Endpoint assay

The use of Ct values also expands the dynamic range of quantitation because data are collected for every PCR cycle. A linear relationship between Ct and initial DNA amount has been demonstrated over flve orders of magnitude, compared with the one or two orders of magnitude typically observed with an endpoint assay. 

Also, the test procedure (protocol) is fundamental because it allows comparing results from different laboratories and from different experimental sets. Moreover, selected test protocol could affect the interpretation of the results, the information content and its application in the safety evaluation process, as stated by Frazer if the biological system is exposed to a test chemical for 24 h and the endpoint assay is immediately conducted, the data produced would be most relevant to the acute toxicity of the test material. If, on the other hand, the system is exposed to material for 24 h and the system is cultured in the absence of the test material for additional 48 h before the endpoint assay is conducted, the data would be more relevant to recovery from toxicity rather than acute toxicity [7]. 

Second, there are specific endpoint assays for which an adverse outcome clearly dictates the action to be taken. These endpoints include either immediate or delayed hypersensitivity reactions, because once the individual is sensitized a dose-response relationship may not apply. 

Endpoint assays such as proliferation or cytotoxicity assays are routinely used for functional assessments. For these assessments, primary cells, transformed cells, or cells transfected with the target receptor are exposed to range of concentrations of the test article. Proliferation or cytoxicity is then measured using a variety of methods such as crystal violet vital dye staining, MTT/MTS incorporation, or a luminescence readout like ATP lite. In addition, assays that analyze phosphorylation of specific transcription factors, or release of specific cytokines and chemokines, are also common. Figure 9.5 illustrates the measure of functional consequences of receptor-test article interaction by quantifying cytokine release. Cells from the species under evaluation were cultured in the presence of serial dilutions of the test article or control reagents, and supernatants harvested for determination of cytokine levels by ELISA (i.e.,... 

Assay A laboratory test to identify and/or measure the amount of a particular substance in a sample. Types of assays include endpoint assays, in which a single measurement is made at a fixed time kinetic assays, in which increasing amounts of a product are... 

Fixed-time assays include the so-called endpoint assays, and measure the change in [S] or [P] that occurs over a fixed, relatively long, period of time. They rely on quantitative (or near-quantitative) conversion of substrate to product, and are used exclusively for substrate quantitation. A linear calibration curve of signal change against initial substrate concentration is used to quantitate substrate. Fixed-time assays do not require [S]0 <0.1 Km, since essentially complete conversion... 

A commercial endpoint assay for total serum cholesterol9 employs three enzymes, cholesterol esterase, cholesterol oxidase, and peroxidase ... 

Manometry. Simple manometric methods may be used in endpoint assays, to measure the total quantity of a gas (such as NH3 or CO2) produced in an enzymatic reaction. Automated manometers may be used in a kinetic mode, to quantitate gas evolution as a function of time. These methods are based on the ideal gas law, which states that the volume occupied by a gas is directly proportional to the number of moles of the gas at constant pressure and temperature. The gas volume is thus measured as a function of time or following completion... 

The oxidized form of ABTS exhibits an absorbance maximum at 420 nm, with a molar absorptivity of 3.48 x 104A7 em The AG assay was developed as an endpoint assay, relying on the complete conversion of substrate to product over a 1-h period at 37 °C and pH 5.9, the optimum pH for the complete system. The specific activity of PROD at this pH is 2.11 I.U./mg. 

Real-time PCR is a simpler and more powerful approach to quantification than endpoint assays. The reaction is monitored each cycle, and the profiles of the curves are used to calculate initial target concentrations. Real-time PCR is described in further detail in later sections of this chapter. 

All of the assays mentioned can be set up in an automated, medium-throughput system however, all require a specific assay to measure the compound concentration at the end of the assay, which places significant limitations on throughput. There have been recent attempts to solve the throughput problems inherent in this type of assay by developing generic endpoint assays for metabolic stability, but there has been no clear solution to date. 

In general, the Factor VII activity estimated by a substrate assay correlated well with Factor VII activity determined by a clot-endpoint assay (P4, S13). However, some differences have been seen. Activation of Factor VII by kaolin or by exposure to cold temperatures (2-8°C) increased the level of clotting Factor VII by 4-7 times but bad no effect on the amount detected by the substrate assay (S9, TIO). It has been postulated that the partially carbox-ylated molecules of Factor VII present in patients plasma during anticoagulant therapy are detected by a substrate assay but not in a clot-endpoint test (P4). These differences between the two assay systems have been used to screen for hereditary Factor VII deficients and to detect the presence of Factor Vila in thrombotic disorders and in components for transfusion therapy (S13). 

Kirsch-Volders, M., Elhajouji, A., Cundari, E., and Van Hummelen, P. (1997). The in vitro micronucleus test A multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat Res 392, 19-30. 

A SINGLE-STEP BIOLUMINESCENT ENDPOINT ASSAY FOR NUCLEIC... 

Here we demonstrate an alternative to multi-step approaches that use ELIDA for endpoint assays of PCR reactions," We show that by optimising PCR reagent formulations to work with d-a-S-ATP instead of dATP, ELIDA reagent can be directly mixed with the resulting PCR reaction giving quantitative results in a one-step assay. 

Gandelman O, Church V, Murray JAH, Tisi LC. A single-step bioluminescent endpoint assay for nucleic acid amplification. This volume... 

The enzyme was stable for 10 min up to 45°C, whereas the activity decreased rapidly to 30% after 10 min at 55°C (Fig. 2). The temperature maximum (Tmax) was found to he 47°C under the conditions tested (30 min endpoint assays), whereupon activity rapidly diminished, possibly because of enzyme thermal instability. 

Spectrophotometry is the mainstay of the main chemical analyzers. However, stand-alone machines are occasionally used to measure colorimetric and endpoint assays. There has been a recent resurgence of interest in the use of visible spectrophotometry to detect bilirubin released following an intracranial bleed not detected by CT scan. 

Crustacea glossary. The content of this site is impressive. http //deltaclon.es/pd HlT302.pdf HBT endotoxin tests limulus amoebocyte chromogenic endpoint assay. This downloadable document gives some information on the use of the blue blood of Limulus (the Hbt LAL test), http //en.wikipedia.org/wiki/Horseshoe crab Horseshoe crab. See also http / /www.ceoe.udel.edu/horseshoecrab/ http / /www.beach-net.com/horseshoe/Bayhorsecrab.html... 

---