Plate-read assay

Here, the type of assay format selected will depend on whether it is performed on the field or in the lab. For field testing, dipstick formats may be more appropriate versus the multiwell microtiter plates read on spectrophotometers. It is simple and easy to use and can be performed in minutes (74, 75). 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

Advantages This assay in conjunction with the use of Multiwell plates, multichannel pipettes, microplate washer and shaker, together with a plate reading spectrophotometer, enables a large number of samples to be processed quickly. Interfacing the plate reader with a computer would provide an obvious additional advantage. 

Use the Bravo liquid handler to transfer 4.5 pi plasma to all wells of the assay plate, and spin down the plate and seal it with aluminum adhesive seal. After at least 17 h of incubation, prepare the Color Developer similar to step 2 in Subheading 3.1 and dispense with Combi 2 pi to all assay wells. After spinning the plate, read on PHERAstar plate reader at OD555 (fccNote 9). 

The turbidimetric principle and automated microtiter plate reading apparatus are more commonly used in the kinetic rather than the end-point mode. Turbidity readings of each reaction mixture are taken at frequent intervals throughout an incubation period. The logarithm of the time (the onset time) taken to reach a specified level of turbidity is inversely proportional and linearly related to the logarithm of the concentration of endotoxin in the material under test. Standardization is necessary with each series of assays. This approach is only practical for routine application using microprocessor-controlled equipment. 

Bushway PJ, Mercola M, Price JH (2008) A comparative analysis of standard microtiter plate reading versus imaging in cellular assays. Assay Drug Dev Technol 6 557-567... 

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. 

These assays are easy to perform. Cells are plated 24h before the assay, preloaded with the dye, and allowed to incubate for a period of time, generally 1 h. Agonist and/or antagonist is added to the cells, the cells are incubated, and then read on the FLIPR. 

However, many recent instruments are still not considered satisfactory, since professional developers in the field of high-throughput screening (HTS) want to use the full performance of the latest generation of robots and computers for automation. This results in new instrumental developments, like the possibility of reading not only 96, but 384 or even 1536 wells plates as well as DNA chips, very rapidly (in a minute or so) and repeatedly without any mechanical failures. Hence, in the eyes of company scientists developing new assays, many present-day instruments still correspond to an intermediate stage of development. For research laboratory scientists, on the other hand, the actual equipment offers excellent performance. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

The major advantage associated with continuous assays is that the initial rate of product formation can be determined with complete confidence, and any unusual behavior of the enzyme would be immediately apparent. The major disadvantage is a question of throughput an instrument such as a platereader would remain dedicated to the reading of a single plate for the duration of the enzyme-substrate incubation period, compared with an equivalent discontinuous assay where an entire plate may be measured in a... 

After terminating the coupling reaction with ascorbic acid, reaction mixture clarified by precipitating protein with ethanol Turbidity decreased by final addition of caffein, sodium benzoate, and Teepol micromethod After the coupling reaction with ascorbic acid is terminated, azo color is extracted and read low blank the extract can be applied directly to a thin-layer plate and be separated rapidly to determine the ratio BMC BDC Micro versions assay artificially activated enzyme... 

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