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Technical Briefs |
a address for correspondence: Clin. Lab., Diaconessenhuis, P.O. Box 90052, 5600 PD, Eindhoven, The Netherlands
1
Diaconessenhuis, Eindhoven and
2
Elkerliek Hospital, Helmond, The Netherlands;
Detection of antinuclear antibodies (ANA), usually by indirect immunofluorescence, is generally regarded as an important test in the diagnosis of systemic autoimmune diseases. If ANA are present, the subsequent identification of autoantibody specificity contributes to the final diagnosis and may also be helpful in disease management. ANA, which show many different serologic specificities, are partly directed against extractable nuclear antigens (ENA). The most common ENAs are SSA, SSB, Sm, RNP, and Scl-70. The presence of these specific ENA antibodies is related to certain autoimmune diseases such as Sjögren syndrome (SSA, SSB), mixed connective tissue disease (RNP), systemic lupus erythematosus (Sm), and scleroderma (Scl-70) (1).
ENA antibodies can be detected in several ways. Double immunodiffusion, often in combination with immunoblotting (IB), is widely used but is time-consuming and requires well-trained and experienced technicians (2). A few years ago several ELISAs were introduced which have proven to be both sensitive and specific for detection of ENA antibodies (2)(3)(4). However, they are expensive and require the availability of an ELISA reader. Recently, a simple dot-blot method (DB) for the detection of ENA antibodies [Biomedical Diagnostics (BMD)] has become available. In the present study we compared this DB with the currently used combination of counterimmunoelectrophoresis and immunoblotting (CIE/IB) for the detection of antibodies against ENA.
Sera from 146 patients were selected for the evaluation of detection of antibodies directed against SSA, SSB, Sm, and RNP. Besides samples with CIE/IB-proven ENA antibodies, samples with negative results were included to check for cross-reactivity and purity of the antigens used in the DB method. Moreover, 35 samples from patients suspected for scleroderma or a related disease were selected for Scl-70 antibody detection. All patient sera used contained ANA as confirmed by indirect immunofluorescence and were selected from a large sample collection to ensure a variety of antibodies. The sera in this collection, some of which were sent from other laboratories, were stored at -70 °C until analysis.
CIE for the detection of antibodies against SSA, SSB, RNP, and Sm was performed as described by Bunn and Kveder (5). Briefly, 10 µL of patient serum or characterized antiserum was loaded into 3-mm wells cut out of an agarose plate. Subsequently, antigen was put in 2-mm-wide troughs at the cathodal site of the wells. After electrophoresis the plates were washed with cold PBS and stained with Coomassie Blue. Antibodies present in patient samples could be detected and identified by the formation of specific precipitation lines between wells filled with patient samples and wells filled with characterized antisera. The antigen sources used were rabbit thymus (for detection of antibodies against SSB, Sm, and RNP) and bovine spleen (for detection of SSA antibodies).
If the results of CIE were doubtful or weak an IB was performed as described by Verheijen et al. (6). Detection of antibodies against Scl-70 was done only by IB. Briefly, protein extracts from HeLa cells were separated on a polyacrylamide gel and transferred to nitrocellulose. Strips of this blot were incubated with patient sera. Antibody–antigen complexes were stained with 4-chloro-1-naphthol in the horseradish peroxidase system. The subsequent identification of antibodies was based on the specific immunoblot pattern.
The DB method of BMD is a qualitative assay, which utilizes strips of
nitrocellulose on which purified antigens are blotted at prelocated
spots (see Fig. 1
). The antigen sources used are bovine and rabbit thymus (SSA,
Sm, and Scl-70) or calf spleen and rabbit thymus (SSB and Sm/RNP). The
test procedure was performed according to directions supplied by the
manufacturer. Test strips were incubated for 10 min with a 50-fold
dilution of patient serum in a PBS-Tween solution supplied by the
manufacturer. Subsequently the test strips were washed by gentle
agitation in a test tube filled with PBS-Tween for 1 min. After the
excess buffer solution was removed with a filter paper, the test strips
were incubated with an alkaline phosphatase–Protein A conjugate for 10
min. The test strips were then washed for 1 min by gentle agitation in
a test tube filled with PBS-Tween. Again excess buffer was removed with
a filter paper. Finally the test strips were stained with
5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium for 5 min.
