Competitive ligand binding assays

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. 

Application of molecularly imprinted polymers in competitive ligand binding assays for analysis of biological samples... 

Andersson, L.I. Application of molecularly imprinted polymers in competitive ligand binding assays for analysis of biological samples. In Molecularly Imprinted Polymers Man-made Mimics of Antibodies and Their Applications in Analytical Chemistry, Sellergren, B., Ed. Elsevier Amsterdam, 2001 341-354. 

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... 

This equation illustrates the components of a competitive protein binding assay system. That is, the reaction system contains both radioactive and non-radioactive free ligand (P and P) and both radioactive and non-radioactive protein bound ligand (P Q and PQ). This type of assay assumes that binding protein will have the same affinity for the labeled or non-labeled material that is being measured. Although this assumption is not always completely valid, it usually causes no problems of consequence with most radioassays or radioimmunoassays. 

Fig. 5.10 Principle of competitive ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). The extent of complex forming is monitored by the addition of a bioactive reporter ligand (R) resulting in the formation of protein-reporter complex (PR). The concentration free R is directly dependent on the concentration and affinity of L R is monitored by ESI-MS at its corresponding m/z trace.

To this end, the pellets remaining from the competitive MS binding assay were, after several washing steps, resuspended in binding buffer and incubated with a great excess of competitor (50 pM (+)-methadone) to liberate the unknown bound ligand (as well as the bound marker). Then the supernatants obtained by centrifugation were analyzed by LC-ESl-MS/MS. In addition to morphine as the marker, naloxone was identified as the hit that had been searched for. Thereby, the relative concentrations of marker (2.93 nM) and hit (2.30 nM) pointed to the fact that the hit had a similar affinity to the //-opioid receptor as the marker [65]. 

Charcoal is widely used to separate antibody-bound ligand (bound) from non-antibody-bound ligand (free) in competitive protein binding assays. Its use was originally described by Miller for vitamin Bi assay and later by Herbert et al. for B12. In 1965, Herbert suggested its use to separate bound and free in the radioimmunoassay of insulin. Since then it has been used for a large number of radioimmunoassays and radioreceptor assays. 

TABLE 3.2 Typical Features of Competitive and Noncompetitive Ligand-Binding Assays... 

Ligand-binding assay - hke RIA, ELISA and other types of competitive antigen -antibody-related methods - are broadly apphed to detect and quantify macromolecules in biomatrices. In addition, ceU-based assays and antibody titer determinations might be additional quantification methods for macromolecules. Several of these methods have already been used and established in clinical chemistry to determine levels of endogenous substrates in the past. Thus, it is a direct approach to use similar methods in the case of measurement of for example, recombinant analogues and other suitable biopharmaceuticals. 

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