CA2353920A1 - Test system for detecting different markers, and production and use thereof - Google Patents
Test system for detecting different markers, and production and use thereof Download PDFInfo
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- CA2353920A1 CA2353920A1 CA002353920A CA2353920A CA2353920A1 CA 2353920 A1 CA2353920 A1 CA 2353920A1 CA 002353920 A CA002353920 A CA 002353920A CA 2353920 A CA2353920 A CA 2353920A CA 2353920 A1 CA2353920 A1 CA 2353920A1
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- nucleic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
Abstract
The present invention relates to a test system comprising at least two recognition species which recognize at least two different markers with formation of a complex, its preparation and use in a suitable detection process.
Description
Test system for the recognition of different markers, its preparation and use The present invention relates to a test system comprising at least two recognition species which recognize at least two different markers with formation of a complex, its preparation and use in a suitable detection process.
The areas of application of- test systems, such as diagnostics, are widespread in biology, biochemistry, medicine and pharmacc>logy. Especially in medicine, a reliable and clear diagnosis of diseases, such as viral infections or cancer, is of extreme importance for increasing the quality of life, since only by early recognition of a dise<~se can a timely and effective treatment take place. Based on the recognition of disease-specific markers or ligands, such as nucleic acid sequences, proteins or antigens, the pathogen or the disease in the biological sample is detected. Diagnostic tests are widespread in which a marker or a class of markers in each case is detected, such as in ELISA or in amplification methods, such as PCR, b-DNA, Southern, Western or Northern blotting. The types of detection used range from simple staining methods and calorimetric methods via fluorescence energy transfer (FRET) and fluorescence quenching up to scintillation proximity assay (SPA).
A significant disadvantage in the use of only one marker or one class of marker is that false-positive test results easily result, which also lead to wrong conclusions regarding a specific disease. A
second test or still further tests rnust often therefore be carried out on the same or complementary analytes in order to be able to make a reliable statement with regard to sickness/health. This leads to more tests, whose results are to be compared with one another, which is at the same time laborious and cost-intensive.
The areas of application of- test systems, such as diagnostics, are widespread in biology, biochemistry, medicine and pharmacc>logy. Especially in medicine, a reliable and clear diagnosis of diseases, such as viral infections or cancer, is of extreme importance for increasing the quality of life, since only by early recognition of a dise<~se can a timely and effective treatment take place. Based on the recognition of disease-specific markers or ligands, such as nucleic acid sequences, proteins or antigens, the pathogen or the disease in the biological sample is detected. Diagnostic tests are widespread in which a marker or a class of markers in each case is detected, such as in ELISA or in amplification methods, such as PCR, b-DNA, Southern, Western or Northern blotting. The types of detection used range from simple staining methods and calorimetric methods via fluorescence energy transfer (FRET) and fluorescence quenching up to scintillation proximity assay (SPA).
A significant disadvantage in the use of only one marker or one class of marker is that false-positive test results easily result, which also lead to wrong conclusions regarding a specific disease. A
second test or still further tests rnust often therefore be carried out on the same or complementary analytes in order to be able to make a reliable statement with regard to sickness/health. This leads to more tests, whose results are to be compared with one another, which is at the same time laborious and cost-intensive.
It is therefore an object of the present ' invention to develop qualitatively better, less complicated and less expensive analyte tests than those already known.
Surprisingly, it has now been found that the linkage of two or more test results at the molecular level in the sense of Boolean linkage allows a qualitatively very good, simple and inexpensive analyte test, the different test results essentially not interfering with one another.
The invention therefore relates to a detection process comprising the following steps:
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes both the first marker and the second. marker, (c) treatment of the samp~_e with a third recognition species which recognizes the second marker, (d) detection of the presence or absence of a complex of the recognition species and markers mentioned.
In addition, the present invention relates to a detection process comprising the following steps:
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes the first marker and a third recognition species, (c) treatment of the sample with a third recognition species which recognizes the second marker and the second recognition species, (d) detection of the presence: or absence of a complex of the recognition species and markers mentioned.
To increase the specificity, it is advantageous in a further embodiment that further recognition species which recognize further markers are employed in further treatment steps, which results in n + 1 recognition species on widening to :n ligands where n is equal to a natural number.
In further preferred embodiments, the detection process is carried out in homogenous, partly homogeneous (modular) or immobi:Lized form. In a homogeneous embodiment, the binding events are produced stepwise and the complex possibly formed is detected in a "proximity assay" . The first and the last components are preferably labelled such that they can only produce a signal when mutually opposite. Preferred detection processes are, for example, LOCI (Ullmann, E. et al.
(1994) Proc. Natl. Acad. Sci. USA 91, 5426), fluorescence energy transfer (FRET) Cardullo, R.A.
(1992) in "Nonradioactive Labeling and Detection of Biomolecules", 414-423, Springer Verlag), fluorescence quenching (Ladokhin, A.S. (1997) "D:istribution analysis of depth-dependent fluorescence quenching in membranes:
a practical guide", Methods Enzymol., 278, 462-473) or scintillation proximity assay (SPA) (Picardo, M. & Hughes, K.T. (1997) "Scintillation proximity assays. High Throughput Screening", Ed. Devlin, J.P.;
Verlag Dekker, New York, N.Y., 307-316).
In a partly homogeneous or modular embodiment, the binding events are produced stepwise and in solution and, as soon as the complex has formed, it is bound to a solid support via one of the components. The complex formed is detected by means of a marker, in particular a non-radioactive marker or radioactive marker, preferably by means of a fluorescence marker, enzymatic marker, redox marker or spin marker (Kessler, C. (Ed.) Nonradioactive Labeling and Detection of Biomolecules (1992), Springer Verlag, 414-423).
In an immobilized embodiment, a recognition species, preferably the first recognition species, is bound to a solid support and subsequently built up by stepwise addition of the other components of the complex. Labelling is preferably carried out by methods which are identical or similar to those in the case of the partly homogeneous embodiment.
A suitable support for th~~ immobilization is especially solid or gelatinous material, in particular chip material and/or thin layers of the material, preferably ceramic, metal, in particular noble metal, glasses, plastics, crystalline m<~terials or (bio)-molecular filaments, in particular cellulose or structural proteins.
