Specific serum IgG pattern against rheumatoid arthritis (RA) fibroblasts
like synoviocytes membrane proteins in RA patients compared to healthy
subjects.
E Solau-Gervais1, D Lefranc2, C Dupont1, V Dutoit2, J Dewailly1, C Fontaine3, S Dubucquoi2. RM
Flipo1, L Prin2,
A mettre dans l’ordre souhaité
Department of Rheumatology (1), Immunology (2) and Orthopaedics surgery (3)
Lille University Hospital, FRANCE
Correspondence and reprints:
E Solau-Gervais
Department of Rheumatology,
Hôpital Roger Salengro,
59035 Lille, FRANCE
Tel.: +(33) 320 444 415
1
Abstract
Objective: Reactivity against some of the proteins in the synovial membrane (SM) of rheumatoid
arthritis (RA) patients is potentially specifically involved in the diagnosis of the disease. However, usual
approaches do not always discriminate the immune response against such targets in RA patients from
the physiological immune response in healthy subjects. This study was undertaken to compare the
reactivity patterns displayed in RA, healthy and ankylosing spondylitis (AS) sera against SM proteins.
Materials and methods: Serum IgG response patterns in patients with RA (n=50) and healthy subjects
(n=27) were compared after exposure to protein extracts of human SM obtained from various joints in
RA patients. To determine IgG antibody responses, post-exposure sera were analysed by Western
blotting technique and the data obtained were then further evaluated using Linear Discriminant Analysis
(LDA).
Results: Patterns obtained with RA sera showed a high degree of reactivity with small joint SM and low
reactivity with knee and hip SM. When sera IgG responses were evaluated on a wrist SM, Chi2 analysis
allowed us to distinguish IgG specific reactivities towards eight antigenic targets specifically linked to
RA disease. LDA allowed us to identify a cluster of discriminant set of immune reactivities towards
putative targets which distinguished RA subjects from healthy and AS subjects with a sensitivity of 82%
and a specificity of 84 %.
Conclusion: Our study demonstrates the existence of a specific immune profile of IgG antibodies in
RA. Analysis of the humoral response against relevant tissue targets provides a promising approach for
identifying new markers in RA.
2
Introduction
Rheumatoid arthritis (RA) is a chronic systemic disease characterised by inflammation of the joints and
the destruction of cartilage and bone. Although RA can affect all peripheral joints – with the exception
of distal interphalangeal joints, erosions tend to occur earlier in the wrists, hands and feet than in knees
or hips 1. Clinically, synovitis is a major characteristic of the RA disease. Synovitis is a consequence of
synovial membrane (SM) hypertrophy resulting from the hyperplasia of resident synovial cells and the
infiltration of T cells and macrophages2 involved in inappropriate and chronic inflammatory responses.
The efficiency of anti-CD20 therapy3 4 and the presence of rheumatoid factor (RF) and anti-citrullinated
peptide antibodies such as filaggrin5, fibrin6 or vimentin7 in RA patients, also underscore the potential
role of B lymphocytes in the physiopathology of RA8.
Nevertheless, even though the presence of RF and anti-citrullinated protein (anti-CCP) antibodies are
required for the diagnosis of RA, almost 50% of patients in the early stages of the disease have negative
RF and anti-CCP antibodies5. As such, several other antigens (collagen9, heat shock protein10) have been
proposed as putative targets. Serological proteomic approaches have been previously used to identify
new immunogenic targets recognized by serum IgG. Using fresh human placenta as a substrate,

enolase11 was found to be a discriminant target in very early RA, and in chondrosarcoma cells, a
human stress protein, BiP (immunoglobulin binding protein), was identified as a putative antigen12 13.
But in these two studies only 24.8% of RA sera recognized the 50 kDa  enolase11 band and 30% of RA
patients possessed serum autoantibodies to Bip12. Consequently, neither of these antigens has
definitively been implicated in the pathophysiology of RA and it seems that the reactivity more than an
antigen is implicated for RA diagnosis and that more than one reactivity could be involved in the
chronically immune destructive process of RA.
