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Ultrasensitive immunoassays

a technology of immunoassays and immunoassays, applied in the field of ultrasensitive immunoassays, can solve the problems of limited detection sensitivity, detection is not possible below a certain number of molecules, and limits the minimun number of antigen molecules, etc., and achieves the effect of low numbers

Inactive Publication Date: 2005-10-20
LANDEGREN ULF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0009] The present invention enables detection of extremely low numbers of antigenic molecules, even down to a single molecule. The invention provides reliable immunoassays in situations where insufficient numbers of antigens are available for conventional assays.

Problems solved by technology

Common to all these immunoassays, is that detection sensitivity is limited by the affinity of typical antibodies.
With the prior art immunoassays, detection is not possible below a certain number of molecules, because the background, i.e. unspecifically bound material, interferes with the results.
Non-specifically trapped antibodies give rise to an undesired background signal and limits the minimun number of antigen molecules that can be detected and it will not be possible to distinguish between false positive and true positive results below a certain number of antigen molecules.

Method used

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  • Ultrasensitive immunoassays

Examples

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example

[0035] Immunoglobulins were modified in a reaction with SPDP (3-(-pyridyldithio)propionic acid N-hydroxysuccinimide ester, from Pharmacia Biotech) according to the manufacturer's suggestions. Oligonucleotides were thiolated, either through the addition of a suitable phosphoramidite according to Connolly (Connolly B A, Nucl. Acid. Res. 1987 15:3131), or 3′aminomodified oligonucleotides were reacted with SPDP, followed by reduction of the dithiopyridyl bond, using dithiothreitol.

[0036] SPDP-modified antibodies were incubated with three equivalents of SH-containing oligonucleotides at 4° C. over night. The reaction mixture was separated using a Zorbax HPLC gel filtration column. Residual free antibody were removed from the isolated conjugate by ion exchange MonoQ FPLC separation.

[0037] The two oligonucleotides used to conjugate the antibodies were Oligo 1: 5′Tr S C3-ATA GAC TGA GCG TGG ACA TTA ATA TGT ACG TAG GCT TAA TTG AGT 3′ and Oligo 2: 5′P ATG TAC GAC CCG TAG ATA TTA TCA TAC TGG...

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Abstract

The present invention relates to an immunological test kit and immunoassay using a first immobilized antibody having affinity for a specific antigen. The invention is characterized by a second and third antibody being specific for different determinants of the antigen and modified with cross-linkable oligonucleotides. For detection, the oligonucleotides are amplified, whereby only such oligonucleotides will be amplified which have been cross-linked to each other. In this way unspecific background is avoided and detection is possible down to single molecules.

Description

TECHNICAL FIELD [0001] The present invention relates to ultrasensitive immunoassays. More specifically, it relates to immunological test kits and processes for immunological detection of a specific antigen. In the present invention, the fields of immunology and molecular genetics are combined. BACKGROUND OF THE INVENTION [0002] Immunoassays represent powerful tools to identify a very wide range of compounds, such as antigens and antibodies. Examples of immunoassays are ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), and RIA (radio immunoassay). Common to all these immunoassays, is that detection sensitivity is limited by the affinity of typical antibodies. [0003] With the prior art immunoassays, detection is not possible below a certain number of molecules, because the background, i.e. unspecifically bound material, interferes with the results. Detection of very low numbers of antigen is becoming increasingly important, especially for diagnostic applications. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/6816C12Q1/682G01N33/532G01N33/543
CPCC12Q1/6816C12Q1/682Y10S435/975Y10S435/81G01N33/54306
Inventor LANDEGREN, ULF
Owner LANDEGREN ULF
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