Plate reader

Several established protocols have been adapted for 96-well plate readers including catalase, hyaluronidase, acetylcholinesterase, protein phosphatases and membrane-bound ATPases (22-26). In several instances these have involved novel protocols that are well suited to the ELISA format. For example, a sensitive, rapid microtitre-based assay for hyaluronidase activity was described by Frost and Stem (23). The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtitre plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtitre plate, artefacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples, with an interassay variation of less than 5%. 

The standard protocol for measuring inorganic phosphate using variations of the Fiske and Subbarow method were described in Section 8.3. There have been a number of applications described recently that have adopted a plate-reader format (see, for example, 25-27). With careful planning most assays that use colorimetric reagents (e.g. those for measuring protein concentrations described in Section 4) can be modified to facilitate the use of a plate reader. 

These analysers use centrifugal forces to mix samples and reagents that are held in wells arranged in concentric circles about the axis of rotation of a spiiming 

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An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Fatty Acid Transporters. Figure 2 Quencher-based real-time fatty acid uptake assay with a fluorescently labeled FFA analogue (C1-Bodipy-C12). Predominantly protein-mediated fatty acid uptake by 3T3-L1 adipocytes (diamonds) was compared with diffusion-driven uptake by fibroblasts (squares) using the QBT Fatty Acid Uptake reagent (Molecular Devices Corp., CA, USA), which contains C1-Bodipy-C12 as substrate in conjunction with a cell impermeable quencher. Uptake kinetics was recorded using a Gemini fluorescence plate reader. Error bars indicate the standard deviations from 12 independent wells. RFU relative fluorescence units. 

An additional requirement not noted in Table 1 is compliance with GLP7 These practices establish a paper trail for all procedures involved in the determination of residues. With regard to immunoassays, GLPs require calibration of measurement devices such as adjustable pipettors and dedicated spectrophotometers. Computer software output, as noted above, must be verified prior to use. This process can be simplified by limiting the application of specialized software to the operation of an instrument and carrying out the residue calculations in a broadly available spreadsheet such as Excel. On a positive note, in recent years, the software accompanying most microtiter plate readers has become generally easier to use and usually incorporates internal spreadsheets that are compatible with external systems. 

Other readouts, such as high content imaging or enzyme kinetic rate determinations, require extended detection intervals in the plate reader and are best automated on a dedicated workcell or semiautomated workstation platform. 

An operation or series of operations that contributes to the validation of screening results. Such operations include validation of liquid handling devices and plate readers, experiment controls, such as determination of the Z factor and use of assay controls, and postexperiment controls, such as data analysis validation and database administration. Results of a screen are validated only after a set of quality controls have been performed. 

Fig. 3. Comparison between IP-One kit versus calcium mobilization assay (384-well format). Human embryonic kidney (HEK) 293 cells expressing a chemokine receptor were evaluated on HTRF IP-One kit (CisBio, Bedford, MA) and fluroescent imaging plate reader (FLIPR) with Calcium 3 kit (Molecular Devices, Mountain View, CA).

Parallel methods using scanning 96/384-well plate UV spectrophotometers are inherently faster [292]. They will become 50-fold faster with the imminent introduction of diode-array plate readers. 

In a different approach three different structurally defined aza-crown ethers were treated with 10 different metal salts in a spatially addressable format in a 96-well microtiter plate, producing 40 catalysts, which were tested in the hydrolysis of /xnitrophenol esters.32 A plate reader was used to assess catalyst activity. A cobalt complex turned out to be the best catalyst. Higher diversity is potentially possible, but this would require an efficient synthetic strategy. This research was extended to include lanthanide-based catalysts in the hydrolysis of phospho-esters of DNA.33... 

Decisive for the success of the assay is the finding that the rate of oxidation constitutes a direct measure of the ee (Figure 8). High throughput was demonstrated by analyzing 100 samples in a 384-well format using a UV/fluorescence plate reader. Each sample contained 1 pmol of... 

Jones, P. B., Herl, L., Berezovska, O., Kumar, A. T. N., Bacskai, B. J. and Hyman, B. T. (2006). Time-domain fluorescent plate reader for cell based protein-protein interaction and protein conformation assays. J. Biomed. Opt. 11, 054024-10. 

Aqueous solubility values for the samples analyzed compared favorably with results obtained by traditional methods. The solubility values for amiodarone HC1, reserpine, and benzanthrone were lower than the LOQ of the, uPLC system used for the evaluation. Results of the evaluation of compound solubility employing no-filtration /iPLC were compared with those obtained by two traditional methods (1) multiscreen filtration followed by a UV plate reader, and (2) the shake flask method followed by a UV plate reader. As shown in Figure 6.31, the solubility values determined by the different methods are comparable for most compounds examined. Figure 6.32 shows the results of evaluations of aqueous solubility at four different pH levels for phenazopyridine and piroxicam samples. 

Compound impurities can lead to biased results when plate readers are used for detection since the total absorbance at a particular wavelength is employed for the determinations. The yt/PLC system employed in these determinations can easily compensate for this problem since only the value for the peak area due to the compound (at the corresponding retention time) is considered for the calculations. For example, in the case of a nifedipine sample, purity was determined to be... 

MultiScreen + UV Plate Reader Shake-flask + UV Plate Reader ... 

FIGURE 6.31 Comparison of solubility results obtained by no-filtration method followed by PLC detection and those obtained by two traditional methods multiscreen filtration followed by UV plate reader, and shake flask method followed by UV plate reader. (Data provided by Steven Hobbs, Courtney Coyne, and Gregory Kazan.)... 

Separation-based assays are preferred in many applications because they allow discrimination of signals due to substrate, product, and interference. When assays that involve fluorescence detection are developed, they are typically carried out by employing plate readers. When separation-based methods are employed for these applications, the influences of interferences (quenchers and other fluorescent compounds) on the final results are minimized because both substrate and product are quantified. With a separation-based approach, the label employed does not need to be placed in close proximity to the site of action of the enzyme, therefore minimizing the effect of the label on the mode of action of the enzyme. Of course, it is often desirable to develop assays that employ substrates free of labels. 

Typically, MS does not play an important role in solubility measurement because UV and nephelometric plate readers provide high throughput and are sufficiently sensitive for most compounds. 

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