Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/552—Glass or silica

Definitions

• the present invention relates to scintillation proxim ⁇ ty * radioimmunoassay, and more particularly to compositions and methods for improving the sensitivity of scintillation proximity radioimmunoassay.
• Radioimmunoassay Because of its specificity, sensitivity, and technical simplicity, radioimmunoassay has found increased use in recent years in clinical, research, and industrial laboratories. Radioimmunoassay is primarily useful in detecting and quantifying picogram quantities of a reactant of interest.
• This reactant may be a ribonucleic acid, a deoxyribonucleic acid, an antibody, an antigen, a metabolite, a biological receptor antagonist, a biological receptor agonist, a drug, a hormone, or a vitamin.
• the detection and quantification of the above reactants can be useful in the diagnosis of several clinical conditions, i.e., pregnancy, hepatitis, genetic abnormality, or infection.
• the presence of the reactant of interest (AG) is detected and quantified by a reaction in which radiolabelled reactant (AG*) and non- labelled reactant (AG) compete to biochemically bind to a ligand (AB) .
• ligand or (AB) is used to describe any molecule which will selectively and specifically biochemically bind to another specific and distinct molecule identified for the purposes of this invention as the reactant of interest or (AG) .
• AG In order to perform a competitive or direct assay, AG must also be available in a radiolabelled form, e.g., AG*.
• radioimmunoassay 125 I "C and 3 H are the most frequently used radiolabels, with 32 P, "Tc and 57 Co also finding limited use.
• AG*-AB Separation is necessary since it is the concentration of AG*-AB which is indicative of the concentration of AG.
• concentration of AG*-AB is determined by measuring the amount of radioactive energy emitted by the complexed reactants after all free-AG* has been removed.
• Separation is accomplished by either absorption or precipitation, or by utilizing ligands bound to a solid- support structure.
• absorption the complexed reactants are absorbed onto activated charcoal, dextran-coated charcoal, talc, etc.
• the absorbing material is thereafter removed from the medium, typically by centrifugation, and washed several times to remove any excess free-AG*.
• precipitation the complexed reactants are precipitated from solution by a protein precipitator such as (NH.)_SO , ethanol dioxane, or polypropylene glycol.
• the precipitate is then separated and washed to remove excess free-AG*.
• the solid-support is withdrawn from the sample and washed to remove excess free- AG*.
• the radioactivities of the remaining aqueous sample and the separated complexed reactants are thereafter measured and compared to determine the concentration of AG. For example, a high concentration of AG in the sample will result in a high concentration of AG-AB, and accordingly, a low concentration of AG*-AB. Therefore, the overall radioactivity of the separated complexed reactants is low. Thus, the radioactivity emitted by the separated complexed reactants is inversely proportional to the amount of AG in the sample being tested.
• Scintillation counting is a generally known and widely used technique for the measurement of low energy beta emitting radionuclides in addition to gamma and alpha emitters. Scintillation counting is utilized to make quantitative measurements of radioactivity by incorporating a radiolabelled analyte into a solution with a scintillator capable of producing photons resulting from the kinetic interaction of nuclear decay products. For example, a tritium-labeled compound is incorporated in a solution containing a scintillator, energy from the beta decay is transferred with reasonable efficiency to the scintillator upon kinetic interaction. This transferred energy excites the scintillator which thereafter emits multiple photons of light proportional to the energy of the beta particle.
• SPRA is similar to other radioim unoassays, relying on the biochemical binding of radiolabelled reactant AG* and non-radiolabelled reactant AG to a ligand AB. However, instead of separating the bound from the unbound reactants, SPRA takes advantage of the relatively weak radioactive energies emitted by the radiolabel to accurately and instantaneously measure the amount of complexed radiolabelled reactant, from which the amount or presence of the non-radiolabelled reactant can be extrapolated.
• the basic principle of SPRA is shown in FIG. 3. As shown in FIG.
• SPRA generally, in a competitive assay, utilizes AG, AG*, AB and microscopic support bodies. Typically, these support bodies are formed as amorphous bodies, referred to as beads. Attached to the surface of these beads are ligands AB. The ligands are typically bound to the surface of the bead covalently. The beads also contain a suitable scintillator integrated within the body of the bead.
• radiolabels commonly used in radioimmunoassay emit relatively weak energies. For example, beta particles emitted
• the radiolabels in order for a radiolabeled compound to be detected by the scintillation counter, the radiolabels must be brought into close enough proximity to the bead to excite the scintillator within. In a dilute suspension of beads and radiolabeled compounds, few of the radiolabels would be in a close enough proximity to a bead to excite the scintillator, and thus one could predict that very few of the radiolabelled compounds would be detected. However, because the beads have attached to their surface AB, AG* readily attaches to the surface of the bead. This reaction brings the radiolabel of AG* into close enough proximity to the scintillator to excite it. Therefore, the number of radiolabeled compounds detected markedly increases. These results are expressed as levels of radioactivity in the sample as measured by a scintillation counting system.
