Scintillation proximity test

Details Scintillation proximity test Scintillation proximity test

2000-05-16

2003-02-25

Jones, W. Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C250S485100, C250S486100, C250S487100, C422S068100

Reexamination Certificate

active

06524786

ABSTRACT:

This invention concerns scintillation proximity tests, that is to say assays or other experiments involving the scintillation proximity principle.
Current scintillation proximity assay (SPA) technology involves the use of scintillant beads made from either cerium-doped yttrium silicate (Y
2
SiO
5
:Ce) (hereafter referred to simply as yttrium silicate or YSi) or polyvinyltoluene (PVT) containing an organic scintillant such as PPO. Assays are carried out in aqueous buffers using radioisotopes such as
3
H,
125
I,
14
C,
35
S or
33
P, that emit low-energy radiation, the energy of which is easily dissipated in an aqueous environment. For example, the electrons emitted by
3
H have an average energy of only 6 keV and have a very short path length (~1 &mgr;m) in water. If a molecule labelled with one of these isotopes is bound to the bead surface, either directly or via interaction with another molecule previously coupled to the bead, the emitted radiation will activate the scintillant and produce light. The amount of light produced, which is proportional to the amount of labelled molecules bound to the beads, can be measured conveniently with a liquid scintillation (LS) counter. If the labelled molecule is not attached to the bead surface, its radiation energy is absorbed by the surrounding aqueous solvent before it reaches the bead, and no light is produced. Thus, bound ligands give a scintillation signal, but free ligands do not, and the need for a time-consuming separation step, characteristic of conventional radioligand binding assays, is eliminated. The manipulations required in the assays are reduced to a few simple pipetting steps leading to better precision and reproducibility.
PCT WO 91/08489 (Packard Instrument Company Inc.) describes a support body for use in scintillation proximity radioimmunoassay, the support body being constructed of a scintillating material, having coupled to its surface a multiplicity of ligands such a antigens, antibodies, etc. capable of selectively binding a reactant of interest. Preferably the support bodies consist of yttrium silicate activated with an inorganic cerium salt such as the oxide, carbonate, or chloride.
WO 94/26413 concerns the study of cellular and biochemical processes in living cells or in components of cells. Specifically described are devices and methods for the study of cellular and biochemical processes, using the scintillation proximity principle.
The simplicity of the scintillation proximity format allows almost complete automation of assays using robotic sample processors and microtitre plate scintillation counters. Consequently, SPA technology is capable of high throughput, which is particularly valuable in the case of drug- or sample-screening assays. SP assays have been carried out routinely in 96-well microtitre plates which are counted 6 wells at a time in specially designed microtitre plate scintillation counters. The search for increasingly higher throughput has led the manufacturers of these counters to produce instruments capable of counting 12 wells at a time, thus doubling throughput. It has also seen the advent of 384-well plates, although at present these can still only be counted 12-wells at a time.
A problem associated with SPA is that of colour quenching, caused by the presence in the assay medium of coloured compounds that absorb the light emitted by the current SPA bead types. Colour quenching attenuates the signal, thereby decreasing signal to noise and hence the sensitivity. Many of the samples being screened by SPA assays are coloured and the majority of these are yellow or brown in colour and absorb light in the blue region of the visible spectrum. Both PVT- and Y
2
SiO
5
:Ce-based SPA beads emit light in the blue region (maximal emission normally in the range 350 nm-450 nm) and so are susceptible to this effect.
An alternative detection system suitable for use in low to ultra-low light level imaging applications in the biological and biomedical sciences is CCD (Charge Coupled Device) Detection which has been used, for example, in assays which involve chemiluminescent, bioluminescent and fluorescence detection. Applications include immunoassays (Hooper et al, J.Biolum.Chemilum., 9, 113-122, (1994)), and the analysis of specific fluorescent dye-labelled nucleic acids by hybridisation following electrophoretic separation of nucleic acid samples (EP 214713 to Astromed Ltd.). Ultra low-light imaging using CCD technology is quantitative and fast and the new generation of imaging instruments which use CCD cameras for detectors can image the whole of a plate at once and so have great potential for increasing sample throughput compared with microtitre well plate scintillation counters. Area imaging, i.e. the simultaneous imaging by CCD of all wells in a microtitre well plate is considered to be particularly advantageous when used in conjunction with high well-density plates containing 96, 384, 864, or more wells, since the time required to make measurements is significantly reduced compared with conventional scintillation counting techniques.
Imaging technology, in particular area imaging, has also been applied to isotopically labelled materials as an alternative to autoradiography. This approach has been most widely used in applications such as the quantitative analysis of proteins by 2-D gel electrophoresis (Patterson, et al, Biotechniques, 15(6), 1076, (1993)) and receptor localisation (Tang, et al, Biotechniques, 18(5), 886, (1995)). An imaging plate, coated with a radiation sensitive agent (e.g. strontium sulphide/samarium/cerium or barium fluorobromide/europium) is exposed to a radiolabelled sample and an image is formed due to radiation incident on the lanthanide metal coating of the plate. Following exposure, the image is read by means of an imaging plate reader.
CCD detection of SPA counts has also been reported (Englert, D., Society for Biomolecular Screening, Second Annual Conference Oct. 14-17, (1996), pp209-221) using PVT-based microspheres. However, the photon count from the SPA wells was not sensitive enough to enable usable results to be obtained, due to low light output of the beads, sub-optimal signal detection capability of the system, as well as quenching by coloured samples. For conventional scintillation counting, instruments can be calibrated to take into account colour quenching. However, in the case of CCD detection using conventional SPA beads and under normal assay conditions, the number of photons detected per disintegration was insufficient to enable determination of quenching levels and quench correction was not possible. To date there appear to be no reports of working assays in which sample detection and measurement was obtained using this technique.
The present invention seeks to overcome the dual problems of low sensitivity of current CCD-based detection as well as colour quench in conventional SPA bead technology. The invention provides use in a scintillation proximity test of a phosphor that has an emission maximum of 480 nm-900 nm, and of a charge coupled device for detecting radiation emitted by the phosphor.
A scintillation proximity test is a test in which a surface carrying a phosphor is contacted with a body of fluid containing a radioisotope. Part of the radioisotope becomes immobilised adjacent the surface; the remainder of the radioisotope remains dispersed or dissolved in the fluid. The mean free path of electrons or other particles or radiation resulting from radioactive disintegrations of the radioisotope are small relative to the dimensions of the body of fluid, whereby that part of the radioisotope immobilised adjacent the surface is capable of exciting phosphor carried by the surface, but that part of the radioisotope dispersed or dissolved in the fluid is generally too far from the surface to be capable of exciting phosphor carried by the surface.
The surface may be massive, as for example a wall of a vessel or wells of a multiwell or microtitre plate; or particulate, as for example threads or beads. The phosphor may be present as a coating applied on

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