RESUMO
Blood sera of patients with autoimmune diseases scleroderma (Scl), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been shown to yield a specific immune response to topoisomerase I, the product of expression of a cDNA fragment cloned into lambda gt11 and monoclonal antibodies (MAB) to the enzyme. The 'topoisomerase test' is not absolutely specific for Scl. The stable positive response of autoimmune sera to anti-topoisomerase monoclonal antibodies has a specific character and is associated with the interaction of the Fab fragment of MAB with the IgG fraction of autoimmune serum. The response observed indicates the induction of anti-idiotypic antibodies against topoisomerase. The anti-idiotype, isolated by HPLC and affinity chromatography demonstrated the following functional activities: (i) the immunological reaction against DNA; (ii) high-affinity DNA-binding with topoisomerase-specific consensus; (iii) ability to compete with the native enzyme for binding with DNA and MAB to topoisomerase; (iv) immunological reaction against MAB to topoisomerase.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , DNA/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Dados de Sequência Molecular , Mapeamento por Restrição , Escleroderma Sistêmico/imunologiaRESUMO
A DNA-nicking activity was detected in the sera of patients with various autoimmune pathologies and was shown to be a property of autoantibodies. The DNA hydrolyzing activity, which was purified by affinity and high-performance liquid chromatography, corresponded in size to immunoglobulin M (IgM) and IgG and had a positive response to antibodies to human IgG. The DNA hydrolyzing autoantibodies were stable to acid shock and yielded a DNA degradation pattern that was different from that of deoxyribonuclease (DNase) I and blood DNase.
Assuntos
Autoanticorpos/metabolismo , Doenças Autoimunes/metabolismo , DNA/metabolismo , Acetatos/farmacologia , Ácido Acético , Autoanticorpos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , DNA Polimerase I/metabolismo , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Ágar , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Imunoglobulina M/isolamento & purificação , Imunoglobulina M/metabolismo , Cinética , Lúpus Eritematoso Sistêmico/imunologia , PlasmídeosRESUMO
The principal existence of natural catalytic antibodies in the autoimmune sera is discussed. In the course of the autoimmune process, the induction of antiidiotypic antibodies against topoisomerase I has been shown in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis. The above antibodies were obtained in preparative amounts. Proceeding from the concept of the idiotypic network, the antibodies were suggested to be natural enzymes and their properties were studied. They appeared to be anti-DNA antibodies, competing with the native topoisomerase I for binding to anti-topoisomerase monoclonal antibodies and possessing highly specific DNA-binding activity (Kd is about 0.1 nM). The antiidiotypic antibodies specifically inhibit the topoisomerase-catalysed relaxation reaction and affect the formation of covalent DNA-protein complex. Possible involvement of antiidiotypic antibodies against topoisomerase in the catalysis of reactions of DNA transformation is analysed. Catalytic antibodies that are natural enzymes possessing DNA-nicking activity have been isolated from the blood sera of patients with different autoimmune pathologies.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Catálise , DNA/metabolismo , DNA Topoisomerases Tipo I/imunologia , DNA Topoisomerases Tipo I/metabolismo , Humanos , Soros ImunesRESUMO
Immunoscreening of the human placenta cDNA-library in the expression vector lambda gt11 using non-isotope detection based on the avidin-biotin system allowed to identify a number of clones encoding human topoisomerase I. The fusion protein from an extract of Escherichia coli cells infected with the recombinant phage lambda gt11 interacts with the monoclonal antibody raised against topoisomerase I from calf thymus; the dissociation constant being 5.7.10(-8) M. The restricted DNA fragments coding for the topoisomerase polypeptide in the composition of the fusion protein were recloned, and expression in the pEX vector was obtained. The functional analysis of the expression products has enabled localization of the epitope of binding the monoclonal antibody. It was demonstrated that the identified fusion protein can be applied for diagnosis of autoimmune diseases.
Assuntos
Clonagem Molecular , DNA Topoisomerases Tipo I/genética , DNA/genética , Expressão Gênica , Placenta/enzimologia , Anticorpos Monoclonais , DNA Topoisomerases Tipo I/isolamento & purificação , Feminino , Vetores Genéticos , Humanos , GravidezRESUMO
A method of purification of rat liver cystathionase by high-performance liquid chromatography (HPLC) utilizing non-ideal gel filtration method is proposed. Resolution factors-flow rate, pH values, ionic strength of the mobile phase-were optimized. Antibodies to the enzyme were purified using an immunosorbent synthesized on the basis of epoxylated Toyopearl-65. Radioimmunoassay and immunoblotting demonstrated antibody monospecificity towards cystathionase. These monospecific antibodies were utilized for detecting enzyme amounts (up to 30 pg) using the avidin-biotin system. Rat cDNA expression library in phage lambda gt11 was screened. The cystathionase cDNA clone was isolated, and the structure of the insert was determined.
