ASSESSING THE PATIENT
Autoantibodies in the
autoimmune rheumatic
diseases
Richard Watts
Abstract
The detection of autoantibodies to a variety of antigenic targets plays
an important role in the diagnosis of autoimmune rheumatic disease.
Rheumatoid factor and antinuclear antibodies have been key in the diag-
nosis of rheumatoid arthritis and systemic lupus erythematosus for many
years. There have been several developments over the past 5 years.
Antibodies against cyclic citrullinated peptides (CCP) have been shown
to be specific for rheumatoid arthritis and may predict the development
of erosive disease. This assay will probably become widely used over
the next few years. There is increasing interest in the use of anti-C1q
antibodies for the detection and monitoring of renal nephritis as this as-
say appears to be more sensitive than anti-DNA antibodies. ELISA based
assays are being increasingly used in place of both radioimmunoassay
and immunofluorescence because they readily automated and less de-
pendent on the use of experienced technicians.
Keywords autoantibodies; ANA; rheumatoid factor; ANCA; anti-CCP
­antibodies; ELISA
The detection of autoantibodies in the serum of patients with
suspected autoimmune rheumatic disease is an important part of
the diagnostic process. The clinical utility of the results is depen-
dent on the quality of the laboratory test. Increasingly, assays are
available commercially and this can bring problems of standard-
ization. Access to a reference laboratory is essential in difficult
cases particularly when there is a discrepancy between the clini-
cal features and the laboratory results.
An ideal diagnostic test has both high sensitivity and specific-
ity, that is, it identifies all patients with the disease (high sensitiv-
ity) and is not present in those who do not have the disease (high
diagnostic specificity). A number of different methods are used
to detect autoantibodies with varying specificity and sensitivity.
The most widely used methods are indirect ­immunofluorescence
(IIF) and enzyme-linked immunosorbent assay (ELISA). ELISAs
can be readily automated and therefore are readily suited for use
in a routine non-specialized laboratory. They may be quantified.
In general, they are more sensitive and less specific than other
methods such as immunoprecipitation or immuno-electrophore­ sis.
Increasingly, ELISA assays are replacing other methods in ­routine
laboratories.
Richard Watts MA DM FRCP is a Consultant Rheumatologist at Ipswich
Hospital NHS Trust, Ipswich, UK, and Senior Lecturer at the School of
Medicine, Health Policy and Practice, University of East Anglia, UK.
Competing interests: none declared.
MEDICINE 34:11
What’s new?
• The major development during the last five years has
been the recognition of the utiliy of antibodies directed
against cyclic citrullinated peptides in the diagnosis
of patients with rheumatoid arthritis. This has led to
a re- evaluation of traditional methods of diagnosing
rheumatoid arthritis
• There is increasing awareness of the possible role of
anti-C1q antibodies in the assessment of patients with
lupus nephritis
• The trend towards the increased use of ELISAs has
continued due to the easy automation of these assays
Rheumatoid factor
Rheumatoid factor (RF) is, primarily, an IgM antibody that is
directed against the Fc part of the patient’s own IgG molecules.
Rheumatoid factors are traditionally detected using an agglutina-
tion assay with gelatin particles coated with denatured IgG, but
increasingly ELISAs are being used. Although 80% of patients
with rheumatoid arthritis are RF positive, these antibodies are
not specific for rheumatoid arthritis and occur in a wide range of
autoimmune rheumatic diseases and infections.
Anti-cyclic citrullinated peptide (CCP) antibodies
The relatively poor specificity of traditional RF assays for RA has
led to the development of new serological markers. The most
promising of these are antibodies directed against citrullinated
antigens.1 The citrulline moiety, which is the key component
of the antigenic determinant that is post-translationally gener-
ated by peptidylarginine deaminases. ELISA assays have been
developed to detect antibodies against cyclic citrullinated pep-
tides. Anti-CCP antibodies are present in 80% of patients with
established RA. The specificity is around 95–98%, with a sensi-
tivity of 65%. Furthermore, a positive anti-CCP test appears to
predict the development of erosive RA and this predictive value
­complements that of RF.
Antinuclear antibodies
Antinuclear antibodies (ANA) are autoantibodies directed against
cellular proteins or nucleic acids.2 Despite the name, a number of
these antigens are partly or wholly confined to the cell cytoplasm.
The most frequently occurring ANA react with DNA-­protein or
RNA-protein complexes. These autoantibodies, which are usually
high titre, high affinity IgG antibodies, are generated by a T-cell
dependent process driven by the autoantigen.
