Assay of Macromolecules

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the 

TABLE 32.4 Examples of Immunoassays Used to Quantitate Macro molecules 

Eiizyme-Iinked immunosorbent assay Radioimmunoassay Immunoradiometric assay Radioreceptor assay 

is based on antibody recognition of an antigenic epitope (i.e., a molecular region on the surface of a molecule capable of binding to the specific antibody). 

As discussed in Chapter 30 and elsewhere (13), interspecies scaling is based upon allometry (an empirical approach) or physiology. Protein pharmacokinetic parameters such as volume of distribution (Pd), elimination half-life (b/2)/ and elimination clearance (CL) have been scaled across species using the standard allometric equation (14)  

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P. Urios and N. Cittanova, Adaptation of fluorescence polarization immunoassay to the assay of macromolecules, Anal. Biochem. 185, 308-312(1990). 

For each in-study run, the standard curve must satisfy criteria described in the standard-curve section however, run acceptance is based primarily on the performance of the QC samples. When using total error for ligand binding assays of macromolecules, the run acceptance criteria recommended in the precision and accuracy section requires that at least four of six (67%) QC results must be within 30% of their nominal values, with at least 50% of the values for each QC level satisfying the 30% limit. The recommended 4-6-30 rule imposes limits simultaneously on the allowable random error (imprecision) and systematic error (mean bias). If the application of an assay requires a QC target acceptance limit different than the 30% deviation from the nominal value, then prestudy acceptance criteria for precision and accuracy should be adjusted so that the limit for the sum of the interbatch imprecision and absolute mean RE is equal to the revised QC acceptance limit. 

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