3±1.1) (Gan et al., Biochem. 29, p. 9432-9440 (1990)).
The polymorphism observed in humans between the sequences of the filaggrin units within the same profilaggrin gene is not apparent in rats and mice. Filaggrins also exhibit a great inter- and intraspecific variability in terms of their sequence. This variability does not however affect their functional properties, nor their overall amino acid composition or their biochemical properties. Similarly, the tissue localizations of profilaggrin and of filaggrins are identical in the various mammals studied.
The autoantibodies (AFAs) are associated with the clinical and biological parameters which define the most active and the most severe forms of the disease, and are thought to precede the appearance of the clinical signs. The detection thereof therefore proves to be an essential step in the diagnosis of RA.
According to document WO-A-98/08946 in the applicant's name, a method has been developed for detecting AFAs, in a biological sample, based on the discovery that citrullination of filaggrin is necessary for detection of AFAs present in sera from patients suffering from RA, this citrullination generating epitopes recognized by the AFAs. This method comprises the following steps:
the biological sample analyzed is brought into contact with a citrulline-containing recombinant filaggrin or a fragment thereof, under conditions suitable for the formation of immune complexes with the AFAs, and
the immune complexes possibly formed with the AFAs are detected.
The results demonstrate that the method is highly sensitive, most of the sera from patients suffering from RA recognizing the citrulline-containing recombinant filaggrin.
It was however observed, in the course of the steps for diagnosing RA in the patients whose sera were found to be positive in this method, that not all were RA cases. This method in fact leads to the detection of false positives, as will be illustrated in the following examples, and cannot therefore be used reliably, due to an insufficient specificity.
The present inventors have now developed a new ELISA assay for diagnosing RA in patients, which has great specificity while at the same time conserving the high sensitivity of the abovementioned method.
According to this method, rheumatoid arthritis (RA)-specific autoantibodies (AFAs) are detected in a biological sample liable to contain said autoantibodies (AFAs) and other autoantibodies not specific for RA, as follows:
firstly, a filaggrin (FNC) or a derivative of filaggrin (FNC) or a filaggrin peptide (PFNC) comprising at least one arginine residue and, secondly, said citrulline-containing filaggrin (FC) or a derivative of said citrulline-containing filaggrin (FC) or said citrulline-containing peptide (PFC) are provided,
said biological sample is brought into contact with, firstly, said filaggrin (FNC) or said derivative of said filaggrin (FNC) or said peptide (PFNC) and, secondly, said filaggrin (FC) or said derivative of said filaggrin (FC) or said peptide (PFC), under conditions suitable for the formation of immune complexes with the autoantibodies (AFAs),
the immune complexes formed between the auto-antibodies (AFAs) or the other antibodies present in the sample and, firstly, said filaggrin (FNC) or said derivative of said filaggrin (FNC) or said peptide (PFNC) and, secondly, said citrulline-containing filaggrin (FC) or said derivative of said citrulline-containing filaggrin (FC) or said peptide (PFC) are detected and quantified, this quantification being expressed by a value respectively XNC and XC,
the value of XC is reduced by the value of XNC.
To implement the method of the invention, the filaggrin (FNC) or the derivative of a filaggrin (FNC) or the peptide (PFNC) is immobilized on the solid support and the filaggrin (FC) or a derivative of the filaggrin (FC) or the peptide (PFC) is immobilized on another solid support. They may be, inter alia, two wells on the microtitration plate or two tips of the Vidas device (registered trademark) marketed by the applicant, but also strips or any other suitable support.
Before describing the invention in greater detail and presenting the preferred variants thereof, some terms used in the description and the claims are defined below.
Citrullination is the reaction of conversion of an arginine residue to a citrulline residue by conversion of the —C(NH)NH2 group of the arginine to —CONH2. The reaction is advantageously carried out in vitro with the peptidylarginine deiminase (PAD) enzyme. This enzyme may be a rabbit muscle PAD; it is however within the scope of those skilled in the art to select another suitable PAD. It is of course possible to envision any other means of citrullination, such as chemical reagents or microorganisms.
According to the invention, when reference is made to a citrulline-containing filaggrin, this means that a proportion of approximately at least 20% of the arginine residues have been converted to citrulline residues, this conversion taking place randomly. Preferably, this degree of citrullination is at least 30%, and advantageously at least 50%. It has been observed that the immunoreactivity of a citrulline-containing filaggrin for which the degree of citrullination is approximately 80% is similar to that of a filaggrin for which the degree of citrullination is approximately 50%. Under these conditions, it is therefore unnecessary to seek to achieve very high degrees of citrullination.
If the citrullination is not carried out randomly, but is region-specific, and in particular if the conversion reaction can be limited to an arginine residue belonging to an epitope of the filaggrin after citrullination, the degree of citrullination can be lower than the abovementioned values, without the immunoreactivity of the filaggrin thus citrullinated being effected by this.
The expression “derivative of a filaggrin (FNC)” or “corresponding citrulline-containing derivative” means: a chimeric filaggrin which consists of a combination of fragment(s) of sequences of human and rat filaggrin and/or a combination of fragment(s) of sequences of human and/or rat filaggrin and of consensus peptide(s).
To implement the method of the invention, the filaggrin is preferably chosen from human filaggrin and a filaggrin of animal origin. It advantageously consists of a recombinant rat filaggrin having the sequence SEQ ID No. 1.
In place of the filaggrin, it is possible to choose a filaggrin peptide (PFNC), the sequence of which comprises at least one arginine residue, and the corresponding citrulline-containing peptide (PFC) for which said arginine residue at least of PFNC has been converted to a citrulline residue. According to a variant of the method of the invention, the peptide contains an epitope of filaggrin after citrullination. Thus, a peptide (PFNC) may in particular be chosen from the following peptides:

