Isotype analysis of the anti-CENP-B anticentromere autoantibody: evidence for restricted clonality.

Utilizing the centromere B fusion protein (CENP-B) and specific, matched monoclonal antiisotype reagents in an enzyme-linked immunosorbent assay, we found that anti-CENP-B autoantibodies were skewed to the IgG1 isotype. The overall kappa:lambda light chain ratio was 2:1, although several individual sera showed a strong predominance of one of the light chains. Isoelectric focusing of light chain-skewed sera showed polyclonal patterns. Our findings are consistent with the anti-CENP-B autoantibody response being a chronic, antigen-driven response.

Human autoantibody to topoisomerase II.

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.

Clinical associations of anticentromere antibodies and antibodies to topoisomerase I. A study of 355 patients.

Anticentromere antibodies (ACA) and anti-topoisomerase I (anti-topo I) were assayed in serum samples from 355 patients: 89 with proximal scleroderma; 54 with CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias), without proximal scleroderma; 154 with primary and secondary Raynaud's disease; and 58 with other rheumatic diseases, without Raynaud's disease. Sera from healthy control subjects were also assayed. Using immunoblotting techniques, anti-topo I was detected in 28% of the patients with proximal scleroderma; using immunodiffusion techniques, this antibody was found in only 20% of the same group of patients. Anti-topo I and ACA were found primarily in patients with scleroderma, CREST syndrome, and Raynaud's phenomenon. ACA identified patients with less severe disease, whereas anti-topo I identified patients with skin and cardiac involvement and patients with malignancies.

Detection of anticentromere antibodies using cloned autoantigen CENP-B.

A solid-phase enzyme-linked immunosorbent assay has been established using a cloned fusion protein, CtermCENP-B [beta-gal], as antigen. The fusion protein carries the major epitope of CENP-B, the major centromeric autoantigen. The enzyme-linked immunosorbent assay was more sensitive than immunofluorescence techniques in detecting anticentromere antibodies in patients with scleroderma or Raynaud's disease, and was weakly positive in 3% of normal controls and in 3% of 70 patients with other connective tissue diseases.

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting.

Antibodies to topoisomerase I in sera from patients with scleroderma.

In a study of sera from patients with scleroderma, it was found that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I.

Three human chromosomal autoantigens are recognized by sera from patients with anti-centromere antibodies.

We have identified 39 individuals with anti-centromere antibodies (ACA) in our patient population, all of whom have Raynaud's syndrome or disease. We have used sera from the ACA-positive patients and from 123 controls (22 normal individuals and 101 additional patients with either Raynaud's disease or Raynaud's syndrome plus an associated connective tissue disease) to screen the proteins of highly purified human (HeLa) mitotic chromosomes by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. Three antigens were recognized by the sera from the ACA-positive patients. These were centromere protein (CENP)-B (80,000 mol wt--recognized by all ACA-positive sera), CENP-A (17,000 mol wt--recognized by 38 of 39 ACA-positive sera), and CENP-C (140,000 mol wt--recognized by 37 of 39 ACA-positive sera). None of these antigens were recognized by any of the 123 control sera, although binding was occasionally seen to other chromosomal antigens. Therefore the ACA response is highly uniform in our patient population. Antibody to CENP-B shows a 100% correlation with anti-centromere staining by indirect immunofluorescence.

High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients.

Patients with rheumatic diseases often have circulating autoantibodies to nuclear components. The clinical significance of the antibodies is controversial, although in some cases they are valuable in the diagnosis of the disease. This report presents results of a study of Scl-70, an autoantigen recognized by sera of many patients with the most severe form of progressive systemic sclerosis. It was possible to show, by three independent criteria, that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I. Therefore, antibody probes of high titer and high affinity are now available for the study of this important nuclear enzyme.

Antibodies to native DNA and serum complement (C3) levels. Application to diagnosis and classification of systemic lupus erythematosus.

The sensitivity and specificity of the presence of antibodies to native DNA and low serum C3 levels were investigated in a prospective study in 98 patients with systemic lupus erythematosus who were followed for a mean of 38.4 months. Hospitalized patients, patients with other connective tissue diseases, and subjects without any disease served as the control group. Seventy-two percent of the patients with systemic lupus erythematosus had a high DNA-binding value (more than 33 percent) initially, and an additional 20 percent had a high DNA-binding value later in the course of the illness. Similarly, C3 levels were low (less than 81 mg/100 ml) in 38 percent of the patients with systemic lupus erythematosus initially and in 66 percent of the patients at any time during the study. High DNA-binding and low C3 levels each showed extremely high predictive value (94 percent) for the diagnosis of systemic lupus erythematosus when applied in a patient population in which that diagnosis was considered. The presence of both abnormalities was 100 percent correct in predicting the diagnosis os systemic lupus erythematosus. Both tests should be included in future criteria for the diagnosis and classification of systemic lupus erythematosus.

Familial discoid lupus erythematosus associated with heterozygote C2 deficiency.

Two siblings with chronic discoid lupus erythematosus and several family members were found with heterozygous C2 deficiency. An association with histocompatibility markers HLA-B18 and HLA-Dw2 was demonstrated, and the slow allotype of factor B was present. Linkage studies in this family suggested a close linkage between the C2 deficiency gene and genes coding for B18, Dw2, and BfS antigens. One HLA-ACB/DBf recombinant was observed showing closer linkage between HLA-D and Bf than between HLA-B and Bf.