Screening for ANA is an important initial stage in the assess-
ment of autoimmune rheumatic disease. High titre ANA (>1:160)
are more likely to be clinically significant in the presence of
autoimmune rheumatic disease. The test is usually performed
by IIF on fresh frozen sections of rodent liver and/or kidney,
441 © 2006 Elsevier Ltd. All rights reserved.
 ASSESSING THE PATIENT
or on ­cultured human cell lines such as HEp-2. HEp-2 cells are
preferred because individual cells and their organelles are easily
observed. They divide rapidly and present antigens, which are
only sparsely present or absent in the resting nuclei of tissue
sections.
Amongst the RNA-protein complex targets, anti-Ro antibodies
are directed against the human antigen and may not be detected
on rodent sections. Several immunofluorescence staining pat-
terns occur, reflecting different antigenic specificities (Figure 1).
The IIF ANA may be falsely negative if the antigen is located
outside the cell nucleus (e.g. anti-Jo-1 and anti-ribosomal P), or
if the antigen is present in a form not recognized by the autoan-
tibody (e.g. anti-Ro directed against epitopes on the Ro molecule
not expressed in cultured HEp-2 cells).
The assay requires an experienced technician to read the slides
and results are prone to subjective interpretation, therefore, in
many non-specialized laboratories ELISA assays are being used
to detect ANA. These assays vary in sensitivity and specificity
both between different ELISA assays and between ELISA and IIF.
IIF remains the gold standard.
Anti-DNA antibodies
The detection of antibodies to double stranded (ds) DNA is cen-
tral to the diagnosis of SLE. Anti-dsDNA antibodies rarely occur
in other diseases. In contrast, antibodies to single-stranded DNA
are found in wide variety of autoimmune and infectious condi-
tions. Anti-dsDNA antibodies can be detected using several meth-
ods. The most specific is IIF using the haemoflagellate Crithidiae
lucillae that has a kinetoplast containing circular dsDNA, and
this is considered the ‘gold standard’. The ELISA is rapid, sensi-
tive, quantifiable although less specific than the Crithidia assay,
but is dependent on the use of pure dsDNA. Radioimmunoas-
says are also available commercially but increasingly laborato-
ries are trying to reduce the use of test that involve radioisotopes
for safety reasons and ELISA anti-dsDNA assays are commonly
­performed.
Diagnostic algorithm for antinuclear antibody positivity
Homogeneous Speckled
PCNA
Histone dsDNA
Ro/SS-A
Sm
La/SS-B
U1 RNP
Drug-induced lupus Systemic lupus Sj gren’s syndrome
erythematosus erythematosus
Figure 1
MEDICINE 34:11
Antibodies to extractable nuclear antigens
Detection of antibodies against the extractable nuclear antigens
(ENA) is useful in the diagnosis of several autoimmune ­rheumatic
diseases.2 The clinical associations of these autoantibodies are
given in Table 1. These antibodies were traditionally detected
using immunodiffusion techniques, although ELISAs are now
very widely used. Enzyme digestion studies showed that these
antigens were sensitive to RNase and trypsin and are composed
of ribonucleoprotein (RNP). The Sm (or Smith antigen; Sm, Ro
and La antigens were each named after the first two letters of
the surname of the patients in whom antibodies to them were
first found), is resistant to RNase and trypsin. The RNP antigenic
determinants are found in association with U1 RNA, whereas
Sm determinants are present on U1,U2 and U4-6 RNA. The other
two important nuclear antigens are Ro and La, these are identical
to the independently identified antigens Sjögren’s syndrome A
(SS-A) and Sjögren’s syndrome B (SS-B).
Antibodies against tRNA synthetases have been identified in
patients with polymyositis. The tRNA synthetases are cytoplas-
mic enzymes that attach tRNA to its corresponding amino acid
during the assembly of polypeptides. Antibodies against several
tRNA synthetases have been described, including antiJo-1 (histi-
dyl), PL-&7 (threonyl), PL-12 (alanyl), OJ (isoleucyl), EJ (glycl)
and KS (asparginyl. AntiJo-1 (histidyl) is the most common hav-
ing been found in up to 30% of patients with myositis. Patients
with these autoantibodies often share the same clinical features;
the tRNA synthetase syndrome -Raynaud’s phenomenon, fever,
interstitial lung disease, arthralgia and myositis. The tRNA syn-
thetases are located in the cytoplasm and hence are often not
detected on routine ANA testing, although a perinuclear staining
pattern on IIF ANA may be a clue to their presence.
Anti-topoisomerase-1 antibodies (Scl-70) are associated with
the diffuse cutaneous variant of scleroderma and a high risk of pul-
monary disease. Topoisomerase-1 is a100kDa nucleolar enzyme
involved in uncoiling DNA prior to replication. Other enzymes
targeted in scleroderma (such as RNA polymerase I, II and III)
Staining pattern
Nucleolar Centromere
Speckled
Homogeneous
U1 RNP
Scl-70
Mixed connective Scleroderma
tissue disease
442 © 2006 Elsevier Ltd. All rights reserved.
 ASSESSING THE PATIENT