• a known amount of AG* and an unknown amount of AG are added to an appropriate aqueous suspension containing a plurality of microscopic support bodies, each support body having bound to its surface a multiplicity of AB molecules.
• AG and AG* indiscriminately and competitively biochemically bind to AB thus bringing the radiolabel of AG* into close enough proximity to the bead to allow the radiation emitted by the radiolabel to excite the scintillator within the bead.
• the scintillator When excited, the scintillator emits detectable photons of light, which, in turn, are detected by a liquid scintillation counting system.
• the detected photons are utilized by the liquid scintillation counting system to calculate a level of radioactivity for the sample being assayed. After an appropriate incubation period, wherein the reaction reaches equilibrium, the radioactivity level of the sample is measured by the liquid scintillation counting system, and the resulting measurement is used to determine concentration of AG in the sample. For most assays a standard curve is developed (radioactivity detected vs. concentration of AG) wherein a high level of radioactivity detected corresponds to a low concentration of AG. Accordingly, in a competitive SPRA the level of detected radioactivity is inversely proportional to the concentration of AG in the sample being tested. Thus, by using SPRA the concentration of AG may be continuously and sensitively monitored without the need for a separation or washing procedure.
• SPRA a drawback in SPRA are the support bodies utilized in conducting the assay.
• the support bodies presently utilized in SPRA are complicated structures which are expensive to manufacture. Moreover, these support bodies inefficiently detect the presence of AG*-AB complexes bound to their surface.
• support bodies useful in SPRA are constructed by first forming a porous amorphous microscopic particle, typically referred to as a bead. These particles are typically constructed from acrylamide, acrylic acid, polymers of styrene, agar, agarose, polycarbonate, polypropylene, polystyrene, cellulose acetate or latex. Thereafter, cyanogen bromide is incorporated into the bead for the effective binding of ligands to the surface.
• a scintillating material In order for these materials to be useful in SPRA a scintillating material must be incorporated within their structure. This is accomplished by first choosing a water insoluble scintillator. A solvent is then chosen which is miscible with both water and the scintillator; for instance, dimethyl sulfoxide (DSMO) is an effective solvent if diphenyloxazole (PPO) is utilized as the scintillator. The beads are then placed into the solvent containing an appropriate concentration of the scintillator. The scintillator and solvent are allowed to permeate throughout the body of the porous beads, and the beads are thereafter removed and placed in water, precipitating the scintillator within the body of the bead.
• DSMO dimethyl sulfoxide
• PPO diphenyloxazole
• the beads are removed from the water and washed to remove any excess solvent and precipitated scintillator from their surface.
• the beads are then suspended in a solution containing both a detergent, such as Tween 20, to prevent the beads from sticking, and gelatin to bind any sites on the surface of the porous bead not bound to a ligand. This lowers the non-specific binding of the beads.
• a bactericide such as sodium azide, is applied to the beads to prevent bacteria growth thereon.
• the finished product a bead randomly incorporating a scintillator throughout its body
• Beads presently used in SPRA as described in the referenced European patent application, only randomly scatter scintillator throughout their entirety.
• the amount and distribution of scintillator in each bead is variable, and accordingly, no two beads will respond similarly when in close proximity to a radiolabel.
• particulate energized matter such as a beta particle or auger electron
• a significant number of radioactive events will not be detected and the assay results will be flawed.
• even in beads containing a large quantity of scintillator this "pass through” effect will occur since the beads have scintillator just randomly scattered throughout.
• a particular object of the present invention is to provide a more accurate, less costly method for conducting SPRA which produces more reproducible results.
• a more particular and highly important object of the present invention is to provide an inexpensive support body for use in SPRA which can be mass produced to give reproducible and more accurate results, which until now have been impossible to obtain.
• a reagent for use in SPRA consisting of a support body constructed from scintillating material, the support body having coupled to its surface a multiplicity of ligands.
• the ligands are capable of selectively biochemically binding to a reactant of interest.
• Another aspect of the present invention is directed to the process of assaying an aqueous suspension containing a known concentration of a radiolabelled reactant and an unknown concentration of a non-radiolabelled reactant.
• the reactant is selected from the group consisting of antibodies, antigens, deoxyribonucleic acids, ribonucleic acids, biological receptors, biological receptor antagonists, biological receptor agonists, steroids, enzymes and vitamins.
• the inventive process includes several steps.
• One step in the inventive method is adding to an aqueous suspension a plurality of scintillating support bodies consisting of a scintillating material and having coupled to their surfaces a multiplicity of ligands.
