Assay, formats

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

In general, there are advantages and disadvantages to both assay formats and both are widely employed in pharmacological research. The specific strengths and weaknesses inherent in both approaches are discussed in more detail in Chapters 4 and 5. As a preface to the consideration of these two major formats, a potential issue with both of them should be considered namely, dissimulations between the concentrations of drugs added to the experimentally accessible receptor compartment and the actual concentration producing the effect. 

There are a number of assay formats available to test drugs in a functional mode. As discussed in Chapter 2, a main theme throughout the various stimulus-response cascades found in cells is the amplification of receptor stimulus occurring as a function of the distance, in biochemical steps and reactions, away from the initial receptor event. Specifically, the further down the stimulus-... 

HTS is usually carried out it multiwell plates and the industry has settled on certain standard formats. One key standard is that most screening assays are carried out in 96 (8 12) or 384 (16 24) well plates with a standard footprint. This standard is important because most automation is optimized for use with these plates and attendant assay volumes in the 100 pL range. Furthermore, source plates containing test compound are typically stored in a similar configuration, allowing for more efficient transfer of test compound from the source plate to the assay plate. Various types of plates are available to match different assay formats, including... 

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay format. 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

The first step in the process involves the development of a suitable HTS assay format. A more detailed discussion of the formats available for HTS by target class are outlined later in this chapter. This section will focus on some of the practical considerations to determine the proper assay technology for an HTS campaign. 

The range of possible assay formats varies between organizations, but a list of the parameters which should be addressed include ... 

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