Eukaryotic expression of recombinant human centromere autoantigen and its use in a novel ELISA for diagnosis of CREST syndrome.
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.
Stahnke
G
G
Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
Meier
E
E
Scanarini
M
M
Northemann
W
W
D000818:Animals; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000096:biosynthesis* / Q000276:immunology; D015153:Blotting, Western; D017675:CREST Syndrome / Q000175:diagnosis*; D002478:Cells, Cultured; D002503:Centromere / Q000276:immunology*; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis* / Q000276:immunology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D009036:Moths; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology
D001323:Autoantibodies; D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins