Ligand binding assay defined

This chapter describes the challenges and practical approaches to ligand-binding assays for biomarker quantification. The main points include (1) defining the purpose of the bioanalytical method application, (2) assay development using the appropriate reagents, (3) assay qualification and validation meeting the intended purpose of an assay, and (4) statistical treatments and interpretation. 

Due to the method principle, ligand-binding assays are inherently non-linear. Thus, four- and five-parameter mathematic models are used to create calibration curves, and consequently a higher number of calibration points is needed to define the curve most accurately. Especially in the asymptotic parts of the calibration curve, a sufficient number of calibrators must be placed to define upper and lower limits of quantification with pre-defined accuracy and precision. Unless it is shown that matrix constituents have no impact on detection signals, calibration curves must be prepared in an authentic matrix. 

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... 

To determine whether pharmacological differences are dictated by differences in nonpolar accessible surfaces of the targets within ligand-binding sites, a nonpolar distance, dnp (i,j), between the affinity-assayed kinases i, j is introduced. The dnp (i,j) is determined by differences in accessible nonpolar surface areas of the respective nonpolar hulls, Hnp ( ), Hnp (j). As rigorously defined in the previous... 

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