Microplate readers, detection

Cell inhibition rate analysis Cells were seeded in 96-well plates at a density of 1.5/ 104 cells/well in quadruplicate and incubated at 37°C in a 5% C02 atmosphere. After 24 hr, cells were treated with 0.1% DMSO (control) or, compound at various concentrations (0.01 - 0.33 mM) for 48 hr. Cell inhibition rate was analyzed after 48 hr using the Cell Counting Kit CCK-8 (Dojindo Laboratories, Japan), according to the manufacturer s instructions. OD values were measured at a wavelength of 450 nm using a Multi-Detection Microplate-Reader (Bio-TEK, Winooski, VT). 

The dynamic range of the TRF-ELISA covers IgG concentrations from 5 to lOOng/mL [108]. Detection limits of more than one magnitude lower (0.1 ng/mL) can be obtained with commercially available time-resolving microplate readers. 

In addition to the further increase in sensitivity, the advantages of microplate-based detection methods are manifold The direct digital readout of a conventional microplate-reader allowed for easy processing of multiple samples the compatibility to standard ELISA procedures ensures a robust and simple handling, and therefore also a significant decrease in method... 

Measure fungal growth by detecting culture absorbency in a microplate reader (600 nm). A control of the growth can be performed after 24 h using a photonic microscope. 

Determinations of antioxidant activity are widely used in phytochemistry, nutrition, food chemistry, clinical chemistry, as well as in human, animal, and plant physiology, etc. Methods adapted to HPLC have appeared only recently but can be expected to have multiple apphcations in the future. ARTS" is an excellent metastable chromogen for the detection and quantification of the HAA and LAA of biological samples. Thus, using a simple photometer (end-point method), a microplate reader (multisample titration method), or HPLC equipment, a broad range of possibilities are available for the characterization of diverse samples (animal- or plant-derived). Some apphcations of special interest could include ... 

Enzyme-Linked Immunosorbent Assay Both competitive and sandwich ELlSAs are available. Although the competitive ELISA is faster because it uses only one incubation with an antibody, it is reported to be less sensitive and exhibits large imprecision. ELISA can be performed on a microplate reader, allowing semiautomation. In the sandwich assay, the primary antibody (antialbumin antiserum) is fixed on the plastic plate, which is then washed. Samples, controls, and calibrators are added, and the complexes detected and quantified by a second antibody conjugated to an enzyme label. 

Microtiter plates (MaxiSorp-immunoplates, NUNC A/S, Roskilde, Denmark) were coated overnight at 23°C with the cohesin test samples (200 pl/well, 270 nM of miniCipC c, wild-type or mutated Coh2CBD t). The plates were blocked for 2.5 h with blocking solution (300 pl/well 3% (w/v) bovine serum albumin in TrisNC buffer) and washed three times with TrisNC buffer (300 pl/well). The cohesin-dockerin interaction was initiated upon addition of dockerin samples (200 pl/well, 94 nM of XynDocA c or XynDocS t), and the plates were incubated for 2.5 h. After five washes, the bound dockerins were detected by means of the fused-xylanase activity substrate solution (240 pl/well 2.9 mM / -nitrophenyl p-D-cellobioside) was added followed by incubation at 60 C. Optical density was detOTnined at 420 nm on a VERSAmax microplate reader (Molecular Devices Corp., Sunnyvale CA). 

In order to achieve high throughput with ORAC determinations, Huang et al.464 have developed a robotic eight-channel liquid handling system coupled with a microplate fluorescence reader, which has improved efficiency at least 10-fold, compared with the discontinued COB AS FARAII analyser. The CV was 15% or less and the limits of detection and quantification were 5 and 6.25, uM, respectively. 

Majority of HTS is performed in microplates most frequently used formats contain 96, 384, and 1,536 wells per plate (Fig. 2). Consequently, HTS assays require detection approaches that could be easily executed in a plate format. Although the number of novel detection approaches adapted in HTS is constantly growing, assays based on spectrophotometric properties are by far the most prevalent, reflecting their technical and technological simplicity and low cost. Absorbance, fluorescence, luminescence, and derivations thereof are common in HTS assays. Broad selection of relatively inexpensive monochromator-, optical filter-, or diode array-based plate readers and suitable generic plates provides a foundation to spectroscopic HTS assays. 

ELISA tests are used widely for the detection of contaminants both in analytical laboratories and in the food industry. Commercial kits are generally used, as they have the advantage of containing all the materials necessary to run the tests (in some cases the preparation of solutions is required). They meet performance specifications set by the manufacturer, which are evaluated by the end user to see if the method is suitable for his or her application (processed food, type of matrix, etc.). Quantitative tests require the use of a microplate or strip reader. Some commercial ELISA kits are available on the market for the detection of soy allergens [12,13] (see Table 17.1 for performance parameters). The column raw and processed food relates to information provided by the manufacturer about the applicability to raw and processed food. 

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