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Patent

Fluorescence quenching with immunological pairs in immunoassays

TL;DR: In this article, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium.
Abstract: Immunoassays employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium. In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The composition is irradiated with light at a wavelength, absorbed by the fluorescing molecule and the amount of fluorescence determined. By employing appropriate standards, the presence and amount of the ligand can be determined.
Citations
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Patent
04 Aug 1980
TL;DR: In this article, two-site or "sandwich" immunometric assay techniques for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies are described and compared to conventional assays using polyclonal antibodies.
Abstract: "Two-site" or "sandwich" immunometric assay techniques for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies are described and compared to conventional assays using polyclonal antibodies. Also described are inhibition assays using complexes of antigens with a monoclonal antibody.

1,540 citations

Patent
12 Oct 2001
TL;DR: In this paper, a method for detecting a nucleic acid is described. The method comprises contacting the nucleic acids with one or more types of particles having oligonucleotides attached thereto, and a detectable change (preferably a color change) is brought about as a result of the hybridization of the oligon nucleotides on the nanoparticles to the nucleus.
Abstract: The invention provides methods of detecting a nucleic acid. The methods comprise contacting the nucleic acid with one or more types of particles having oligonucleotides attached thereto. In one embodiment of the method, the oligonucleotides are attached to nanoparticles and have sequences complementary to portions of the sequence of the nucleic acid. A detectable change (preferably a color change) is brought about as a result of the hybridization of the oligonucleotides on the nanoparticles to the nucleic acid. The invention also provides compositions and kits comprising particles. The invention further provides methods of synthesizing unique nanoparticle-oligonucleotide conjugates, the conjugates produced by the methods, and methods of using the conjugates. In addition, the invention provides nanomaterials and nanostructures comprising nanoparticles and methods of nanofabrication utilizing nanoparticles. Finally, the invention provides a method of separating a selected nucleic acid from other nucleic acids.

1,043 citations

Patent
10 May 1996
TL;DR: In this paper, the authors proposed a hybridization of the target and complement sequences to shift the probe to an open conformation, which is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label.
Abstract: Unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences comprise a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. Also, universal stems and kits useful for constructing said probes. Also, assays utilizing said probes and kits for performing such assays. Also, such probes capable of allelic discrimination.

842 citations

Patent
06 Nov 2000
TL;DR: In this article, a high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention of the algorithm.
Abstract: Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

697 citations

Patent
24 May 2002
TL;DR: In this paper, a semiconductor nanocrystal compound is described capable of linking to an affinity molecule, which can be used to determine the presence of a detectable substance in a material.
Abstract: A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation—when excited by an electromagnetic radiation source or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affinity molecule. The compound is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. subsequent exposure to excitation energy will excite the semiconductor nanocrystal in the probe causing the emission of electromagnetic radiation. Further described are processes for respectively: making the luminescent semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

697 citations

References
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Patent
17 Dec 1973
TL;DR: In this paper, a novel immunoassay is provided, as well as particular reagents, for determining the presence of a ligand, where a Reagent is employed having at least two epitopes, one of the epitopes being common with the ligand and the other epitope being a foreign epitope.
Abstract: A novel immunoassay is provided, as well as particular reagents, for determining the presence of a ligand. A Reagent is employed having at least two epitopes, one of the epitopes being common with the ligand, and the other epitopes being foreign to the ligand. The two epitopes are positioned in the Reagent so that antibody bound to one of the epitopes sterically inhibits the binding of antibody to the second epitope. In carrying out the assay, the sample, the Reagent, and antibodies to the two epitopes are combined. Because of the steric inhibition to the simultaneous binding of the two antibodies to the Reagent, the amount of antibody bound to the epitope of the Reagent foreign to the ligand will be related to the amount of ligand present in the assay medium. By analyzing directly or indirectly for the antibody bound to the epitope foreign to the ligand, and comparing the results to known standards, qualitative or quantitative determinations of the amount of ligand may be made. Various detector systems may be employed for determining the amount of antibody to the foreign epitope which is unbound or bound. These systems include stable free radicals, enzymes, radioactive labels, fluorescers, and the like.