• the ligands are capable of selectively biochemically binding to the reactant, regardless of the presence of a radiolabel.
• the radiolabeled reactant emits radiation energy capable of exciting a scintillating support body upon biochemically binding to a ligand.
• the scintillating support body emits detectable photons of light when excited.
• Another step in the inventive process is detecting the photons of light emitted by the plurality of scintillating support bodies in the aqueous medium. The photons of light detected are indicative of the concentration of the non-radiolabeled reactant.
• a further aspect and a preferred embodiment of the present invention is directed to a process for assaying an aqueous sample to determine the concentration of a reactant of interest using scintillation proximity radioimmunoassay.
• a first step in the inventive process is providing a plurality of scintillating support bodies consisting of yttrium silicate and from about 0.1 to about 10.0% by weight of an inorganic salt of cerium.
• a multiplicity of ligands are then coupled to the surface of the support bodies. The ligands are capable of selectively biochemically binding a reactant of interest, regardless of whether it is radiolabelled.
• a known quantity of the reactant is chemically coupled to a radiolabel, and is thereafter mixed in an aqueous suspension with an unknown quantity of non-radiolabelled reactant and a plurality of support bodies.
• Both radiolabelled and non-radiolabelled reactants compete to biochemically bind to the ligands coupled to the surface of the support body.
• the radiolabelled reactant emits radiation energy capable of exciting the scintillating support body upon biochemically binding to a ligand.
• the scintillating support body emits detectable photons of light when excited.
• the photons of light emitted by the plurality of scintillating support bodies are detected.
• the photons of light detected are indicative of the concentration of the non-radiolabeled reactant.
• the support bodies, reagents, and assay processes of the present invention may be utilized in conjunction with any ligand-reactant combination that specifically biochemically binds together and in which the reactant may be radiolabelled without effecting its specificity for the ligand.
• Ligand- reactant combinations with which the present invention is especially useful include: (1) receptor-agonist; (2) receptor-antagonist; (3) enzyme-substrate or co-factor; (4) RNA-DNA; and (5) DNA-DNA. It is understood that in the present invention either element may serve as a ligand or a reactant.
• FIG. 1 is a schematic representation of the specific biochemical binding of the reactant of interest (AG) to a ligand (AB) which, in turn, is coupled to the surface of a scintillating support body, represented as a sphere;
• FIG. 2 is a schematic representation of a direct scintillation proximity radioimmunoassay, wherein the radiolabeled reactant of interest (AG*) binds to a ligand (AB) , thus bringing the radiolabel of AG* into a close enough proximity to the scintillating support body, represented as a sphere, to allow the emitted radiation of the radiolabel to excite the scintillating support body, thereby causing the scintillating support body to emit photons of light which are detected by a detector;
• FIG. 1 is a schematic representation of the specific biochemical binding of the reactant of interest (AG) to a ligand (AB) which, in turn, is coupled to the surface of a scintillating
• FIG. 3 is a schematic representation of a competitive scintillation proximity radioimmunoassay, wherein the non- radiolabeled reactant (AG) and the radiolabeled reactant (AG*) compete to biochemically bind to the ligand (AB) which is coupled to the surface of the scintillating support body, represented as a sphere, AG* upon binding to AB brings the radiolabel of AG* into a close enough proximity to the scintillating support body to allow the radiolabel to excite the scintillating support body, thereby causing the scintillating support body to emit photons of light which are detected by a detector; and
• FIG. 4 is a graphic representation of trials conducted to determine the counting efficiencies of scintillating support bodies having diameters of 3, 10 and 100 microns.
• scintillating support bodies 10, 100 and 200 are represented as suspended in an aqueous solution.
• the preferred suspending agent is glycerol.
• the scintillating support bodies consist of a scintillating material. Because SPRA is performed in an aqueous medium, the preferred scintillating material must be insoluble in water.
• the preferred scintillating materials of the present invention are base glasses which when appropriately activated or doped respond as scintillators and emit detectable photons of light when excited by the kinetic interaction of nuclear decay particles, or calcium fluoride.
• the preferable activating materials or dopants are selected from the group comprising Mn, Cu, Pb, Sn, Au, Ag, Sm, and Ce, as well as all other rare earths commonly used to activate scintillators with the most preferred activating material being Ce (Cerium) .
• the scintillating support bodies of the present invention consist of yttrium silicate glass activated with from about 0.1 to about 10.0 percent by weight of an inorganic cerium salt. Cerium is added to the yttrium silicate as an inorganic salt, preferably as the oxide, carbonate or chloride.
• the scintillating support bodies of the present invention are most preferably formed as amorphous particles referred to for the purposes of this invention as beads.