By testing distinct human tissue targets, consensual immune patterns of self recognition have been
demonstrated in healthy subjects14 15. In multiple sclerosis, we have found discriminant immune patterns
distinguishing the IgG responses towards self antigens derived from brain homogenates from healthy
subjects16.
3
Using a large panel of antigens from fibroblasts like synoviocytes (FLS) cultured from synovial
membranes, we compared by immunoblotting, the serum IgG repertoires from patients with rheumatoid
arthritis, ankylosing spondylitis (AS) and healthy subjects. The analysis of our data has lead to the
identification of specific serological patterns that discriminate the immune response of RA patients from
AS patients and healthy subjects.
4
Materials and methods
Patients:
Sera were obtained from 101 patients and kept frozen at -80°C until used. Forty six patients were
diagnosed with clinically definite RA according to ACR criteria17 and thirty nine patients were
diagnosed as ankylosing spondylitis (AS) according to the New York criteria. Sera from 16 healthy
subjects were tested as normal controls. All patients were followed up in the Department of
Rheumatology. Table I summarizes the epidemiological characteristics of patients with RA.
The synovial samples were obtained from other RA patients after synovectomy or arthroplasty. All of
these patients had been diagnosed with clinically definite RA according to ACR criteria17. Wrist and
finger synovial membranes (SM) were obtained after synovectomy. Knee and hip SM were obtained
after arthroplasty. All subjects had given their written informed consent.
Cell isolation and culture:
Synovial membranes were minced with scissors and digested with Dulbecco's modified Eagle's minimal
essential medium (DMEM) (Sigma, Poole, UK) with 600 U/ml of collagenase type XI (Sigma, Poole,
UK) for 90 mn at 37°C. Cells were washed in DMEM with 10 % foetal calf serum (FCS) (SeraLabHarlan, CrawleyDown, UK) and seeded into 25 cm2 tissue cultured flasks. Confluent cultures of
adherent fibroblasts like synoviocytes (FLS) were passaged at 1:2 ratio in 150 cm2 flasks. Cells were
detached from the flasks with 0,25% trypsin (Life Technologies, Paisley, Scotland). The cells used
during the study were limited to between passage 3-8. FLS were cultured in DMEM supplemented with
10% FCS.
Rheumatoid factor and anti-cyclic citrullinated antibody
5
IgM rheumatoid factors (BMD, Marne la Vallée, Paris) and anti-cyclic citrullinated antibodies (Inova,
San Diego, USA) were measured by Elisa, using commercially available tests and following
manufacturers’ instructions.
SDS-PAGE
The FLS cells were homogenized in Laemmli buffer and heated at 95°C for 10 min. Eighty (80) l of
this lysate was loaded per well onto a 10 to 20% gradient polyacrylamide gel beside a molecular mass
marker (Amersham Pharmacia Biotech). Just before electrophoresis, the homogenates were reduced
with 10 mM DTT (Sigma-Aldrich). Electrophoresis was performed for 12 hours in Tris/glycine buffer at
100 V.
Blotting and analysis procedures
Proteins were transferred onto 0.45-m PVDF membranes (GE healthcare) at 0.8 mA/cm2 and later
saturated with 5% non-fat dried milk. Each well was cut into 30 strips, 3-4 mm wide. Western blotting
was conducted with total sera, diluted 1/100 in TBS (100 mM Tris (pH 8.0), 0.3 M NaCl) containing
0.5% Tween 20 (w/v) and 5% non-fat dried milk. After incubation for 1 night at 4°C, the IgG Abs were
revealed with an anti-human FcHRP-conjugated Ab (1/10,000; Sigma-Aldrich). Fluorograms were
prepared using an ECL kit (Amersham Pharmacia Biotech). Densitometric analyses were performed on
nonsaturated autoradiographs using the Quantity One software (Bio-Rad, Hercules, CA) apparatus to
localize and compare the IgG immune profile patterns. The Ab reactivities were superimposed and
aligned using Diversity database 2.2 software (Bio-Rad). Immune profiles were analysed when two
independent assays had produced identical patterns.