The recognition species and/or markers employed in the detection process according to the invention are, in particular, a synthetic substance, a natural substance and/or a natural substance derivative, preferably a peptide, peptoid, protein, saccharide or a nucleic acid. A receptor or a functional part thereof, for example, is particularly preferred, in particular a functional part which originates from the extracellular domain of a membrane-based receptor, an antibody or a functional part thereof, in particular an Fv fragment (Skerra & Pliickthun (1988), Science 240, 1038), a single-chain Fv fragment (scFv; Bird et al. (1988), Science 242, 423; Huston et al. (1988), Proc. Natl.
Acad. Sci. U.S.A. 85, 5879) or an Fab fragment (Better et al. (1988), Science 240, 1041), an aptamer, for example a DNA or RNA aptamer or cLerivatives thereof, for example aptamers provided with protective groups customary in nucleic acid chemistry, a cell constituent, in particular a lipid, glycoprotein, filament constituent, lectin, liposome, mitogen, antigen, secondary metabolite or hapten, a cell, in particular a lymphoid cell, or a vir°us, in particular a virus constituent, especially a cap,sid, or a viroid or a derivative, in particular an acetate, or their active parts, or a single-stranded or double-stranded nucleic acid, in particular DNA, RNA, p-RNA (Pitsch, S. et al., Helv. Chim. Acta. (1993), 76, 2161; Pitsch, S. et al., Helv. Chim. Acta, (1995), 78, 1621), p-DNA
(DE 198 37 387.2), PNA (peptide nucleic acid; Nielsen, P.E. et al. (1991) Science, 254, 1497), CNA
(Aminocyclohexylnucleic acid; PCT/EP98/06002) or an aptamer (see, for example, Bock, L.C. et al. (1992) Nature, 355, 564) or hybrids of the substances mentioned.
According to the present invention, aptamers, on account of their binding properties to specific molecules which are different from nucleic acids, such as proteins, do not belong to the nucleic acids, but to antibody derivatives. DNA aptamers or RNA aptamers are preferred.
The nucleic acids according to the invention including aptamers can also be modified. For this, the methods known from nucleic acid chemistry to the person skilled in the art can be used. Modifications are preferred which lead to stabilization of the nucleic acids (see, for example, Ullmann,. E. & Peyman, A.
(1990) Chemical Reviews, 90, 543, No. 4).
Customarily, the recognition of a marker by a recognition species takes place by means of non-covalent interactions, in particular by means of hydrogen bonds, salt bridges, stacking, formation of metal ligands, charge-transfer comp:Lexes, Van-der-Waals forces or hydrophobic interactions. For example, a nucleic acid is recognized as a marker by a completely or partly complementary nucleic acid or a synthetic substance, such as a chemical, a natural substance and/or a natural substance derivative are recognized as antigenic substances by an appropriate antibody or antibody derivative. According to the detection process according to the invention, the markers can belong to any desired class of substance, but preferably of at least two different classes.
According to the detection process according to the invention, additionally at least one recognition species is labelled, preferably all recognition species are labelled, especially at lea:~t two recognition species are differently labelled. As already illustrated above in greater detail, the marker, depending on whether a homogeneous,. partly homogeneous (modular) or immobilized embodiment is concerned, can be non-radioactive or radioactive, preferably LOCI, FRET, fluorescence quenching, SPA, a fluorescence marker, enzymatic marker, redox mar~:er or spin marker.
In a further preferred embodiment, the marker and/or the signal can be amplified, which leads to an increase in the sensitivity of the: detection process.
The amplification of the marker relates, in particular, to the amplification of nucleic acids, for example by PCR, NASBA, LCR, SDA, Q~ replication or RT-PCR
(Kessler, C. (1992) supra). The signal amplification is achieved, for example, by 'cross-:Linking' of binding components, antibody or nucleic acid trees (e. g.
b-DNA), catalytic substrate reaction (e. g, alkaline phosphatase, peroxidase, a-galactosidase} or signal cascades.
In addition to the mentioned in-vitro amplification, in-vivo amplification is also possible, e.g. detection of r-RNA, indirect detection of antigens.
In principle, the markers can be divided into two classes. In the case of 'positive markers', the absence of these markers is detected, for example, by means of the absence of a signal.. Positive markers refer in general to markers present in a healthy organism, e.g. m-RNA. Negative marlcers are in general designated as the substances of a pathogen or of an ill organism, which can be determined by means of the detection process according to the invention .
In the detection process according to the invention, either two or more negative, two or more positive or two or more positive and negative markers can be detected. The detection thus takes place either via the occurrence or via the absence of a signal.
Likewise, a displacement of a signal, e.g. by the displacement of a molecule from a complex or from its binding conformation (e. g. Molecular Beacons, S. Tyaki, _ - 7 -Kramer F.R., Nature Biotechnology 14, 303-308, 1996;
R.P. Ekins, Clinical Chemistry, 44%9, 2015-2030, 1998) is possible in a competitive assay. For this, a substance is added to the test sy~>tem which displaces one of the markers to be detected, the molecular complex built up from markers and recognition species and thus also the signal associated therewith disappearing. By means of a titration, the concentration of the displaced marker can thus be determined in a simple manner.
The detection process according to the invention can now be present in at least one of the following alternative embodiments, which are particularly preferred:
1. At least one marker is a natural or unnatural, single-stranded or double-stranded nucleic acid and each further marker is a synthetic substance, natural substance or natural ~~ubstance derivative other than a nucleic acid, preferably an antigen.
2. The first marker and each further marker is a natural or unnatural, single-~;tranded or double stranded nucleic acid or alternatively a synthetic substance, a natural substance or a natural substance derivative other than a nucleic acid, preferably an antigen.
Surprisingly, it has now been found that the linkage of two or more test results at the molecular level in the sense of Boolean linkage allows a qualitatively very good, simple and inexpensive analyte test, the different test results essentially not interfering with one another.