Disease associations of commonly detected autoantibodies

Autoantibody Disease Prevalence Disease specificity Associated clinical features

dsDNA SLE 70% High Lupus nephritis

Sm SLE 5% (Caucasian); 30-50% High Vasculitis, CNS lupus
(African-Caribbean)
Ro (SS-A) SLE 40% Low Photosensitivity, subacute cutaneous LE,
congenital heart block, neonatal LE
Sjögren’s syndrome 80% High Extra-glandular disease, vasculitis,
lymphoma
La (SS-B) SLE 15% Low As for Ro
Sjögren’s syndrome 50% High As for Ro
U1RNP SLE 30% Low Raynaud’s phenomenon, swollen fingers,
arthritis, myositis (overlap syndrome –
MCTD)
rRNP SLE 15% High CNS lupus (psychosis, depression)
PCNA (cyclin) SLE 5% High
Phospholipid SLE 35% High Thrombosis, fetal loss, thrombocytopenia
C1q SLE 40% Moderate Lupus nephritis
Topoisomerase 1 Systemic sclerosis 30% High Diffuse cutaneous variant of scleroderma
Centromere Systemic sclerosis 30% Moderate Limited cutaneous variant of
scleroderma, absence of lung disease
RNA-polymerases Systemic sclerosis 20% High Diffuse cutaneous variant of scleroderma,
visceral involvement
PM-SC1 Systemic sclerosis 5% High Scleroderma/polymyositis overlap
Jo-1 Polymyositis 30% High Polymyositis with fibrosing alveolitis
(anti-synthetase syndrome)
SRP Polymyositis 4% High Severe myositis
Mi-2 Polymyositis 10% High Dermatomyositis
CCP Rheumatoid arthritis 80% High Rheumatoid arthritis
PR-3 Vasculitis 90% High Wegener’s granulomatosis
MPO Vasculitis 50% Moderate Microscopic polyangiitis, Churg Strauss
syndrome

SRP, signal recognition particle; PR-3, proteinase 3, MPO, myeloperoxidase; CCP, cyclic citrullinated peptide.

Table 1

are also nucleolar and are associated with an increased risk of

cardiac and renal disease with a poor prognosis. Anti-cen­tromere
antibodies (detected by IIF on HEp-2 cells) are specific for the
limited cutaneous variant of scleroderma and react with the
kinetochore of metaphase chromosomes.
Antiphospholipid (anticardiolipin) antibodies
Antibodies to phospholipids particularly cardiolipin are responsi-
ble for the false positive Wasserman reaction and lupus anticoagu-
lant seen in patients with the primary antiphospholipid antibody
syndrome (APS) and in some patients with SLE. The interaction of
phospholipid and a co-factor β2-glycoprotein is necessary for the
detection of the antigenic site to be available for binding of anti-
body. Antiphospholipid antibodies are associated with an increased
risk of vascular thrombosis, recurrent fetal loss, livedo reticularis
and thrombocytopaenia. A positive IgG anticardiolipin test present
on more than one occasion at least 6 weeks apart is most specific
for APS, but some patients are only IgM antibody positive.
Anti-C1q antibodies
C1q is the first component of the classical pathway of comple-
ment activation, hereditary deficiency is a risk factor for SLE.
Antibodies against C1q are present in the serum of patients with
SLE (up to 47%) and are associated with renal involvement.
Monitoring anti-C1q may be useful to detect renal flares.3
Antineutrophil cytoplasmic antibodies
Antineutrophil cytoplasmic antibodies (ANCA) were first detected
in the sera of patients with systemic vasculitis in the early 1980s.
ANCA are specific for granule proteins of neutrophils and mono-
cytes. Using IIF on ethanol fixed human neutrophils two major
staining patterns are observed: cytoplasmic and perinuclear. Two
main antigenic targets have been identified ­ proteinase 3 (PR3),
which is associated with cANCA and ­ myeloperoxidase (MPO)
which is associated with pANCA. The combination of cANCA
and PR3 is highly specific (>90%) for Wegener’s granu­lomatosis