Statistical analysis
The data were expressed in binary mode (0 = absence of reactivity against antigenic band; 1 = presence
of reactivity against antigenic band) so that IgG Ab patterns could be analysed using either the Chi2 or
6
Fisher’s exact test. This approach allowed us to select the most relevant band showing qualitatively
different immune recognition patterns between healthy subjects, AS and RA. In a second phase, linear
discriminant analysis (LDA) was used to balance the discriminating Ags between the populations of
individuals. All Ags with a p value < 0.05 in the previously mentioned statistical tests were selected for
LDA 18. Using a stepwise logistic regression model, and supported by the LDA method, we were able to
isolate a subgroup of SM Ags on the basis of their power to discriminate between the different
populations involved in the study. On the basis of the presence (x 1) or absence (x 0) of each selected
Ag, and the coefficient previously defined by LDA, a score was calculated for each subject as a
representative value of the individual immune profile, using the following formula: score = Ag1 coef1 x
(0(absent) or 1(present)) + Ag2coef2 x (0(absent) or 1(present)) + Ag3coef3. . . . Initially, the analysis
focused on discriminating between RA patients and healthy subjects. Threshold values were determined
using a receiver operating characteristic (ROC) curve. In a second phase, the analysis focused on AS. A
 test was used to evaluate concordance with clinical data.
Bidimensionnal electrophoresis
The FLS were lysed in buffer (7M urea, 2M thiourea (sigma), 50 mM C7BzO (Calbiochem), 4% triton
100X (sigma), 1X anti-protease (Roche), and 1% DTT (Sigma)). Following incubation at room
temperature for 1 hour under shaking, the cell lysate was then centrifuged at 20000g for 20 min at 4°C,
and the supernatant was recovered. The protein concentration was measured by 2D-QUANT KIT (GE
Healthcare).
Immuno-electro-focalisation (IEF) was performed with an IPGphor III (GE) using precast 18 cm pH 310 linear IPG gel strips (GE). Equal amounts (1 mg) of total proteins were mixed with rehydration
solution to a total volume of 300 µL with 2% v/v pharmalytes pH 3-10 (GE). Sample load was realized
by in-gel rehydratation and IEF was performed at 150 V for 1 h; 300 V for 1 h; 1000 V for 1 h; and
7500 V for 10h. The current was limited to 50 µA per gel strip. After IEF separation, the IPG strips were
stored at -70°C until further use or immediately equilibrated in equilibrium buffer (50 mM tris-HCl, pH
7
8.8, 6M urea, 30% v/v glycerol, 5% w/v SDS), and 2.5% w/v DTT for 2x10 min then including 2.5%
w/v iodoacetamine for 1x10 min. Equilibrated IPGs were transferred to a polyacrylamide gradient gel (T
= 9–16%) containing bis-acrylamide (C = 2.6%). Electrophoresis was performed using a Protean II xi 2D cell (Bio-Rad). 90V applied until the front line of bromophenol blue reached the bottom of the gel.
For Western blotting, gels were electroblotted onto PVDF membranes (GE Healthcare) and treated as
described earlier.
For MALDI-TOF MS analysis of proteins, gels were fixed with 50% v/v ethanol, 3% v/v
orthophosphoric acid for 2h. After 3 washes in distilled water, the gels were stained with Coomassie
brillant blue G-250 (Sigma).
