Ligand binding assays

It should be noted that receptor affinity does not always correlate to downstream response, for example, a compound may bind to the receptor as an antagonist 

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The methods now used to measure 25-OH-D are competitive protein-ligand binding assays that use either serum globulin (diluted rat serum) (27)28) or a vitamin D-deficient rat kidney... 

The basic clinical tool used at the present time Is the competitive ligand binding assay for 25-OH-D. Although concentrations are low In the serum of patients with osteomalacia and v . tamln D deficiency rickets, we have recently noted the Interesting paradox that levels can be only 1/2 normal In the face of oyert bone disease (32). This had led us to propose that substrate levels of 25-OH-D3 available to the hydroxylase In kidney which Is responsible for the conversion of 25-OH-D3 to the tissue active metabolite, l,25(OH)2D3, may be rate limiting for this enzyme. 

Techniques that measure mRNA expression and ligand binding assays that measure receptor expression have been used extensively to identify those nAChR subtypes that are expressed in dopamine neurons. In situ hybridization studies using mouse (Marks et al. 1992 Grady et al. 1997) and rat (Le Novere et al. 1996) brain have detected the mRNAs for all of the known nAChR subunits, except a2 and p4, in... 

Continuous-flow Ligand Binding Assays Based on Mass Spectrometry... 

S. A. Cell-free ligand binding assays for nuclear receptors. Methods Enzymol. 2003, 364, 53-71. 

Receptor-Ligand Binding Assay Drug Discovery ... 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

As with chromatographic methods, microbiological and ligand-binding assays should be shown to be selective for the analyte. The following recommendations for dealing with two selectivity issues should be considered ... 

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. 

Further radiolabeled ligand-binding assays have been reported for a wide variety of analytes, namely morphine and leu-enkephaline [30], (S )-propanolol [33], atra-zine [47], 17-jS-estradiol [59], 2,4-dichlorophenoxyacetic acid (2,4-D) [32], yohimbine and corynanthine [60], and corticosterone/cortisol [29]. Other assays are summarized in Table 3. 

COMPARISON OF IMMUNOHISTOCHEMISTRY WITH BIOCHEMICAL LIGAND-BINDING ASSAYS... 

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. 

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