The reaction was terminated by washing the test strips with deionized
water. The strips were then air-dried. Only strips on which the
positive control position was stained as a clearly marked blue spot
were able to be evaluated and used for this study. The absence of the
blue spot on the positive control position occurred only once during
the study.
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The comparison between CIE/IB and DB for the individual ENA antibodies
is presented in Table 1
. The agreement between CIE/IB and DB was 99%, 100%, 100%,
99%, and 97% for SSA, SSB, RNP, Sm, and Scl-70, respectively. The
samples that showed a discrepancy for SSA and Sm proved to be only
weakly demonstrable in the test method with the positive result. The
corresponding patients were diagnosed with a systemic autoimmune
disease or were seriously suspected of having one because of their
clinical performance. The medical file of the patient with the
CIE/IB-positive Scl-70 did not reveal specific symptoms for
scleroderma.
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The sensitivity and specificity of the DB was calculated by comparison
with the CIE/IB and also are presented in Table 1
. Overall the
sensitivity and specificity of DB for detection of specific ENA
antibodies were excellent with the exception of sensitivity for Scl-70
and Sm, which was reasonable. However, as no gold standard for ANA
characterization exists, one has to be careful with the interpretation
of these data. The same accounts for the five test results that showed
a discrepancy. These might be the result of nonspecific binding but
might also indicate a (weak) ENA antibody. With the exception of the
sample that was positive for Scl-70 by CIE/IB only, this would concur
with the suspected or diagnosed presence of an autoimmune disease in
these particular patients.
The DB test is advantageous for time management as described previously for a similar test system for ENA antibodies (7). A single test required ~30 min. The test procedure could easily be performed in batch assays as every 5 min a new DB test could be started. Visual scoring of the test-strip reactions was mastered quickly after a few assays. After a short training period, technicians were able to perform the entire test procedure. Moreover, in comparison with CIE/IB the time required to report results was substantially shortened from days to hours.
A major drawback of the DB method is the blotting of RNP antigen in
combination with Sm antigen (see Fig. 1
). This implies that if both the
Sm spot and the Sm/RNP spot are positive the presence of Sm antibodies
alone cannot be distinguished from the combined presence of Sm and RNP
antibodies. For all nine samples that, according to DB, contained Sm
antibodies both spots were positive. As this only indicates the
possible presence of RNP antibodies these results could not be included
in the study. Several ELISAs for ENA characterization are now also
available for routine use (2)(3)(4). Compared with the DB,
the high costs of these systems render them less attractive although
they exhibit excellent sensitivities and specificities and also are
less time-consuming than CIE/IB.
We conclude that DB may be an attractive method for those laboratories that wish to perform ENA antibody characterization without substantial investments in equipment and (or) the training of laboratory staff.
Footnotes
References
The following articles in journals at HighWire Press have cited this article:
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  R J Lock and D J Unsworth Antibodies to extractable nuclear antigens. Has technological drift affected clinical interpretation? J. Clin. Pathol., March 1, 2001; 54(3): 187 - 190. [Abstract] [Full Text] [PDF]
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 A. A.M. Ermens, A. J.M. Bayens, A. Crooymans, A. A.M. Broekman-van Hout, and H. L.P. van Duijnhoven Evaluation of a Simple Dot-Blot Method for the Detection of Anti-Neutrophil Cytoplasmic Antibodies Directed against Proteinase 3 and Myeloperoxidase Clin. Chem., October 1, 2000; 46(10): 1717 - 1719. [Full Text] [PDF]
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