The invention therefore relates to a detection process comprising the following steps:
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes both the first marker and the second. marker, (c) treatment of the samp~_e with a third recognition species which recognizes the second marker, (d) detection of the presence or absence of a complex of the recognition species and markers mentioned.
In addition, the present invention relates to a detection process comprising the following steps:
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes the first marker and a third recognition species, (c) treatment of the sample with a third recognition species which recognizes the second marker and the second recognition species, (d) detection of the presence: or absence of a complex of the recognition species and markers mentioned.
To increase the specificity, it is advantageous in a further embodiment that further recognition species which recognize further markers are employed in further treatment steps, which results in n + 1 recognition species on widening to :n ligands where n is equal to a natural number.
In further preferred embodiments, the detection process is carried out in homogenous, partly homogeneous (modular) or immobi:Lized form. In a homogeneous embodiment, the binding events are produced stepwise and the complex possibly formed is detected in a "proximity assay" . The first and the last components are preferably labelled such that they can only produce a signal when mutually opposite. Preferred detection processes are, for example, LOCI (Ullmann, E. et al.
(1994) Proc. Natl. Acad. Sci. USA 91, 5426), fluorescence energy transfer (FRET) Cardullo, R.A.
(1992) in "Nonradioactive Labeling and Detection of Biomolecules", 414-423, Springer Verlag), fluorescence quenching (Ladokhin, A.S. (1997) "D:istribution analysis of depth-dependent fluorescence quenching in membranes:
a practical guide", Methods Enzymol., 278, 462-473) or scintillation proximity assay (SPA) (Picardo, M. & Hughes, K.T. (1997) "Scintillation proximity assays. High Throughput Screening", Ed. Devlin, J.P.;
Verlag Dekker, New York, N.Y., 307-316).
In a partly homogeneous or modular embodiment, the binding events are produced stepwise and in solution and, as soon as the complex has formed, it is bound to a solid support via one of the components. The complex formed is detected by means of a marker, in particular a non-radioactive marker or radioactive marker, preferably by means of a fluorescence marker, enzymatic marker, redox marker or spin marker (Kessler, C. (Ed.) Nonradioactive Labeling and Detection of Biomolecules (1992), Springer Verlag, 414-423).
In an immobilized embodiment, a recognition species, preferably the first recognition species, is bound to a solid support and subsequently built up by stepwise addition of the other components of the complex. Labelling is preferably carried out by methods which are identical or similar to those in the case of the partly homogeneous embodiment.
A suitable support for th~~ immobilization is especially solid or gelatinous material, in particular chip material and/or thin layers of the material, preferably ceramic, metal, in particular noble metal, glasses, plastics, crystalline m<~terials or (bio)-molecular filaments, in particular cellulose or structural proteins.
The recognition species and/or markers employed in the detection process according to the invention are, in particular, a synthetic substance, a natural substance and/or a natural substance derivative, preferably a peptide, peptoid, protein, saccharide or a nucleic acid. A receptor or a functional part thereof, for example, is particularly preferred, in particular a functional part which originates from the extracellular domain of a membrane-based receptor, an antibody or a functional part thereof, in particular an Fv fragment (Skerra & Pliickthun (1988), Science 240, 1038), a single-chain Fv fragment (scFv; Bird et al. (1988), Science 242, 423; Huston et al. (1988), Proc. Natl.
Acad. Sci. U.S.A. 85, 5879) or an Fab fragment (Better et al. (1988), Science 240, 1041), an aptamer, for example a DNA or RNA aptamer or cLerivatives thereof, for example aptamers provided with protective groups customary in nucleic acid chemistry, a cell constituent, in particular a lipid, glycoprotein, filament constituent, lectin, liposome, mitogen, antigen, secondary metabolite or hapten, a cell, in particular a lymphoid cell, or a vir°us, in particular a virus constituent, especially a cap,sid, or a viroid or a derivative, in particular an acetate, or their active parts, or a single-stranded or double-stranded nucleic acid, in particular DNA, RNA, p-RNA (Pitsch, S. et al., Helv. Chim. Acta. (1993), 76, 2161; Pitsch, S. et al., Helv. Chim. Acta, (1995), 78, 1621), p-DNA
(DE 198 37 387.2), PNA (peptide nucleic acid; Nielsen, P.E. et al. (1991) Science, 254, 1497), CNA
(Aminocyclohexylnucleic acid; PCT/EP98/06002) or an aptamer (see, for example, Bock, L.C. et al. (1992) Nature, 355, 564) or hybrids of the substances mentioned.
According to the present invention, aptamers, on account of their binding properties to specific molecules which are different from nucleic acids, such as proteins, do not belong to the nucleic acids, but to antibody derivatives. DNA aptamers or RNA aptamers are preferred.
The nucleic acids according to the invention including aptamers can also be modified. For this, the methods known from nucleic acid chemistry to the person skilled in the art can be used. Modifications are preferred which lead to stabilization of the nucleic acids (see, for example, Ullmann,. E. & Peyman, A.
(1990) Chemical Reviews, 90, 543, No. 4).
Customarily, the recognition of a marker by a recognition species takes place by means of non-covalent interactions, in particular by means of hydrogen bonds, salt bridges, stacking, formation of metal ligands, charge-transfer comp:Lexes, Van-der-Waals forces or hydrophobic interactions. For example, a nucleic acid is recognized as a marker by a completely or partly complementary nucleic acid or a synthetic substance, such as a chemical, a natural substance and/or a natural substance derivative are recognized as antigenic substances by an appropriate antibody or antibody derivative. According to the detection process according to the invention, the markers can belong to any desired class of substance, but preferably of at least two different classes.
According to the detection process according to the invention, additionally at least one recognition species is labelled, preferably all recognition species are labelled, especially at lea:~t two recognition species are differently labelled. As already illustrated above in greater detail, the marker, depending on whether a homogeneous,. partly homogeneous (modular) or immobilized embodiment is concerned, can be non-radioactive or radioactive, preferably LOCI, FRET, fluorescence quenching, SPA, a fluorescence marker, enzymatic marker, redox mar~:er or spin marker.
In a further preferred embodiment, the marker and/or the signal can be amplified, which leads to an increase in the sensitivity of the: detection process.
