Relative quantitative assay

Relative quantitative assay is similar in approach, but generally involves the measurement of endogenously occurring analytes. Since in this case even a zero or blank calibrator will contain some amount of analyte, quantification can only be done relative to this zero level. Examples of this include immunoassays for cytokines such as interlueken-6 or certain gene expression assays (e.g., RT-PCR). 

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]. 

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. 

Quasi-quantitative assays traditionally include measures of enzymatic or ligandbinding activity, as in flow cytometry and anti-drug antibody assays [9]. One of the common characteristics of these assays is the lack of a true reference standard, where reference standards are poorly characterized, do not completely represent native protein, or differ from native proteins in terms of potency or immunoreactivity. As stated above, if the analytical response is continuous across the range in question, the analytical results can be expressed in terms of a characteristic of known test samples. The following is one example an ELISA qualified as a quasi-quantitative assay because it could not be validated as a relative quantitative assay. 

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... 

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... 

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... 

A very important advantage of the relative bioavailability assay is that experimental parameters may be selected which are easily quantitated. Thus, Tso et al. (11) determined the relationship between dietary calcium concentration (X, g/kg) and... 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

The most common enzyme label in IAs and ILAs is horseradish peroxidase (HRP) due to its high turnover number, the sensitivity of its colorimetric and luminometric assays, its suitability for diverse conjugation procedures, and relative small molecular size (40 kDa compared to 100 kDa for alkaline phosphatase). The use of labeled enzymes as tracers allows qualitative and quantitative assay procedures that are not dependent on instrumentation. Thus absorbance, luminescent, electrochemical, or multistage assay systems could be performed (Fig. 10) [23]. 

Host-cell protein ELISA have the advantage of quantitating host protein impurities. The disadvantage, however, is that the quantitation is of a group of impurities. Western blot analysis, on the other hand, provides the analyst with a relative level of an individual impurity compared to other impurities. If the level of one or more host protein impurities appears to be excessive based on the intended use of the drug product then it may be necessary to identify those impurities. This can provide assurance that the impurity is innocuous and it can also define the physicochemical properties of the impurity such that the process can be modified to reduce its presence in future production lots. The identification can also lead to the development of a quantitative assay for monitoring the individual impurity in every lot. 

Hall Ode termined the half-wave potential of sodium cephalothin. The E value was dependent upon both pH and concentration. The equation for the pH dependence at a concentration of 0. 5 mM in Mcllvaine, buffers was E = -0.992-0.078 pH. All data obtained were versus the saturated calomel electrode at 25 C. The method was not considered sufficiently accurate for use as a quantitative assay since the relative standard deviation was about 5% ... 

By studying the steroid content of arterial and venous umbilical cord blood, it is possible to obtain a considerable amount of information on steroid metabolism in both the fetus and the placenta. Quantitative assays can indicate uptake or production by either body, and as the blood is relatively easily obtained in quantity it also provides a ready source of material from which new compounds can be extracted and identified. In abnormal conditions a study of its steroid content can provide early information on steroid production by the infant adrenal at birth. 

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. 

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