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particular subtype of NPY receptor, such as SMS-KAN cells expressing NPY-Y2 receptors and SKNMC cells expressing NPY-Yj receptor. 125I-NPY binding to membranes by filtration assay is compared with SPA assay using the WGA-PVT SPA bead and NPY agonists (NPY analogs) for competing the binding to NPY-Y2 receptor (Fig. 9). Though the signal-to-noise ratio is higher in a filtration assay than in an SPA assay, the profiles of competition of 125I-NPY binding by NPY analogs were similar, the IC50s obtained were similar, and the rank order of potency for the agonists was the same in both assays. This suggests that the SPA assay can substitute for the conventional radioligand-binding assay; as it is a homogeneous assay, the throughput can be increased, and the assay can also be automated.

Solubilized receptor approaches involving antibodies or biotinylation have also been used. Biotinylation of receptor can be achieved by chemical methods [39] or the enzymatic method [40,41]. In the enzymatic biotinylation, a unique proprietary biotinylation sequence (Avidity, Denver, CO) is engineered at the

Figure 9 Comparison of NPY receptor competition binding assays by (A) filtration and (B) SPA methods. The IC50 values obtained using NPY-Y2 R membranes for various agonists and antagonists were similar in both methods, and the rank order potency of the compounds was the same in both methods, suggesting that the SPA method can substitute for the traditional filter binding assay.

Figure 9 Comparison of NPY receptor competition binding assays by (A) filtration and (B) SPA methods. The IC50 values obtained using NPY-Y2 R membranes for various agonists and antagonists were similar in both methods, and the rank order potency of the compounds was the same in both methods, suggesting that the SPA method can substitute for the traditional filter binding assay.

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