    
    
        the S47S peptide, the sequence of which is represented in SEQ ID No. 2, corresponding to amino acids 71 to 119 of the sequence of a human filaggrin unit and containing 6 arginine residues,
        the E12H peptide, the sequence of which is represented in SEQ ID No. 3, as determined with reference to the nucleotide sequences of the human profilaggrin gene described by Gan S. Q. et al. (Biochemistry, 29: 9432-9440, (1990)) and comprising 1 arginine residue,
        the E12D peptide, the sequence of which is represented in SEQ ID No. 4, as determined with reference to the nucleotide sequences of the human profilaggrin gene described by Gan S. Q. et al. (Biochemistry, 29: 9432-9440, (1990)) and comprising 3 arginine residues,
        the G22Q peptide, the sequence of which is represented in SEQ ID No. 5, corresponding to a consensus sequence of a rat filaggrin unit comprising 4 arginine residues,
        the G26E peptide, the sequence of which is represented in SEQ ID No. 6, corresponding to a consensus sequence of a rat filaggrin unit comprising 10 arginine residues.
    
    


As mentioned above, while a filaggrin (FNC) is provided for implementing the method of the invention, the citrulline-containing filaggrin (FC) used has the peptide sequence of filaggrin (FNC), in which at least 20%, preferably at least 30%, and better still at least 50%, of the arginine residues of said FNC are converted to citrullines.
The autoantibodies (AFAs) are circulating antibodies, and a biological sample to be analyzed according to the method of the invention may be chosen from blood, plasma and serum.
The detection and quantification of the immune complexes formed are carried out by any techniques well known to those skilled in the art, and in particular using an enzyme for the detection and its calorimetric or fluorescent substrate for the quantification (examples: alkaline phosphatase/4-methylumbelliferyl phosphate, peroxidase/ortho-phenylenediamine).
As will be illustrated in the examples, it is thus possible to bring the immune complexes formed into contact with a conjugate consisting of a labeled antibody directed against human immunoglobulins, under conditions suitable for the formation of labeled immune complexes, and then the labeled immune complexes are detected and quantified. The conjugate is advantageously an anti-human immunoglobulin antibody, whole or fragmented (examples: Fab′, Fab′2 fragments), labeled with peroxidase or with alkaline phosphatase, and the formation of labeled immune complexes is then detected and quantified by colorimetry or fluorimetry, the quantification being expressed as optical density or fluorescence values XNC and XC.
A positive assay revealing the presence of RA-specific autoantibodies (AFAs) in the sample analyzed is characterized by the value XC being greater than the value XNC.
The invention is illustrated below and its characteristics and advantages are brought to light.


