Method for determining an analyte

• US 20050084855A1
• Filed: 05/31/2002
• Published: 04/21/2005
• Est. Priority Date: 06/01/2001
• Status: Active Grant

• Current Assignee: Arrowhead Research Corporation
• Original Assignee: Arrowhead Research Corporation
• Inventors: Albrecht, Christian, Spinnler, Katrin, Blank, Kerstin, Gilbert, Ilka, Mai, Thao, Brink, Gunnar, Steipe, Boris, Oesterhelt, Filipp, Gaub, Hermann
• Granted Patent: US 7,700,371 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

The present invention relates to a method for determining an analyte in a sample. According to said method, a probe is transferred from a stamp member onto an analyte that is bound to a support member. The invention is particularly suitable for microarrays. The invention also relates to an apparatus for performing said method.

Claims

1. Method for determining an analyte in a sample, characterized in that a sample that might contain the analyte being determined is brought into contact with a first surface (i.e. support member) so that the analyte possibly contained in the sample is bound to the support member;

a second surface (i.e. stamp member) to which a probe that can bind the analyte is bound is brought closer to the support member so that the probe and the analyte can interact, the binding of the probe to the analyte and the binding of the analyte to the support member being more stable under an externally applied tensile force than the binding of the probe to the stamp member;

the stamp member and support member are separated from each other; and

it is determined whether the probe is bound to the support member and/or how much probe is bound to the support member.

2. Method according to claim 1, characterized in that molecules that can bind the analyte (i.e. receptors) are immobilized on the support member, the binding of the analytes to the receptor being more stable under an externally applied tensile force than the binding of the probe to the stamp member.

3. Method according to claim 2, characterized in that the receptors and/or probes are selected from the group comprising polyclonal or monoclonal antibodies, antibody fragments or antibody derivatives that can recognize and bind the analyte(s).

4. Method according to claim 3, characterized in that the receptors and/or probes used are antibody fragments or derivatives selected from Fv-, Fab-, Fab′

- or F(ab′

)₂ fragments, “

single-chain antibody fragments”

, bispecific antibodies, chimeric antibodies, humanized antibodies and fragments containing CDRs (complementarity determining regions) that recognize an epitope of the analyte.

5. Method according to claim 1, characterized in that the receptors and/or probes used are nucleic acids such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), synthetic nucleic acids such as LNA (locked nucleic acid) and PNA (peptide nucleic acid), and three-dimensional structures made of nucleic acids, preferably aptamers.

6. Method according to claim 1, characterized in that the analyte is selected from the group comprising proteins, polypeptides, peptides, nucleic acids, modified nucleic acids, molecules similar to nucleic acids such as peptide nucleic acid (PNA) or locked nucleic acid (LNA), three-dimensional nucleic acid structures, aptamers, carbohydrates and modified variants thereof.

7. Method according to claim 6, characterized in that the analyte is a protein, polypeptide or a peptide.

8. Method according to claim 1, characterized in that the probe binds specifically to the analyte.

9. Method according to claim 1, characterized in that the probe is selected from the group comprising antibodies, antibody fragments and antibody derivatives.

10. Method according to claim 1, characterized in that the probe carries a marker.

11. Method according to claim 1, characterized in that the probe is immobilized at the stamp member by a sensor complex.

12. Method according to claim 11, characterized in that the sensor complex comprises a first binding partner that is bound to the stamp member and a second binding partner that is bound fixedly to the probe, wherein the first binding partner and the second binding partner can bind non-covalently to each other.

13. Method according to claim 11, characterized in that the first binding partner and the second binding partner are nucleic acids.

14. Method according to claim 13, characterized in that the first binding partner and the second binding partner are selected from the group comprising single-stranded DNA and/or single-stranded RNA.

15. Method according to claim 14, characterized in that the 5′

end of the first binding partner is bound to the stamp member and the 3˜

end of the second binding partner is bound to the probe, or that the 3′

end of the first binding partner is bound to the stamp member and the 5′

end of the second binding partner is bound to the probe.

16. Method according to claim 1, characterized in that the stamp member comprises a plurality of areas separated from each other, in which probes with different specificity for different analytes are bound.

17. Method according to claim 2, characterized in that the support member comprises a plurality of areas separated from each other, in which receptors with different specificity for different analytes are bound.

18. Method according to claim 16, characterized in that the support member has a plurality of areas separated from each other, in which receptors with different specificity for different analytes are bound in such a way that they are disposed symmetrically to the areas on the stamp member, so that when the stamp member and the support member approach each other, the areas on the probes and receptors that have specificity for the same analyte become aligned.

19. Method according to claim 16, characterized in that both the support member and the stamp member have areas in which receptors are immobilized, as well as areas in which probes are immobilized, the respective areas being so arranged that when the stamp member and the support member are brought into contact, areas with receptors for an analyte and areas with probes for the same analyte are positioned facing each other.

20. Method according to claim 16, characterized in that the stamp member and/or support member have sub-areas on which a plurality of spots are arranged.

21. Method according to claim 20, characterized in that the stamp member and/or support member have sub-areas with a plurality of spots, the spots on a sub-area each having different receptors or probes that can bind to an analyte.

22. Method according to claim 20, characterized in that the stamp member and/or the support member have at least one spot where receptors capable of binding directly with the probe are immobilized, said spots being used to monitor coupling.

23. Apparatus for determining an analyte in a sample, comprising two surfaces each having sub-areas to which receptors and probes capable of binding with different analytes are bound, said receptors and probes being respectively so arranged that when the two surfaces come into contact, the receptors and/or probes capable of binding with the same analyte face each other, wherein the probes are each immobilized by a sensor complex and bear a marker.

24. Apparatus according to claim 23, characterized in that the apparatus comprises a stamp member and a support member, and that a first binding partner is bound fixedly to the stamp member and a second binding partner is bound fixedly to the probe, wherein the first binding partner and the second binding partner can bind non-covalently to each other.

25. Apparatus according to claim 24, characterized in that the first binding partner and the second binding partner are nucleic acids.

26. Apparatus according to claim 25, characterized in that the first binding partner and the second binding partner are selected from the group comprising single3o stranded DNA and/or single-stranded RNA.

27. Apparatus according to claim 26, characterized in that the 5, end of the first binding partner is bound to the stamp member and the 3′

end of the second binding partner is bound to the probe, or that the 3˜

end of the first binding partner is bound to the stamp member and the 5′

end of the second binding partner is bound to the probe.

28. Apparatus according to claim 23, characterized in that the apparatus also comprises a device for bringing close together and separating the two surfaces.

29. Apparatus according to claim 23, characterized in that the apparatus also comprises a device for detecting whether and/or in what quantity the probe is bound to the support member.

30. Apparatus according to claim 29, characterized in that the apparatus has a device for detecting the marker.

31. Method according to claim 23, characterized in that the stamp member comprises a plurality of areas separated from each other, in which probes with different specificity for different analytes are bound.

32. Apparatus according to claim 23, characterized in that the support member comprises a plurality of areas separated from each other, in which receptors with different specificity for different analytes are bound.

33. Apparatus according to claim 31, characterized in that the support member has a plurality of areas separated from each other, in which receptors with different specificity for different samples are bound in such a way that they are disposed symmetrically to the areas on the stamp member, so that when the stamp member and the support member approach each other, the areas on the probes and receptors that have specificity for the same sample become aligned.

34. Apparatus according to claim 23, characterized in that the areas each have a plurality of spots on each of which different receptors and probes capable of binding with an analyte are arranged.