In-gel digestion and MALDI-TOF MS analysis
Excised plugs from CBB-stained gels, were destained with 200 mL 50% ACN in 10 mM NH4HCO3
and dried in a SpeedVac concentrator. Protein was digested overnight at 377C with sequencing-grade
trypsin (5 mg/mL; Promega Madison, WI, USA) in 50 mM NH4HCO3. The resulting peptides were
extracted twice with 25 mL 50% ACN/ 0.1% TFA. The collected extracts were lyophilized, and were
resuspended in 10 mL 0.1% TFA and desalted on ZipTip C18-microcolumns (Millipore, Bedford, MA,
USA). Elution was performed with CHCA (5 mg/mL) directly onto the MALDI target (2 mL of the
solution was applied to a plated sample holder and introduced into the mass spectrometer after drying).
MALDI-TOF MS was used to obtain PMF for proteins using a Voyager DE-STR instrument (Applied
Biosystems, Framingham, MA, USA). Ions were accelerated at 20 kV and reflected at 21.3 kV. Spectra
were acquired in the delayed extraction, reflectron R mode. Scans (100–300) were averaged to produce
final spectra. Spectra were externally calibrated using the monoisotopic MH1 ion from three peptide
standards (trypsin autodigestion products: 842.510, 1045.564 and 2211.1046 Da).
8
Database search based on PMF spectra
The obtained peptide mass fingerprints spectra were analyzed by searching the National Centre for
Biotechnology
Information,
nonredundant
protein
database
with
ProFound
(http://prowl.rockefeller.edu/cgi-bin/Pro Found), Version 3.2. The parameters for each search were
varied in order to achieve the best possible results. The standard parameters were as follow: Homo
sapiens, 0–250 kDa Mr (depending on the region where the spot occurred in the gel), tryptic digest with
a maximum number of one missed cleavage. Peptide masses were stated to be monoisotopic, and
methionine residues were assumed to be partially oxidised. The identity of proteins was annoted using
the Swiss-Prot and TrEMBL databases.
9
Results
Selection of tissue target
We first tried to analyse the antigenic profile of RA sera compared to healthy subjects using synovial
membrane as a tissue target. We initially compared the sera IgG responses to different joint SM (knees,
hips, fingers and wrists) and the reactivity against wrist and finger SM was strong although it was weak
against knee and hip SM (data not shown). These distinct patterns were noted even though the protein
concentration (10 g/well), as previously measured by Bio-rad protein assay, and the protein
distribution per lane, as evaluated by Ponceau red staining, were quite similar. Then, a comparative
study of self IgG patterns obtained with sera collected from healthy subjects, AS and RA, revealed interindividual differences with some conserved sets of proteins bands, as well as a higher numbers of
protein bands recognized by IgG from RA sera, as illustrated in figure 1.
To standardize the technique and as RA synovial membrane was not a homogenous tissue, we decided
to use FLS as tissue target. Antigenic reactivity of RA sera was first tested on FLS after 3 to 5 passages.
Most of bands of reactivity was similar in the three conditions but the most important reactivity was
observed at the fifth passage (Fig 2). After the fifth passage, the reactivity decrease (data not shown).
So, the following experiments have been done with FLS at the fifth passage. Cultured FLS of different
synovial membranes (one hip, one finger, two wrists, one ankle and one knee) were used at substrate to
compare bands of reactivity of three RA sera with a reproducibility of 80% (Fig 3).
Distinct serum IgG reactivity patterns in RA patients
A comparative study of self IgG patterns obtained with sera collected from 16 healthy subjects, 39 AS
and 46 RA, revealed inter-individual differences with some conserved sets of proteins bands, as well as
a higher numbers of protein bands recognized by IgG from RA sera, as illustrated in figure 4.
With healthy sera, an analysis of the different patterns obtained with regard to the molecular mass of the
10
proteins revealed the presence of only 4 to 17 bands of reactivity per strip for each healthy subject, with
a median of 7 bands of reactivity per subject (Fig 4).
