Ligand binding assay specificity

Branham et al. [11] used an ER-ligand binding assay to measure the RBA for 46 chemicals considered to be potential phytoestrogens and mycoestrogens. The classes of chemicals examined (flavones, isoflavones, flavanones, coumarins, chalcones, and mycoestrogens) are shown in Figure 18.8. Representative chemicals from each of these classes showed some affinity for ER, ranging widely in RBAs from 43 to 0.00008 E2 = 100). No further work was done to describe specific SARs for these classes. 

Note that controversies over the technical and clinical validation of immunohistochemistry have not been completely resolved. Whether or not this method should completely replace biochemical ligand-binding assays remains controversial. Despite this cautionary statement, it is true that the specificity of immunohistochemistry is theoretically valid because it is based on the use of well-characterized monoclonal antibodies raised against epitopes restricted to the ERs. 

Allegretto, E. Detection of RARs and RXRs in cells and tissues using specific ligand-binding assays and ligand-binding immunoprecipitation techniques. In Retinoid Protocols, edited by C.P.F. Redfern, Totowa, NJ, Humana Press, pp. 219-232, 1998. 

Kll. Korenman, S. G., Radio-ligand binding assay of specific estrogens using a soluble uterine macromolecule. J. Clin. Endocrinol. 28, 127-130 (1968). 

Findlay, J.W.A. (2009) Specificity and accuracy data for ligand binding assays for macromolecules should be interpreted with caution. The AAPS Journal, 10, 433 434. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

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