Indicator assay design

Credible pharmaceutical product expiration dates are obtained by rigorous, scientifically designed studies using reliable, meaningful, and specific stability-indicating assays, appropriate statistical concepts, and computers to analyze the resulting data [2]. A comprehensive review of all aspects of pharmaceutical product stability has been published by Lintner [3] and more recently by Connors et al. [4]. 

Aspartate transaminase (AST) or the equivalent serum glutamic oxaloacetic transaminase (SGOT) and alanine transaminase (ALT) or the equivalent serum glutamic pyruvic transaminase (SGPT) are useful indicators of liver conditions such as alcoholic liver disease (Cohen and Kaplan, 1979). A multiplexed, paper-based microfluidic assay designed for quick, semiquantitative measurement of AST and ALT in a hn-ger-stick specimen has been tested clinically (Pollock et al., 2012). Such paper-based techniques have also been supplemented by optical detection (Swanson et al., 2015). Microfluidic channels combined with electrochemical sensors have also used to measure ALT and AST (Song et al., 2009). 

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

Indicator displacement assays (IDAs) - or, in the specific case of fluorescent indicators, F-IDAs - are based on the next alternative concept described here. A receptor with an affinity for a given analyte is loaded with an indicator, usually a fluorescent or colored dye, whose spectral properties undergo a change upon complexation with the receptor. Treatment of this indicator-receptor complex with the analyte results in the displacement of the indicator from the receptor and a restoration of the indicator s original spectral properties, indirectly reporting analyte coordination (Fig. 27). For effective detection, two main requirements have to be fulfilled (1) the receptor/indicator interaction must be reversible and weaker than the interaction of the receptor with the designated analyte and (2) the indicator must show significantly different optical properties when bound to the receptor and when freely dissolved in solution. 

There is also some merit in choosing substrate concentrations that result in incremental increases in v of similar magnitude. O Table 4-2 indicates suitable substrate concentrations, expressed as fractions or multiples of the Km value, leading to consistent increases in y, and this could be used as a template when designing a kinetic assay. 

FIGURE i 6 Diagram indicating that the total variance in the analysis results equals the sum of the method variance and the process variance. A capable measurement system has a method variance that is less than 30% of the design width (difference between upper specification level and lower specification level). The production process is considered to be under full control when the average assay value is centered at Six Sigma values away from the lower and upper specification levels. 

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