Application Notes. MicroBeta INTRODUCTION.

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1 Drug Discovery Research Clinical Screening MicroBeta Application Notes Colour quench correction in Scintillation Proximity Assays Using ParaLux Count Mode K.T. Hughes 1, J.C. Ireson 1, N.R.A. Jones 2 and P. Kivelä 2 ( 1 Amersham International plc., Forest Farm, Whitchurch, Cardiff. CF4 7YT, Wales UK, 2 PerkinElmer Life Sciences, P.O.Box 10, FIN-20101, Turku, Finland) INTRODUCTION The MicroBeta TriLux is a multi-detector instrument for liquid scintillation and luminescence counting. It can simultaneously count up to 12 samples at a time in microtitration plates or in tubes. There are three MicroBeta models; 16 and 32 shelf capacity instruments, and a version fitted with an external sample loading tray that can be part of a fully automatic robot system. Each detector in the MicroBeta comprises two photo-multiplier tubes (PMTs), one is positioned above the sample and one below. The PMTs are connected in coincidence circuitry, a robust and well established method for liquid scintillation counting. Scintillation Proximity Assay (SPA) represents an innovative approach to high throughput screening because it allows the rapid and sensitive assay of a wide variety of molecular interactions in a truly homogeneous system. SPA and MicroBeta counting systems can be readily automated. Much SPA involves the screening of natural products or colored compounds, and as a consequence of the non-separation method, this can result in many different and highly colored samples within one assay. For the purposes of high throughput screening, it is essential that an instrument can accurately assess the level of color quench (or counting efficiency) and correct the observed count rate (CPM) to the true activity (DPM). is bound to the bead, it is brought into close enough proximity to enable it to stimulate the scintillant to produce light. Unbound radioactivity is too distant from the scintillant and the energy released is dissipated before reaching the bead. In general, the beads sink towards the bottom of the microtitration plate well and are therefore much closer to the lower PMT in the MicroBeta. The magnitude of color quench is dependent on the path length of the light in the color and therefore the effect of color is much smaller in the lower PMT than in the upper one. In this sense, SPA represents a unique sample type for which a novel counting method has been developed. ParaLux Count Mode takes full advantage of the lower PMT in two ways. Firstly, the difference in count rates between coincidence and non-coincidence events in the lower PMT is used to determine the counting efficiency and secondly, considerably higher counting efficiency can be achieved by including non-coincident counts observed solely in the lower PMT in the overall count rate. All existing methods of quench correction ascertain counting efficiency by determining the position of the isotope spectrum. For ex- In SPA scintillant is incorporated into small fluomicrospheres. These microspheres or beads are designed in such a way as to bind specific molecules. If a radioactive molecule

2 No. of counts No. of counts No. of counts ample, the MicroBeta uses the Spectral Quench Parameter of the Isotope (SQP(I)) which is the multi-channel analyzer (MCA) channel number that equates to the mid point of the isotope spectrum. The shift in position of the isotope spectrum (in terms of MCA channel number or energy) is therefore a limiting factor. In the case of tritium, the movement of the isotope spectrum is quite small and therefore the SQP(I) has a limited sensitivity to changes in counting efficiency and also has a finite dynamic range. 50 SQP(I) 0 Fig. 1 The SQP(I) quench indicating parameter is based on the position of the isotope spectrum Fig. 2A Carbon-14 color quench series. SQP(I) provides a good indicator because there is dynamic movement of the spectra with change in counting efficiency Fig. 2B Tritium color quench series. Minimal shift in spectrum position results in the SQP(I) being relatively insensitive to changes in counting efficiency, especially with heavily color-quenched samples. PARALUX COUNT MODE ParaLux Count Mode represents a genuine improvement in counting efficiency and quench parameter. Twin MCA technology is applied to construct simultaneously two spectra of each sample (see figure 3.). MCA-1 is connected in conventional coincidence circuitry. This accepts all beta decay that is of sufficient energy to produce many photons that are in turn detected by both PMTs. MCA-2 stores the beta decay that is detected by the lower PMT only. Counts registered below channel 150 in MCA-2 are discarded because these include unwanted background counts that are a feature of all PMTs. It is possible to change this channel setting to optimize for efficiency or background. (See Operating Instructions.) ParaLux includes two different counting modes, high efficiency and low background. Both modes use a new quench parameter, the Asymmetric Quench Parameter of the Isotope (AQP(I)). This is calculated from the ratio of the counts in the two MCAs. Figure 4 shows that, for tritium, AQP(I) commands a much wider scale than spectrum position based quench parameters and is therefore more sensitive to changes in counting efficiency, especially with heavily quenched samples. In high efficiency mode, the contents of MCA-1 and MCA-2 are added together. With a default window setting, this results in an increase in counting efficiency of 10 % for uncolored samples and up to 300 % for colored samples. The count rate in low background mode is acquired from MCA-1 only and is therefore identical to conventional coincidence counting. In high throughput screening there may be a variety of different colours present in an assay and it is therefore necessary that a quench curve constructed from one color is suitable for quench correction of all other colors. Figure 5. shows that AQP(I) curves of various colors do overlap, and that therefore quench correction can be made with a single curve. 2

