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Evaluation of a dot-blot method for identification of antibodies against extractable nuclear antigens and anti-cytoplasmic antibodies.

To the Editor:

Antibodies against extractable nuclear antigens (ENAs) are useful diagnostic markers for various autoimmune diseases. Anti-SSA and anti-SSB antibodies are found in Sjogren Syndrome (SS), anti-Sm antibodies are found in systemic lupus erythematosus (SLE), anti-RNP antibodies are found in mixed connective tissue disease (MCTD), and anti-Scl-70 antibodies are found in scleroderma. Antibodies against cytoplasmic Jo-1 and M2 are helpful markers for the diagnosis of, respectively, polymyositis and primary biliary cirrhosis. The antibodies can be identified by either counter immunoelectrophoresis (CIE) or immunoblotting. These techniques, however, are time-consuming, technically demanding, and do not provide for fast turnaround times. Over the last few years, alternative techniques such as ELISA and dot blot (DB) have been introduced. A DB technique from Biomedical Diagnostics recently has become available and has been evaluated for autoantibodies against ENAs by Ermens et al. (1). In the present study, we evaluated the same DB technique for autoantibodies against ENAs and against the cytoplasmic antigens Jo-1 and M2. The DB technique was compared with CIE for the detection of antibodies against ENAs and Jo-1 and with indirect immunofluorescence (IF) for the detection of antibodies against M2.

The DB technique was performed according to the manufacturer's instructions. CIE was performed as described by Walravens et al. (2). To detect antibodies against mitochondria by IF, we incubated sera for 30 min at room temperature with rat kidney and rat stomach sections at a 1:20 serum dilution, and then washed and incubated the sera with fluorescein isothiocyanate-labeled sheep anti-human immunoglobulin. Tissue sections were examined under ultraviolet light, using an Orthoplan Leitz Wetzler microscope. Mitochondrial antibodies appeared as discrete granular staining, predominantly in the distal tubules of the kidney and in the parietal cells of the stomach.

Table 1 shows the results of the method comparison study. The agreement between CIE and DB for antibodies against ENAs was 99%, 98%, 99%, 100%, and 0%, for SSA, SSB, RNP, Sm, and Scl-70, respectively. The medical records of the patients with CIE+/DB- SSA, CIE+/DB- SSB, CIE-/DB+ SSB, and CIE-/DB+ RNP revealed symptoms typical for systemic lupus erythematosus, SS, mixed connective tissue disease, and SS, respectively. The three patients with CIE-/DB+ results for Scl-70 had symptoms typical for scleroderma. These data confirm the previously reported (1) excellent sensitivities and specificities of DB techniques for the detection of antibodies against ENAs. The agreement between CIE/IF and DB for the detection of the cytoplasmatic antigens Jo-1 and M2 was 100% and 93%, respectively. The patients with IF+/DB- M2 had autoimmune hepatitis and chronic hepatitis C.

IF detects various subtypes of anti-mitochondrial antibodies, but lacks the ability to detect specific antigens, such as the M2 subtype, which is highly specific for primary biliary cirrhosis. Therefore, DB is a valid alternative to CIE and IF for the detection of antibodies against ENA, Jo-1, and M2 with markedly faster turnaround time. The DB method is also more sensitive for the detection of anti-Scl-70 antibodies.


(1.) Ermens AAM, Bayens AJM, van Gemert ACM, van Duijnhoven LP. Simple dot-blot method evaluated for detection of antibodies against extractable nuclear antigens. Clin Chem 1997; 12:2420-2.

(2.) Walravens MJF, Vanherrewegen H, Lacquet F, Godefridis G, Korevits G, Stevens E, et al. Counterimmunoelectrophoresis with serum prediffusion: an improved method for the detection and identification of antibodies against extractable nuclear and cytoplasmic antigens. J Immunol Methods 1996;201:89-98.

Alex Mewis Godelieve Marien Norbert Blanckaert Xavier Bossuyt * Department of Clinical Pathology University Hospitals Leuven Kapucijnenvoer 33 B-3000 Leuven, Belgium

* Author for correspondence. Fax 32 16 332896;e-mailxavier.bossuyt@uz.kuleuven.ache.
Table 1. Comparison between DB and CIE (a) for detection of antibodies
against SSA, SSB, RNP, Sm, Scl-70, or Jo-1, and between DB and IF for
detection of antibodies against M2.

 Concordant Discordant


SSA 126 57 68 1 0
SSB 126 98 26 1 1
RNP 126 112 13 0 1
Sm 126 100 26 0 0
Scl-70 3 0 0 0 3
Jo-1 50 31 19 0 0
 IF-/DB- IF+/DB+ IF+/DB- IF-/DB+
M2 32 2 28 2 0

 Sensitivity Specificity

SSA 0.98 (0.92-1) 1 (0.94-1)
SSB 0.96 (0.81-0.99) 0.99 (0.94-1)
RNP 1 (0.75-1) 0.99 (0.96-1)
Sm 1 (0.86-1) 1 (0.95-1)
Jo-1 1 (0.82-1) 1 (0.89-1)

(a) CIE was considered the reference method for calculation of
sensitivity and specificity (including 95% confidence intervals) for
SSA, SSB, RNP, Sm, and Jo-1.
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Title Annotation:Letters
Author:Mewis, Alex; Marien, Godelieve; Blanckaert, Norbert; Bossuyt, Xavier
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Feb 1, 1999
Previous Article:"Citation classics in Clinical Chemistry": contributions by AACC members duly noted.
Next Article:Thoughts on After the Genome IV.

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