Recombination-based assay

Stults, N. L., et al. (1992). Use of recombinant biotinylated aequorin in microtiter and membrane-based assays Purification of recombinant aequorin from Escherichia coli. Biochemistry 31 1433-1442. 

Functional in vitro assays can be whole-cell assays or animal tissue assays. For in vitro cell-based assays, recombinant molecular targets can be expressed in cell lines and linked to second-messenger systems to measure functional responses. Examples of functional... 

Recombinant adenoviruses are versatile tools for gene delivery into mammalian tissue-culture cells. Adenoviruses can infect both dividing and nondividing cells, with efficiencies approaching 100%. Here we describe the use of adenoviruses to express reporter proteins in high-throughput cell-based assays. 

At present, the use of primary cells in the pharmaceutical industry is not as common as the use of recombinant cell-based assays. A rough estimate shows about 20% of all initial screening is carried out using primary cells and the rate is about the same for secondary screening. The use of primary cells is expected to increase in the next few years as emerging technologies start appearing on the market. 

Since Rluc and GEP recombinantly fused proteins can be expressed in living cells, BRET is an interesting tool for monitoring molecular interactions in cell-based assays. BRET has been particularly used for the study of GPCRs by probing receptor oligomerization or activation. ... 

An interesting and more recent expression system for the development of cell-based assays is based on BacMams. These recombinant baculoviruses containing mammalian cell-active expression cassettes seem to be an efficient strategy to speed up assay development. These viruses are produced in insect cells and transiently express but do not replicate in transduced mammalian cells. The expression level can be well controlled by titrating the amount of virus. Highly reproducible transient expression levels, which are a prerequisite to use transient transfection for HTS, might thus be an attractive alternative for some approaches. 

Another much used yeast-based assay is the yEGFP assay (first described by Bovee et al., 2004). In this assay recombinant yeast cells are constructed that express the human estrogen reporter a (ER a) and yeast enhanced green fluorescence protein), the estrogenic potential is then measured by a Fluorometer. 

As target resolution increases, the mechanism of action in drug discovery has become much more complex. Assay development methods have used molecular biology techniques to keep pace. Cells expressing recombinant proteins can act as biosensors for real-time analysis of target inhibition in the cell instead of relying on traditional cytotoxicity. These target-, rather than phenotypic-, based assays permit... 

This has been issued to describe analysis of the expression construct in eukaryotic and prokaryotic cells used for the production of r-DNA-derived protein products [29]. Both the expression construct and the purified final recombinant protein should be characterized. It should thereby be verified that the correct coding sequence has been incorporated and maintained during culture. A master cell bank (MCB) and a working cell bank (WCB) should be considered. Nucleic acid analyses, PCR-based assays and DNA-sequencing are methods to be used. A consistent quality of the final product should be guaranteed. 

Ligand-binding assay - hke RIA, ELISA and other types of competitive antigen -antibody-related methods - are broadly apphed to detect and quantify macromolecules in biomatrices. In addition, ceU-based assays and antibody titer determinations might be additional quantification methods for macromolecules. Several of these methods have already been used and established in clinical chemistry to determine levels of endogenous substrates in the past. Thus, it is a direct approach to use similar methods in the case of measurement of for example, recombinant analogues and other suitable biopharmaceuticals. 

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