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US 6,878,515 B1Granted
Ultrasensitive immunoassays
OwnerUlf Landegreninventor
Life of this patent
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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.
Claims8
A test kit comprising
- a) a first immobilized reagent having affinity to a specific macromolecule, and
- b) a second and a third affinity reagent specific for different determinants of said macromolecule, and modified with conjugatable oligonucleotides which conjugate through
- i) hybridization of an oligonucleotide complementary to the conjugatable oligonucleotides;
- ii) hybridization of the conjugatable oligonucleotides to each other;
- or
- iii) ligation of the oligonucleotides,
- wherein a signal is generated by nucleic acid amplification only when said second and third affinity reagents are closely bound on said macromolecule;
- wherein said macromolecule is a protein.
5 more elements
An immunoassay for detection of a specific antigen, comprising:
- a) contacting a sample suspected of containing said specific antigen with a first antibody linked to a solid support, said first antibody being specific for a first epitope on the antigen;
- b) washing off excess sample;
- c) incubating with a solution of a second and a third antibody specific for a second and a third epitope of said antigen, and modified with conjugatable oligonucleotides, wherein said oligonucleotides conjugate to each other when said second and third antibody are both bound to said antigen through
- i) hybridization of an oligonucleotide complementary to the conjugatable oligonucleotides;
- ii) hybridization of the conjugatable oligonucleotides to each other;
- or
- iii) ligation of the oligonucleotides;
- d) washing off excess solution;
- e) amplifying said conjugated oligonucleotides;
- and
- f) detecting the amplified products.
8 more elements
Dependencies are inferred from the claim text.