789 citations

Patent
21 Jun 1971
TL;DR: In this article, the authors proposed novel assays for biologically active substrates employing purified target receptors, including fluorescent and radiochemical assays, employing labeled receptor in one embodiment and labeled substrate in another.
Abstract: This invention relates to novel assays for biologically active substrates employing purified target receptors. The assays comprise fluorescent assays for quenching and enhancement as well as radiochemical assays, employing labeled receptor in one embodiment and labeled substrate in another. Methods for the preparation of these purified receptors are also disclosed.

765 citations

Patent
08 Jul 1976
TL;DR: A fluorometric system to determine the kind and amount of substances derived from a biological fluid or tissue in which the substances to be detected (e.g., antigen, antibody, hormone or enzyme) are coated onto a substrate surface in fluorescent form is described in this paper.
Abstract: A fluorometric system to determine the kind and amount of substances derived from a biological fluid (eg, serum or urine) or tissue in which the substances to be detected (eg, antigen, antibody, hormone or enzyme) are coated onto a substrate surface in fluorescent form Multiple coating areas of different samples may be employed The fluorometric system includes a source of filtered light to excite fluorescence, optical systems for conducting the excitation light to such coating, and optical systems for receiving emitted fluorescence and for detecting the same The system efficiency and optical characteristics disclosed avoid photo-bleaching; limit fading; and are especially adapted to provide accurate surface reading fluorometry

122 citations

Patent
02 Oct 1973
TL;DR: In this paper, a method for determining qualitatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) is provided, which employs a reagent which involves bonding a compound having structural similarity to the compound to be determined (ligands analog) to a fluorescing compound.
Abstract: A sensitive method for determining qualitatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) is provided. The method employs a reagent which involves bonding a compound having structural similarity to the compound to be determined (ligand analog) to a fluorescing compound. The unknown compound is referred to as a ligand, the conjugate of the structurally similar compound and fluorescer is referred to as ligand analog-fluorescer, and compounds which recognize a specific structure and bind to such structure are referred to as receptors and are normally antibodies. The fluorescer which is chosen will have either a change in quantum yield or a change in its emisson and/or absorption spectra or all of them, when bound to antibody, as compared to being unbound. For the purposes of the assay, all that is required is that there be a change in the emisson intensity at some wavelength or band of wavelengths. The rate at which fluorescer antibody binds to the fluorescer portion of the ligand anaog-fluorescer or the amount of fluorescer antibody bound to the fluorescer portion of the ligand analog-fluorescer at equilibrium will be related to the amount of ligand antibody bound to the ligand analog portion of the ligand analog-fluorescer. Therefore, by combining antibodies to both ligand and fluorescer, with ligand analog-fluorescer and an unknown, one can determine the amount of ligand present in the unknown by relating the emission intensity at a particular wavelength or band of wavelengths to standards.

108 citations

Patent
11 Apr 1972
TL;DR: In this article, a radioimmunoassay of 125I Angiotensin I generated by the action of renin in incubated plasma samples is presented, where antibody-specific antibody is employed as the specific antibody, and 125I Antibody to Angio-I is used as the labeled antigen.
Abstract: Apparatus for determination of Angiotensin I comprises a vial of 125I Angiotensin I containing anion exchange resin, a vial of Angiotensin I standard, at least one vial of Angiotensin I Antiserum, and the following reagents: tris(hydroxymethyl)aminomethane, powdered charcoal, barbital buffer mixture, 8-hydroxyquinoline sulfate, bovine serum albumin powder, and Dimercaprol solution. The method involves the radioimmunoassay of Angiotensin I generated by the action of renin in incubated plasma samples. Antibody to Angiotensin I is employed as the specific antibody, and 125I Angiotensin is employed as the labeled antigen, and synthetic Angiotensin I is employed as a reference standard. Powdered charcoal is used to separate free Angiotensin I from antibody-bound Angiotensin I.

24 citations