The amplification of the marker relates, in particular, to the amplification of nucleic acids, for example by PCR, NASBA, LCR, SDA, Q~ replication or RT-PCR
(Kessler, C. (1992) supra). The signal amplification is achieved, for example, by 'cross-:Linking' of binding components, antibody or nucleic acid trees (e. g.
b-DNA), catalytic substrate reaction (e. g, alkaline phosphatase, peroxidase, a-galactosidase} or signal cascades.
In addition to the mentioned in-vitro amplification, in-vivo amplification is also possible, e.g. detection of r-RNA, indirect detection of antigens.
In principle, the markers can be divided into two classes. In the case of 'positive markers', the absence of these markers is detected, for example, by means of the absence of a signal.. Positive markers refer in general to markers present in a healthy organism, e.g. m-RNA. Negative marlcers are in general designated as the substances of a pathogen or of an ill organism, which can be determined by means of the detection process according to the invention .
In the detection process according to the invention, either two or more negative, two or more positive or two or more positive and negative markers can be detected. The detection thus takes place either via the occurrence or via the absence of a signal.
Likewise, a displacement of a signal, e.g. by the displacement of a molecule from a complex or from its binding conformation (e. g. Molecular Beacons, S. Tyaki, _ - 7 -Kramer F.R., Nature Biotechnology 14, 303-308, 1996;
R.P. Ekins, Clinical Chemistry, 44%9, 2015-2030, 1998) is possible in a competitive assay. For this, a substance is added to the test sy~>tem which displaces one of the markers to be detected, the molecular complex built up from markers and recognition species and thus also the signal associated therewith disappearing. By means of a titration, the concentration of the displaced marker can thus be determined in a simple manner.
The detection process according to the invention can now be present in at least one of the following alternative embodiments, which are particularly preferred:
1. At least one marker is a natural or unnatural, single-stranded or double-stranded nucleic acid and each further marker is a synthetic substance, natural substance or natural ~~ubstance derivative other than a nucleic acid, preferably an antigen.
2. The first marker and each further marker is a natural or unnatural, single-~;tranded or double stranded nucleic acid or alternatively a synthetic substance, a natural substance or a natural substance derivative other than a nucleic acid, preferably an antigen.
3. A natural or unnatural, single-stranded or double-stranded nucleic acid as a marker is recognized by a natural or unnatural, single-stranded or double-stranded nucleic acid as recognition species.
4. A synthetic substance, a natural substance or a natural substance derivative i.s recognized by a synthetic substance, a natural substance or a natural substance derivative, preferably by an antibody or an antibody derivative, as recognition species.
5. At least one recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid and each further recognition species is a synthetic substance, different natural _ 8 -substance or different natural substance derivative other than a nucleic acid, preferably an antibody or an antibody derivative.
6. The first recognition species and each further recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid or alternatively a synthetic substance, different natural substance or different: natural substance derivative other than a nucleic acid, preferably an antibody or an antibody derivative.
7. At least one recognition species is a hybrid of a natural or unnatural, single-~~tranded or double stranded nucleic acid and another natural or unnatural, single-stranded or double-stranded nucleic acid.
8. At least one recognition speciE;s is a hybrid of a synthetic substance, a natur<~l substance or a natural substance derivative anal another synthetic substance, another natural substance or another natural substance derivative.
9. At least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
10. A first recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid, a second recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a synthetic substance, a natural substance or a natural substance derivative, preferably an antibody or antibody derivative.
11. A first recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid, a second recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and another natural - g -or unnatural single-stranded or double-stranded nucleic acid, and the third recognition species is a further different natural or unnatural, single-stranded or double-stranded nucleic acid.
12. A first recognition species is a synthetic substance, a natural substance or a natural substance derivative, preferably an antibody or antibody derivative, a second recognition species is a hybrid of a synthetic substance, a natural substance or a natural substance derivative, preferably an antibody or antibody derivative, and another synthetic substance, another natural substance or another natural substance derivative, preferably another antibody or antibody derivative, and a third recognition species is a further different synthetic substance, a further different natural substance or a further different natural substance derivative, preferably a further different antibody or a further different antibody derivative.
Another subject of the preaent invention is a test system comprising at least two recognition species, which recognize at least two different markers with formation of a complex, preferably the recognition species or markers already described above. In a preferred embodiment, at least one recognition species is immobilized on a support, such as preferably already described above in greater detail.
The test system according to the invention can be employed in the following preferred embodiments:
1. At least one recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid and at least one: other recognition species is another natural or unnatural, single-stranded or double-stranded nucleic acid.
2. At least one recognition species is a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative, and at least one other recognition species is another synthetic substance, different natural substance or different. natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
3. At least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a :synthetic substance, different natural substance or different natural substance derivative other th<~n a nucleic acid, preferably an antibody or antibody derivative.
4. At least one recognition speciE:s is a hybrid of a natural or unnatural, single-:>tranded or double-stranded nucleic acid and another natural or unnatural single-stranded or double-stranded nucleic acid.
5. At least one recognition species is a hybrid of a synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative, and another synthetic substance, different natural substance or' different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
The test system according to the invention can be produced, for example, by assembling the recognition species necessary for the individual embodiments, or by immobilizing at least one recognition species on a support, such as preferably already described above, by the process generally known to the person skilled in the art.
The test system according to the invention can be employed in the detection process according to the invention, as described in greater detail above. In particular, it is used for the detection of the presence and/or absence of at least two different markers in a sample. It is preferably present in the form of a diagnostic or an analyte. It is therefore used, in particular, for the detection of disorders or for environmental analysis, in .particular for the detection of toxins and/or allergens>.
The following figures and examples are intended to describe the invention in greater detail, without restricting it.
DESCRIPTION OF THE FIGURES
Fig. 1 shows schematically the detection two of analytes (A and B) in an assay in the immobilized embodiment.
Fig. 2 shows schematically the detection two of analytes (antigens A and B) in an assay the in immobilized embodiment.
Fig. shows schematically the detection two 3 of analytes (nucleic acid A and B) in an assay in the immobilized embodiment.
