Troubleshooting assays

When rerunning an assay while troubleshooting, change only one factor at a time. 

This chapter describes how to be more successful in HPLC operation by summarizing a series of standard operating procedures representing the best practices of experienced HPLC analysts. It offers some brief guidelines on maintenance/troubleshooting and on means of enhancing HPLC assay precision. 

Other aspects of sample preparation to consider, depending on the objective of the CZE method, are sample denaturation, reduction and/or derivatization, and the use of an IS. IS are widely used in CE separations to aid in normalization of slight inter-assay variability and in method troubleshooting. 

Experience with CE method transfer in the biotech/pharmaceutical industry over the past 10—20 years has demonstrated that training is a key element that requires special attention for CE methods. A training video with troubleshooting examples can be very useful. Tips and hints should also be shared during the method transfer process. Other key elements for a successful transfer include selection of the proper testing strategy and assay acceptance criteria. 

Troubleshooting examples as part of the assay transfer material is very helpful. 

The protocol outlined in this chapter has been optimized for processing a large number of mouse embryos simultaneously. Since a lot of protocols include optional or even unnecessary steps largely for historical reasons, this protocol intends to maximize the efficiency of assays and minimize the temporal and financial burden, which are often the serious issues in a large-scale experiment. For reference, the alternative procedures, which were omitted or simplified in this protocol, are described in Subheading 4 as potential troubleshooting tips. 

Table B1.1.1 Troubleshooting Guide for the Modified Lowry Protein Assay... 

Table B1.1.3 Troubleshooting Guide for Biuret Protein Assay...

The main disadvantage of all Bradford-type protein assay reagents is that they are not compatible with surfactants at concentrations routinely used to solubilize membrane proteins. With some exceptions, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. Table B1.1.4 is a brief troubleshooting guide for this technique. 

Since the response of the detector (and the separation) is a function of a flow rate, it is essential that the standard response curve be determined at the same flow rate as the tablet assay. If retention times differ significantly from the runs of the standards, there is a need to troubleshoot the HPLC to determine where the problem resides. Refer to Chapter 3 for a discussion of retention time precision. 

The fluorescence-based assay described herein is used to screen large libraries of compounds, in 96-well format, for the ability to inhibit HIV protease and to accurately determine the affinity of identified inhibitors for the enzyme. Much of the discussion in this section will be widely applicable to fluorescence-based assays and enzyme assays in general. Because numerous potent inhibitors of HIV protease have been identified, part of this procedure outlines the analysis required to deal with these potent compounds. Like any enzyme assay, preparing this assay for routine use can be divided into three parts (1) determination of an appropriate enzyme concentration for assay, (2) determination of the substrate concentration dependence (Km and Vmax), and (3) determination of inhibitor concentration dependence (IC50 and K, values). Parts one and two do not need to be repeated every time inhibitor assays are run—only once to check new batches of enzyme or to troubleshoot any problems with the assay. 

Wu JT. Quantitative Immunoassay A practical guide for assay establishment, troubleshooting, and clinical application. Washington D.C AACC Press, 2000. 

The existing IPC method may serve to quickly monitor key processing steps. It may be necessary to develop a new assay if the existing one does not enable the type of investigations that troubleshooting requires. The accuracy and reliability of the new method are more important than convenience. 

When inadequate in vivo exposure or PK properties are observed, pharmaceutical profiles can be used to troubleshoot the cause of poor in vivo exposure or PK [26]. Assays for solubility, permeability, and stability (metabolic, plasma, acid) can help to track down the inadequate properties responsible for poor in vivo performance. Property optimization synthesis can then be initiated. Subsequent series analogs can be assayed to rank order compounds by properties for subsequent in vivo tests, in order to give the highest likelihood of success. Often animal studies are expensive and time-consuming, especially if they are performed using the animal activity model. Simple in vitro profiling assays can provide information for improved decisions and efficiency. 

Use in vitro assays to troubleshoot the cause of poor in vivo exposure or PK... 

Interpretation, critical parameters and troubleshooting in RNase protection assays... 

It is recommended that all instalments (including microplate readers and washers) undergo validation, calibration, and routine maintenance programs established and documented to meet regulatory requirements. This eliminates instruments as a cause of poor assay performance and streamlines the assay troubleshooting process. 

Immunoassays developed along the recommendations provided above should perform consistently and generate reliable data over time. However, occasional failures happen variations in laboratory environmental conditions, reagent quality, operator skill, and instalment performance, for example, make systematic troubleshooting skills invaluable in discerning the cause for assay failures. 

Immunoassay troubleshooting is a sequential, cumulative process whereby one first looks at the calibration curve, then QC s, and finally the sample results. If the curve is not acceptable, the assay fails. If the curve is acceptable, then one reviews QC performance if QC performance is unacceptable, the assay fails. If QCs are acceptable, then one examines individual sample results. If the assay fails as the result of an apparent technical error, the error is corrected and the assay is repeated. If the cause for the failure is not apparent or the assay has failed consistently, then a troubleshooting process should be initiated prior to further analysis (and depletion) of study samples. A summary of the calibrator/QC/sample result troubleshooting process is depicted in Fig. 3.14. 

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