Conjugates, and use thereof in detection methods
The invention relates to methods for the quantitative and qualitative detection of an analyte in an assay as well as appropriate reagents therefor, especially a carrier molecule to which one or several analyte-specific binding partners are bound and which is provided with additional binding points for a specific binding partner X or Y that is associated with a component of a signal-forming system. Also disclosed is the use of such a conjugate particularly in homogeneous binding tests.
1 . A conjugate which consists of a carrier molecule that is associated with one or more analyte-specific binding partners, wherein the conjugate possesses additional binding sites for a specific binding partner X or Y.
2 . The conjugate as claimed in claim 1 , wherein the carrier molecule consists of dextran, cyclodextrin or dendrimers or wherein the carrier molecule consists of antibodies, albumin molecules, enzymes, or mixtures thereof, that are covalently bonded together.
3 . The conjugate as claimed in claim 1 , wherein the additional binding sites are biotin, digoxigenin, fluorescein, dinitrophenol or single-stranded nucleic acid chains.
4 . The conjugate as claimed in claim 1 , wherein the carrier molecule is covalently bonded to one or more analyte-specific binding partners.
5 . The conjugate as claimed in claim 1 which possesses at least 2 binding sites for a specific binding partner X or Y.
6 . The conjugate as claimed in claim 1 , wherein the carrier molecule consists of antibodies that are covalently bonded together.
7 . A microparticle bonded to a conjugate as claimed in claim 1 , wherein the microparticle is bonded to a specific binding partner X or Y of the conjugate.
8 . The microparticle as claimed in claim 7 which, as a component of a signal-generating system, is associated with photosensitizers or with chemiluminescent substances.
9 . A reagent which contains at least one of: (1) one or more conjugates as claimed in claim 1 and (2) one or more microparticles as claimed in claim 7 .
10 . A test kit which contains a reagent as claimed in claim 9 .
11 . A method of quantitatively or qualitatively detecting an analyte in a sample in a homogeneous or heterogeneous binding test, comprising
(1) contacting at least one of
(a) a conjugate as claimed in claim 1 ,
(b) a microparticle as claimed in claim 7 , and
(c) a reagent as claimed in claim 9
with the sample,
(2) binding said conjugate, microparticle or reagent to the analyte, and
(3) quantitatively or qualitatively detecting the analyte in the sample.
12 . A method of quantitatively or qualitatively detecting an analyte in a sample, comprising
(1) contacting a conjugate as claimed in claim 1 with the sample, wherein the conjugate possesses specific binding sites for the specific binding partner X, which is associated with a component of a signal-generating system,
(2) binding said conjugate to the analyte, and
(3) quantitatively or qualitatively detecting the analyte in the sample.
13 . The method of quantitatively or qualitatively detecting an analyte in a sample as claimed in claim 12 , further comprising
(1) contacting a second conjugate as claimed in claim 1 with the sample, wherein the second conjugate possesses specific binding sites for the specific binding partner Y, which is associated with a component of a signal-generating system, and
(2) binding said second conjugate to the analyte.
14 . The method as claimed in claim 12 , wherein the number of binding sites for the specific binding partner X is at least 2.
15 . The method as claimed in claim 13 , wherein the number of binding sites for the specific binding partner Y is at least 2.
16 . The method as claimed in claim 13 , wherein X and Y are the same specific binding partner or different specific binding partners.
17 . The method as claimed in claim 12 , wherein X is avidin, streptavidin, an anti-digoxigenin antibody, an anti-dinitrophenol antibody, a single-stranded nucleic acid chain, an anti-hapten antibody, an enzyme, an enzyme substrate or an antibody, wherein the antibody is able to bind particular polypeptides, oligopeptides or enzymes specifically.
18 . The method as claimed in claim 13 , wherein Y is avidin, streptavidin, an anti-digoxigenin antibody, an anti-dinitrophenol antibody, a single-stranded nucleic acid chain, an anti-hapten antibody, an enzyme, an enzyme substrate or an antibody, wherein the antibody is able to bind particular polypeptides, oligopeptides or enzymes specifically.
19 . The method as claimed in claim 12 , wherein components of the signal-generating system are brought, as a result of the binding of the analyte-specific binding partners, to a distance from each other which permits an interaction between these components, and the magnitude of this interaction is measured.
20 . The method as claimed in claim 12 , wherein components of the signal-generating system are brought, as a result of the binding of the analyte-specific binding partners, to a distance from each other which permits no interaction, or only very slight interaction, between these components, and the residual magnitude of this interaction is measured.
21 . The method as claimed in claim 12 , wherein the components of the signal-generating system are microparticles.
22 . The method as claimed in claim 12 , wherein components of the signal-generating system are microparticle-associated photosensitizers and microparticle-associated chemiluminescent substances.
23 . The method as claimed in claim 12 , wherein the method is an immunoassay.
24 . The method as claimed in claim 12 , wherein the method is a homogeneous binding test.
25 . The conjugate as claimed in claim 5 which possesses more than 5 additional binding sites for a specific binding partner X or Y.
26 . The conjugate as claimed in claim 5 which possesses more than 10 additional binding sites for a specific binding partner X or Y.
27 . The conjugate as claimed in claim 5 which possesses more than 15 additional binding sites for a specific binding partner X or Y.
28 . The conjugate as claimed in claim 5 which possesses more than 18 additional binding sites for a specific binding partner X or Y.
29 . The conjugate as claimed in claim 6 , wherein the antibodies that are covalently bonded together are mouse or goat IgG antibodies.
30 . The microparticle as claimed in claim 7 , wherein the microparticle is a latex particle.
31 . The method as claimed in claim 19 , wherein the interaction is an energy transfer.
32 . The method as claimed in claim 20 , wherein the interaction is an energy transfer.
33 . A binding test as claimed in claim 21 , wherein the microparticles are latex particles.