EXAMPLE 1
Production of a Recombinant Rat Filaggrin
A) Cloning of the Rat Profilaggrin Gene
The studies by Haydock and Dale, 1986 (J. Biol. Chem., 261, 12520-12525) and by Rothnagel et al., 1987 (J. Biol. Chem., 262, 15643-15648) have shown that the rat profilaggrin gene is made up of 20±2 repeat units of approximately 1200 base pairs (bp) and has no intron in its coding region, each profilaggrin unit being made up of a filaggrin domain and a binding sequence.
Furthermore, the coding sequence for two incomplete profilaggrin units has been published by Haydock and Dale, 1990 (DNA Cell Biol., 9, 251-261).
Based on these data, oligonucleotides intended for the PCR amplification of the coding sequence for a complete rat profilaggrin unit were designed. The rat genomic DNA matrix was prepared from whole blood of a Wistar rat. Three different PCR products were obtained with three different pairs of oligonucleotides. These PCR products were cloned into a vector pCAPs (PCR cloning kit, Boehringer) and sequenced. They exhibit a high degree of homology (of more than 99%) with one another and also a 95% and 98% homology with the two incomplete profilaggrin units published by Haydock and Dale, 1990.
These data confirm the nature of the DNA obtained and suggest that the available sequences are representative of the existing rat filaggrin domains, a certain inter- and intra-individual variability being expected.
One of the sequences obtained was cloned into an expression vector pMR (Cheynet et al., 1993, Prot. Exp. Purif., 4, 367-372) as a fusion with a coding sequence for 6 histidines in the 5′ position. The construct identified as pOL041 was used to transform E. coli bacteria. It encodes a 399 amino acid protein with a theoretical molecular weight of 42 210, having the sequence SEQ ID No. 1.
B) Expression and Purification of the Recombinant Rat Filaggrin
Culturing:
To place the transformed E. coli strain (BL21 or DH5α) in culture, an isolation from the library conserved at −80° C. is carried out on agar medium with 0.1 mg/ml ampicillin.
From an isolated colony, a preculture is prepared in liquid medium (yeast extract, 2% glucose and 0.1 mg/ml ampicillin) in Erlenmeyer flasks placed at +37° C. and with orbital shaking (280 rpm).
This preculture then makes it possible to seed 5 l Erlenmeyer culture flasks, each containing 1 l of the same medium optionally substituted with PMSF at a final concentration of 0.5 mM. These Erlenmeyer flasks are cultured at +37° C. with shaking at 280 rpm, until an OD at 600 nm of 0.8 is obtained. Once this OD value has been reached, the culturing is continued for a period of 3 hours.
The culture is then stopped and centrifuged for 25 minutes at 6-000 g. The bacterial pellets are then collected and conserved at −25° C.
Purification:
The bacterial pellet is lysed by the action of lysozyme in the presence of protease inhibitor in a buffer consisting of 0.1 M NaH2PO4/0.6 M NaCl/0.2% Tween/1 mM Na azide/6 mM imidazole, pH 7.3, and by sonication. This lysate is then clarified for 30 minutes at 23 700 g and the supernatant is conserved.
The recombinant filaggrin is purified by metal (nickel) chelate affinity chromatography at +4° C.
Briefly, after re-equilibration of the gel in lysis buffer, the lysis supernatant is injected onto the column, followed by washing (and monitoring of the UV 280 nm detection signal) with the buffer consisting of 0.1 M NaH2PO4/0.6 M NaCl/1 mM Na azide/6 mM imidazole, pH 7.3, until the UV 280 nm detection signal has returned to base line.
