Detection conjugate and method for analysis

• US 8,865,414 B2
• Filed: 12/17/2009
• Issued: 10/21/2014
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Medizinische Hochschule Hannover
• Original Assignee: Medizinische Hochschule Hannover
• Inventors: Hennig, Christian, Hansen, Gesine
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US13/139,870
• Publication Number:
  US 20110311966A1
• Time in Patent Office: 1,769 Days
• Field of Search: 435/6.1, 435/7.1, 536/22.1, 536/23.1, 536/24.3, 530/350, 530/387.1
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

The invention provides a combination of reagents as a test kit containing a detection conjugate having a binding portion and a reagent for deactivating of the fluorochrome portion by interaction with the linker. The binding portion especially is an antibody portion. The detection conjugate has a fluorochrome portion connected to the antibody portion, and a linker connected to the fluorochrome portion, wherein the linker comprises an oligonucleotide. The fluorochrome portion can be deactivated by hydrolysis of the linker or by specific hybridization of a quencher having an oligonucleotide to the linker.

Claims

1. A detection conjugate for detection of analytes in samples, comprising

a first reagent having a detection conjugate having a binding portion specific for an analyte, a fluorochrome portion connected to the binding portion, and an oligonucleotide linker connected by its first end to the fluorochrome portion, which oligonucleotide linker at least in a section is an oligonucleotide having a quencher section within its nucleic acid sequence, and

a second reagent having a quencher that is connected to that end of an oligonucleotide hybridizeable to the quencher section which upon hybridizing with the oligonucleotide linker is located at the first end thereof,

wherein the binding portion is selected from the group consisting of antibodies, antibody fragments forming a paratope, lectins, peptides specific for predetermined MHC I, peptides specific for predetermined MHC II, and MHC tetrameres.

2. The detection conjugate according to claim 1, wherein the oligonucleotide linker is connected to the binding portion with its second end opposite the first end.

3. The detection conjugate according to claim 1, wherein the oligonucleotide linker has a first oligonucleotide linker section that is bound to the binding portion, wherein the first oligonucleotide linker section in its nucleic acid sequence has a fluorochrome section, and in that the fluorochrome portion is connected to a second oligonucleotide linker section hybridizeable to the fluorochrome section.

4. The detection conjugate according to claim 3, wherein the second oligonucleotide linker section that is connected to the fluorochrome portion has the quencher section.

5. The detection conjugate according to claim 4, wherein at least two detection conjugates are contained, each having fluorochrome sections with identical or similar nucleotide sequences to which the same second oligonucleotide linker section is hybridizeable under physiological conditions.

6. The detection conjugate according to claim 1, wherein at least two detection conjugates are contained, each having quencher sections of unique nucleotide sequences.

7. The detection conjugate according to claim 1, wherein at least two detection conjugates are contained, each having quencher sections having identical or similar nucleotide sequences to which the same oligonucleotide connected to the quencher is hybridizeable under physiological conditions.

10. The detection conjugate according to claim 1, wherein independent from one another, the oligonucleotide linker and/or the oligonucleotide connected to the quencher is a single-stranded oligo ribonucleic acid (RNA), oligo deoxyribonucleic acid (DNA), or oligo peptidenucleic acid (PNA).

11. The detection conjugate according to claim 3, wherein independent from one another, the first oligonucleotide linker section of the oligonucleotide linker and/or the second oligonucleotide linker section hybridizeable thereto which is connected to the fluorochrome portion, is a single-stranded oligo ribonucleic acid (RNA), oligo deoxyribonucleic acid (DNA), or oligo peptidenucleic acid (PNA).

12. The detection conjugate according to claim 1, wherein two or more oligonucleotide linkers are connected to the binding portion.

13. A method for analysis of a biological sample with the detection conjugate according to claim 1, comprising:

contacting the sample with the first reagent having a detection conjugate having a binding portion specific for a first analyte, contacting the sample with the second reagent, and contacting the sample with a detection conjugate having a binding portion specific for a second analyte.

14. A method for analysis of biological samples, comprising the production of the detection conjugate according to claim 1 in contact with the sample.

8. A detection conjugate for detection of analytes in samples, comprising

a first reagent having a detection conjugate having a binding portion specific for an analyte, a fluorochrome portion connected to the binding portion, and an oligonucleotide linker connected by its first end to the fluorochrome portion, which oligonucleotide linker at least in a section is an oligonucleotide having a quencher section within its nucleic acid sequence, and

a second reagent which is a nuclease,

wherein the binding portion is selected from the group consisting of antibodies, antibody fragments forming a paratope, lectins, peptides specific for predetermined MHC I, peptides specific for predetermined MHC II, and MHC tetrameres.

