Endogenous analytes

Advantages Let us assume for the moment that the requirements for most trace element analysis methods are also met here These are that a known amount of an enriched Isotope of the element In question (spike) has been added to the sample that subsequent chemical processing renders the spike and endogenous analyte In the same chemical form and that contamination Is under control The second assumption, equilibration of analyte and spike, can be a critical one. In that lack of equilibration could contribute systematic errors ... 

VALIDATION CHALLENGES FOR ENDOGENOUS ANALYTE AND BIOMARKER IMMUNOASSAYS... 

Detection and Measurement of Interference Due to Heterophilic Antibodies Often, a method is used to quantify an endogenous analyte associated... 

For a majority of biomarker assays, standard calibrators are prepared in an analyte-free alternative matrix instead of the de facto sample matrix of patient samples. For such methods, it is crucial to demonstrate that the concentration response relationship in the sample matrix is similar to that of the alternate matrix. Spike-recovery experiments with the reference standard may be inadequate to evaluate the matrix effect, as the reference standard may not fully represent the endogenous analyte. Instead, parallelism experiments are performed through serial dilutions of a high-concentration sample with the calibrator matrix. Multiple individual matrix lots (>3 lots) should be tested to compare lot-to-lot consistency. In the instance that the limited amounts of sample are available, apooled matrix strategy can be used with caution as discussed by Lee et al. [15]. The samples can be serially diluted with the standard matrix (standard... 

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. 

We never accept stability data generated on manufactured/recombinant molecules spiked into matrix to be necessarily fully representative of the stability of endogenous analytes. It is not uncommon to find different commercially available QC material to have different stability properties for the same analyte because the source of the material spiked in may be different. For biomarkers, we believe that true stability data can only be derived from experiments using endogenous molecules in the matrix of interest. This, of course, is not the case for exogenously administered macromolecules. 

The prevalidation phase may be regarded as a method optimization phase where the calibration model is confirmed range, LLOQ, and ULOQ are defined and matrix interference is evaluated. The use of anchor points for the calibration curve may be feasible. As mentioned above, determination of the LLOQ may be difficult because of the presence of the endogenous analyte. Dilutional linearity may be evaluated as well. It is also considered useful to assess the biomarker in healthy and diseased individuals. For this purpose, at least 25 individuals should be tested [9] to account for intrasubject variability caused by circadian and seasonal fluctuations and intersubject variability. 

This property was exploited in the chromatographic separation and subsequent M S analysis of endogenous analytes from human semm and small drug molecules in biofluids (see Section 9.6). 

In some cases a suitable control matrix may not be available, e.g., soils from different sources around the world (and thus widely variable). In some cases it may be necessary to resort to use of a matrix containing known analyte concentrations that lie well below the LLOQ required for the particular analysis at hand, or analyte-stripped or surrogate matrices (Section 9.4.7c), but all of these approaches are problematic and may require additional vahdation procedures. Unavailability of a truly blank matrix was until very recently a major intrinsic problem in quantitation of endogenous analytes but novel new procedures have been recently developed for this particular application (Section 11.7). 

The nse of surrogate matrices was a major concern in biomedical analyses in the context of quantitation of endogenous analytes, bnt this particular problem was solved recently by an ingenious approach (Li 2003a Jemal 2003) discussed in Section 11.7. 

Internal, external, or mass defect-based calibration can be performed (42). External calibration is done with mixtures of standards measured separately from analyzed unknown samples. In contrast, internal calibration requires the presence of calibrants in the sample. Importantly, if the m/z of endogenous analytes are known, they can be used to calibrate the mass spectrometer. This type of calibration is preferable because no increase in complexity of sample occurs and influences from different parameters, such as sample thickness and analyte environment, are accounted for. As an example, trypsin autocleavage molecular ions are used for calibration in proteomics research (43), as is mass defect-based calibration, where peptides of enzymatically cleaved proteins are used for calibration and protein identifications (42,44). The latter approach is also useful for determination and subsequent elimination of nonpeptide species from peak lists. 

A single fluorescent protein coupled with an MRE makes up single FP-based biosensors. The MRE can be either exogenous or endogenous. Analyte binding to the MRE causes conformational changes of the fluorescent protein consequently altering its fluorescent properties. 

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