Definitive quantitative assay

Definitive quantitative assay uses calibrators fitted to a known model to provide absolute quantitative values for unknown samples. Typically, such assays are only possible where the analyte is not endogenous, for example, a small-molecule xenobiotic drug. 

Demonstration of selectivity and specificity of an assay for a biomarker must be considered in the context of whether the assay is a definitive quantitative assay or a relative quantitative assay (see Section 1.2). It can be difficult to show absolute specificity and selectivity for a relative assay due to the uncertainty of the exact species being measured and unknown components of the matrix. At a minimum, reproducible measurements of endogenous samples with and without spiking of the reference analyte should be demonstrated. On the other hand, likely truncated forms of a well-characterized biomarker measured by a definitive quantitative assay can be tested for specificity, as in the following example. 

Biomarker assays (as other bioassays) may be classified into definitive quantitative assays, relative quantitative assays, quasi-quantitative assays, and qualitative assays with varying degrees of validation requirements (Table 5.5-3) [14, 15]. For definitive quantitative assays, a well-defined or characterized standard of the biomarker is available. In the case of relative quantitative assays, calibration is performed with a standard that is not well characterized, not available in pure form, or not representative of the endogenous biomarker. Results from these assays are... 

Parameter Definitive Quantitative Assay Relative Quantitative Assay Quasi- Quantitative Assay Qualitative Assay... 

Hirsch, and A.W. Hsie. A quantitative assay of mutation induction at hypoxanthine-guanine phospho-ribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system) Development and definition of the system. Mutat. Res. 45 91-101, 1977. 

Definition Limit of quantitation is a parameter of quantitative assays for low levels of compounds in sample matrices, such as impurities and degradation products in food additives and processing aids. It is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The limit of quantitation is expressed as the concentration of analyte (e.g., percentage, milligram per kilogram, parts per billion) in the sample. 

As stated above, since capacitation in its truest sense represents the ability of the sperm to fertilize an egg and fertilization is a multistep process, development of simple quantitative assays to measure this extratesticular maturational event have been difficult and must be carefully tested when introduced for use in a new experimental model. In fact, there are no direct assays of capacitation and all of the assays to evaluate this process are based on different definitions. These difficulties, no doubt, are due to our ignorance of the molecular underpinnings of this event, and knowledge gained in this area will be instrumental in the development of new and specific assays. A few representative assay approaches are listed below to provide an overview of the advantages and disadvantages of the more common assays presently in use. 

This chapter deals with the assessment of definitive and relative quantitative assays. A good reference on the analytical validation of a quasi-quantitative assay is a white paper on immunogenicity by Mire-Sluis et al. [6] and the guidance on assay development and validation for high-throughput screening developed by scientists from NIH and Eli Lilly Company [7]. 

It should be understood that in most cases, the calibration material provided may not be 100% pure (most are not supplied with a Certificate of Analysis). However, notwithstanding the recommendations below, this is often not a major issue since the majority of biomarker assays will, by definition, be relative quantitative assays that do not measure absolute concentration. In addition, in many assay kits, particularly more recent ones, the calibration material may not be well characterized or purified, or it may not be fully representative of the endogenous analyte to be measured. Therefore, we would recommend procuring material from at least one other third-party manufacturer against which concentrations can be checked to ensure consensus. If there is disparity, another source may be required. It is also a good idea to check with the kit manufacturer as to where they have sourced their calibration material, since they often do not manufacture it themselves. On more than one occasion with certain assays we... 

As previously described, most biomarker assays are relative quantitative methods and not absolute (sometimes called definitive) quantitative methods. These differences are solely related to how well defined the calibration standard material is, and how well it is characterized or represents the endogenous analyte to be measured. 

The stages of validation of biomarker assays include establishment of the biomarker (development), so-called prevalidation, prestudy validation, and in-study validation [13-15]. The following short discussion will focus on the GLP-like definitive and relative quantitative assays. As the development and validation of an assay for novel biomarkers is quite diverse, the application of strict validation procedures appears problematic. Therefore, upon establishment of the prototype assay in the development phase, a formalized validation plan should be developed that... 

O Neill, J.P., Brimer, P.A., Machanoff, R., Hirsch, G.P., Hsie, A.W. 1977. A quantitative assay of mutation induction at the hypoxanthine-quanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT System) Development and definition of the system. Mutat. Res. 45 91-101. Huberman, E. 1976. Cell-mediated mutagenicity of different genetic loci in mammalian cells by carcinogenic polycyclic hydrocarbons. In Screening Tests in Chemical Carcinogenesis, eds. R. Montesano, H. Bartsch,... 

Assessment and definition of sensitivity are often described for quantitative analysis but are of equal importance for qualitative devices of the dip-stick type that are very popular for farm- or field-based screening assays. Because of the somewhat subjective nature of visually assessed assays, the assay s sensitivity must be validated using a number of observers to determine at what level a test is deemed positive. The number of false positives and false negatives must be carefully determined in order to balance consumer safety and potential economic loss to animal producers. 

Calculation of the Endocellulase Activity from the Intrinsic Viscosity Values. The enzymic degradation of polymeric substrates can occur at different bonds in the same substrate molecule, and the enzymic activity has to be defined here as the initial number of moles of glyco-sidic bonds split per second (53). This definition corresponds to the definition of the katal, symbolyzed kat. This unit is defined as the catalytic amount of any catalyst (including any enzyme) that catalyzes a reaction rate of one mole per second in an assay system (54), and it is recommended by the International Union of Pure and Applied Chemistry (55) for the quantitative evaluation of catalytic activities. 