Fig. 4 shows schematically the complex of markers and recognition species according to Example 1.
Fig. shows schematically the complex of markers and recognition species according to Example 2.
EXAMPLES
Simultaneous detection of a deoxyribonucleic acid and of a labelled antibody/antigen Starting compounds:
The reagents needed for the example, such as Texas Red~-labelled oligonucl.eotide conjugate (24-mer DNA; Interactiva; DNA 1), a biotinylated oligonucleotide conjugate (24-mer DNA; Interactiva;
DNA 3); a synthetic oligonucleot~ide (57-mer DNA;
Interactiva; DNA 2), which has sequences complementary to the two other DNAs, streptavidin-conjugated anti-human IgG F(ab')2 (goat; Rockland) and a fluorescein-labelled human IgG F(ab')2 fragment (Rockland) as antigen, are all commercially obtainable (Reagent Specification DNA 1 Texas Red-5'-AAA-TGC-ATG-TCG-TCG-TGA-(recognition TGT-AAA-3' species 1) DNA 2 5'-ATT-GTC-ATA-ATC-A'7.'C-TTG-AGA-CGC-TTT-(marker 1) TTT-TTT-TTT-ACA-TCA-C:GA-CGA-CAT-GCA-TTT-3' DNA 3 Biotin-5'-GCG-TCT-CAA-CAT-GAT-TAT-GAC-(recognition AAT-3' species 2) 'Antibodies Streptavidin-conjugated anti-human IgG
(recognition F(ab')2 (goat) species 3 Antigen Fluorescein-labelled human IgG F(ab')2 (marker 2) fragment Table 1: Recognition species and markers used.
Example 1 1 nmol of Texas Red-labelled oligonucleotide conjugate (DNA 1), 1 nmol of biotin-labelled oligonucleotide conjugate (DNA 3) a.nd one pmol of the 57-mer oligonucleotide (DNA 2) were taken up in 150 ~,l of hybridization buffer (5 x SSC, 0.020 SDS) in each case. The constituents were heated at 60°C for 30 min, mixed with one another and incubatE:d at 37°C for 3 h.
They were allowed to cool to room temperature (RT), 1 nmol of the streptavidin ant=L-human IgG F(ab')2 conjugate and 1 nmol of the fluorescein-labelled IgG F(ab')2 fragment were added and the mixture was allowed to stand at RT overnight. The complex formed was detected by means of a yellow band in a non-denaturing gel (15o strength TEB gel, BioRad).
Example 2 1 nmol of biotin-labelled oligonucleotide conjugate (DNA 1), 1 nmol of biotin-labelled oligonucleotide conjugate (DNA 3) and one pmol of the 57-mer oligonucleotide (DNA 2) were taken up in 150 ~1 of hybridization buffer (5 x SSC, 0.020 SDS) in each case. The constituents were heated at 60°C for 5 min, mixed with one another and incubated at 37°C for 3 h.
The solution was allowed to cool to room temperature (RT) and added to a streptavidin-coated microtitre plate (BIOTEZ, order No. 040298920). The supernatant solution was removed by pipette a.nd the support was washed 5x with 500 ~1 of 0 . 9 o NaCl solution. 200 ~.1 of a solution of 200 ~1 of streptavidin anti-human IgG F(ab')2 (goat) solution (1.6 mg,~ml) preincubated at RT for 2 h and 40 ~l of the fluorescein-labelled IgG F(ab')2 fragment solution (5.0 mg/ml) were then added and the mixture was incubated at RT for 1-2 h.
~rne supernatant solution was in turn removed by pipette and the support was washed 5x with 500 ~,1 of 0.9o NaCl solution. The formation of the complex was detected by measuring the fluorescence of the fluorescein (~max,A: 494 nm, ~,aX,E: 525 nm) .
(Reagent Specification DNA 1' Biotin-5'-AAA-TGC-ATG-TCG-TCG-TGA-TGT-(recognition AAA-3' species 1) DNA 2 5'-ATT-GTC-ATA-ATC-ATC-TTG-AGA-CGC-TTT-(marker 1) TTT-TTT-TTT-ACA-TCA-CGA-CGA-CAT-GCA-TTT-3' (DNA 3 Biotin-5'-GCG-TCT-CAA-CAT-GAT-TAT-GAC-(recognition AAT-3' species 2) Antibodies Streptavidin-conjugated anti-human IgG
(recognition F(ab')2 (goat) species 3 Antigen Fluorescein-labelled human IgG F(ab')2 (marker 2) fragment Table 2: Recognition species and marl~ers used.
Another subject of the preaent invention is a test system comprising at least two recognition species, which recognize at least two different markers with formation of a complex, preferably the recognition species or markers already described above. In a preferred embodiment, at least one recognition species is immobilized on a support, such as preferably already described above in greater detail.
The test system according to the invention can be employed in the following preferred embodiments:
1. At least one recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid and at least one: other recognition species is another natural or unnatural, single-stranded or double-stranded nucleic acid.
2. At least one recognition species is a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative, and at least one other recognition species is another synthetic substance, different natural substance or different. natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
3. At least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a :synthetic substance, different natural substance or different natural substance derivative other th<~n a nucleic acid, preferably an antibody or antibody derivative.
4. At least one recognition speciE:s is a hybrid of a natural or unnatural, single-:>tranded or double-stranded nucleic acid and another natural or unnatural single-stranded or double-stranded nucleic acid.
5. At least one recognition species is a hybrid of a synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative, and another synthetic substance, different natural substance or' different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
The test system according to the invention can be produced, for example, by assembling the recognition species necessary for the individual embodiments, or by immobilizing at least one recognition species on a support, such as preferably already described above, by the process generally known to the person skilled in the art.
The test system according to the invention can be employed in the detection process according to the invention, as described in greater detail above. In particular, it is used for the detection of the presence and/or absence of at least two different markers in a sample. It is preferably present in the form of a diagnostic or an analyte. It is therefore used, in particular, for the detection of disorders or for environmental analysis, in .particular for the detection of toxins and/or allergens>.
The following figures and examples are intended to describe the invention in greater detail, without restricting it.
