Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1991-09-23
1993-09-21
Kepplinger, Esther L.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436524, 436528, 436534, 436804, 25230117, G01N 33543
Patent
active
052468690
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for the simultaneous assay of two or more radioactively labeled ligands.
The U.S. Pat. No. 4,568,649 describes a proximity assay for ligands whereby biomolecules capable of specifically binding the ligands are attached or coated onto scintillating support medium. Such biomolecules are e.g. immunoglobulins, receptors or nucleic acids. The support medium can be e.g. in the form of small beads.
According to the assay, the solution containing a known amount of radioactively labeled ligand and an unknown amount of unlabeled, but otherwise similar investigated ligand, is incubated with the beads. Alternatively, an amount of radioactively labeled ligand can be incubated with the beads. If the isotope used as the label is low-energetic, e.g. tritium, the emitted electrons or beta-particles have ranges less than a few micrometers and only the label that gets bound and thus gets in close proximity to the scintillating material produces bursts of light called scintillations. These can be detected with a liquid scintillation counter. The unbound label remains too far to produce scintillations. The competition between labeled and unlabeled ligands results in that the more there is investigated, i.e. unlabeled, ligand present in the solution, the less of the labeled ligand will get bound and the less scintillations are produced.
A great advantage of the described assay is that it does not require separation of the unbound and bound label fractions. A disadvantage is that while being suspended in the solution the beads can settle down affecting adversely in binding and output of scintillation light. Dispensing the beads, e.g. by pipetting, can also be problematic.
As an improvement a support structure has been proposed in which scintillating compounds called scintillators are processed in or onto fibers forming a filter mat. The mat also retains liquid by capillary forces.
Although no separation steps are necessary in proximity assays, the samples still require dispensing into different sample containers in different ligand assays. If several ligands are to be assayed from the same sample, the total sample volume may limit the number of assays that can be performed. In a busy laboratory the manipulation of many sample containers and the availability of counting facilities may limit throughput. Although usage of other isotopes, such as .sup.14 C and .sup.35 S, as extra labels could be possible in principle, beta-particle ranges of their emissions are long and can produce scintillations even without the label being bound. A consequence would be increase in the background count rate.
The objective of the present invention is to develop a method that does not have the drawbacks mentioned above. The method described by the invention is characterized in that ligands are assayed simultaneously in the same sample by using proximity assay so that for determining the ligands support materials with different scintillation characteristics are employed, each said support material having attached onto it molecules specifically binding one of said ligands.
According to one advantageous embodiment the support material consists totally or partially of scintillator.
With the invention several ligands can be assayed simultaneously. Furthermore, a great advantage is that each ligand can be labeled with the same low-energetic isotope, such as tritium. The contributions of each ligand to the measured scintillation count rate can be determined if each binding molecule type is attached onto different scintillating support material, which differ in some measurable characteristic of the scintillation light. The support material can be in the form of e.g. beads or fibers.
One scintillation characteristic that can be utilized in the present invention is the different light output of different scintillators upon excitation by electrons of the same energy. This manifests as a characteristic pulse height distribution or spectrum when counted in a liquid scintillation counter. Consequently, according to another advanta
REFERENCES:
patent: 4016250 (1977-04-01), Saxena
patent: 4568649 (1986-02-01), Bertoglio et al.
George T. Reynolds, "Solid and Liquid Scintillation Counters", Nucleonics, vol. 10, No. 7, pp. 46-53, (1952).
"Latest Developments in Scintillation Counting", Nucleonics, vol. 10, No. 3, pp. 32-41, (1952).
Bosworth et al., "Scintillation Proximity Assay," Nature, vol. 341, pp. 167-168, (1989).
Oikari Timo
Potter Colin
Warner Gerald
Kepplinger Esther L.
Wallac Oy
Wolski Susan C.
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