Interference with Assays

Physical interferences often arise when the test compound or metabolite has the same color as the endpoint color of the reaction used to measure the analyte. Any compound that imparts an unusual color to blood or urine may cause interference, depending on the analytical technology (e.g., disulphine blue) (Halloran and Torrens 

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Detergent must not be very expensive Detergent should be readily removable after treating membrane fraction Detergent must not interfere with assays such as lipids, protein colorimetric determinations and enzymatic activity many of the nonionic detergents contain high phosphate levels that interfere with certain lipid analyses and certain of the polyoxyethylene derivatives affect protein colorimetric and enzyme assays... 

Detergent should be inexpensive Detergent should be easily removable Detergent should not interfere with assays such as lipid and protein colorimetric assays and enzyme assays... 

However, these compounds and the fragments are not without their intrinsic problems and should not be used as is. Some examples of potentially problematic compounds include those with chemically reactive groups, dyes, and fluorescent compounds which interfere with assays, frequent hitters/promiscuous binders, and inorganic complexes (55). It is important, then, to a priori filter out such compounds or reagents which are practically useless from a drug discovery point of view. 

In some cases, particularly when working with samples containing low PGase activity, it may be necessary to run assays for extended reaction periods. One of the concerns with extended assays is the potential for microbial growth to interfere with assay results. This may be avoided by including an antimicrobial agent in the reaction mixture. The stipulation that... 

Although the peptide aminomethylcoumarin (AMC) substrate approach has been widely used for measuring the activities of many proteases, a disadvantage of this approach arises from the ultraviolet excitation and emission wavelengths required for detecting free AMC where there is a possibility for fluorescence interference. RllO-labeled substrates are less encumbered by interference due to a spectral red shift, but many compounds in small molecule chemical libraries exhibit fluorescent properties that may interfere with assays based on fluorescence detection (Simenov et al. 2008). 

Nerve growth Stimulates nerve Interferes with assay... 

Becanse it contains cardenohdes, Asclepias can have digi-talis-hke effects and potentiate digitalis toxicity (See monograph on Cardiac glycosides). Interference with assays of plasma digoxin concentrations is also possible (1). 

Cefoxitin 1-2g iVq.ehr S0% q.6hr q.S-12hr q.l2hr May produce faise increase in serum creatinine ieyel by interference with assay 1 g after dialysis 1 g/day Dose for GFR 10-50 ml/min... 

With knowledge of the target protein stability window and other properties, additives can be kept to a minimum. This can help to avoid problems of interference with assays or other procedures and will avoid the need for an extra purification step to remove additives at a later stage in purification. Examples of buffers and additives, together with their use, are shown in Table 9. 

According to Craig, this method has been applied successfully tc the determination of cycloserine in biological fluids, such as blood, urine, cerebrospinal fluid and to the determination of crystalline cycloserine. No naturally occurring amino acids have been found to interfere with assay results. The minimum assayable level is about 100 ppm in tissue or solid samples such as animal feeds, and about 25 ppm in liquid samples. 

Approximate ranges of subsurface bacterial biomass and metabolic activity from the literature are shown in Table II. There have been few studies of relative measures of biomass or activity at contaminated versus uncontaminated sites. It is clear that certain functional classes of bacteria appear to increase in organic-rich environments (11-121 There are substantial problems associated with determining microbial activity in contaminated environments due to the diversity of types of microorganisms and chemical interferences with assay procedure. 

These have a tendency to disrupt membranes and therefore interfere with assays that are carried out in whole cells or membrane preparations, m which only one part of the cell s activity is being monitored e.g., uptake by the cell of a particular compound or receptor binding using a radiolabeled ligand. If the cell is damaged by a substance that has no direct action on these systems, the assay may break down and give a misleading result. 

These will interfere with assays that are based on oxidative/reductive processes. 

The extended target validation led to increased numbers of target screens with in vivo active compounds. Hence, even more time could be spent on hit validation, namely the introduction of control tests to eliminate, for example, readout interfering compounds (hits that were only found due to their optical properties or chemical interference with assay components). 

During routine analysis, bloody specimens have not proven to interfere with assay performance. However, grossly bloody specimens (>80 pL whole blood in 1 mL transport media) may interfere with performance. 

Cloned receptors offer the advantages of high expression, homogeneity, control of pathogenic factors, and the potential to eliminate endogenous substances that may interfere with assay sensitivity. When possible, the use of cloned receptors is desirable. 

Preparation of samples for use in RRAs varies. If the substance of interest is in biological fluids (e.g., urine, spinal fluid, plasma) it may be possible to add fluid directly to the RRA. In many instances, however, components (salts, proteins) interfere with assay integrity. In this case, it is necessary to devise a preparation scheme suitable for use with the RRA and which does not alter the quantities of the substance of interest, or to incorporate an appropriate internal standard in the method (e.g., radioactive tracer amounts of the compound of interest preferably labeled with an isotope different from that incorporated in the ligand). In the case of natural product analysis, standard aqueous or organic extraction procedures are followed and the sample is freeze-dried or dried to provide the final preparation. Aqueous fractions can be redissolved in assay buffers whereas dimethylsulfoxide (DMSO) is a useful solvent for organic extracts since many RRAs tolerate < 1 % DMSO in the final assay. 

At the practical level, it should be borne in mind that RRAs can be subject to a number of confounding factors. Chief among these is the possibility that substances contained in samples may interfere with assay specificity. Samples processed with acids or bases, if not properly neutralized, may yield falsepositive data since RRAs are pH dependent. It would be ill advised to add organic extracts directly to an RRA since assays are performed in aqueous buffers that are not miscible with all organic solvents. 

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