Eukaryotic expression of recombinant human centromere autoantigen and its use in a novel ELISA for diagnosis of CREST syndrome
| Autor: | Wolfgang Northemann, Gisela Stahnke, Elke Meier, Mirco Scanarini |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
CREST Syndrome
Chromosomal Proteins Non-Histone Immunology Blotting Western Centromere Centromere protein B Sf9 Enzyme-Linked Immunosorbent Assay macromolecular substances Biology Moths Autoantigens law.invention Antigen Affinity chromatography law Complementary DNA Immunology and Allergy Animals Humans Cloning Molecular Cells Cultured Autoantibodies cDNA library Autoantibody Molecular biology Recombinant Proteins DNA-Binding Proteins Recombinant DNA Centromere Protein B |
| Zdroj: | Journal of autoimmunity. 7(1) |
| ISSN: | 0896-8411 |
| Popis: | The cDNA coding for the human full-length centromere protein B (CENP-B) was isolated from a liver cDNA library by oligonucleotide screening and extended at the 5' and 3' ends by linker addition. The cDNA was inserted into a modified baculovirus transfer vector which mediated high-level expression of recombinant human CENP-B with a histidine-hexapeptide as affinity ligand at its N-terminus in infected Spodoptera frugiperda (Sf9) insect cells. Based on the histidine-hexapeptide moiety, the recombinant CENP-B was purified to homogeneity by single-step affinity chromatography using metal chelating matrix. An ELISA established with the eukaryotically expressed and purified recombinant human full-length CENP-B demonstrated its excellent specificity, sensitivity and reproducibility for the measurement of autoantibodies directed to the human CENP-B (ACA-B) representing a diagnostic marker for CREST syndrome, an autoimmune rheumatic disease. In this study, all pathological sera from patients (n = 80) with serologically and clinically diagnosed CREST were positively assayed for ACA-B, whereas 399 sera obtained from blood donors and 82 out of 84 sera from patients with autoimmune rheumatic disorders which were unrelated to CREST were negative in the ELISA. |
| Databáze: | OpenAIRE |
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