Chemiluminescence Immunoassay for S-Adenosylhomocysteine Detection and Its Application in DNA Methyltransferase Activity Evaluation and Inhibitors Screening

Xiaogang, L.; Meng, M.; Zheng, L.; Zhihuan, X.; Song, P.; Yin, Y.; Eremin, S.A.; Rimo, X.

Авторы: Xiaogang Li, Meng Meng, Zheng Lei, Zhihuan Xu, Song Pei, Yin Yongmei, Eremin Sergei A., Rimo Xi
Журнал: Analytical Chemistry
Том: 88
Номер: 17
Год издания: 2016
Издательство: American Chemical Society
Местоположение издательства: United States
Первая страница: 8556
Последняя страница: 8561
DOI: 10.1021/acs.analchem.6b01579
Аннотация: Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), a common product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH, and characterized the polyclonal antibodies in each group. The optimal antibody product was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate to bind with SAH antibodies. Successively, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG was conjugated with SAH antibodies and HRP catalyzed luminol oxidation by H2O2, resulting in a high chemiluminescent signal. The method could detect as low as 9.8 ng mL-1 SAH with little cross-reaction (3.8%) with SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.05 to 4.0 U/mL. The inhibition study showed that, in presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine and 5-Aza-2'-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 μM and 0.48 μM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNAMTase activity and screening its inhibitors.
Добавил в систему: Еремин Сергей Александрович

	ИСТИНА	Войти в систему Регистрация
	ИПМех РАН
	Главная Поиск Статистика О проекте Помощь

ИСТИНА

ИПМех РАН

Chemiluminescence Immunoassay for S-Adenosylhomocysteine Detection and Its Application in DNA Methyltransferase Activity Evaluation and Inhibitors Screeningстатья

Прикрепленные файлы