Bioassays protein binding assays

In laboratories which use an NSILA bioassay, however, somatomedin activity has been expressed in units of insulinlike activity, generally p,U/ml, a convention which has to some extent continued as radioligand assays were introduced. Expressed in these units, normal human samples have a mean value of 350 66 pU/ml by competitive protein-binding assay (Z4). In the same laboratory, a mean normal value ( SD) of 148 45 ng/ml was determined by IGF-I RIA (Z5). The close correspondence between reference values obtained by RIA in different laboratories, when expressed as ng/ml of SM-C or IGF-I, suggests that mass units, if uniformly adopted, would allow better comparison of results. With the increasing availability of highly pu-... 

Shaw, W., Slade, B. A., Harrison, J. W., and Nino, V., Assay of serum folate Difference in serum folate values obtained by L. casei bioassay and competitive protein binding assay. Clin. Biochem. (CHtawa) 7, 165-178 (1973). 

Purity for a small molecule is a relatively simple concept. Normally, an HPLC method is sufficient to measure the content and impurity levels of a small molecule drug. A macromolecule, such as a protein, has a much more complex behavior. Determining protein concentration by UV absorption spectroscopy can give a measure of the total protein in the product, but it will not necessarily differentiate between active protein and inactive protein (i.e., denatured or otherwise degraded). A validated method or methods to determine the biological activity of the molecule is needed. So, whereas protein concentration is usually tested as part of the specifications, it is also normally accompanied by one or more methods that measure or correlate to biological activity. This is the bioassay. These methods can be animal-based or cell-based, protein interaction assays, binding methods such as surface plas-mon resonance or ELISA (enzyme-linked immunosorbent assay) and immunoblot methods. 

The ELISA is a solid surface binding method widely used for the detection, quantification, and determination of the binding characteristics of different proteins and other biological molecules. ELISA methods can be used as a potency assay in cases when the function of a therapeutic protein is only to bind a target molecule in vivo or as a second release assay in addition to the functional potency bioassay to evaluate ability of the therapeutic protein to bind to a designated target. Binding assays are often used as a characterization assay in structure-function studies. 

ACTH has been measured by bioassay, receptor assay, or immunoassay. Most bioassays are based on the effects of ACTH on adrenocortical cells (e.g, the stimulation of steroidogenesis or depletion of ascorbic acid). Responses are usually observed in the adrenal glands of hypophysec-tomized animals in situ or in isolated adrenal cells in vivo. In genera these bio assays are expensive and difficult to perform and are Hmited to research laboratories. Receptor assays use solubilized binding proteins obtained from normal or neoplastic adrenocortical ceUs. Only biologically active ACTH is measured, and a 10-pg/mL detection limit has been reported in unextracted plasma. These assays have limited application in the clinical laboratory partly because of the complexity and lability of receptor preparations. 

Bioassays are influenced by protein-reactive compounds [34] and tolerate only limited numbers of non-related molecules. False-positive and false-negative results from screening of compound mixtures have been reported. For example, octa- and hexapeptide mixtures significantly depress binding of the hormone neuropeptide Y to its receptor when measured in a standard competition assay [35]. 

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