With RA sera, the same analysis of the different patterns revealed the presence of a significantly higher
number bands of reactivity (p=0.001) than observed with healthy subjects, with 7 to 24 bands of
reactivity per strip for each RA patients, and a median value of 15 bands of reactivity per patients
(Fig 4).
In a third phase, we evaluated the IgG Ab responses of 39 sera from AS patients and of 16. Each AS
patient’s sera revealed 10 to 32 bands of reactivity with a median of 18.
Detection of discriminant antigenic bands:
Alignment of the patterns obtained with the 16 healthy controls, 46 RA and 39 AS, allowed us to
identify 84 protein bands of reactivity.
o Between healthy subjects and RA
Chi2 analysis and Fisher’s test allowed us to distinguish thirteen protein bands specifically linked to RA
disease. Figure 5 shows the frequency of each of these selected protein bands in healthy controls and
RA.
We then applied LDA to define more precisely differences in IgG reactivity between healthy controls
and RA patients. The LDA took into account five IgG reactivities revealed as discriminant among the
eighteen specifically linked to RA. These 5 IgG reactivities were named p20, p28, p34, p38 and p42
their molecular weights were 95, 72, 68, 57 and 53 kDa respectively. Coefficient values associated with
the presence or the absence of each IgG reactivity assigned by LDA enabled the calculation of a score
for each subject using the following equation: 0.466 (0/1 p20) + 0.605 (0/1 p28) + 0.629 (0/1 p34) +
0.495 (0/1 p38) + 0.618 (0/1 p42). The results revealed an excellent degree of concordance (k=0.93). A
receiver operating characteristic curve (ROC curve) determined a cut-off at 0.55 and distinguish RA
from healthy subjects with a specificity of 89 % and a sensitivity of 93 %.
o Between healthy subjects and AS
11
When healthy subjects and AS patients were compared with Chi2 analysis and Fisher’s test, twenty two
protein bands specifically linked to AS disease were distinguished (Figure 5). When we applied LDA,
the equation took into account six specific reactivities, and the coefficient values assigned by LDA
associated with the presence or the absence for each reactivity was: -31.098 (0/1 p26) –1.289 (0/1 p28)
+ 18.799 (0/1 p41) – 6;847 (0/1 p44) – 1.715 (0/1 p46) -1.708 (0/1 p47). The results revealed an
excellent degree of concordance (k=0.89). A receiver operating characteristic curve (ROC curve)
determined a cut-off at -21,69 and distinguish RA from healthy subjects with a sensitivity of 89 % and a
specificity of 100 %.
We used a similar approach to try to separate RA from healthy subjects and AS. An LDA taking into
account protein bands selected by Chi2 analysis or Fisher test revealed 22 antigenic bands. The LDA
defined a two-equation system that projected each case studied onto a two-axis graph (Fig. 7). Threshold
values at 0.15 on the x-axis, and at 0.35 at the y-axis delineate three areas. Area I contains 44 of 46
(95%) patients with RA. Area II contains 39 of 39 (100%) patients with AS. Area III contains 14 of 16
(87%) healthy controls.
Characterization of discriminant antigens
To further characterize discriminant Ags, a proteomic approach was adopted. First, RA sera able to
recognize all protein bands previously defined as antigenic candidates in 1-DE, were selected and used
for 2-DE. The presence of multiple antigenic spots have been revealed by 2-DE followed by
immunoblotting assays. The superposition of antigenic spots and protein spots revealed by a standard
colloidal Coomassie blue-stained 2-DE enabled the selection of proteins for further in-gel digestion and
MALDI-TOF analysis. Representative antigenic spots are shown in Fig. 7. They were matched on a
preparative gel for further characterization with MALDI-TOF as previously described, on the basis of
peptide mass matching.
12
Discussion
In this report, we describe the presence of a specific IgG pattern against rheumatoid wrist SM proteins in
RA compared to healthy controls and AS patients. A comparative analysis of the 61 patterns obtained
with all of the sera studied showed that despite inter-individual changes, a limited set of conserved
bands of reactivity was noted.