DESCRIPTION OF THE FIGURES
Fig. 1 shows schematically the detection two of analytes (A and B) in an assay in the immobilized embodiment.
Fig. 2 shows schematically the detection two of analytes (antigens A and B) in an assay the in immobilized embodiment.
Fig. shows schematically the detection two 3 of analytes (nucleic acid A and B) in an assay in the immobilized embodiment.
Fig. 4 shows schematically the complex of markers and recognition species according to Example 1.
Fig. shows schematically the complex of markers and recognition species according to Example 2.
EXAMPLES
Simultaneous detection of a deoxyribonucleic acid and of a labelled antibody/antigen Starting compounds:
The reagents needed for the example, such as Texas Red~-labelled oligonucl.eotide conjugate (24-mer DNA; Interactiva; DNA 1), a biotinylated oligonucleotide conjugate (24-mer DNA; Interactiva;
DNA 3); a synthetic oligonucleot~ide (57-mer DNA;
Interactiva; DNA 2), which has sequences complementary to the two other DNAs, streptavidin-conjugated anti-human IgG F(ab')2 (goat; Rockland) and a fluorescein-labelled human IgG F(ab')2 fragment (Rockland) as antigen, are all commercially obtainable (Reagent Specification DNA 1 Texas Red-5'-AAA-TGC-ATG-TCG-TCG-TGA-(recognition TGT-AAA-3' species 1) DNA 2 5'-ATT-GTC-ATA-ATC-A'7.'C-TTG-AGA-CGC-TTT-(marker 1) TTT-TTT-TTT-ACA-TCA-C:GA-CGA-CAT-GCA-TTT-3' DNA 3 Biotin-5'-GCG-TCT-CAA-CAT-GAT-TAT-GAC-(recognition AAT-3' species 2) 'Antibodies Streptavidin-conjugated anti-human IgG
(recognition F(ab')2 (goat) species 3 Antigen Fluorescein-labelled human IgG F(ab')2 (marker 2) fragment Table 1: Recognition species and markers used.
Example 1 1 nmol of Texas Red-labelled oligonucleotide conjugate (DNA 1), 1 nmol of biotin-labelled oligonucleotide conjugate (DNA 3) a.nd one pmol of the 57-mer oligonucleotide (DNA 2) were taken up in 150 ~,l of hybridization buffer (5 x SSC, 0.020 SDS) in each case. The constituents were heated at 60°C for 30 min, mixed with one another and incubatE:d at 37°C for 3 h.
They were allowed to cool to room temperature (RT), 1 nmol of the streptavidin ant=L-human IgG F(ab')2 conjugate and 1 nmol of the fluorescein-labelled IgG F(ab')2 fragment were added and the mixture was allowed to stand at RT overnight. The complex formed was detected by means of a yellow band in a non-denaturing gel (15o strength TEB gel, BioRad).
Example 2 1 nmol of biotin-labelled oligonucleotide conjugate (DNA 1), 1 nmol of biotin-labelled oligonucleotide conjugate (DNA 3) and one pmol of the 57-mer oligonucleotide (DNA 2) were taken up in 150 ~1 of hybridization buffer (5 x SSC, 0.020 SDS) in each case. The constituents were heated at 60°C for 5 min, mixed with one another and incubated at 37°C for 3 h.
The solution was allowed to cool to room temperature (RT) and added to a streptavidin-coated microtitre plate (BIOTEZ, order No. 040298920). The supernatant solution was removed by pipette a.nd the support was washed 5x with 500 ~1 of 0 . 9 o NaCl solution. 200 ~.1 of a solution of 200 ~1 of streptavidin anti-human IgG F(ab')2 (goat) solution (1.6 mg,~ml) preincubated at RT for 2 h and 40 ~l of the fluorescein-labelled IgG F(ab')2 fragment solution (5.0 mg/ml) were then added and the mixture was incubated at RT for 1-2 h.
~rne supernatant solution was in turn removed by pipette and the support was washed 5x with 500 ~,1 of 0.9o NaCl solution. The formation of the complex was detected by measuring the fluorescence of the fluorescein (~max,A: 494 nm, ~,aX,E: 525 nm) .
(Reagent Specification DNA 1' Biotin-5'-AAA-TGC-ATG-TCG-TCG-TGA-TGT-(recognition AAA-3' species 1) DNA 2 5'-ATT-GTC-ATA-ATC-ATC-TTG-AGA-CGC-TTT-(marker 1) TTT-TTT-TTT-ACA-TCA-CGA-CGA-CAT-GCA-TTT-3' (DNA 3 Biotin-5'-GCG-TCT-CAA-CAT-GAT-TAT-GAC-(recognition AAT-3' species 2) Antibodies Streptavidin-conjugated anti-human IgG
(recognition F(ab')2 (goat) species 3 Antigen Fluorescein-labelled human IgG F(ab')2 (marker 2) fragment Table 2: Recognition species and marl~ers used.
Claims (36)
1. Detection process comprising the following steps:
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes both the first marker and the second marker, (c) treatment of the sample with a third recognition species which recognizes the second marker, (d) detection of the presence or absence of the first and the second marker in the sample, by the detection of the presence or absence of a complex of the recognition species and markers mentioned.
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes both the first marker and the second marker, (c) treatment of the sample with a third recognition species which recognizes the second marker, (d) detection of the presence or absence of the first and the second marker in the sample, by the detection of the presence or absence of a complex of the recognition species and markers mentioned.
2. Detection process comprising the following steps:
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes the first marker and a third recognition species, (c) treatment of the sample with a third recognition species which recognizes the second marker and the second recognition species, (d) detection of the presence or absence of the first and the second marker in the sample, by the detection of the presence or absence of a complex of the recognition species and markers mentioned.
(a) treatment of a sample comprising a first and a second marker with a first recognition species which recognizes the first marker, (b) treatment of the sample with a second recognition species which recognizes the first marker and a third recognition species, (c) treatment of the sample with a third recognition species which recognizes the second marker and the second recognition species, (d) detection of the presence or absence of the first and the second marker in the sample, by the detection of the presence or absence of a complex of the recognition species and markers mentioned.
