Radioisotopic and Luminescence Counting in 384- and 96-Well Microplates
Alfred J. Kolb* and Kenneth Neumann
Packard Instrument Company
800 Research Parkway
Meriden, CT 06540
http://www.netsci.org/Science/Screening/feature07.html
Abstract
The 96-well microplate is established as the universal format for high throughput screening (HTS) in drug discovery. However, there is a growing interest in reducing assay volumes and increasing sample density. One of the most readily available formats to accomplish this is the 384-well microplate. The TopCount® HTS is a recent innovation based on the proven TopCount Microplate Scintillation and Luminescence Counter. The TopCount HTS has been developed to count radioisotopic and luminescent samples in both 96-well and 384-well microplates.
The use of the 384-well microplate in HTS has been discussed for several years, but without instruments it remained an unproved concept. In this report, experiments using scintillation proximity assays (SPA) and luminescence assays show that the 384-well microplate can be readily adapted to the assays and equipment in today's HTS laboratory. This includes the use of the MultiPROBE® liquid handling system which can be used to accurately position and pipette reagents, including SPA beads, into the 384- or 96-well plates. Preliminary experiments suggest that in-plate binding assays will also be readily adaptable to the 384-well format.
Introduction
Over the last few years there has been a growing demand for assay miniaturization in high throughput screening. The successful adaptation of the 96-well microplate serves as a practical example of the power of miniaturization. Assays that once required 2 to 3 ml volumes are now done in 1/10 this volume. However, before the 96-well plate became a standard in HTS there were many questions about the suitability of this small format to screening assays and the availability of all the necessary peripheral equipment to support it. These problems were quickly solved. Today we are faced with the same questions about the 384-well format.
Because the acceptance of the 384-well microplate was unproved, instrument companies were unwilling to commit R&D resources to develop such equipment. Scientists could not develop 384-well assays without analytical instruments and instrument companies would not develop equipment without some indication that there would be assays to measure. This unproductive cycle has been broken with the development and introduction of the TopCount HTS. This instrument is an advance over the popular TopCount Microplate Scintillation and Luminescence Counter which analyzes samples in 24-well and 96-well microplates. The HTS has the capability to measure radioisotopes or luminescence in both 96- and 384-well microplates. This development will allow laboratories to continue using the 96-well format while scaling down assays to the 384-well format. The 40 plate external stacker allows the unattended processing of over 15,000 samples in 384-well plates or almost 4,000 samples in 96-well plates. Twelve wells are measured simultaneously and 96- and 384-well plates can be mixed in the stacker. This will add significantly to the efficiency, flexibility and future adaptability of the screening laboratory.
There are two questions most often asked regarding the use of the 384-well microplate. First, can assays be scaled down to 100 µL, 50 µL or even less and still have a strong signal and a good signal to noise ratio? Second, will equipment be available for operations such as pipetting and plate washing? The answer to both of these questions is yes. In this article, examples will be given of reduced volume scintillation proximity assays (SPA, Amersham International) and the luminescent luciferase enzyme assay (LucLite®, Packard Instrument Company). Preliminary experiments indicate that in-plate binding assays will also be an application that can be scaled down from 96-well plates.
A major advantage of the 384-well microplate is that much of the current equipment in use today can be adapted to this format. An example illustrated here is the use of the MultiPROBE liquid handling robot (Packard) to prepare a SPA assay in the 384-well plate. The 384-well plate has exactly one-half the spacing between wells compared to a 96-well plate. This allows the use of the MultiPROBE, as well as hand-held, multichannel pipettes. Samples are pipetted into every other well, the pipette is then indexed and the alternate wells are processed.
With regard to other 384-well plate equipment, microplate readers for fluorescence and absorbance have been introduced and the adaptation of plate washers to the 384-well format is also inevitable. Because the 384-well plate has the same outside dimensions as the 96-well plate, much of the current plate handling equipment should be readily adaptable. Microplate centrifuges, incubators, plate carousels, shakers and robotic arms should all fit this new format, thus eliminating the need to replace new and expensive equipment.
Results and Discussion
A common first impression of the 384-well microplate is that the well volume is too small to be useful. However, the 384-well plate used with the TopCount, the OptiPlate®-384, has several promising properties as shown in Table 1.
| Characteristic | OptiPlate-96 | OptiPlate-384 | % Ratio (384/96) |
|---|---|---|---|
| Useable Volume (µL) | 360 | 110 | 31% |
| Total Surface Area (mm2) | 240 | 145 | 60% |
| Bottom Area (mm2) | 33 | 9 | 27% |
Table 1. Characteristics of the OptiPlate-96 and
OptiPlate-384. Volumes and measurements listed are 90% of the total
to better represent usable portions of the plates. Total surface
area means the area of the sides and bottom of the well.
These white, opaque polystyrene plates have square wells and hold a total volume of about 120 µL. The usable volume is approximately 100 µL. Since many assays are already being run at 100 µL, these assays can be directly transferred to the 384-well plate or scaled down further. The surface area of the inside of the well is 60% of the 96-well plate. This should make in-plate binding assays relatively easy to convert to the 384-well format and still retain a good isotopic or luminescent signal. The bottom area of the 384-well plate is about 25% of a 96-well plate. This should support the growth of a sufficient number of adherent cells for reporter gene and other cell based assays while offering considerable savings in cell culture reagents and physical space in the culture facility.
