A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin

被引:1
|
作者
Rasooly, Reuven [1 ]
Do, Paula [1 ]
He, Xiaohua [1 ]
Hernlem, Bradley [1 ]
机构
[1] ARS, Foodborne Toxin Detect & Prevent Res Unit, Western Reg Res Ctr, USDA, Albany, CA 94710 USA
关键词
alpha-amanitin; qPCR; MTT; adenovirus; GFP; cell-based assays; food safety; BETA-AMANITIN; STRUCTURAL BASIS; MUSHROOM; PHALLOIDES; TRANSCRIPTION; AMATOXINS; URINE; CHROMATOGRAPHY; INHIBITOR; DIAGNOSIS;
D O I
10.3390/ijms242216402
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
alpha-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita. Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of alpha-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by alpha-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of alpha-amanitin through the inhibition of cellular activity. This method can detect 10 mu g/mL of alpha-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, alpha-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous alpha-amanitin heated at 90 degrees C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of alpha-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors.
引用
收藏
页数:12
相关论文
共 50 条
  • [1] A Sensitive Cell-Based Assay to Measure the Doxycycline Concentration in Biological Samples
    Kleibeuker, Wendy
    Zhou, Xue
    Centlivre, Mireille
    Legrand, Nicolas
    Page, Mark
    Almond, Neil
    Berkhout, Ben
    Das, Atze T.
    HUMAN GENE THERAPY, 2009, 20 (05) : 524 - 530
  • [2] Cell-based assay for β-secretase activity
    Oh, M
    Kim, SY
    Oh, YS
    Choi, DY
    Sin, HJ
    Jung, IM
    Park, WJ
    ANALYTICAL BIOCHEMISTRY, 2003, 323 (01) : 7 - 11
  • [3] A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance
    Ouellette, Steven B.
    Noel, Brett M.
    Parker, Laurie L.
    PLOS ONE, 2016, 11 (09):
  • [4] Development of a novel, sensitive cell-based corin assay
    Lambertz, Pascal
    Theisen, Laura
    Laengst, Natalie
    Garvie, Colin W.
    MacDonald, Bryan T.
    Yu, John
    Elowe, Nadine H.
    Zubov, Dmitry
    Kaushik, Virendar K.
    Wunder, Frank
    BIOCHEMICAL PHARMACOLOGY, 2019, 160 : 62 - 70
  • [5] Cell-based assay for monitoring transglutaminase activity
    Jeon, JH
    Jang, GY
    Kim, CW
    Shin, DM
    Cho, SY
    Kwon, JC
    Lee, HJ
    Choi, KH
    Kim, IG
    ANALYTICAL BIOCHEMISTRY, 2004, 333 (02) : 399 - 401
  • [6] A sensitive and robust Western Blot cell-based assay to measure the activity of botulinum neurotoxin serotype A
    Fernandez-Salas, Ester
    Wang, Joanne
    Molina, Yanira
    Nelson, Jeremy
    Garay, Patton
    Lewis, Ramilla
    Paul, David
    Aoki, K. Roger
    TOXICON, 2008, 51 : 11 - 11
  • [7] A cell-based screening assay for Natural Killer cell activity
    Blom, W. Marty
    van Nielen, Wim G. L.
    de Groene, Els. M.
    Albers, Ruud
    INTERNATIONAL IMMUNOPHARMACOLOGY, 2009, 9 (06) : 746 - 752
  • [8] Cell-based assay for the measurement of complement convertase activity
    Stasilojc, Malgorzata
    Kuzniewska, Alicja
    Stasilojc, Grzegorz
    Rodriguez de Cordoba, Santiago
    Okroj, Marcin
    IMMUNOBIOLOGY, 2023, 228 (05) : 49 - 49
  • [9] A cell-based assay to measure the activity of the complement convertases
    Stasilojc, Malgorzata
    Stasilojc, Grzegorz
    Kuzniewska, Alicja
    Rodriguez de Cordoba, Santiago
    Okroj, Marcin
    EUROPEAN JOURNAL OF IMMUNOLOGY, 2024, 54 : 90 - 90
  • [10] A Cell-Based Assay to Measure the Activity of the Complement Convertases
    Stasilojc, Malgorzata
    Stasilojc, Grzegorz
    Kuzniewska, Alicja
    de Cordoba, Santiago Rodriguez
    Okroj, Marcin
    KIDNEY INTERNATIONAL REPORTS, 2024, 9 (07): : 2260 - 2268