3. Detection process according to Claim 1 or 2, characterized in that further recognition species which recognize further markers are employed in further treatment steps.
4. Detection process according to one of Claims 1-3, characterized in that a recognition species, preferably the first recognition species, is immobilized on a support.
5. Detection process according to Claim 4, characterized in that the support is selected from a solid or gelatinous material, in particular chip material and/or thin layers of the material, preferably ceramic, metal, in particular noble metal, glasses, plastics, crystalline materials or (bio)molecular filaments, in particular cellulose or structural proteins.
6. Detection process according to one of Claims 1-5, characterized in that the recognition species and/or the marker mentioned is a synthetic substance, a natural substance and/or a natural substance derivative, preferably selected from a peptide, peptoid, protein, saccharide or a nucleic acid.
7. Detection process according to Claim 6, characterized in that the synthetic substance, a natural substance or a natural substance derivative is selected from a receptor or a functional part thereof, in particular from the extracellular domain of a membrane-based receptor, an antibody or a functional part thereof, in particular an Fv fragment, a single-chain Fv fragment (ScFv) or an Fab fragment, a cell constituent, in particular a lipid, glycoprotein, filament constituent, lectin, liposome, mitogen, antigen, secondary metabolite or hapten, a cell, in particular a lymphoid cell, or a virus, in particular a virus constituent, especially a capsid, or a viroid, or a derivative, in particular an acetate, or their active parts, or a single-stranded or double-stranded nucleic acid, in particular a natural nucleic acid in the form of a DNA or RNA or an unnatural nucleic acid, preferably p-RNA, p-DNA, PNA or CNA, or hybrids of the substances mentioned.
8. Detection process according to one of Claims 1-7, characterized in that the recognition of a marker by a recognition species takes place by means of non-covalent interactions, in particular by means of hydrogen bonds, salt bridges, stacking, formation of metal ligands, charge-transfer complexes, Van-der-Waals forces or hydrophobic interactions.
9. Detection process according to one of Claims 1-8, characterized in that at least one recognition species is labelled, in particular all recognition species are labelled, preferably at least two recognition species are differently labelled.
10. Detection process according to Claim 9, characterized in that the marker is a non-radioactive marker or radioactive marker, preferably an LOCI
marker, FRET marker, fluorescence quenching marker, SPA
marker, fluorescence marker, enzymatic marker, redox marker or spin marker.
marker, FRET marker, fluorescence quenching marker, SPA
marker, fluorescence marker, enzymatic marker, redox marker or spin marker.
11. Detection process according to one of Claims 1-10, characterized in that the marker and/or the signal is amplified.
12. Detection process according to one of Claims 1-11, characterized in that the detection is carried out competitively according to step (d) of the process.
13. Detection process according to one of Claims 1-12, characterized in that at least one marker is a natural or unnatural, single-stranded or double-stranded nucleic acid and each further marker is a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antigen.
14. Detection process according to one of Claims 1-12, characterized in that the first marker and each further marker is a natural or unnatural, single-stranded or double-stranded nucleic acid or alternatively a synthetic substance, a different natural substance or a different natural substance derivative other than a natural nucleic acid, preferably an antigen.
15. Detection process according to one of Claims 1-14, characterized in that a natural or unnatural, single-stranded or double-stranded nucleic acid as a marker is recognized by a natural or unnatural, single-stranded or double-stranded nucleic acid as recognition species.
16. Detection process according to one of Claims 1-15, characterized in that a synthetic substance, a natural substance or a natural substance derivative is recognized by a synthetic substance, a natural substance or a natural substance derivative, preferably by an antibody or an antibody derivative, as recognition species.
17. Detection process according to one of Claims 1-16, characterized in that at least one recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid and each further recognition species is a synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or an antibody derivative.
18. Detection process according to one of Claims 1-16, characterized in that the first recognition species and each further recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid or alternatively a synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or an antibody derivative.
19. Detection process according to one of Claims 1-16, characterized in that at least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and another natural or unnatural, single-stranded or double-stranded nucleic acid.
20. Detection process according to one of Claims 1-16, characterized in that at least one recognition species is a hybrid of a synthetic substance, a natural substance or a natural substance derivative and another synthetic substance, another natural substance or another natural substance derivative.
21. Detection process according to one of Claims 1-16, characterized in that at least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
22. Detection process according to one of Claims 1-16, characterized in that a first recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid, a second recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a synthetic substance, a natural substance or a natural substance derivative, preferably an antibody or antibody derivative.
23. Detection process according to one of Claims 1-16, characterized in that a first recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid, a second recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and another natural or unnatural, single-stranded or double-stranded nucleic acid, and the third recognition species is a further different natural or unnatural, single-stranded or double-stranded nucleic acid.
24. Detection process according to one of Claims 1-16, characterized in that a first recognition species is a synthetic substance, a natural substance or a natural substance derivative, preferably an antibody or antibody derivative, a second recognition species is a hybrid of a synthetic substance, a natural substance or a natural substance derivative, preferably an antibody or antibody derivative, and another natural substance or another natural substance derivative, preferably another antibody or antibody derivative, and a third recognition species is a further different synthetic substance, a natural substance or a natural substance derivative, preferably a further different antibody or antibody derivative.
25. Test system for the detection of the presence or absence of at least two different markers in a sample comprising at least two recognition species which recognize at least two different markers with formation of a complex, at least two of the recognition species being differently labelled.
26. Test system according to Claim 25, characterized in that at least one recognition species is immobilized on a support.
27. Test system according to Claim 25 or 26, characterized in that at least one recognition species is a natural or unnatural, single-stranded or double-stranded nucleic acid and at least one other recognition species is another natural or unnatural, single-stranded or double-stranded nucleic acid.
28. Test system according to Claim 25 or 26, characterized in that at least one recognition species is a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative, and at least one other recognition species is a synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
29. Test system according to Claim 25 or 26, characterized in that at least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and a synthetic substance, a different natural substance or a different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
30. Test system according to Claim 25 or 26, characterized in that at least one recognition species is a hybrid of a natural or unnatural, single-stranded or double-stranded nucleic acid and another natural or unnatural, single-stranded or double-stranded nucleic acid.