The recombinant filaggrin (rFlg), adsorbed onto the gel, is eluted with the buffer consisting of 0.1 M NaH2PO4/0.6 M NaCl/1 mM Na azide/0.5 M imidazole, pH 7.15.
The fraction thus obtained is concentrated on a 10 kDa membrane.
The final purification step is carried out by gel filtration chromatography on a Superdex 200 support, in 20 mM Tris/0.3 M NaCl buffer, pH 11. The concentrate is injected onto the column, the fractions being selected by monitoring the UV signal at 280 nm and 214 nm. This step makes it possible to isolate and recover the immunologically active recombinant filaggrin of interest, in the second elution peak (MW≈42 000).
EXAMPLE 2
Citrullination of the Recombinant Filaggrin
After assaying the total proteins by the Bradford method (Biorad reagents), the citrullination is performed, by the action of peptidylarginine deiminase, for 4 hours at +37° C. with magnetic stirring, at a rate of 4 enzyme units/mg of total proteins, in the presence of 0.1 M EDTA/1 M DTT/0.5 M CaCl2. The amount of total proteins is then re-assayed on the deiminated fraction.
The citrulline-containing filaggrin is purified by Ni-NTA chromatography and then gel filtration chromatography.
The degree of citrullination detected is 53%.
Examples 3 and 4 below illustrate the diagnosis of RA in a method of detection of the prior art and in a method of detection according to the invention, so as to allow the comparison thereof.
The method of detection of the prior art consists of an ELISA assay, of the type such as that described in WO-98/08946 in the applicant's name, using citrulline-containing filaggrin and based on the detection of immune complexes formed between the AFA antibodies possibly present in the sample and the citrulline-containing filaggrin.
The method of detection according to the invention is an ELISA assay using citrulline-containing filaggrin and non-citrulline-containing filaggrin and which is based on the parallel detection of the immune complexes formed, firstly, with the citrulline-containing filaggrin and, secondly, the non-citrulline-containing filaggrin, and on the difference in values between the two.
EXAMPLE 3
A) Samples
The assays were carried out on the following samples:
63 control sera affected by the following pathological conditions: algodystrophy, gout, malignant hemopathy, Paget's disease, neuralgia, mechanical pathology, periarteritis nodosa, polymyositis, lupus-related rheumatism, psoriatic rheumatism, scleroderma, ankylosing spondylitis, spondylarthropathy, Gougerot's syndrome, Sharp's syndrome, vasculitis.
65 sera from patients suffering from clinically diagnosed rheumatoid arthritis.
B) Material
Nunc Maxisorp 468667 plate
Incubator at 35-39° C.
Axia microwasher U4402
Biotek (commercial trademark) plate reader
C) Protocol
Deposition in wells: 100 μl per well of 2.5 μg/ml of filaggrin for 2 hours at 37° C. in PBS buffer, pH 7.2;
3 washed with 300 μl per well of PBS/0.05% Tween buffer, pH 7.2;
blocking for 1 hour at 37° C. with 250 μl per well of PBS/1% BSA buffer, pH 7.2;
3 washed with 300 μl per well of PBS/0.05% Tween buffer, pH 7.2;
Incubation for 1 hour at 37° C. with 100 μl per well of serum diluted to 1/100 in buffer consisting of 10 mM Tris, 350 mM NaCl, 1% TritonX100, 1% BSA, 10% rabbit serum, pH 7.6;
3 washed with 300 μl per well of PBS/0.05% Tween buffer, pH 7.2;
incubation of an anti-human IgG conjugate diluted in buffer consisting of 10 mM Tris, 350 mM NaCl, 1% TritonX100, 1% BSA, 10% rabbit serum, pH 7.6; 100 μl per well;
3 washed with 300 μl per well of PBS/0.05% Tween buffer, pH 7.2.
Enzymatic Revelation:

    
    
        100 μl of ortho-phenylenediamine (OPD) per well
        incubation for 10 min at 18-25° C.
        blocking with 100 μl H2SO4 
        reading at 492 nm.
    
    


The samples are assayed in parallel on a plate coated with non-citrulline-containing filaggrin and on another plate coated with citrulline-containing filaggrin.
The samples are assayed unduplicated.
The results are expressed as optical density. The signals indicated at an optical density of 3 are equal to or greater than 3.
D) Results
The results are given in tables 1 and 2 below.
With the results obtained on citrulline-containing filaggrin according to WO-A-98/08946 (table 1), the performance levels are as follows:

















100% specificity
threshold
  34% sensitivity
(22/65)




at 1.337





 98% specificity
threshold
  38% sensitivity
(22/65)




at 0.937





 95% specificity
threshold
61.5% sensitivity
(40/65)




at 0.335









With the results obtained on citrulline-containing filaggrin/non-citrulline-containing filaggrin according to the invention (table 2), the performance levels are as follows:

















100% specificity
threshold
38% sensitivity
(25/65)




at 0.731





 98% specificity
threshold
69% sensitivity
(45/65)




at 0.009





 95% specificity
threshold
77% sensitivity
(50/65)




at −0.004









In both cases, at 95% specificity, 3 sera are false positives.
All the samples detected on the citrulline-containing filaggrin phase are detected on the differential system with 100% and 98% specificity. A single serum is additionally detected with the differential system. At 95% specificity, 7 samples are additionally detected with the differential system.
The differential system is more effective than the citrulline-containing filaggrin phase on this population, with 69% sensitivity for 98% specificity.
For this population, the performance levels of the assays normally used are as follows:
GIFOR (immunofluorescence on section of rat esophagus)

















98% specificity
threshold
49% sensitivity
(32/65)




at 2





Gblot






98% specificity
threshold
58% sensitivity
(38/65)




at 2.25









The performance levels obtained by ELISA on rat filaggrin are better than the techniques currently used, GIFOR and GBlot.







TABLE 1







Results as OD at 492 nm of the ELISA assays on


citrulline-containing filaggrin (FC)












Negative controls
FC
RA sera
FC















58
0.050
78
0.051



57
0.056
76
0.059



10
0.058
72
0.077



33
0.060
74
0.077



51
0.062
81
0.079



53
0.062
79
0.088



44
0.064
73
0.091



54
0.066
71
0.098



47
0.069
80
0.102



49
0.069
75
0.109



38
0.070
65
0.113



39
0.070
86
0.121



40
0.071
66
0.124



50
0.072
83
0.132



29
0.073
69
0.141



45
0.073
77
0.147



25
0.076
84
0.151



55
0.076
85
0.156



28
0.079
90
0.157



42
0.083
67
0.158



46
0.083
68
0.158



60
0.085
70
0.202



43
0.087
89
0.207



31
0.088
88
0.276



34
0.088
92
0.299



37
0.090
93
0.387



41
0.090
87
0.409



16
0.092
64
0.434



35
0.092
82
0.472



48
0.092
95
0.496



26
0.093
98
0.499



52
0.093
96
0.524



17
0.097
94
0.535



61
0.098
100
0.611



27
0.111
99
0.659



19
0.113
91
0.667



20
0.113
103
0.726



4
0.116
101
0.729



22
0.116
102
0.801



36
0.123
97
0.853



18
0.124
104
0.939



24
0.124
105
1.203



14
0.128
106
1.240



12
0.129
107
1.526



23
0.130
108
1.694



7
0.133
109
1.900



5
0.134
110
2.076



9
0.135
111
2.387



30
0.142
114
2.593



59
0.146
112
2.610



6
0.158
113
3.000



21
0.159
115
3.000



11
0.166
116
3.000



32
0.167
117
3.000



15
0.169
118
3.000



62
0.193
119
3.000



56
0.222
120
3.000



3
0.253
121
3.000



13
0.269
122
3.000



1
0.335
123
3.000



8
0.369
124
3.000



63
0.937
125
3.000



2
1.337
126
3.000





127
3.000





128
3.000
















TABLE 2







Results as OD at 492 nm of the ELISA assays on


citrulline-containing filaggrin (FC) and non-


citrulline-containing filaggrin (FNC)














