Solid-phase Platform Research Articles

Development of a scintiplate assay for recombinant human α 2B-adrenergic receptor

A high-throughput solid-phase platform for ligand-binding assays using microtiter plates (Scintiplates) has been developed using the scintillation proximity assay principle. The system has been developed using human α 2B-adrenergic receptor ( α 2 B -AR) expressed from Semliki Forest virus vectors in CHO cells. α 2B-AR bind natural (adrenaline and noradrenaline) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Antagonist radioligands were used for the binding experiments, and the values obtained for the binding constants with the Scintiplate system are in good agreement with those obtained by the traditional filter-binding assay system. The Scintiplate assay offers the advantages of a high-throughput format over the filter-binding assay and is amenable for screening many compounds rapidly for generation of leads.

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).