Suspensions or slurries pose a problem since by definition they are not homogeneous. The problem is how to obtain reliable quantitative results from suspensions. One method for dealing with suspensions is to prepare individually weighed samples and stress them at concentrations greater than the final analytical concentration. Prior to analysis the samples are then diluted to the final analytical concentration with a solvent that completely dissolves the sample. For example, if the final analytical concentration desired is 0.3mg/mL, suspensions could be prepared at a concentration of approximately 1 mg/mL by adding 3mL of the appropriate solvent to samples of approximately 3 mg in 10-mL volumetric flasks. Prior to assay, the samples can then be diluted to volume with a solvent capable of completely dissolving the sample. 

The rapid diffusion of A -tetrahydrocannabinol into the plastic of containers and into the rubber stoppers normally used as closures for plasma vials (70 - 96%) and the significant binding to glass at low tetrahydrocannabinol concentrations (20 and 40% at 0.1 and 0.05 Ug/ml, respectively, in 50 ml volumetric flasks) definitely demand careful techniques in the handling, storage, and assay of this compound from aqueous and biological fluids. In fact, the results of any pertinent study where these conditions were not held in account should be quantitatively suspect. 

Biotinidase deficiency and biotin holocarboxylase synthetase deficiency can be definitively diagnosed by direct enzymatic assay. Biotinidase activity in plasma or serum is usually determined by using the artificial substrate, biotinyLp-aminobenz< >ate. If biotinidase activity is present, then biotin is cleaved, releasing jD-aminobenzoatc. The / -aminobenzoate then is reacted with reagents that result in the development of purple color that can be quantitated colorimetrically. In the absence of biotinidase activity,/ -aminobenzoate is not liberated. Biotinidase activity in patients with an isolated carboxylase deficiency or biotin holocarboxylase synthetase deficiency is normal. 

Manual entry of data encompasses tests ranging from simple pass/ fail tests or sample descriptions through recording quantitative results from complex assays depending upon the configurable definition of the test. All prompts issued by the system can be quickly and easily changed on-line to suit the requirements of individual laboratories. 

Determination of Assay Sensitivity with Replication and Statistics. Sensitivity can be defined as the ability of a test to discriminate between adjacent levels or concentrations of test analyte. There are other definitions of sensitivity, but the one specified is sufficiently general to serve several needs in residue analysis. For example, the definition recognizes that test sensitivity can vary with the point on the standard curve. If one of the points used is zero, then the sensitivity estimate can be either the level of smallest quantitation or the level of detectability of the method. The... 

Precision is a fundamental measure of assay performance and it should be assessed whenever possible [55]. This will allow a minimum understanding of how much confidence can be placed in the analytical data. In fully characterized assays designed to support definitive nonclinical and clinical studies, between-run and within-run assay precision of 15% or less (20% at the quantitation limit) is acceptable. Because of the shortened time scale for assay development and characterization in discovery these guidelines are excessively restrictive. Precision values of 20 to 30% are acceptable, as these are still less than intersubject variability associated with many nonclinical experiments. With this level of imprecision, data quality is sufficient to answer fundamental scientific questions such as relative levels of drug absorption, relative values of absolute bioavailability, and relative degree of drug clearance. 

This guideline refers to terms and definitions of parameters included in validation experiments, whereas Q2B describes the way in which validation can be performed. Attributes covered in Q2A include specificity (for identification tests) accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range (for impurity tests) and accuracy, precision, specificity, linearity, and range for assay measurements (e.g., content, potency, and dissolution testing). 

Quantitative methods are assays that result in meaningful numeric measurements for a characteristic of a product. Quantitative methods are used in assessing whether final product meets specifications. They are also used to measure product quality (or quantity) in various stages of manufactuiing and the results are often used in quality control charts. Validation is an objective process used to determine whether a quantitative method is performing as expected and is appropriate for its intended use. This chapter provides the motivation behind validation, some terms and definitions used in validation, a consolidated statistically sound approach to validation, along with appropriate statistical analysis, and reporting of validation results. A hypothetical but realistic example is presented and is used to illustrate the validation process. 

To determine dose (or assay) the quantity of the compound is assessed in the most specific way by HPLC or specific capillary electrophoresis methods using authenticated reference/working standards for quantitative evaluation are suitable for many products. In the case of viral or cell therapy, total protein determination or number of viable particles or (specific) cells might be appropriate dose definitions. In addition, DNA hybridization assays or the determination of total DNA can be applied. Alternatively, for some products a dose definition may be based on the potency of the applied amount. 

A very important observation at this point is that an IDMS assay is in principle a physical measurement since it is a measurement of ratio of isotopes and not of a ratio of elements (as in classical analytical chemistry). Indeed two numbers of atoms are compared in a ratio determination and these atoms belong to the same element. Hence ail the chemical interferences, normal in a chemical assay, do not affect the result anymore. Combined with the fact that the requirement of being quantitative - essential and difficult in classical chemistry assay - must not be fulfilled (after spiking), this means that IDMS ranks higher in the hierarchy of methods than normal elemental assay methods since it is far less subject to potential chemical error sources. In other words its inherent potential for good precision and accuracy (i.e. small overall uncertainty) and - at least as important -the transparency of the uncertainty propagation in (Eqs. 4 and 5) give it the character of what some have called a "reference method" or even a definitive method". 

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