Assay robustness

The use of QSAR descriptors for prediction and understanding of the activity of antimicrobial peptides has previously been limited to comparisons between peptides that differ in only a small number of amino acids. This has been primarily due to the cost and difficulty of producing large numbers of peptides as well as the cost of assaying their activity. However, with the recent advance in high-throughput peptide synthesis technique in combination with rapid assay of activity with the luminescence-based assay, robust amounts of data have begun to be available (35). 

Currently available activity assays measuring ENPPl activity require a substantial amount of enzyme [7], special instrumentations [2, 7], or radioactive materials [8]. Some of them utilize nitrophenyl-based substrates [8, 9], which are useful to study the enzyme reaction, but do not provide sufficient assay sensitivity or relevance to physiology. Here, we describe two new ENPPl activity assays that use two biologically relevant substrates, ATP and ADP easy stepwise procednres lead through the development and optimization of these assays. The latter is an important step for adopting the assay for every new batch of the ENPPl enzyme produced in-house or purchased commercially to ensure assays robustness and sensitivity. 

Incubation time, if shortened, may have an effect on the assay range and may make the assay more likely to suffer from drift. Incubation times, if increased, may help with assay drift and improve the assay range. However, increasing the overall assay time can decrease the potential sample throughput. Incubation temperatures may affect the assay range and the assays robustness. 

After cell line identification, extensive optimization of assay conditions is often necessary to maximize the biological response. Multiple assay end points, sample incubation times, cell seeding density, cell viability, and the need for cell culture conditions, such as culture media, supplements, passaging, and fetal calf serum, are all conditions that may need to be assessed. For the anti-CD40 therapeutic Nab bioassay, Baltrukonis etal. [23] found assay robustness was significantly impacted by the source of culture media and lots of fetal bovine serum. 

The effect of excessive fecal bile acids may be an unappreciated cause of chronic diarrhea in several clinical settings (Porter et al. 2003). BUe acids are minimally absorbed in the proximal small intestine, and the bUe acid pool flows to the distal ileum, where the acids are reabsorbed by the enterohepatic transport system and then returned to the liver by the portal vein (John-Baptiste et al. 2012 Westergaard 2007). Remaining bUe acids are excreted in feces. As a result, increases in fecal bile acids are indicative of malabsorption, which can cause diarrhea, but which stiU requires further validation due to lack of assay robustness. 

For high-throughput screens, 2 prime statistical analysis can provide an indication of overall assay robustness based on the average and standard deviation of a positive and negative control. Z-prime values above 0.5 indicate a robust and acceptable assay. 

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. 

ATP-STD NMR is described as a simple, cost-effective and robust assay that can be used to identify inhibitors of virtually any nucleotide-binding target. 

Successful management of a central HTS operation requires a discipline of process control that extends beyond the automation systems. The progression of HTS projects must be carefully guided and tracked through several milestones designed to ensure that a steady and predictable stream of robust assays are available for transfer to the automation platforms and that sufficient bulk reagents and consumable supplies will arrive in time to support the HTS campaigns. 

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

An assay or screen is said to exhibit ROBUSTNESS when it has a high discriminatory power and produces a low number of FALSE NEGATIVE and FALSE POSITIVE results. 

For the detection of slow-acting biological agents (which may not produce symptoms for several days), the system response time would depend on the frequency of sampling and analysis. The frequency of sampling and analysis would be determined by factors such as the cost of the assay, the frequency with which critical reagents need to be replaced, the robustness of the detector, and so on. The minimum response time would be determined by the time required to collect a sample, prepare it for analysis, conduct the assay, and report the results. In the event of an alarm from a detector with a significant false-alarm rate, additional time would be required to determine its validity and to decide on an appropriate response. 

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