MEDICINE 34:11 443 © 2006 Elsevier Ltd. All rights reserved.

 ASSESSING THE PATIENT
whilst pANCA-MPO occurs in microscopic polyangiitis and Churg
Strauss syndrome but is less specific.4 There are a ­number of other
antigens associated with pANCA (e.g. lactoferrin) but with less
clinical significance. ­Ideally both IIF and ELISA for PR3 and MPO
should be performed on each sample.
Autoantibodies in the monitoring of disease activity
Serial detection of autoantibodies has a limited role in the
­assessment of disease activity. Rising levels of dsDNA ­antibodies
in patients with SLE may correlate with increasing disease activ-
ity. Similarly an increase PR3-ANCA levels occurs in patients with
active Wegener’s granulomatosis. However, in both ­ situations
the rise in antibody level can precede clinical relapse by many
months and in some patients fall immediately prior to the relapse,
suggesting tissue deposition. The detection of a rise in titre should
serve to heighten suspicion and an increase in the frequency of
review, rather than a reflex increase in ­immunosuppression.
When to request autoantibody tests
The detection of autoantibodies is a key part of the assessment
of the patient with suspected autoimmune rheumatic disease. In
over 95% of patients with untreated active SLE, ANA can be
detected using HEp-2 cells. Patients with autoimmune disease
may present with a systemic multisystem illness or be non-
­specifically unwell. Such patients should be tested for the pres-
ence of autoantibodies. However, screening for ANA should not
be used indiscriminately. The probability of a positive result is
dependent on the prior probability of the disease in question.
Where that is low there is an increased chance of a false positive
result. The positive predictive value of ANA in SLE is around
11%. ANA are not specific for autoimmune rheumatic disease
and can occur in other conditions such as organ specific autoim-
mune disease (primary autoimmune cholangitis, primary biliary
MEDICINE 34:11
cirrhosis), chronic infection (subacute bacterial endocarditis),
viral infection and lymphoproliferative disease. Detection of an
autoantibody in an unexpected situation should prompt a careful
clinical review for features of the suggested disease. Equally the
absence of an autoantibody should not lead to the discarding of
the diagnosis if the clinical features are sufficiently clearcut and
supported by other evidence such as histology. About 15% of
healthy adults and 8% of children have detectable ANA, usually
in low titre. The frequency of ANA in normal people is higher in
women and increases with age so that around 25% of women
over the age of 60 years are positive.
Most routine laboratories offer testing for the antibodies
described above. Specialist reference laboratories may also offer
a broader range of tests in line with their research interests. ◆
References
1 Zendman A J W, van Venrooji W J, Pruijn G J M. Use and significance
of anti-CCP antibodies in rheumatoid arthritis. Rheumatology 2006;
45: 20–5.
2 Damoiseaux J G, Cohen Tervaert J W. From ANA to ENA how to
proceed? Autoimmun Rev 2006; 5: 10–17.
3 Trendelenburg M. Antibodies against C1q in patients with SLE.
Springer Semin Immunopathol 2005; 27: 276–85.
4 Hagen E C, Andrassy K, Csernok E et al. The diagnostic value of
standardised assays for ANCA in idiopathic systemic vasculitis:
results of an international collaborative study. Kidney Int 1998; 53:
743–53.
Further reading
Giles I, Isenberg D A. Antinuclear antibodies; an overview. In: Wallace D J,
Hahn B H, eds. Dubois’ lupus erythematosus. 5th ed. Baltimore:
Williams and Wilkins, 2001.
444 © 2006 Elsevier Ltd. All rights reserved.