Our immunoblot results reveal peculiar serum IgG reactivity patterns towards synovial membrane
antigens in RA patients compared to healthy subjects. Linear discriminant analysis determined a
combination of four discriminant IgG reactivities, with a sensitivity of 82% and a specificity of 84%,
when RA sera were compared to healthy controls and AS sera. While western blotting may not be
routinely applicable, this result shows that the specific diagnosis of RA is concomitant with reactivity
towards not one but several antigens. Already in a previous study, using a similar approach, we were
able to identify discriminating antibody profiles associated with neurological autoimmune diseases16.
This study described specific immune profiles associated with multiple sclerosis but distinct from
Sjogren’s disease and healthy subjects, and within the multiple sclerosis group, 3 different IgG patterns
associated with clinical forms of the disease. As far as rheumatic diseases are concerned, protein
expression patterns has been studied twice. The two authors stress differential expression profiles of RA
and osteoarthritis synovial tissues and highlight the interest of studies at the protein level 19 20. Previous
studies using Western Blot analysis have also found immune reactivities towards Bip 12 or alpha
enolase11 21 in rheumatoid arthritis, and towards triosephosphate isomerase (TPI) in osteoarthritis22. In
RA, the synovial membrane plays a pivotal role in the pathophysiology of the disease, with the
proliferation of fibroblast-like-synoviocytes23 (FLS) and the infiltration of the subintimal layer by
mononuclear cells24. Moreover, synovitis is the first and a major symptom potentially leading to
cartilage destruction. However, synovial membrane has only been used on a few occasions to analyse
immunogenic reactivities. Blass and al., using total protein preparations from RA SM, detected the
13
68kDa antigen25, subsequently identified as Bip26. Previously, we used SM to show that antibodies in
synovial fluids react to several proteins in RA. This study highlights the fact that other tissues, unlike
SM, are unable to identify antibodies27. Since looking for only one IgG reactivity in RA failed to clearly
distinguish RA from healthy controls, more than one IgG reactivity seems to be involved in RA
pathophysiology. A recent study, using antigen microarray, shows that a profile of autoantibodies
provides diagnostic and prognostic information28.
For technical reasons, most of the previous studies used SM from the knee joints19 20 25 27, even though
clinically detectable synovitis in RA is essentially present in small joints. Immunohistologic studies on
synovial membranes have found no differences between wrist and knee29, but in term of proteomic
analysis, no comparison has been made of the different joint sites. In our study, the IgG reactivity
profiles against protein extracts from large joints such as hip and knee, and small joints such as wrist
and hands, were different. Analysis of Ponceau red protein distribution revealed similarities between the
two articular sites but the difference in IgG reactivity suggests that the SM proteins, which are targets
for immune reactivity in RA, are different, perhaps modified by post-translational events, as suggested
by protein deimination like vimentin or filaggrin in RA. Since small joint SM is obtained by
synovectomy, and hip or knee joint SM from arthroplasty, the inflammatory status of the SM might be
higher in small joints, generating more modified epitopes and consequently new immune reactivities.
Firstly, we prospectively compared antigenic profiles in RA sera from patients at the onset of the disease
and 6 months later. We found the same patterns, which suggested that reactivities remained stable over
time. Secondly, we compared early RA (mean disease duration: 2 months) and advanced RA (mean
disease duration: 109 months) and found that most of the protein bands were present in the two groups
but only two were present in early RA only. As this second analysis was not conducted prospectively,
the result only suggests that the distorsion of self-reactive IgG antibody repertoires is still in progress.