Negative









controls
FNC
FC
FC − FNC
RA sera
FNC
FC
FC − FNC

















1
0.780
0.335
−0.445
64
0.777
0.434
−0.343


2
1.747
1.337
−0.410
65
0.202
0.113
−0.089


3
0.433
0.253
−0.180
66
0.204
0.124
−0.080


4
0.268
0.116
−0.152
67
0.222
0.158
−0.064


5
0.271
0.134
−0.137
68
0.221
0.158
−0.063


6
0.294
0.158
−0.136
69
0.195
0.141
−0.054


7
0.262
0.133
−0.129
70
0.255
0.202
−0.053


8
0.492
0.369
−0.123
71
0.131
0.098
−0.033


9
0.231
0.135
−0.096
72
0.109
0.077
−0.032


10
0.149
0.058
−0.091
73
0.115
0.091
−0.024


11
0.256
0.166
−0.090
74
0.099
0.077
−0.022


12
0.218
0.129
−0.089
75
0.131
0.109
−0.022


13
0.351
0.269
−0.082
76
0.072
0.059
−0.013


14
0.209
0.128
−0.081
77
0.157
0.147
−0.010


15
0.248
0.169
−0.079
78
0.055
0.051
−0.004


16
0.158
0.092
−0.066
79
0.090
0.088
−0.002


17
0.162
0.097
−0.065
80
0.104
0.102
−0.002


18
0.188
0.124
−0.064
81
0.079
0.079
0.000


19
0.174
0.113
−0.061
82
0.466
0.472
0.006


20
0.173
0.113
−0.060
83
0.124
0.132
0.008


21
0.219
0.159
−0.060
84
0.137
0.151
0.014


22
0.171
0.116
−0.055
85
0.137
0.156
0.019


23
0.179
0.130
−0.049
86
0.101
0.121
0.020


24
0.172
0.124
−0.048
87
0.372
0.409
0.037


25
0.123
0.076
−0.047
88
0.236
0.276
0.040


26
0.139
0.093
−0.046
89
0.137
0.207
0.070


27
0.153
0.111
−0.042
90
0.057
0.157
0.100


28
0.120
0.079
−0.041
91
0.514
0.667
0.153


29
0.113
0.073
−0.040
92
0.058
0.299
0.241


30
0.181
0.142
−0.039
93
0.134
0.387
0.253


31
0.126
0.088
−0.038
94
0.240
0.535
0.295


32
0.204
0.167
−0.037
95
0.156
0.496
0.340


33
0.096
0.060
−0.036
96
0.122
0.524
0.402


34
0.122
0.088
−0.034
97
0.424
0.853
0.429


35
0.125
0.092
−0.033
98
0.063
0.499
0.436


36
0.156
0.123
−0.033
99
0.208
0.659
0.451


37
0.122
0.090
−0.032
100
0.117
0.611
0.494


38
0.101
0.070
−0.031
101
0.207
0.729
0.522


39
0.101
0.070
−0.031
102
0.165
0.801
0.636


40
0.098
0.071
−0.027
103
0.072
0.726
0.654


41
0.117
0.090
−0.027
104
0.142
0.939
0.797


42
0.110
0.083
−0.027
105
0.231
1.203
0.972


43
0.112
0.087
−0.025
106
0.087
1.240
1.153


44
0.089
0.064
−0.025
107
0.118
1.526
1.408


45
0.097
0.073
−0.024
108
0.268
1.694
1.426


46
0.107
0.083
−0.024
109
0.136
1.900
1.764


47
0.092
0.069
−0.023
110
0.095
2.076
1.981


48
0.113
0.092
−0.021
111
0.127
2.387
2.260


49
0.089
0.069
−0.020
112
0.256
2.610
2.354


50
0.091
0.072
−0.019
113
0.555
3.000
2.445


51
0.081
0.062
−0.019
114
0.128
2.593
2.465


52
0.112
0.093
−0.019
115
0.518
3.000
2.482


53
0.081
0.062
−0.019
116
0.338
3.000
2.662


54
0.083
0.066
−0.017
117
0.315
3.000
2.685


55
0.092
0.076
−0.016
118
0.295
3.000
2.705


56
0.233
0.222
−0.011
119
0.224
3.000
2.776


57
0.065
0.056
−0.009
120
0.184
3.000
2.816


58
0.058
0.050
−0.008
121
0.184
3.000
2.816


59
0.153
0.146
−0.007
122
0.181
3.000
2.819


60
0.089
0.085
−0.004
123
0.171
3.000
2.829


61
0.095
0.098
0.003
124
0.160
3.000
2.840


62
0.184
0.193
0.009
125
0.145
3.000
2.855


63
0.206
0.937
0.731
126
0.081
3.000
2.919






127
0.062
3.000
2.938






128
0.056
3.000
2.944









EXAMPLE 4
This example makes it possible to compare a diagnostic test according to the invention, the ArFA-ELISA assay, and several diagnostic tests of the prior art which are commercially available or which use known methods, namely the AKA by immunofluorescence, AhFA-IB, AhFA-ELISA and CCP-ELISA assays.
A) Samples
The assays were carried out on 711 sera, 240 of which came from patients suffering from rheumatoid arthritis (RA) and 471 of which came from non-RA patients. Among the non-RA patients, 157 were suffering from an inflammatory disease chosen from progressive lupus erythematosus (21), systemic scleroderma (9), psioratic rheumatism (43), ankylosing spondylitis (40) and other inflammatory diseases (44), and 314 were suffering from a non-inflammatory disease chosen from arthrosis (104), compressive neuropathy (25), Paget's disease (68), reflex sympathetic dystrophy (29), serious bone diseases (49) and other non-inflammatory diseases (39).