To illustrate the capabilities of the 384-well microplate, two common assays were selected for initial testing. These assays were the simple, mix and measure SPA, and the homogeneous LucLite luciferase assay.
Figure 1 shows the results of a SPA (125I cyclic-AMP) where the assay components, including SPA beads, were pipetted with the MultiPROBE or a hand held pipette. The total assay volume was 100 µL in the OptiPlate-384. The 96-well control plates (OptiPlate-96) were prepared in 200 µL. All samples were counted on the TopCount HTS for 3 min. As would be expected, the results of either method gave the same displacement curves. Another important assay parameter is the signal to noise (Bo/NSB, maximum binding/non-specific binding) ratio which is a measure of the assay window as shown in Figure 2. A comparison of this same SPA showed that the use of the MultiPROBE with the 384-well plate gave a Bo/NSB of greater than 15 compared to a value of less than 13 for a 96-well plate prepared manually.
A further test of the 384-well plate for SPA was carried out at Zeneca Pharmaceuticals, Macclesfield, England (data kindly supplied by Dr. John Major and Dr. Mark Beggs) and is shown in Figure 3. In this example, an 125I SPA dose response curve was run in a 50 µL total volume and counted for 1 min. The data points gave a good fit with the same midpoint as measured in the 96-well assay. The error bars around the midpoint (indicated by the vertical bars around the closed circle) show the excellent fit of the data. The assay window for the 50 µL and 25 µL volumes was well above the minimum window required for acceptance as a screening assay. The dose response curve run at 25 µL and also counted for 1 min. exhibited more scatter than was considered acceptable (data not shown). This illustrates a limit in miniaturization for some assays because of diminishing signal. Low activity samples could be counted longer, but this would limit throughput. There are ongoing developments in 384-well microplate design that promise to significantly increase the assay signal and thus make lower volumes practical.
The luciferase reporter gene is a popular luminescence assay for transcriptional regulation. The standard flash chemistry has recently been improved upon with the introduction of LucLite. This assay kit extends the half-life of the luciferase luminescent signal to over five hours. This eliminates critical timing steps and allows the batch processing of many thousands of samples. This assay was measured in the TopCount HTS in the single photon (luminescence) counting mode. Known amounts of luciferase enzyme were used to compare the signal in the 96-well plate (100 µL) and the 384-well plate (50 µL). These results are shown in Figure 4. The two curves are parallel and signals from several hundred cps to many hundred thousand cps can be measured. The 384-well plate offers several cost advantages with the use of fewer cells per assay, lower culture reagent costs, less labor and more efficient use of cell culture facilities.
Conclusions
The 384-well microplate is a very promising format for HTS. It will allow significant reductions in reagent and disposal costs and can use currently available assay methods and equipment. This saves the considerable expense required to completely re-equip a laboratory with new automated pipettes, robots and peripheral devices. It could cost many hundreds of thousand dollars to enlarge a robot with more plate carousels, larger incubators, a longer track or a larger room. Instead, the same or similar equipment and the same robot real estate could be used to accommodate four times the samples by converting to 384-well plates.
Screening technology and assay miniaturization is a very dynamic field. While this makes HTS an exciting discipline to work in, it also creates anxiety when it comes to selecting between various methods. A question that is commonly asked is whether it is worth converting to 384-well plates or would it be better to wait for the next step in assay miniaturization? Since the next step in miniaturization is not clearly defined, the answer to this question is also not clear. The danger in waiting for the next step could mean that two or more years of enhanced productivity would be lost by not incorporating the 384-well format. Advances in nanoliter dispensing technology, single photon imaging and highly sensitive fluorescent assays are all under development and many of these instruments and assay methods will still be suitable for use in the 96 and 384-well formats. With these prospects for the future, it would seem that there is greater risk in ignoring the 384-well format than there is in incorporating it as a part of an ongoing HTS program.
TopCount, MultiPROBE, LucLite and OptiPlate are trademarks of Packard Instrument Company.
Figure 1
Figure 1. 125I cAMP Assay Prepared and
Analyzed in OptiPlate-96 and -384. A cAMP SPA standard curve was
prepared according to manufacturers recommendations. The
OptiPlate-96 plates (closed diamonds) had a total assay volume of
200 µL and the OptiPlate-384 (closed squares) had 100
µL. All pipetting steps, including the SPA beads were
performed with the MultiPROBE liquid handling robot.
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Article
Figure 2
Figure 2. Bo to NSB Ratios for a 125I cAMP SPA
in OptiPlate-96 and -384 Using Manual and Automated Pipetting. The
signal to noise ratios (Bo/NSB, maximum
binding/non-specific binding) for a cAMP SPA were calculated for
96- (hatched bars) and 384-well OptiPlates (solid bars) that were
prepared by manual or automated pipetting with the MultiPROBE.
Figure 3
Figure 3. 125I SPA Inhibition Curve in a
OptiPlate-384. A % Inhibition curve of an 125I SPA was
prepared in a total volume of 50 µL and the midpoint (closed
circle) calculated at 0.698 nM.
Figure 4
Figure 4. Luciferase Enzyme Assay in OptiPlate-96 and
-384. A luciferase enzyme assay was prepared using LucLite reagents
in white 96-well (closed diamonds) or 384-well (closed squares)
OptiPlates in 100 µL and 50 µL, respectively. Samples
were counted in the TopCount using the SPC (single photon counting)
mode. Gross CPS is luminescence counts per second uncorrected for
background.
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