Assay methods plate

Conventional plate assays require several hours ineubation and eonsequently the possibility of using rapid mierobiologieal assay methods has been studied. Two such methods are ... 

J. Strahan, Development and application of an enzyme-linked immunosorbent assay method for the determination of multiple sulfonylurea herbicides on the same microwell plate, in Environmental Immunochemical Methods, ed. J.M. Van Emon, C.L. Gerlach, and J.C. Johnson, American Chemical Society, Washington, DC, pp. 65-73 (1996). 

Grendahl and Sung [96] tested a simple, reliable, and inexpensive assay method for the quantitation of serum levels of miconazole and other imidazole drugs. This assay, which is similar to Kirby-Bauer test, was sensitive to 0.2 pg of drug/mL and linear from <0.2 to 10 pg/mL. Concentration of inoculum and agar depth in test plates was not as critical as the type of medium, amount of inoculum or type of drug used. 

FIGURE 14.2 The bio-bar-code assay method, (a) Probe design and preparation, (b) PSA detection and bar-code DNA amplification and identification (reproduced from [17] with permission) (see Plate 14 for color version). 

The ratios of lectin-glycoprotein dissociation constants were measured by microtiter plate binding assay method. At pH>7.5, a values reached a maximum of about 50, indicating an extremely efficient separation between SBP and AGP. 

In the case of radioactive isotopes, however, the newcomer in the field is faced with a bewildering array of instruments and manufacturers. Developments in this field have been rapid and papers a few years old probably describe assay methods which are now out of date. Currently there is a vast choice amongst ion chambers, proportional counters, Geiger-Miiller counters, crystal scintillation counters, liquid scintillation counters and semi-conductor counters — all with admirable characteristics for particular purposes. If a worker s interests lie solely in gas chromatographic examination of products then he can obtain a counter specially designed to monitor column effluents. Similarly there are various scanners especially for paper chromatograms or thin layer plates. 

For greatest accuracy of the estimates of the total protein concentration in unknown samples, it is essential to include a standard curve in each run. This is particularly true for the protein assay methods that produce nonlinear standard curves (e.g., Lowry method, Coomassie dye-binding method). The decision about the number of standards used to define the standard curve and the number of replicates to be done on each standard depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required of the results. In general, fewer points are needed to construct a standard curve if the color response curve is linear. For assays done in test tubes, duplicates are sufficient however, triplicates are recommended for assays performed in microtiter plates due to the increased error associated with microtiter plates and microtiter plate readers. 

Sorenson, K. and Brodbeck, U. 1986. A sensitive protein assay method using micro-titer plates. Experientia 42 161 -162. 

FIGURE 12.7 New methods of creating IC50 assay-ready plate via direct transfer. Top achieving 11 point, half log serial dilution by first creating a three-concentration source plate with a traditional approach and obtaining a full span assay-ready plate. Bottom visual inspection of the plate. 

One of the more impressive advantages of LC/MS is that less resolution and selectivity are required from the LC separation. For assay methods involving complex matrices such as plasma, the separation of analyte from matrix components is not as critical, so the separation can be much shorter. Assay methods requiring 15 to 20 min with LC/UV or fluorescence can be shortened to only a few minutes. Methods that previously required large numbers of theoretical plates and long (25-cm) analytical columns can now be implemented with less resolution and shorter (1- to 15-cm) columns. 

Optical assay methods, including the scintillation readout for radioactive assays, suffer from interference through absorbance by colored test compounds, which give rise to a so-called inner filter effect or color quenching . Additional sources of artefacts in fluorescence assays are auto-fluorescence of compounds, plates, and reagents, as well as scattering of excitation Hght. 

Diaz, J. L., Oltersdorf, T., and Fritz, L. C. (2000) Monitoring interactions of Bcl-2 family proteins in 96-well plate assays. Methods Enzymol. 322, 255-266. 

Besides choosing the appropriate Ab, the assay method can be designed or manipulated to improve assay specificity using (a) protein precipitation, (b) liquid/liquid or solid-phase extraction, (c) HPLC separation of the analyte from the interfering compounds, (d) sample dilution with buffer or control matrix, or (e) an affinity solid phase (e.g., antibody-coated microtiter plate or polystyrene beads) to capture the analyte followed by wash steps. Affinity-purified antibodies and protein blockers are used in EIA to decrease nonspecific binding in plate assays. Increasing incubation time to reach equilibrium also improves binding specificity. 

Resistance to bacitracin does not emerge rapidly in originally susceptible strains. The thermal and storage stability of bacitracin is enhanced by the presence of an equimolar concentration of Zn " and the antibacterial action is potentiated by an excess of Zn " ". It has been suggested that Zn + is also associated with the mechanism of antibacterial action. No chemical assay methods are yet available for bacitracin. For the determination of potency the cylinder plate method with Micrococcus flavus (ATCC 10 240) as test organism is used. ... 

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