Assay selectivity

On the base of alcohol oxidase and flavocytochrome b, the enzymatic kits for selective assay of ethanol, methanol, formaldehyde and L-lactate were developed. 

For drug substances and drug products, applications for enantiomers and racemates should include a stereochemically specific identity test and/or a stereochemically selective assay. The choice of control tests should be based on the method of manufacture and stability characteristics and, in the case of the finished product, its composition. 

Several collaborating laboratories (usually five participating laboratories) test the proposed substance using a variety of techniques. The relative reactivity or relative absorbance of the impurities present in a substance must be checked when a nonspecific assay method is employed, e.g. by colorimetry or ultraviolet spectrophotometry. It is particularly important to quantify the impurities when a selective assay is employed. In such a case, it is best to examine the proposed substance by as many methods as practicable, including, where possible, absolute methods. For acidic and basic substances, titration with alkali or acid is simple but other reactions which are known to be stoichiometric may be used. Phase solubility analysis and differential scanning calorimetry may also be employed in certain cases. 

Summation of the results of the determinations of water, organic solvents, mineral impurities and the organic components amounts to loo %. The results of methods employed to analyze the substance other than these given above are not used for the calibration of the assigned value but are reported to support the results obtained by the defining methods. For most reference substances intended as assay standards the assigned content is normally expressed as is so that it is essential (when establishing the CRS) to determine the content of water and residual solvents for a non-specific assay and also, for a selective assay, to determine the content of impurities. 

However, if a class-selective assay is desirable (for multi-analyte assays), the handle should be located at or near a position that differentiates members of the class and exposes features common to the class. Using the pyrethroid example, an ideal immunogen should retain the phenoxybenzyl moiety and link the protein from the distal acid end (Figure 9). Using such an immunogen hapten, a class-specific immunoassay was developed that was highly cross-reactive with the type I pyrethroids permethrin, phenothrin, resmethrin and bioresmethrin. ... 

Figure 9 Structure of the immunogen hapten used to generate antibodies for a type I pyrethroid class-selective assay. Pyrethroids lacking an a-cyano group are generally termed type I. This hapten exposed the features most common to type I pyrethroids, the phenoxybenzyl group, the cyclopropyl group and the lack of a cyano group, resulting in antibodies that recognized permethrin, phenothrin, resmethrin and bioresmethrin, but not cypermethrin...

A drawback to using class-selective assays or assays with... 

An assay used to determine the relative potency of active or lead compounds towards an alternative target. A selectivity assay (or panel of assays) may include targets of the same family or unrelated targets. 

Chemical Leads Primary Assay IC50 < 1 pM Selectivity Assay 10-fold selectivity Cellular Assay IC50< 1 pM Acceptable in vitro ADME Acceptable PK Profile Potentially patentable... 

Characterized Hits/Series Primary Assay IC50 < 10 pM Selectivity Assay 5-fold selectivity Cellular Assay IC50< 10 pM... 

Additional selectivity assays for a wider range of molecular targets will also be introduced. These could include, for example, a panel of kinases representing the various classes of kinases or a panel of GPCRs. 

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

Drag development is a time-consuming and costly process. Recently, the need of very sensitive and selective assays for the complete characterization of New Chemical Entities (NCE) has become very stringent. 

In this chapter, we describe how the database is built, including com-poimd selection, assay selection, and testing. In Sec. 2, we detail the various... 

The ADR tools in BioPrint assist the user in accessing and implementing the ADR data included in BioPrint. One can quickly access the ADRs listed for a selected compound, the ADRs related to a selected body system, and the ADRs statistically associated with in vitro assay data (ADR associations). The ADR associations can be searched both by assay and by ADR. When searching by assay, ADRs associated with the selected assays and IC50 values are returned along with the risk assessment. The risk information includes the number of compounds that list the ADR, and the number of compounds... 

Affinity selection assays (compound library is applied to a protein target receptor all compounds that do not bind are removed compounds that do bind are then identified)... 

Card, A., Caldwell, C., Min, H., et al. (2009) High-throughput biochemical kinase selectivity assays panel development and screening applications. J Biomol Screen 14(1), 31-42. 

Henderson AD, Richmond W, Elkeles RS (1993) Hepatic and lipoprotein lipases selectively assayed in postheparin plasma. Clin Chem 39 218-223... 

Bogerd, H.P., Fridell, R.A., Benson, R.E. and Cullen, B.R. (1996) Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by an in vivo randomization-selection assay. Mol. Cell. Biol., 16, 4207-A214. 

---