Enzyme Assay Design for High-Throughput Screening

The article describes a high-throughput method for the liberation of water-soluble cell contents by exploiting the phenomenon of irreversible membrane electropermeation (HT-irEP). The method is exemplified in recombinant proteins and plasmid liberation from yeast Saccharomyces cerevisiae on the detectable level. Obtained extracts are pure enough to be readily applied for further analytical analysis such as enzyme assay, PCR, and so on. From the same HT-irEP extract, one can measure activity of the target protein and perform amplification of the corresponding gene from the DNA vector by PCR for recombinant protein with intracellular expression. Therefore, the method is suitable for the high-throughput screening (HTS) of yeast libraries where extracellular expression of recombinant protein is problematic. The method can be easily automated and integrated into existing HTS systems.

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Transfected Cell Lines as Tools for High Throughput Screening: A Call for Standards

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710100600302 ◽

2001 ◽

Vol 6 (3) ◽

pp. 133-136 ◽

Cited By ~ 10

Author(s):

Len Pagliaro ◽

Morten Præstegaard

Keyword(s):

Cell Lines ◽

High Throughput ◽

High Throughput Screening ◽

Target Identification ◽

Complex Variables ◽

Screening System ◽

Library Size ◽

Global Issues ◽

Screening Strategies ◽

Assay Design

During 1999, Journal of Biomolecular Screening presented a series of Point-Counterpoint articles that addressed a question posed by editor Bill Janzen: "What is the future of HTS?" These articles discussed many of the global issues involved in HTS, such as target identification and library size, as well as the scientific and technical challenges facing the field. In this perspective we address a related, but very focused, issue that is increasingly important for many of us in the HTS community: the use of stably transfected cell lines as an integral part of screening strategies. Transfected cell lines provide powerful tools for assay design, but at the same time they introduce complex variables into the screening system. Although it is difficult to develop precise definitions and standards for biologicals such as cell lines, we propose that the development of guidelines for the nomenclature and use of transfected cell lines is essential for their use in HTS.

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Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2008.104711 ◽

2008 ◽

Vol 54 (10) ◽

pp. 1725-1728 ◽

Cited By ~ 118

Author(s):

X Kate Zhang ◽

Carole S Elbin ◽

Wei-Lien Chuang ◽

Samantha K Cooper ◽

Carla A Marashio ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Enzyme Assay ◽

Internal Standard ◽

Enzyme Reaction ◽

Dried Blood Spots ◽

Krabbe Disease ◽

Molar Ratio ◽

Lysosomal Storage ◽

Blood Spots

Abstract Background: Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories. Methods: We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with a lysosomal storage disorder (LSD) were tested using the modified assay. Results: In our study, the median enzyme activity measured in adults was generally increased 2–3–fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples. Conclusions: The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.

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