Reagent Assays

While we recommend the application of control cell fines as reagent, assay, and EQA monitoring tools, it is important to emphasize that appropriate tissue controls are also continued to be used in parallel, as tissue is still considered the gold standard in laboratory assay control. For reliable results, it is also important that tissue or cell line controls are fixed and processed in the same manner as diagnostic material submitted for evaluation. 

CIEEL is of particular interest for the development of modern chemiluminescent bioassays. The most popular clinical bioassays utilize thermally persistent spiro-adamantyl-substituted dioxetanes with a protected phenolate moiety. These designed 1,2-dioxetanes include an energy source, a fluorophore, and a trigger grouping, and are therefore structurally similar to bioluminescent substrates such as firefly luciferin. Three main commercial dioxetanes 75 are available as one-reagent assays for alkaline phosphatase and are sold under the name of AMPPD (R1 = R2 = H), CSPD (R1 = Cl, R2 = H), and CDP-Star (R1 = R2 = Cl) <2006S1781, 2003ANA279>. These substrates are sensitive to 10 21 mol of alkaline phosphatase in solution. 

Inclusion of controls for reagents, assay parameters and non-specific binding. 

IAs are classified by several groupings. If the antigen-antibody complex is separated from the unbound reactants before quantitation, it is referred to as a heterogeneous assay. For homogeneous assays, the detection of the bound complexes can be differentiated from the unbound reactants, and no separation process is needed. Another classification is competitive and noncompetitive IA. The measure of occupied or unoccupied Ab determines whether an IA is competitive or noncompetitive, not whether the label is on the Ag or Ab. A broader classification is limited-reagent assay and excess-reagent assay (7,8). They will be discussed in more detail. 

Many conventional RIAs follow limited-reagent assay protocols. The following scheme depicts the AgAb reaction ... 

Table 1 Limited Versus Excess-Reagent Assays...

Bound fraction is separated from free the signal [AgAb ] or [Abl-Ag-Ab2 ] complex (the Ab fraction occupied by the analyte) is measured. The amount of analyte is proportional to the bound complex in a hyperbolic function. Methods similar to those used for transforming or linearizing limited-reagent assays can be applied to these excess-reagent functions. 

Generally, polyclonal antibodies are easier to produce, and high-affinity polyclonal antibodies can be obtained. Monoclonal antibodies are more specific to a certain epitope. They provide continuous production of exactly the same defined reagent and are more preferable for excess-reagent assays. The double sandwich technique has used two antibodies from monoclonals or combinations of mono- and polyclonals, with specificity against two different epitopes of the analyte. One antibody functions as a capturing antibody for the analyte and the other as the label carrier (118). 

Fig. 3 Double-antibody sandwich technique. The capturing antibody (Abl) is immobilized onto the solid phase. The analyte from the sample is captured by forming Abl-Ag immunocomplex. After washing off the extraneous materials from the sample, the reporting antibody (Ab2, which has an enzyme label in this illustration) is introduced. The double-antibody sandwich is formed Abl-Ag-Ab2E. Compounds not recognized by both Abl and Ab2 will be washed away. A chromogenic substrate is added to produce the product for detection. The occupied Ab2 by the Ag is measured this is an excess-reagent assay. As the sample analyte concentration increases, the signal responses increases proportionally.

The double-antibody sandwich technique is applicable to large molecules. Small analytes have difficulty forming the double-antibody sandwich immunocomplex. The smallest analytes reported that have been used in a double sandwich assay are peptides of around 10 amino acid residues (158). The double-antibody sandwich technique measures the occupied antibodies using an excess-reagent assay protocol. A limited-reagent assay protocol can also be designed, as shown in Fig. 4. In this format, the antibodies are immobilized onto the solid phase,... 

Fig. 4 Antibody immobilization in a limited-reagent assay format. The antibody is immobilized onto the solid-phase support. Labeled antigen and sample are introduced, and they compete with one another to form immunocomplex (Ab-Ag or Ab-AgE) with the limited antibody sites on the solid-phase support. After washing, the substrate is added to produce the detecting product. The antibody unoccupied by the sample analyte is measured as the concentration increases, the signal responses decreases.

Acceptance criteria for accuracy and precision of standards and QCs must be determined during method validation, and are analogous to acceptance criteria for chromatographic methods. IAs may not be as inherently precise as chemical methods, because IAs measure a reaction rather than a physicochemical property of the analyte. In cases where internal standards are not used for recovery correction, two to three replicate assays may be conducted on a single sample to improve precision. Despite all of the available mathematical transformations, it is important to remember that this is not a linear system and caution must be used as the concentrations approach either the upper or lower end of the standard curve. For example, variability becomes too large to be acceptable as the B/B0 value goes beyond <0.1 or >0.9 for most limited reagent assays. 

Pyridoxal phosphate is a cofactor for both of the aspartate and alanine aminotransferases (AST ALT) and is often supplied as a separate component of reagent assay kits. The inclusion of pyridoxal phosphate can increase the measured activities of both aminotransferases activity by a small percentage (Stokol and Erb 1998), but this also can alter statistical differences observed between treatment groups in rat studies (Evans and Whitehorn 1995). 

The concentration of a protein can be determined by various colorimetric methods including the old standard Folin—phenol reagent assay of Lowry (79) or the new standard Coomassie blue R-250 method of Bradford (80) or bicin-choninic acid (BCA) assay. Silver stain (108) and gold stain are also widely used to identify and quantitate proteins with a 10-50 times greater sensitivity than Coomassie blue staining. Once a reliable estimate of the protein concentration is obtained, the UV absorptivity of the known protein can be used to determine unknown concentrations of the same protein. From the Beer—Lambert law, A(x) = o% lJI) = where is the absorbance (or optical density) at the... 

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