J. Watson and J.V. Selkirk
SmithKline Beecham Pharmaceuticals, UK
FlashPlate is a 96-well white polystyrene microplate with scintillant-coated wells designed for highvolume, in-plate radiobinding assays. This study investigates the use of FlashPlate as a platform for the measurement of G-protein activation by means of an assay of [35S]-GTPγS binding in Chinese hamster ovary cells. Results are compared to those obtained using a conventional filtration method.
When an external signal is received by a membrane bound receptor, a series of cellular events takes place which results in the activation of an effector molecule. Many receptors transmit this signal via G-protein activation. This response can be used as a functional measure of receptor-ligand association.
The basic mechanism behind G-protein activation involves the exchange of bound GDP for GTP. Using a radioactive, non-hydrolyzable form of GTP, such as [35S]-GTPγS, G-protein activation can be assessed by measuring the accumulation of bound radioactivity.
We investigated the use of FlashPlate technology (Basic FlashPlate, SMP200) to measure [35S]-GTPγS binding, and briefly compared it to the conventional filtration method. The study examined 5-HT activation of human 5-HT1B receptors expressed in Chinese hamster ovary (CHO) cells.
A Chinese hamster ovary cell line (ACC098) expressing the human 5-HT1B receptor was used as the receptor source. Prior to [35S]-GTPγS binding assay, the CHO cells were prepared as described by Thomas, et al ., 1995, and frozen in aliquots until required.
[35 S]-GTPγS binding was carried out as described by Watson, et al ., 1996. In brief, 108 cells were pre-incubated at 30C for 30 minutes in HEPES [20 mM], pH 7.4, in the presence of MgCl2 [3 mM], NaCl [100 mM], ascorbate [0.2 mM] and GDP [10 mM] with or without test drugs. The reaction was started by adding 10 μl of [35S]-GTPγS [100 pM]. This was followed by a further incubation at 30C for 30 minutes. The reaction was stopped by rapid filtration, followed by five washes with ice-cold HEPES buffer. Bound radioactivity was determined by liquid scintillation spectrometry. Non-specific binding was determined by 10 μM unlabeled GTPγS.
Membrane immobilization onto FlashPlate
CHO cells expressing the h5-HT1B receptor were diluted to a desired membrane protein concentration in 25 mM HEPES buffer containing 2.5 mM CaCl2 and 1 mM MgCl2, at pH 7.4. A suspension of diluted receptor (230 μl) was pipetted into each well of a FlashPlate microplate, and the plates were centrifuged at 800 x g for 10 minutes at 4C. The supernatant was removed and the plates were left overnight at 4C. (Later studies have shown that 3 hours is sufficient.) Prior to use, 250 μl of 0.5% BSA in the above HEPES buffer was added to each well and left for 30 minutes at room temperature, in order to block nonspecific binding sites.
Binding Assay on FlashPlate
This method is essentially similar to the filtration method, with some modifications. The same assay buffer was used for both methods.
50 μl of test drug added to appropriate well
50 μl of assay buffer (basal) or 50 μl of GTPγS (NSB) added to appropriate well
50 μl of GDP added to all wells
100 μl of buffer added to all wells
The FlashPlate was then incubated for 30 minutes at 30C. To start the reaction, 50 μl [35S]-GTPγS (200 pM) was added, and the plates were shaken for 30 seconds. After a further 30 minutes, the reaction was stopped by aspirating the contents of each well. The FlashPlate was then immediately placed on a Packard TopCount Microplate Scintillation and Luminescence Counter to measure bound radioactivity.
Several experiments were performed to determine the optimum conditions for binding in FlashPlate coated with CHO cell membranes expressing the h5-HT1B receptor. The maximum response produced by 5-HT [10 μM] was achieved when approximately 20 μg of membrane protein was used to coat each well and 10 μM GDP was used in the assay (Figure 1a). This is further supported by Figures 1b and 1c, which show complete 5-HT concentration response curves. The best signal-to-noise ratio was seen at the above-mentioned protein and GDP concentrations (Figures 1b and 1c, respectively). Figure 1d represents a concentration response curve for GTPγS and shows that concentrations in excess of 10 nM are adequate to estimate non-specific binding.
Intra-assay variation was investigated by measuring the stimulation of [35S]-GTPγS binding produced by 5-HT [10 μM] in 80 of the FlashPlates 96 wells. Figure 2a shows the data in terms of absolute counts per minute; Figure 2b shows the same data with each row expressed as a percentage of its individual basal value. Preliminary results on inter-assay variation over three experiments indicate only up to 6% error on mean values (Figure 3a). Further experiments would be required to provide more detail on variability. For comparative studies, concentration response curves to 5-HT, GR127935 and SB-224289 were carried out using FlashPlate and the filtration method (Figures 3a and 3b, respectively). In both as says 5-HT appears as a full agonist, GR127935 acts as a partial agonist and SB-224289 appears as an inverse agonist. The maximum response of 5-HT and GR127935 differs between the two methods (Table 1), but the intrinsic activity of GR127935 is comparable. The maximum response produced by SB-224289 is similar for both assay systems. The comparison between the two methods is summarized in Table 2.
This studys aim was to investigate the efficacy of PerkinElmers Basic FlashPlate as a tool for measuring [35S]-GTPγS binding, using CHO cells expressing the human 5-HT1B receptor as the receptor source. After optimal conditions were assessed, significant stimulation of binding was achieved in the presence of an agonist. The FlashPlate system was also sensitive enough to pick up partial and inverse agonists. (Assessment of optimal conditions will be essential when designing other assays on FlashPlate, for different receptors expressed in different cell lines.)
The FlashPlate-based method agreed closely with the conventional filtration method in terms of potencies and intrinsic activities of the compounds tested, but differences in maximum responses were seen. Intra- and inter-assay variation present in the FlashPlate system may play a role in this, and might be attributable to non-uniform coating of membrane protein to wells. The pipetting procedure adopted, or the volume of residual reaction mixture after aspiration may also be factors. This may not be a major concern if the window of response is adequate for the given application. In cases where intra- and inter-assay variation is a problem, it might be addressed by performing assays in duplicate or greater multiples.
Significantly, total assay time was reduced from 12 hours for the filtration method to 4 hours on FlashPlate. Labor time was also reduced significantly. The FlashPlate method also allows greater s cope for automation, which would further reduce labor and increase throughput.
In conclusion, there is great potential for using FlashPlate technology to study [35S]-GTPγS binding in receptor-expressing cell lines.
1. Thomas, D.R., et al . (1995) J. Rec. Sign Trans. Res. 15:199
2. Watson, J., et al . (1996) Eur. J. Pharmacol. 314:365-372