Characterization of the antigen reactive with anti‐scl‐70 antibodies and its application in an enzyme‐linked immunosorbent assay

Abstract

The characteristics of the Scl-70 antigen (topoisomerase I) have been analyzed by means of autoantibodies. This antigen is a DNA-binding protein, dissociable from DNA at 0.3M NaCl and bound to a fraction of DNA that is very sensitive to nucleases. The molecular weight of the antigen is 105,000 daltons, whether dissociation conditions are used or not. Using chicken erythrocytes, and taking advantage of the strong interaction of the antigen with hydroxyapatite, we have designed a simple and fast purification protocol that allows the determination of anti-topoisomerase I antibodies by enzyme-linked immunosorbent assay.The presence of autoantibodies against nuclear structures is characteristic of many autoimmune diseases (1). Nevertheless, it is not yet known why these autoantibodies are produced, or what role they play in the pathologic process.Approximately 20% of patients with scleroderma have autoantibodies to a nuclear antigen designated the Scl-70 antigen (2). This antigen was first defined as a 70-kd chromatin antigen (3), but since then, it has been postulated that it could be an 86-kd By means of autoantibodies, we have analyzed the molecular characteristics of the Scl-70 antigen, the type of interaction with DNA, and the topoisomerase activity. A fast purification protocol, which allows the quantification of anti-Scl-70 antibodies by enzymelinked immunosorbent assay (ELISA), is also described. MATERIALS AND METHODSSera. Sera were collected from 9 patients who had been diagnosed as having scleroderma (7). In 5 of these sera, anti-Scl-70 antibodies were detected by immunodiffusion, using reference sera obtained from Dr. Eng Tan (Scripps Clinic, La Jolla, CA) as control. The patients' sera were able to bind a 70-kd protein from an extract obtained as described by Douvas et al (3). In addition, sera with antinuclear antibodies from 10 patients (4 with systemic lupus erythematosus, 3 with mixed connective tissue disease, 2 with Sjogren's syndrome, and 1 with rheumatoid arthritis) and 9 normal human sera were used in blotting and ELISA. Finally, anti-DNA and anti-Sm antibodies were used as controls in the immunofluorescence studies.Immunofluorescence. Indirect immunofluorescence studies were performed with cytocentrifuged cells or nuclei and with cells grown on coverslips, using human melanoma cell lines HeLa and HMcB. Cells were fixed with acetone for 5 minutes or made permeable by freezekhawing and treatment with 0.5% NP-40 for 5 minutes. Afterwards, cells or nuclei were treated for 30 minutes with: (a) phosphate buffered saline (PBS) as a control; (b) 10 mM phosphate buffer, pH 7.2, containing 0.3,0.5, 1 , or 2M NaCl; (c) DNase

Analysis of human antitopoisomerase-I idiotypes.

“…The major protein isolated from calf thymus is 70 kD (2). Antibodies to topo-I bind one or more ofsix epitopes in the enzyme ( 13) and inhibit the enzyme's activity in vitro (3,4,7,(9)(10)(11)(12).…”

A flexible, efficient, checkerboard immunoblot system for the detection and semiquantitation of specific antinuclear antibodies

“…The preparation in lane 3 contains several bands, consistent with degraded Scl-70 antigen (20). The native form of 105 kD is not seen in this preparation.…”

Characteristic Nucleolar-Reticular Immunofluorescence Staining Pattern on Touch Prints of Hamster Liver: A Marker for Scl-70 Antibody

“…Guldner et al [12] stressed that be sides the fine granular nuclear immunofluorescence, characteristic of Scl-70 antibody, there is always a concomitant fluorescence of nucleoli. DNA topoisomerase I was identified as one of the antigen com ponents against which autoantibodies form in sclero derma patients [14,22,31]. The localization of the 100-kD antigen or its degradation products in nuclei seems similar to the intracellular distribution of DNA topoisomerase I, i.e.…”

“…Antibody directed against a thermolabile, chro matin-associated nonhistone nucleoprotein of a mo lecular weight of 70 kD [10,33] revealed a high speci ficity for a severe form of SSc with diffuse skin involvement [9,11,13,24,32,33]. Later studies have determined antigens of higher molecular weights (100, 95, 89, 86, 80 kD) which are target antigens of the Scl-70 antibody [12,14,20,23,31,34], The anti gens of lower molecular weights may be regarded as degradation products of Scl-100 [ 12,14,20].…”