3 MCA-1, Coincidence PM Coincidence circuit PM MCA-2, Bottom PMT AQP(I) = MCA-1 / MCA-2 High Efficiency CPM = MCA-1 + MCA-2 Low Background CPM = MCA-1 Fig. 3 Principle of ParaLux count mode ParaLux Count Mode, AQP(I) H-3 Efficiency /% Coincidence, SQP(I) Fig.4 Comparison of tritium SPA color quench series counted with conventional coincidence circuitry and ParaLux high efficiency mode. Relative efficiency Yellow Red Orange Green Blue AQP(I) Fig.5 ParaLux low background count mode. Tritium SPA quench curves. AQP(I) provides overlapping color quench curves. This permits the use of a single quench series for DPM calculations. EXPERIMENTAL Two experiments were devized to compare ParaLux Count Mode with the traditional spectrum position, SQP(I), quench correction method. To simulate real assay conditions, two receptor binding assays using 3 H and 125 I labels were chosen. The activity in each was representative of count rates used in high throughput laboratories. The % quench figures quoted are based on coincidence counting. 3

4 [ 3 H] CHO M1 RECEPTOR ASSAY A quench curve was set up using a 3 H color quench kit (Amersham, TRKQ 7080) in a Wallac plate and allowed to settle overnight. The assay was twelve replicates of total binding wells from a 3 H receptor assay in the presence of various levels of quench. The assay consisted of 50 µl of buffer or tartrazine, 50 µl l-quinuclidinyl [phenyl-4-3 H] benzilate ([ 3 H]QNB, Amersham, TRK 604) (5.1 nm final concentration), 50 µl CHO cell membranes transfected with rat muscarinic M1 receptors (100 µg) and WGA- PVT SPA beads (Amersham) (2 mg) in 50 mm Tris-HCl buffer, ph 7.4, containing 0.1 % (w/ v) BSA and 5 mm MgCl 2. The plate was shaken for four hours and then left overnight for the beads to settle prior to counting. Table 1. Twelve replicates of total binding wells from [ 3 H] CHO M1 receptor assay 4

5 [ 125 I] ET-1 RECEPTOR ASSAY A quench curve was set up using an 125 I color quench kit (Amersham, RPAQ 4030) in a Wallac plate and allowed to settle overnight. The assay was twelve replicates of total binding wells from an 125 I receptor binding assay in the presence of various levels of quench. The assay consisted of 50 µl of buffer or tartrazine, 50 µl [ 125 I]endothelin-1 (Amersham, IM 223) (39 pm final concentration), 50 µl porcine lung membranes (5 µg), and WGA-PVT SPA beads (Amersham) (1 mg) in 50 mm Tris-HCl buffer, ph 7.4, containing 0.1 % (w/v) BSA, 10 mm MnCl 2, and 1mM EDTA. The plate was shaken for four hours and then left overnight for the beads to settle prior to counting. Table 2. Twelve replicates of total binding wells from [ 125 I] ET -1 receptor assay 5

6 RESULTS See tables 1 and 2 CONCLUSION Precision in QCCPM values are clearly improved in heavily colored samples in both Low Background and High Efficiency ParaLux Count mode. ParaLux Count Mode is an authentic advance in SPA counting. Compared to other techniques counting efficiency can be increased by several hundred percent. The new, ultrasensitive quench parameter, AQP(I), provides superior quench correction at considerably higher levels of color. Time-consuming and costly dilution and recounting of difficult samples can thereby be avoided. Improved quench correction precision could lead to overall shorter count times. 6

7 Counting Scintillation Proximity assay (SPA) on the Wallac MicroBeta TriLux Warning: For research only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Instructions for the safe handling and use of Scintillation Proximity Assay beads are provided in the safety warning and precautions section of the pack leaflet accompanying the product. 1. COUNTING WITHOUT COL- OUR QUENCH CORRECTION When performing SPAs that are not quenched due to the presence of colored compounds, the results are usually expressed as counts per minute (CPM) This is achieved by utilizing the CPM counting mode of the counter. The performance of photomultiplier tubes is never identical and so a calibration procedure is needed before counting unknown samples. This procedure is called Normalization and involves counting the same sample in each of the detectors. In this way the relative efficiency of each of the detectors can be determined. Once the normalization samples have been counted in the MicroBeta, all the relevant data are automatically stored for future use within a Counting Protocol. Any normalization protocol can be linked to any number of counting protocols. Notes Normalization data should be generated for different isotopes and plate types being used. Isotopic and optical crosstalk is insignificant for the radioisotopes employed in bead based assays ( 3 H, 125 I, 33 P and 35 S) performed in the recommended microtitration plates (part numbers and ), and therefore is required no correction) The observed count rate increases as the beads settle, therefore assays should be counted under stable conditions (either in suspension or settled). Yttrium silicate beads settle rapidly (within a few minutes), therefore these assays should be counted with the beads settled. PVT beads settle over a longer period of time (usually several hours). The rate of settling depends on the type of application and buffer components present. In some assays the beads may be prevented from settling by addition of, for example, glycerol, to adjust the density of the buffer. Assays employing 33 P and 35 S must be counted with the beads settled to maximize the signal and reduce the non-proximity effect; for assays employing PVT beads this can be achieved by settling for several hours or gentle centrifugation (typically ten minutes at 800 xg). ParaLux Count Mode normalization protocols must include background subtraction. The background sample should be a black plate that is counted before the plate containing the normalization samples. Instrument background will then be automatically subtracted from the unknown samples and therefore background subtraction should not be selected in the counting protocol. See SPA Spectra and Operating Instructions for window setting and protocol editing. 7

