Abstract |
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.
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Authors | G Stahnke, E Meier, M Scanarini, W Northemann |
Journal | Journal of autoimmunity
(J Autoimmun)
Vol. 7
Issue 1
Pg. 107-18
(Feb 1994)
ISSN: 0896-8411 [Print] England |
PMID | 8198697
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Autoantibodies
- Autoantigens
- CENPB protein, human
- Centromere Protein B
- Chromosomal Proteins, Non-Histone
- DNA-Binding Proteins
- Recombinant Proteins
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Topics |
- Animals
- Autoantibodies
(blood)
- Autoantigens
(biosynthesis, immunology)
- Blotting, Western
- CREST Syndrome
(diagnosis)
- Cells, Cultured
- Centromere
(immunology)
- Centromere Protein B
- Chromosomal Proteins, Non-Histone
(biosynthesis, immunology)
- Cloning, Molecular
- DNA-Binding Proteins
- Enzyme-Linked Immunosorbent Assay
(methods)
- Humans
- Moths
- Recombinant Proteins
(biosynthesis, immunology)
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