4.5 Article

Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

期刊

TOXICOLOGY IN VITRO
卷 69, 期 -, 页码 -

出版社

PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.tiv.2020.104996

关键词

Catalase; Toxicity screening; Hydrogen peroxide; Oxidative stress; Antioxidant defense

资金

  1. ERDF funds through the Operational Programme Competitiveness Factors - COMPETE2020
  2. Foundation for Science and Technology under FCT-Post-doctoral Fellowship (SPP) [SFRH/BPD/116061/2016, PTDC/DTP-DES/1082/2014(POCI-01-0145-FEDER-016657), PTDC/BTM-SAL/29297/2017-POCI-01-0145-FEDER-029297, PTDC/MED-FAR/29391/2017-POCI-01-0145-FEDER-029391, PTDC/BIA-MOL/28607/2017, POCI-01-0145-FEDER-028607]
  3. project Summer Course in Interdisciplinary Research, Development and Innovation in Cellular and Molecular Metabolism - Portuguese Foundation for Science and Technology (FCT) [15-20/7/245]
  4. Directorate General for Higher Education (DGES) [UIDB/04539/2020]
  5. NIH [R01HD070096-01A1]
  6. Fundação para a Ciência e a Tecnologia [SFRH/BPD/116061/2016, PTDC/BIA-MOL/28607/2017] Funding Source: FCT

向作者/读者索取更多资源

Oxidative stress biomarkers are powerful endpoints in toxicological research. Cellular reductive/oxidative balance affects numerous signaling pathways involving H2O2. Detoxification and control of H2O2 levels results mainly from catalase activity. The aim of this work was to develop a precise, simple, cost-effective microassay to measure catalase activity in small tissue samples and cell extracts. We developed a protocol that quantifies H2O2 decomposition by intrinsic catalase in biological samples. Catalase activity was calculated based on rate of decomposition of H2O2, following absorbance at 240 nm. We developed a multi-well spectroscopic approach, reducing sample quantity requirements and allowing simultaneous assessment of large number of samples. The protocol is sensitive across a wide range of catalase activity (11.5-7575 U). The assay presents a 95% confidence interval with an intra-assay coefficient of variation of 3.7%, an inter-assay coefficient of variation of 6.2% and good correlation with a commercial kit. The assay was established and validated for different biological samples, including sheep hepatic tissue and human tumor and non-tumor cell lines. This high-throughput method is robust, sensitive, time-saving and cost-effective, generating highly reproducible results with precision and good correlation with a commercial kit reinforcing the method's validity for research and toxicological applications.

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