Purification of a human centromere antigen (CENP-B) and application of DNA immunoprecipitation to quantitative assay for anti-CENP-B antibodies.
Centromere protein-B (CENP-B) was purified from HeLa nuclear extract by using a combination of Q-sepharose ion change and oligonucleotide sepharose column chromatography. CENP-B was purified more than 10,000 times and was analyzed by immunoblotting and DNA immunoprecipitation. Purified CENP-B was used as an antigen to develop DNA immunoprecipitation for rapid and specific detection of anti-CENP-B antibody in human sera. In this analysis, none of the tested sera immunoprecipitated DNA alone. All of the 40 anticentromere antibody (ACA)-positive sera immunoprecipitated CENP-B-alphoid DNA fragment complex, whereas 10 healthy control sera and 10 other autoantibody positive sera did not. Five of the ACA-positive sera, which did not show reactivity of CENP-B in immunoblotting analysis, immunoprecipitated CENP-B-DNA complex. In some sera antigenicity to CENP-B may be weakened by denaturation.
Muro
Y
Y
Department of Dermatology, Nagoya University School of Medicine, Japan.
Masumoto
H
H
Okazaki
T
T
Ohashi
M
M
D001323:Autoantibodies / Q000302:isolation & purification*; D001324:Autoantigens; D002467:Cell Nucleus / Q000737:chemistry; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000276:immunology / Q000302:isolation & purification*; D004247:DNA / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins; D006801:Humans; D011233:Precipitin Tests
D001323:Autoantibodies; D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004247:DNA