Purification of a human centromere antigen (CENP-B) and application of DNA immunoprecipitation to quantitative assay for anti-CENP-B antibodies

Abstract

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.