8 2. COUNTING WITH COLOR QUENCH CORRECTION SPA is an homogenous technique with no separation step. When performing assays with colored samples, the quenching effect of the color must be corrected for. This is achieved by utilizing the DPM counting mode of the instrument. This procedure is called Standardization and involves a series of samples containing different levels of color and a constant quantity of radioactivity. Simultaneously, the instrument carries out detector normalization. Once the standardization samples have been counted in the MicroBeta, all the relevant data are automatically stored for future use within a Counting Protocol. Any standardization protocol can be linked to any number of counting protocols. Notes Quench curves are set up using a series of coloured standards (for example, tartrazine) and the same quantity of radioactivity in each well of the curve. For assays involving the use of PVT beads and 3 H- or 125 I- labels, the use of the appropriate color quench kit from Amersham (TRKQ 7080 for 3 H and RPAQ 4030 for 125 I) is strongly recommended. Instruments equipped with ParaLux Count Mode (software version 4.3 or greater) will automatically select ParaLux when SPA and DPM are selected. The color quench curve should be set up with the same weight of beads, assay buffer and total volume as employed in the screening assay. A separate quench curve should be set up for each screen and on each instrument. A separate quench curve should be set up for each type of microtitration plate being used. It is essential to ensure that a smooth colour quench curve is obtained. A single outlying point may be edited out. Generally, the best precision of correction is obtained with the automatic smoothing spline curve fit. During programming of the counter, the counter will ask for the DPM value of the standards to be specified. In most screening assays the results are expressed as a relative measurement e.g. % inhibition or B/B 0, and therefore quench corrected CPM (QCCPM) values may be obtained by inserting the count rate of the unquenched standard. However, if there is a requirement for absolute DPM measurements, the unquenched standard should be counted in another appropriately standardized instrument and the absolute DPM value inserted. See Amersham s color quench kit pack leaflet for further details. MicroBeta reports QCCPM as DPM. A new header (for example QCCPM where QCCPM= DPM) can be created in the Output section of the protocol editor. ParaLux Count Mode standardization protocols must include background subtraction. The background sample is an empty plate and should be the same type that is used in the assay. This is counted before the standardization samples. Instrument background will then be automatically subtracted from the unknown samples and therefore background subtraction should not be selected in the counting protocol. Generally, using the SQP(I) quench indicator, the MicroBeta TriLux can correct with acceptable accuracy up to approximately 70% quench. (All replicates are corrected to within 10% of DPM in coincidence counting mode.) Improved precision with highly colored samples (up to 90% quench) can be obtained with ParaLux Count Mode. GENERAL PROCEDURE Instrument performance can be checked for counting efficiency and detector uniformity by following instructions detailed in the color quench kit pack leaflet. AQP(I) variation between detectors should be less than ± 8 units. Decide whether the quench curve will be installed using settled beads or beads in suspension. (See color quench kit pack for details.) See SPA Spectra and Operating Instructions for window setting and protocol editing. 8

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11 OPERATING INSTRUCTIONS Illustrations are from Windows Workstation vers

12 MICROBETA TRILUX SPA SPECTRA Tritium YS Tritium PVT Default window Iodine-125 YS Iodine-125 PVT Default window Sulphur-35 PVT Phosphorus-33 PVT World Headquarters: PerkinElmer Life Sciences, 549 Albany Street, Boston, MA USA (800) In Europe: PerkinElmer Life Sciences, B-1930 Zaventem, Belgium, P.O.Box 10, FIN Turku, Finland, MicroBeta is a registered trademark of PerkinElmer, Inc. Amersham is a trademark of Amersham International, plc. Products for SPA (Scintillation Proximity Assay) are available from Amersham International, plc. Scintillation Proximity Assay Technology is covered by US Patent No , European Patent No and by Japanese Patent Application No. 84/ , Jan 2001 Printed in Finland by Offset House Oy Naantali 2001