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

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), the product o...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2016-09, Vol.88 (17), p.8556-8561
Main Authors: Li, Xiaogang, Meng, Meng, Zheng, Lei, Xu, Zhihuan, Song, Pei, Yin, Yongmei, Eremin, Sergei A, Xi, Rimo
Format: Article
Language:English
Subjects:
Antibodies
Binding sites
Chemiluminescence
Deoxyribonucleic acid
DNA
Goats
Immunoassay
Inhibitors
Laboratory apparatus
Luminescence
Polyclonal antibodies
Screening
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Summary: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), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL–1 SAH with little cross-reaction (3.8%) to 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 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the 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 DNA MTase activity and screening its inhibitors.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.6b01579