Microplate standards

SLAS (2004) http //www.thefreelibrary.com/ ANSI Approves Microplate Standards Drafted by SBS Committee New...-a0112221338... 

The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

Standards that define the footprint, the height, the flanges and the well positions of 96-, 384-, and 1,536-well microplates. 

The measurement of optical density in AChE-biotests after analytical procedure can be done on special microplate photometer, for example, MicroReader 4 (Hyperion Inc., USA). The measurement of optical density was at X = 490 nm. For installation of microplate photometer parameters, the software of the device is used. According to our data, the best results were found with the use of modified 96-cells plate (Budantsev and Budantseva, 2005). They differ from a standard plastic plate by absence of cells bottom. 

To a set of labeled tubes, add 2 ml of OPA reagent (Thermo Fisher) and 200 pi of the appropriate standard or sample. Mix well. If using a microplate format, scale back these quantities 10-fold to fit in the microwells. 

Early laboratory robots were unreliable, but today, these systems perform quite well. Today s robots simply move plates from one robot-friendly position to another, such as the entrance pad of a plate reader. These simplified movements combined with the low weight of a plate allow engineering to simplify the robot designs. As seen in industrial application of robots, robots that are defined and used for a specific application will work day in and day out quite well. It is always best to keep the automation as simple as possible to get the highest level of performance. This is usually accomplished by minimizing the number of moveable parts associated with the automation. Stackers have also become more reliable. This was due, in part, to the standardization of the microplate by an effort of the Society for Biomolecular Screening (Danbury, CT, U.S.A.) in association with the American National Standards Institute (ANSI, Washington, DC, U.S.A.), but also due to the use of simpler stacker mechanisms. Today, there are many choices for devices, workstations, and fully automated systems. The selection as to which automated devices to purchase for HTS should be driven by a clear set of specifications that define the use of the automation. The choices can be expensive, and therefore, replacement may not be possible, so it is important to choose well. 

As phenolics are well-known antioxidant agents, the Folin-Ciocalteau total phenolics assay was performed to assess phenolic content in ginseng roots. This assay was performed as described by Singleton et al. [20] with modifications [21]. Briefly, extract dissolved in 70% methanol was combined with Folin-Ciocalteau reagent, incubated for 5 min, and then 7.5% NaHCOs was added. Samples were transferred into microplate wells in triplicate, incubated in darkness for 2 h, and then absorbance read at 725 nm. Samples were blanked against a treatment with only solvent. A standard curve of quercetin was produced and data were expressed as quercetin equivalents. 

Free radical scavenging activity was assessed using the DPPH (1, 1-di-phenyl-2-picrylhydrazyl) assay as described by Harbilas et al. [22] with incubation time increased to 65 min. Briefly, 250 tiL of 100 timol/L DPPH dissolved in methanol was added to 40 p.L of extract (tested at 5 concentrations) in a microplate well. A standard curve of ascorbic acid (positive control) was included as a reference and all data were blanked against a treatment with only methanol. Absorbance was read with a microplate reader at 517 nm. The inhibitory concentration for 50% scavenging (IC50) of each extract was calculated and compared to the IC50 of the ascorbic acid standard curve. 

There are two yeast expression hosts that have an established track record for high-level production of heterologous proteins, namely Saccharomyces cerevisiae and Pichia pastoris. HTP expression screening using microplate formats has been reported for both these yeasts by Lang and coworkers (Holz et ah, 2002, 2003 Boettner et ah, 2002). In both cases standard protocols have been miniaturized with cells cultured in either 1.5 ml cultures in 96-deep-well plates for S. cerevisiae or 2 ml cultures in 24-deep-well plates for P. pastoris. Soluble... 

In addition to selecting an appropriate assay, it is also necessary to have a pooling strategy. It is more efficient to test many compounds per well on the microplate, rather than one. If one could test 100 compounds per well, then the standard 96-well plate would enable almost 10,000 compounds to be evaluated in one experiment. (Currently, multiwell plates containing more than 96 wells are routinely being used.) To facilitate effective pooling, the library of compounds is usually divided into a number of nonoverlapping subsets. 

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

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... 

The obstacles in the development of a robust routine-suited real-time IPCR method with the typical 1000-fold IPCR sensitivity increase were finally overcome by the use of multimeric antibody-DNA reagents (see Section 2.1.4), a microplate-compatible AbiPrism 7000 real-time cycler (Applied Biosystems), and TopYield Modules (Nunc see Section 2.3.1.). In a study carried out with several model antigens and practical examples, real-time IPCR revealed similar or better sensitivity as conventional IPCR, with significantly lowered standard derivations and a good tolerance for biological matrices such as human plasma [62, 67]. 

As the standardized 96-well microplate format (and multitudes thereof) has found universal prevalence as the platform of choice in routine laboratory applications (ELISA and PCR), the full compatibility of the IPCR... 

For any standard ELISA application requiring enhanced sensitivity in combination with almost complete retaining of all ELISA advantages, such as microplate format, common laboratory equipment and the immunological specificity for target antigens, however, Immuno-PCR is worth consideration. 

A statistical index of precision calculated as ([standard deviation x 100] mean). The CV is a measure of the variability in a group of measurements. Since the CV is unitless, it can be used to compare CVs from different experiments . It is also a quality control tool. For example, in the algal microplate toxicity test, algal cell density in control wells at the end of the test exposure period must have a CV not exceeding 20% to meet test acceptability criteria. Volume 1(1,2,3,10). 

The practical limitation on library oversampling depends on screening instrumentation, for which flow cytometry must be considered the gold standard. Commercially available flow cytometers analyze multicolor fluorescence signals and sort desired cells within user-defined gates at a rate of 50,000 cells per second [44], A salient comparison would be to microplate-based robotic screening. Considering each cell in a combinatorial library to be functionally equivalent to a microplate well, a flow cytometer can screen the equivalent of several million 1536-well microplates per day. 

---