• cerium activated yttrium silicate glass is purchased from one of several manufacturers, or is prepared by methods well known in the art of glass manufacturing. Glass particles in the range of from about 3 to about 100 microns in size are preferably utilized in the practice of the present invention. However, particles having a diameter of about 10 microns are most preferred in the practice of the present invention. According to one experiment, cerium- activated yttrium silicate glass beads of about 3, 10, and 100 microns in diameter were tested for counting efficiencies in SPRA. These trials were conducted using a liquid scintillation counter, 3 H, 14 C, and 32 P.
• the scintillating support bodies of the present invention because they are constructed entirely from activated scintillating material, increase the accuracy of SPRA. Unlike other support bodies presently used in SPRA, which have scintillator scattered randomly throughout, alpha particles, beta particles, or auger electrons will not pass through the scintillating support body of the present invention without impacting and exciting the scintillating material. Accordingly, the frequency at which radioisotopes are in close proximity to the support body is more accurately detected and recorded.
• the reactant of interest AG is represented in FIGS, l and 3 as elements 11 and 210, respectively.
• AG is preferably selected from the group consisting of antibodies, antigens, deoxyribonucleic acids, ribonucleic acids, biological receptors, biological receptor antagonists, biological receptor agonists, steroids, enzymes, and vitamins.
• antigens or antibodies are the most preferred reactants of the present invention.
• the radiolabel is preferably selected from the group of radioisotopes consisting of 3 H, 125 I, "C, 131 I, 75 Se, 3 P and "Co. However, most preferably, the radiolabels are 3 H and 125 I.
• the radiolabelled reactant (AG*) is represented as elements 120 and 220 of FIGS. 2 and 3 respectively. AG* is preferably produced by chemically coupling AG to a radiolabel. It will be apparent to those skilled in this art that the methods and procedures for coupling radiolabels and reactants are well known and do not require further elaboration.
• the radiolabel AG* emits either particulate or electromagnetic radiation.
• the radiolabel utilized in the practice of the present invention emits short range particulate radiation, i.e., alpha particles, beta particles, auger electrons or the like.
• the paths of these short range energies are indicated by arrows 130, 140 or 230, 240 of FIGS. 2 and 3, respectively.
• Arrows 130 and 230 represent alpha particles, beta particles or auger electrons whose paths terminate in the aqueous suspension without impacting and exciting the scintillating material of the scintillating support body.
• Arrows 140 and 240 represent the paths of alpha particles, beta particles, or auger electrons which impact the scintillating material of the scintillating support body, thereby exciting the scintillator.
• the scintillator thereafter emits detectable photons of light, represented by arrows 150 or 250 of FIGS. 2 and 3, respectively.
• the emitted photons are received by a detector 190, 290, preferably the photomultiplier tube of a liquid scintillation counter.
• the radiolabel of AG* is brought into close enough proximity to excite the scintillating support body by the ligands (AB) coupled to the surface of the support body, the ligands (AB) are represented as elements 16, 160 or 260 of FIG. 1, 2 and 3, respectively.
• Ligands (AB) useful in the practice of the present invention indiscriminately and competitively biochemically bind to AG and AG*.
• the ligand is preferably selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, biological receptor, biological receptor antagonist, biological receptor agonist, enzyme, vitamin, steroid and hormones.
• Fig. 2 represents a direct assay of AG* 120.
• the scintillating support body 100 is coupled with a multiplicity of AB 160, capable of selectively biochemically binding to AG*.
• AG* 120 is mixed with a plurality of scintillating support bodies 100, causing AG* to bind AB 160.
• the resulting complex represented as element 170, brings the radiolabel of AG* 120 into close enough proximity to the scintillating support body to allow the radiation emitted 140 by the radiolabel, to excite the scintillating support body, thereby causing the scintillating support body to emit photons of light 150.
• the photons 150 emitted are detected by a detector 190. From the detected photons of light a radioactivity level is determined.
• the radioactivity level measured is directly related to the formation of the AG*-AB complex 170. As the number of AG*-AB complexes 170 increases, the radioactivity level measured also increases proportionately, which in turn, is indicative of the amount of AG* 120 in the sample being assayed.
• a known amount of AG* 220 and an unknown amount of AG 210 are added to an aqueous suspension containing a plurality of scintillating support bodies 200, each having a multiplicity of ligands (AB) 260 bound to its surface.
• AG* 220 and AG 210 thereafter compete to bind to AB 260.
• the reactants AG 210 and AG* 220 bind to AB 160 in proportion to their relative concentrations in the suspension, regardless of the presence of radiolabel.
• AG* 220 binds to AB 260 to form the complex AG*-AB 270.

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• 1990
  • 1990-11-14 WO PCT/US1990/006646 patent/WO1991008489A1/en not_active Application Discontinuation
  • 1990-11-14 EP EP19910901232 patent/EP0502123A4/en not_active Withdrawn

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