Labeled-Ligand Assays

The 25-OH-D Is further metabolized In the kidney to 1,25 dlhydroxycholecalclferol (1,25(OH)2D) which Is considered to be the major physiologically Important, tissue-active metabolite of vitamin D. It circulates In extremely low concentrations (< 100 pg/ml of serum). Assay of 1,25(OH)2D Is extremely tedious. It Is done by competitive binding technique using a combined Intestinal cell cytosol and chromatin binding system, biosynthetic 3h-1,25(OH)2D3 as labeled ligand and synthetic 1,25(0H)2D3 as standard (31). 

A related approach is realized in filter binding assays. Here the reaction solution is filtered, e.g., through nitrocellulose where proteins are absorbed, while small molecules can pass. One example of this technique is the quantification of protein bound and free nucleotides (with radioactive labeled ligands). 

Harris, A., Cox, S., Bums, D., and Norey, C., Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands, /. Biomol. Screen., 8,410,2003. 

Fig. 5.15 Analytical set-up for on-line label-free assay based on ESI-MS. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). PI Carrier/HPLC pump. P2 HPLC pump delivering receptor solution. P3 HPLC pump delivering dissociation solution. PA HPLC pump for final LC-MS analysis of released ligands. 1 Mixing union. 2 Microcoil reactor. VI injection valve.

As binding assays provide a means to characterize the affinity of test compounds to defined targets, they play a very important, not to say an essential role in the drug discovery process. Next to the advantage of effective quantitation, the use of a marker, i.e. a labeled ligand - either with a radioisotope or a fluorophore - has, however, also serious immanent disadvantages. As the performance of mass spectrometry continues to improve, it appears therefore obvious to conduct... 

The key to these assays is to find a suitable method by which the bound labeled ligand can be measured, separate from the unbound fraction. Three separate methods have been used to measure hERG binding filtration through glass fiber filters [36, 37], scintillation proximity assay (SPA) technology [38] and fluorescence... 

Three new assays have been developed that utilize non-labeled ligands and a fluorescent marker. In the first method, 1-aminoanthracene (AMA) is preequilibrated with the PBP and then displaced by more strongly binding ligands, which causes a decrease in AMA fluorescence. One disadvantage of this assay... 

Development of these assays can be difficult. A radio-labeled ligand that binds to the target receptor with high affinity is required, along with sufficient receptor and sufficient radioactivity to cause the SPA bead to signal. These aspects may limit the ability of a specific assay to be miniaturized into a 1536-well plate format. However, for some receptors and ligands, it has proven an effective way to screen. 

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

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