9. The detection conjugate according to claim 8, wherein the oligonucleotide linker has a recognition sequence for a restrictase and the nuclease is a restrictase specific for the recognition sequence.

24. The detection conjugate according to claim 8, wherein the oligonucleotide linker has no quencher section.

15. A method for analysis of biological samples, comprising the steps of:

contacting a biological sample with a detection conjugate having a binding portion having a first specificity for a first analyte, a fluorochrome portion connected to the binding portion, and a linker connected by its first end to the fluorochrome portion,

detecting radiation emitted by the fluorochrome portion, and

deactivating the fluorochrome portion,

wherein

the linker is an oligonucleotide linker which at least in a section is an oligonucleotide having in its nucleic acid sequence a quencher section, and wherein

the deactivation of the fluorochrome portion occurs by contacting the detection conjugate with a quencher which is connected to that end of an oligonucleotide hybridizeable with the quencher section which upon hybridizing to the oligonucleotide linker is located at the first end thereof,

wherein the binding portion is selected from the group consisting of antibodies, antibody fragments forming a paratope, lectins, peptides specific for predetermined MHC I, peptides specific for predetermined MHC II and MHC tetrameres.

16. The method according to claim 15, comprising the subsequent contacting of the sample with a detection conjugate of a first analyte specificity, contacting of the sample with a quencher specific for the first detection conjugate, and contacting of the sample with a detection conjugate of a second analyte specificity.

17. The method according to claim 16, wherein the fluorochrome portion and the oligonucleotide linker of detection conjugates used in subsequent contactings are identical to each other and each time the quencher is the same having the same oligonucleotide.

19. The method according to claim 15, wherein the oligonucleotide linker has a first oligonucleotide linker section which is bound to the binding portion and in its nucleotide sequence has a fluorochrome section, and in that the binding conjugate is produced by contacting the fluorochrome section with a fluorochrome portion which is connected to a second oligonucleotide linker section hybridizeable to the fluorochrome section.

20. The method according to claim 19, wherein the sample is simultaneously contacted with at least two detection conjugates, the fluorochrome sections of which are each unique.

21. The method according to claim 15, wherein the sample is simultaneously contacted with at least two detection conjugates, the quencher sections of which are each unique.

22. The method according to claim 15, wherein the analytes are fixed on a carrier substrate.

23. The method according to claim 22, wherein the carrier substrate is part of a flow channel, in which the analytes are arranged for being circumfused by aqueous compositions.

25. The detection conjugate according to claim 15, wherein the oligonucleotide linker has a first oligonucleotide linker section that is bound to the binding portion, wherein the first oligonucleotide linker section in its nucleic acid sequence has a fluorochrome section, and in that the fluorochrome portion is connected to a second oligonucleotide linker section hybridizeable to the fluorochrome section.

27. The method according to claim 19, comprising the subsequent contacting of the sample with a detection conjugate of a first analyte specificity, contacting of the sample with a quencher specific for the first detection conjugate, and contacting of the sample with a detection conjugate of a second analyte specificity.

18. A method for analysis of biological samples, wherein deactivating the fluorochrome portion occurs by contacting the detection conjugate with a nuclease and subsequent removal of unbound components, the method comprising the steps of:

contacting a biological sample with a detection conjugate having a binding portion having a first specificity for a first analyte, a fluorochrome portion connected to the binding portion, and a linker connected by its first end to the fluorochrome portion,

detecting radiation emitted by the fluorochrome portion,

deactivating the fluorochrome portion,

wherein

the linker is an oligonucleotide linker, and

deactivating the fluorochrome portion occurs by contacting the detection conjugate with a second reagent which is a nuclease,

wherein the binding portion is selected from the group consisting of antibodies, antibody fragments forming a paratope, lectins, peptides specific for predetermined MHC I, peptides specific for predetermined MHC II, and MHC tetrameres.

26. The method according to claim 18, wherein the oligonucleotide linker has a recognition sequence for a restrictase and the nuclease is a restrictase specific for the recognition sequence.

28. The method according to claim 18, wherein the analytes are fixed on a carrier substrate.

29. The method according to claim 18, wherein the carrier substrate is part of a flow channel, in which the analytes are arranged for being circumfused by aqueous compositions.