Translatability assay selection

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

The mDHFR protein complementation assay has been used to map a signal transduction network that controls the initiation of translation in eukaryotes (Remy and Michnick, 2001). A total of 35 different pairs of full-length proteins were analyzed and 14 interactions were identified using the survival selection of cells grown in the absence of nucleotides. In addition, the use of the fMTX reagent in combination with fluorescence microscopy was used to localize the protein complex within cells (Remy and Michnick, 2001). 

The voluminous number of publications in this area over the last 10 years have exceeded 10,000, thus attesting to the intensity of effort directed towards applying molecular methods towards investigation of infectious diseases. Many of these investigations have translated into applications for diagnostic use in the clinical laboratory. The reader is referred to selected reviews on the subject (F2, N3, Wl). The availability of assays for the rapid diagnosis of pulmonary tuber-... 

In summary LC-MS/MS has doubtlessly a high inherent potential for selectivity and accuracy. However, application of this technology is not automatically or necessarily translated into accurate results. Its pitfalls have to be recognized and must be addressed systematically. In particular interferences from in-source transformation of metabolites, differential matrix effects of analyte and internal standard and isobaric transitions can lead to inaccurate results of LC-MS/MS analyses. Further technological developments will probably help to make LC-MS/MS assays more robust towards such interferences, but clinical chemists have to remain watchful for inaccuracies also with powerful and fascinating technologies... 

However, some receptors are constitutively expressed in the nucleus and this type of receptor would not be amenable to a nuclear translocation assay. The activities of nuclear receptors may be dependent upon complex interactions with a number of coregulatory proteins, commonly known as coactivators or corepressors, and modifications by post-translational means. Cell type-specific expression levels of receptors and coregulators may contribute to some, but not all, of the molecular bases for gene and functional selectivity of receptor activity. Therefore selecting a cell line that expresses both the target receptor and the necessary cofactors may be required to design an appropriate assay. 

In contrast, with its intrinsically higher selectivity, tandem mass spectrometry offers a much more effective and practical approach for multicomponent quantitation. This translates into faster method development and, ultimately, rapid and reliable multicomponent quantitation. The routine application of multicomponent quantitation to 5 or 10 compounds in a cassette can be developed in 1 or 2 days, offering typical quantitation range of 5 to 5000 ng/mL. The variability of the assay procedure ranges from 10 to 20%, which is less than the typical intersubject pharmacokinetic variability of 25 to 30%. 

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