Assay reproducibility

Hydrophobic interactions are very sensitive to temperature. Retention of most proteins increases with temperature, but for some the opposite is true, and the magnitude of response is highly individual in any case.34-36 If you elevate temperature sufficiently (56°C and above) you may begin to denature proteins in the sample. This may expose more hydrophobic sites and alter selectivity to a greater degree. Whether or not you exploit temperature as a selectivity factor, good temperature control is essential for assay reproducibility. 

CE has recently emerged as a powerful tool in carbohydrate analysis with enhanced resolution for isobaric isomers, shorter analysis times, and high sensitivity with EIE detection, as well as better assay reproducibility and robustness over the traditional methods. 

Figure 9 Distribution of active adenosine ligands identified in in vitro assays (Reproduced with permission of the authors from Ref. 6)...

FIGURE 14. Principle of the time-resolved (gated) luminescence assay. Reproduced with permission from Reference 2, Copyright 2005 the Royal Society of Chemistry... 

Temperature. The rate of Jaffe complex formation and the absorptivity of the complex are temperature dependent, measurable differences being observed even between 25 °C and 37 °C. Consequently, temperature control is an important component of assay reproducibility. 

TIP Autoinjectors provide reliable injection of the desired volume of sample to HPLCs, GCs, and other instrumentation. This equipment allows assays to be readily standardized, eliminating many potential questions about assay reproducibility. The stability of sample preparations in the injector queue must be considered when examining data from an extended run. 

A unique property of LC/API/MS is the extent to which the analyte signal is affected by the sample matrix or the existence of co-eluting analytes. This property can have a profound influence on sensitivity and assay reproducibility. Because of matrix-ion suppression, it is not possible to estimate extraction recovery by comparison of the signal from a neat sample to an extracted sample. This is because the reduction in signal represents the combined effects of recovery and ion suppression. As first shown by Buhrman et al., quantitative assessment of extraction efficiency is made by spiking the neat sample into an extracted blank and comparison of the result to a similar sample spiked before extraction [120]. Conversely, the extent of ion suppression is obtained by the comparison of the signals for a neat unextracted sample to the same neat solution spiked into an extracted matrix blank. 

Reproducibility which applies to all 5 concepts, may refer to within-assay or between-assay variation. Sometimes within-assay reproducibility is designated as repeatability (Palmer and Cavallero, 1980)... 

Fast, D.M., Kelley, M., Viswanathan, CT., O Shaughnessy, J., King, S.P., Chaudhary, A., Weiner, R., DeStefano, A.J., and Tang, D. (2009) Workshop report and follow up AAPS workshop on current topics in GLP bioanalysis assay reproducibility for incurred samples implications of Crystal City recommendations. The AAPS Journal, 11,238 241. 

Although there are many issues to consider in the conduct of appropriate and relevant ISR assessments, particular attention should be paid to the number of samples selected for reassay. To provide adequate coverage across a study in its entirety, 5 10% of the total sample size should be reassayed, with the 5% minimum limited to larger studies. It is also important to note that acceptance limits for ISR comparisons should be commensurate with the methodology (e.g., chromatographic vs. ligand binding assays) and demonstrated assay performance wide acceptance limits are not recommended. Further details for these issues and other considerations pertinent to ISR can be found in the 2009 workshop report and follow-up publication from the February 2008 AAPS Workshop on assay reproducibility for incurred samples [8]. 

Good assay reproducibility and recovery were observed for neat (undiluted) normal human serum and serum from rheumatoid arthritis patients for quantitation of a fully human anti-TNF-a monoclonal antibody [86]. It has also been reported that excess therapeutic antibodies present in serum were tolerated better in an assay for the detection of antitherapeutic antibodies based on the MSD ECL device [91]. Furthermore, the ECL procedure has been reported to be stable over a broad range of magnetic bead concentrations, probe concentrations, and hybridization conditions for a nucleic acid binding assay, making these assays more versatile and easier to transfer from one laboratory to another [88]. 

Figure 11 (a) Effect of dNTPaS in 2-AP stopped-fiow fiuorescence assays. Reproduced with permission from M. Bakhtina ... 

Table IV. Radiometric Microbiological Assay Reproducibility of Serum Vitamin B12 Levels (pg/mL)...

Table V. Radiometric Microbiological Assay Reproducibility Study of Vitamin B12 Content in Food (30)...

Scheme 1.22. The reaction mechanisms of (a) H2O2 and (b) glucose assays (reproduced from [89])...

Scheme 12.26 Working principle of a switch-off product-coupled tandem assay (reproduced by permission from [56] copyright (2004) Wiley-VCH)...

Fig.1 Microfluidic detection of ATP with bioluminescence assay (Reproduced with permission from Springer [1])...

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