14
This distortion has been already noted in multiple sclerosis30. In contrast, in healthy subjects, the
stability of natural self-reactive antibody repertoires during aging has been demonstrated 31
We chose another inflammatory disease, AS, which is RF negative. Although mean inflammatory
parameters, such as ESR, were not different in the two groups, the immune profile of AS was clearly
different to that of RA. This suggests that the IgG reactivity that we found in RA was specific to the
pathological process of RA and not present in all inflammatory joint diseases. When we compared
healthy subjects to AS we found only one specific reactivity with a molecular weight of 83 kDa. In the
literature, there is little data on immune recognition of autoantigens in AS, and most of the studies failed
to show an increased prevalence of autoantibodies.32 33 Recently, the presence of a 28 kDa protein was
found in 42% of AS sera34. Although altered patterns of antibody reactivity has been described as being
unrelated to the target organ15, we cannot exclude that we may have “missed” the accurate antigen in AS
on account of our using RA and not AS SM as a substrate.
The choice of synovial membrane as a substrate to analyse reactivities in RA sera was motivate by the
fact that this tissue is involved in articular and bone destruction. The difficulty in using whole tissue is
due to the variability in the quantity and quality of proteins in each sample. In light of this promising
data, and with the standardisation of the technique, RA FLS, which play a major role in enzyme and
cytokine production, could be an interesting candidate for use as a substrate. Recent publications on the
FLS proteome demonstrate the presence of proteins characterized as potential autoantigens in RA 35 and
show that FLS reflect the heterogeneity of synovial tissues36. The analysis of IgG profiles of RA
compared to healthy subjects with FLS as a substrate is presently under investigations.
To our knowledge, the observations described in this work are the first to highlight the existence of a
specific serum IgG profile against RA SM in RA patients compared to AS and healthy subjects. These
15
RA-specific proteins need to be characterized and evaluated in a broader population of RA patients as
well as with patients with other inflammatory diseases.
16
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Legends:
Table I: Clinical, biological and radiological characteristics of rheumatoid arthritis and ankylosis
spondylitis patients.
Figure 1:
A] Sera from 5 RA patients were tested against knee rheumatoid synovial membrane and wrist
rheumatoid synovial membrane. RA serum revealed 12 to 18 bands with wrist SM while only 1 to 5
bands were obtained with knee samples
B] Representative patterns obtained with one RA serum against four different synovial membranes (SM)
from hand, wrist, knee and hip showed strong reactivity against wrist and hand SM and weak reactivity
with knee and hip. Ponceau red (PR) staining showed a similar protein profile.
Figure 2: Immune profile stability in 3 RA sera against the same wrist rheumatoid synovial membrane.
Sera were obtained from patients at the onset of the disease (1, 2, 3) and after a disease duration of 6
months (1’, 2’, 3’). Six months later, samples revealed the same number of serological reactivities, with
an identical pattern towards SM antigens.
Figure 3: Immunoreactive patterns against wrist synovial antigens with sera from healthy subjects, RA
and AS. Representative data of IgG profiles obtained with 5 healthy controls, 5 RA and 5 AS. As
revealed by Western blotting, the different pattern obtained with RA sera showed more bands of
reactivity than observed with healthy subjects. In AS patients, the analysis of the different patterns
revealed fewer bands of reactivity than obtained with RA.
Table 2: Statistical analysis of the frequency of IgG reactivity against wrist synovial membrane
antigens, *: p<0.05, **: p<0.001
Figure 4: LDA distinguishes the IgG immune profiles of healthy subjects, RA and AS patients.
A] Illustrative Western blot strip depicting the synovial membrane Ags that support discriminant
immune reactivity on rheumatoid synovial antigens with sera from RA, AS patients and healthy controls
(statistical p values for Fisher’s exact test). This statistical analysis attributes a coefficient value to each
discriminant antigen.
B] These coefficient values, associated with the presence or absence of each discriminant Ag, allowed
us to calculate graphic coordinates for each individual. The graph shows that the calculated scores of
each subject are distributed in distinct areas according to the clinical status, with an excellent degree of
concordance with clinical data (k =0.91 )
C] Receiver operating characteristic (ROC) curve shows a sensitivity of 82% and a specificity of 84%.
19