BACKGROUND: Anti-DNA topoisomerase I (anti-topo I) antibodies have been broadly studied in systemic sclerosis (SSc). The use of different native and molecularly cloned antigens has shown a predominant IgG response, and a variable frequency of positive IgM and IgA tests. We report herein the serological findings of SSc using a recombinant topo I obtained through a standard bacterial system. METHODS: Anti-topo I antibodies were studied in 45 SSc patients and 85 healthy controls through ELISA and western blot. Escherichia coli XL1-blue strain and pT7-7 vector were used to amplify a DNA topo I cDNA clone, and to obtain the recombinant polypeptide. The latter was purified by affinity chromatography, and the enzymatic and antigenic properties were evaluated through specific tests. A native antigen was included for comparison. RESULTS: The SSc group disclosed positive IgM (20%), IgG (86.6%), and IgA (26.6%) anti-topo I tests with the recombinant polypeptide, and a purified calf thymus antigen yielded similar results. IgG autoantibodies were frequently associated with skin involvement, esophageal dysfunction, and restrictive lung disease. The recombinant protein showed a molecular weight of 86.6 kDa, a positive topo I activity using a supercoiled pBR322 DNA relaxation test, and its carboxyl terminus region was recognized by specific antibodies. CONCLUSION: This report confirms that different immunoglobulin classes with anti-topo I activity may occur in SSc. IgG was the predominant serological feature with both, the recombinant and native antigens. The study also demonstrates the association between high levels of these autoantibodies and some clinical manifestations of SSc.
BACKGROUND: Anti-DNA topoisomerase I (anti-topo I) antibodies have been broadly studied in systemic sclerosis (SSc). The use of different native and molecularly cloned antigens has shown a predominant IgG response, and a variable frequency of positive IgM and IgA tests. We report herein the serological findings of SSc using a recombinant topo I obtained through a standard bacterial system. METHODS: Anti-topo I antibodies were studied in 45 SSc patients and 85 healthy controls through ELISA and western blot. Escherichia coli XL1-blue strain and pT7-7 vector were used to amplify a DNA topo I cDNA clone, and to obtain the recombinant polypeptide. The latter was purified by affinity chromatography, and the enzymatic and antigenic properties were evaluated through specific tests. A native antigen was included for comparison. RESULTS: The SSc group disclosed positive IgM (20%), IgG (86.6%), and IgA (26.6%) anti-topo I tests with the recombinant polypeptide, and a purified calf thymus antigen yielded similar results. IgG autoantibodies were frequently associated with skin involvement, esophageal dysfunction, and restrictive lung disease. The recombinant protein showed a molecular weight of 86.6 kDa, a positive topo I activity using a supercoiled pBR322 DNA relaxation test, and its carboxyl terminus region was recognized by specific antibodies. CONCLUSION: This report confirms that different immunoglobulin classes with anti-topo I activity may occur in SSc. IgG was the predominant serological feature with both, the recombinant and native antigens. The study also demonstrates the association between high levels of these autoantibodies and some clinical manifestations of SSc.
Authors: N Bizzaro; E Tonutti; D Villalta; D Bassetti; R Tozzoli; F Manoni; S Pirrone; A Piazza; P Rizzotti; M Pradella Journal: Clin Chem Date: 2000-10 Impact factor: 8.327