31. Test system according to Claim 25 or 26, characterized in that at least one recognition species is a hybrid of a synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative, and another synthetic substance, different natural substance or different natural substance derivative other than a nucleic acid, preferably an antibody or antibody derivative.
32. Process for the production of a test system according to one of Claims 25-31, characterized in that the individual recognition species are assembled.
33. Process according to Claim 32, characterized in that at least one recognition species is immobilized on a support.
34. Use of the test system according to one of Claims 25-31 for the detection of the presence and/or absence of at least two different markers in a sample.
35. Use of the test system according to Claim 32 in the form of a diagnostic or in the form of an analyte.
36. Use of the test system according to Claim 32 or 33 for the detection of a disorder or for environmental analysis, in particular for the detection of disease pathogens, markers of diseases, toxins and/or allergens.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19859912.9 | 1998-12-23 | ||
| DE19859912A DE19859912C2 (en) | 1998-12-23 | 1998-12-23 | Test system for the detection of different markers, its production and use |
| PCT/EP1999/010333 WO2000039581A2 (en) | 1998-12-23 | 1999-12-22 | Test system for detecting different markers, and production and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2353920A1 true CA2353920A1 (en) | 2000-07-06 |
Family
ID=7892569
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002353920A Abandoned CA2353920A1 (en) | 1998-12-23 | 1999-12-22 | Test system for detecting different markers, and production and use thereof |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1141725A2 (en) |
| JP (1) | JP2002533724A (en) |
| AU (1) | AU773046B2 (en) |
| CA (1) | CA2353920A1 (en) |
| DE (1) | DE19859912C2 (en) |
| WO (1) | WO2000039581A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10018626B2 (en) | 2012-03-09 | 2018-07-10 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| US10495638B2 (en) | 2009-10-09 | 2019-12-03 | Invisible Sentinel, Inc. | Device for detection of analytes and uses thereof |
| WO2020079638A1 (en) * | 2018-10-17 | 2020-04-23 | Molecular Devices (Austria) GmbH | Real time western blot assays utilizing fluorescence resonance energy transfer (fret) |
| US10705084B2 (en) | 2009-07-31 | 2020-07-07 | Invisible Sentinel, Inc. | Analyte detection devices, multiplex and tabletop devices for detection of analytes, and uses thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1947459A3 (en) * | 2000-08-24 | 2008-09-24 | Nanogen, Inc. | Differential immunoassay |
| US6673562B2 (en) * | 2000-08-24 | 2004-01-06 | Spectral Diagnostics, Inc. | Differential immunoassay |
| US6893822B2 (en) | 2001-07-19 | 2005-05-17 | Nanogen Recognomics Gmbh | Enzymatic modification of a nucleic acid-synthetic binding unit conjugate |
| US8012770B2 (en) | 2009-07-31 | 2011-09-06 | Invisible Sentinel, Inc. | Device for detection of antigens and uses thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4320109A (en) * | 1979-06-29 | 1982-03-16 | The University Of Southern California | Immunoradiometric assay employing terminal radionuclide labeling and synthesis of conjugates for such assay |
| JPH081438B2 (en) * | 1986-05-13 | 1996-01-10 | 三洋化成工業株式会社 | Enzyme immunoassay |
| US5296347A (en) * | 1991-02-08 | 1994-03-22 | Ciba Corning Diagnostics Corp. | Bridge immunoassay |
| EP0579767B1 (en) * | 1991-04-11 | 2000-08-23 | Biosite Diagnostics Inc. | Novel conjugates and assays for simultaneous detection of multiple ligands |
| GB9624750D0 (en) * | 1996-11-28 | 1997-01-15 | Univ London | Capture assays |
-
1998
- 1998-12-23 DE DE19859912A patent/DE19859912C2/en not_active Expired - Fee Related
-
1999
- 1999-12-22 CA CA002353920A patent/CA2353920A1/en not_active Abandoned
- 1999-12-22 WO PCT/EP1999/010333 patent/WO2000039581A2/en not_active Application Discontinuation
- 1999-12-22 AU AU25390/00A patent/AU773046B2/en not_active Ceased
- 1999-12-22 JP JP2000591429A patent/JP2002533724A/en active Pending
- 1999-12-22 EP EP99968373A patent/EP1141725A2/en not_active Withdrawn
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10705084B2 (en) | 2009-07-31 | 2020-07-07 | Invisible Sentinel, Inc. | Analyte detection devices, multiplex and tabletop devices for detection of analytes, and uses thereof |
| US10495638B2 (en) | 2009-10-09 | 2019-12-03 | Invisible Sentinel, Inc. | Device for detection of analytes and uses thereof |
| US10018626B2 (en) | 2012-03-09 | 2018-07-10 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| AU2013230917B2 (en) * | 2012-03-09 | 2018-11-29 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| EP3578980A1 (en) * | 2012-03-09 | 2019-12-11 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| US10732177B2 (en) | 2012-03-09 | 2020-08-04 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| AU2018264112B2 (en) * | 2012-03-09 | 2021-05-27 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| AU2013230917C1 (en) * | 2012-03-09 | 2021-09-16 | Invisible Sentinel, Inc. | Methods and compositions for detecting multiple analytes with a single signal |
| WO2020079638A1 (en) * | 2018-10-17 | 2020-04-23 | Molecular Devices (Austria) GmbH | Real time western blot assays utilizing fluorescence resonance energy transfer (fret) |
| US11604186B2 (en) | 2018-10-17 | 2023-03-14 | Molecular Devices (Austria) GmbH | Real time western blot assays utilizing fluorescence resonance energy transfer (FRET) |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000039581A2 (en) | 2000-07-06 |
| EP1141725A2 (en) | 2001-10-10 |
| AU2539000A (en) | 2000-07-31 |
| AU773046B2 (en) | 2004-05-13 |
| JP2002533724A (en) | 2002-10-08 |
| DE19859912C2 (en) | 2001-06-21 |
| WO2000039581A3 (en) | 2000-11-23 |
| DE19859912A1 (en) | 2000-07-06 |
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