The filaggrin is a recombinant rat filaggrin; the non-citrulline-containing filaggrin is obtained in accordance with example 1, and the citrulline-containing filaggrin is obtained by citrullination (40% degree of citrullination of arginine residues) of the non-citrulline-containing filaggrin in accordance with example 2.
B) Protocol
The detection of the antibodies according to the AKA by immunofluorescence and AhFA-IB assays was carried out according to the methods described in C. Vincent et al., Ann. Rheum. Dis. (1989) 48, 712-722, and C. Vincent et al., J. Rheumatol. (1998) 25, 838-846.
The AhFA-ELISA assay is used according to the method described by L. Nogueira et al., Ann. Rheum. Dis (2001) 60, 882-887.
The CCP-ELISA assay is carried out with the ImmunoscanTM RA material (Euro-diagnostica, Arnhem, The Netherlands) used according to the manufacturer's recommendations.
The ArFA-ELISA assay according to the invention was used in accordance with protocol C) described in example 3 with the exception of the fact that, in the present example, the samples were assayed in quadruplicate. The average variation between the assays for the same samples was less than 6%.
The results obtained for each of the assays are given in table 3 below.










TABLE 3






95%
98.5%
99%



specificity
specificity
specificity




















‘AKA’
0.52
0.45
0.40




(0.45-0.58)
(0.38-0.51)
(0.34-0.46)



AhFA-IB
0.59
0.48
0.37




(0.53-0.65)
(0.42-0.55)
(0.31-0.43)



AhFA-
0.53
0.38
0.37



ELISA
(0.46-0.59)
(0.32-0.44)
(0.31-0.43)



CCP-ELISA
nd
0.58
0.50





(0.52-0.65)
(0.44-0.57)



ArFA-
0.76
0.67
0.65



ELISA
(0.70-0.81)
(0.60-0.73)
(0.59-0.71)









It emerges from this table that the assay according to the invention is the most reliable of the assays examined in the detection of RA.

## Claims

1. A method for diagnosing rheumatoid arthritis (RA) in a human patient, the method comprising:
obtaining a biological sample from the patient,
providing a filaggrin peptide that has no citrulline residues (PFNC) consisting of the sequence set forth in SEQ ID NO:5,
providing a citrulline-containing peptide (PFC) having the peptide sequence of said PFNC, wherein at least one arginine residue has been converted to a citrulline residue,
contacting a first portion of said biological sample with said PFNC under conditions suitable for the formation of immune complexes with anti-filaggrin-autoantibodies (AFAs),
contacting a second portion of said biological sample with said PFC under conditions suitable for the formation of immune complexes with AFAs,
detecting and quantifying the formation of immune complexes formed between antibodies present in the sample and said PFNC and PFC, this quantification being expressed by a value XNC and XC, respectively,
determining that AFAs specific to RA are present in the biological sample only if the value of XC is greater than the value of XNC, and
diagnosing the patient as having RA if it is determined that AFAs specific to RA are present in the biological sample.

2. The method as claimed in claim 1, wherein said PFC is obtained by the action of peptidylarginine deiminase.

3. The method as claimed in claim 1, wherein the biological sample is selected from the group consisting of blood, plasma and serum.

4. The method as claimed in claim 1, wherein the immune complexes formed are brought into contact with a conjugate comprising a labeled antibody directed against human immunoglobulins, under conditions suitable for the formation of labeled immune complexes, and then the labeled immune complexes are detected and quantified.

5. The method as claimed in claim 4, wherein the conjugate is an anti-human immunoglobulin antibody labeled with alkaline phosphatase or with peroxidase, and then the formation of labeled immune complexes is detected and quantified by colorimetry or fluorimetry, the quantification being expressed as optical density or fluorescence values XNC and XC.