Assay format, biomarker assays

The aim of this section is to cover approaches to assay selection, to provide an overview of the possible assay formats and to give a description of the most frequently used assay formats for potency determinations. Bioassays are developed and used for different purposes during drug discovery and development. Even though they can have the same assay format they have a different purpose in a product development cycle starting from target selection to clinical biomarkers. 

The other limitation of the CFU assay in either format is that it remains an in vitro environment where distant interactions, like liver metabolism (either detoxifying the compounds or producing toxic metabolites) or immunomodulation, are absent. Investigating the meciianism of toxicity in animals for some carefully chosen compounds after being ciiaracTerized in the two previous assays, can provide valuable information for the whole series, allow further refinement of the in vitro assays (e.g., addition of S9 for metabolism) and give an early indication of which biomarkers could be used in later GLP studies. 

The high FRET efficiency obtained in HTRF assays enables a large variety of biological events to be probed. Protein/protein interactions, enzymatic activities (e.g. kinases or proteases), or a large number of biomarkers have been detected with a very low detection limit. Like the other homogeneous fluorescent technologies like FP or FCS, HTRF can be easily automated and miniaturized down to a 1536 weUs plate format for the HTS of large libraries of chemical compounds. ... 

Antibodies to OP-tyrosine will be made. These antibodies will be used to diagnose OP exposure in a biosensor assay with saliva, sweat, or urine. New biomarkers of OP exposure will be identified using mass spectrometry and the new OP-tyrosine antibodies. The identification of new biomarkers for low-dose OP exposme is expected to lead to an understanding of how neurotoxicity is caused by OP doses that are too low to inhibit acetylcholinesterase. For example, it is possible that disruption of microtubule polymerization by OP-adduct formation may explain cognitive impairment from OP exposure. 

One approach to determination of whole-body lipid peroxidation has been measurement of exhaled hydrocarbons by GLC, especially ethane. Hydrocarbon gases are, however, minor end-products of peroxidation and their formation depends on the decomposition of peroxide. Recent studies have demonstrated that isoprostane is a good biomarker of lipid peroxidation in the human body. Isoprostanes are specific products arising from the peroxidation of unsaturated fatty acid residues in lipids and detection of them and their metabolites in urine is a useful assay of whole-body lipid peroxidation. Isoprostanes can be accurately and sensitively measured by mass spec-trometric techniques. 

Several of the papers presented demonstrate that the same antibody can be used in a variety of different formats (31-321. This characteristic will become increasingly important. Certainly the same assay can be used both for analysis of environmental samples and in the analysis of human body fluids as a biomarker approach (29.351 in the latter application immunoassay offers numerous advantages. 

During the last decade, commercially available kits have made available for plasma/serum and urinary biomarkers of bone formation and bone resorption. Some of the assays have been shown to be applicable in rats and dogs, but not all assays work across the species, and there is inherent variability with these markers. Some of the tests are listed in Table 6.6, and a number of references are provided. These tests will have a place in animal models, and they may be useful efficacy markers in the development of novel therapeutic agents. 

Despite the investment in proteomics, the introduction of new protein tests in human medicine has not dramatically increased over the last decade and, to some extent, has been disappointing (Pognan 2004) except in animal clinical chemistry, where the number of available assays has improved due to availability of suitable antisera and commercial investment. If key proteins, which are highly diagnostic, are identihed by proteomics, then commercially available reagent kits in suitable formats for liquid chemistry analyzers, enzyme-linked immunosorbent assays, or chip technology are required to ensure wider application of these potential biomarkers. 

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