Endpoint quantification

Kobayashi H, Higashiyama M, Minamigawa K, et al. Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification. Jpn. J. Cancer Res. 2001 92 203-210. 

Applications Potentiometry finds widespread use for direct and selective measurement of analyte concentrations, mainly in routine analyses, and for endpoint determinations of titrations. Direct potentiometric measurements provide a rapid and convenient method for determining the activity of a variety of cations and anions. The most frequently determined ion in water is the hydrogen ion (pH measurement). Ion chromatography combined with potentiometric detection techniques using ISEs allows the selective quantification of selected analytes, even in complex matrices. The sensitivity of the electrodes allows sub-ppm concentrations to be measured. 

Category C (possible human carcinogen) was evidenced by a dose-related increase in the incidence of leiomyosarcomas in the urinary bladder, a significant dose-related trend for combined hepatocellular adenomas and carcinomas in males, and a significantly higher incidence of combined lung adenomas and carcinomas in females. For the purpose of risk characterization, the RfD approach should be used for quantification of human cancer risk. The chronic exposure analysis revealed <100% RfD, and it is assumed that the chronic dietary endpoint is protective for cancer dietary exposure [64]. 

LIF methods can be applied in-line, at-line and on-line for real-time monitoring as discussed throughout this chapter. In-line or in situ intrinsic LIF is by far more prevalent in real-time applications such as PAT as it is nondestructive and simple to deploy along with attractive analytical merits. In-line application can be accomplished by direct insertion in situ probes or flow cells. This type of monitoring is utilized for realtime analyte quantification monitoring and detection of process endpoints and faults. 

The value of PET for predicting clinical outcomes is complex, since the relevant end points include LV function, symptoms, reduced hospitalizations, and mortality. The utility of PET for assessing viability will vary for each of these endpoints. Most studies on changes in LV function as related to myocardial viability imaging have been performed in patients with moderately impaired systolic performance without quantifying the size of the viable myocardium or scar. In these studies the positive predictive accuracy decreases significantly in patients with more severe LV dysfunction (LVEF <30-35%) [110]. In patients with severe LV dysfunction, quantification of the amount of viable myocardium as more than 31% of the left ventricle accurately predicts improvement in LVEF after revascularization [111, 112]. 

The two main online applications of hyperspectral imaging are blending endpoint determination (Figure 19) and capsule control (Figure 20). Other applications may be possible, such as content uniformity, determination of dissolution properties, and water quantification, but are not described in this chapter. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

All test methods adopted or under development by national or international standard organizations (e.g., OECD, ISO...) are suitable for WASTOXHAS. However, the choice of endpoints to be evaluated, among a large number now available, and their quantification methods, are directly related to criteria defining limit values. Examples of different chemical limits can be found in Lapa et al. (2002b). 

Human specimens. In our laboratory, we first developed a good laboratory practice (GLP)-validated procedure for quantification of intact rafAON in control human plasma. The rafAON assay validation endpoints were standard curve, between-run precision and accuracy, within-run precision and accuracy, effects of dilution and freeze thaw, stability of rafAON at -80° C, and 4°C in plasma for various times, specificity, integrity of rafAON during plasma sample collection and processing, and lipid interference. The reader is referred to a previous citation for further details (17,27). 

Besides enzyme activity, enzyme induction assays can also utilize mRNA and enzyme protein level as endpoints. Gene expression studies now can be performed using branch-chained DNA and microarray techniques. Protein level quantification in general is performed using isoform-specific antibodies and Western blotting. Enzyme activity represents the most relevant endpoint for drug-drug interaction evalua-... 

There are inducers which are also enzyme inhibitors. Ritonavir is an example of an inducer of CYP3A4 but also a potent inhibitor of the P450 isoform. Ritonavir is not an inducer using enzyme activity as an endpoint but can be shown to be an inducer based on gene expression (quantification of CYP3A4... 

A fiber optic probe was used by Blanco et al. for analysis of spasmoctyl samples with the active compound otilonium bromide and cellulose, maize starch, sodium starch glycolate, and glyceryl palmitostearate as excipients. Another study from this group covered the identification, qualification of the substance, and the quantification of the active component. A library search with a comparison to the near-infrared spectra of 163 pharmaceuticals was involved. An on-line monitoring for the determination of the endpoint of polymorph conversions in pharmaceutical processes was recently described further investigations into this field were published and are noted previously. ... 

One of the difficulties in the quantification of the stressor-response profile is that many of the extrapolations are qualitative in nature. Phylogenetic extrapolations are rarely quantified or assisted with structure-activity relationships. Quantification of population level effects is likewise difficult and in some cases probabilities of extinction have been used as the quantified variable, not a subtle population endpoint. 

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. 

We have used four different endpoints to assess caspase activation PARP cleavage, DEVD-afc cleavage, proteolytic processing of caspase-7, and quantification of active caspase-3 and active caspase-7 with the ELISAs. Of these methods, only the active caspase-3 and active caspase-7 ELISAs are capable of quantifying active caspase-3 and active caspase-7 with any certainty. DEVD-afc cleavage is often reported as caspase-3 activity. Clearly... 

There are, of course, many other in vitro, ex vivo, and in vivo endpoints that can be employed depending on target cell type, understanding of potential for GI effects related to the primary pharmacology of the target or lesions identified from preclinical models, such as fecal microflora composition, charcoal transit time, ileum contraction, or quantification of short-circuit current with Ussing chambers. For excellent review of endpoints for assessment of GI toxicity, the reader is referred to Kapp (2008). 

Turbidimetric and nephelometric assay Nephelo-metry and turbidimetry, because of their speed and ease of use, are most widely used. These techniques can be used either by measuring the amount of Ag-Ab complex formation (endpoint methods) or by measuring the rate of complex formation (kinetic methods). The kinetic methods are more rapid because measurements are accomplished within 20 s, and are more precise because sample blanks are not necessary. Kinetic assays are, however, somewhat less sensitive because low-affinity antibodies do not have time to react. The occurrence of Ag-Ab formation is related to the amount of light scattering and is used as the basis for antigen quantification. This approach has been accompanied by the development of... 

Citotoxicity assays using murine neuroblastoma cells (Neuro-2A) have been proposed for the assessment of ciguatoxins and brevetoxins. The simplest method is based in the ability of metabolically active cells to reduce the tetrazolium compound MTT. After incubation with serial dilutions of test samples, cells are exposed to MTT and formation of color due to MTT reduction is read using a spectrophotometer. A new cytotoxicity assay is based on a c-fos-luciferase reporter gene that uses luciferase-catalyzed light generation as an endpoint and a microplate luminometer for quantification. After... 

The field of gene expression analysis has only recently become of broader interest to users of mass spectrometry. The reason for this lag in interest was simply the lack of methods allowing the absolute quantification of NAs in general, or mRNA in particular. Mass spectrometry only allows an endpoint analysis, and even then a standard for normahzation (such as a second allele) is required. Hence, the approaches used have focused on the semi-quantitative analysis of allelic expression which, as described above, is a rather narrow-albeit expanding-field of research. 

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