Assays secondary

CRC, Counterscreen, Orthogonal Assay, Secondary Assay IC50... 

Because the heavy ethyleneamines are very complex materials, assays by titration in aqueous and nonaqueous media are often performed (151). The result is usually expressed as an amine number or amine value, a measure of the total basic nitrogen content of the product. Titrimetric procedures are also available to define primary, secondary, and tertiary amine content (152). 

Schild analysis, like all pharmacological tools, necessarily is predicated on the idea that the drugs involved have one and only one pharmacological activity. This often may not be the case and selectivity is only a function of concentration. If the concentrations used in the assay are below those that have secondary effects, then the tool will furnish the parameter of interest with no obfuscation. However, if a secondary effects are operative in the concentration range required to measure antagonism then the resulting parameter may be tainted by this secondary activity. 

Some biomarkers only provide a measure of exposure others also provide a measure of toxic effect. Biomarkers of the latter kind are of particular interest and importance and will be referred to as mechanistic biomarkers in the present text. Some mechanistic biomarker assays directly measure effects at the site of action as described in Section 2.4 (see Chapter 4, Table 4.2, for examples). Inhibition of acetylcholinesterase is one example. Others measure secondary effects on the operation of nerves or the endocrine system (examples given in Table 4.2 and Chapters 15 and 16). 

The procedure employed for the establishment of the chemical reference substances used in these assays has been previously published (Sandrin et al. 1997). The CRSs for the microbiological assays of antibiotics are first submitted to the chemical tests of the monograph. If the results are satisfactory, a collaborative microbiological assay is carried out, using the International Standard as calibrator. Thus, these reference substances are considered to be secondary reference substances since they are calibrated against existing standards. Potency is expressed in International Units. If an International Standard does not exist, European Pharmacopoeia Units are used. 

Primer design is one of the most important aspects of a robust PCR assay. In general, primers should be designed such that they are not able to form secondary structures such as stemloop or hairpin configurations. A primer must not be complementary at the 3 end, as this will cause primer dimers to form. All primers should have similar melting temperatures and should not contain stretches of individual nucleotides. There are software programs available to assist in primer design, but it is crucial that primers are tested in the assay, especially in a multiplex system. 

The layout of samples and controls configured on a plate during an assay. For example, for a primary screen in 384-well plates, columns 1, 2, 23, and 24 are controls, and columns 3-22 are for individual test compounds, whereas for secondary screening, each row will contain a single compound at varying concentrations. 

A screen applied to confirm independently actives from the primary screen. A secondary screen may employ an assay that differs in type from the primary screen, e.g., biochemical assay vs cell based assay, or it may be of the same type with different readout. 

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

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