Method for simultaneous multiple probes/multiple targets screening procedure

• US 20040248325A1
• Filed: 07/12/2004
• Published: 12/09/2004
• Est. Priority Date: 07/19/2001
• Status: Abandoned Application

• Current Assignee: Signet Laboratories Incorporated (Revvity, Inc.)
• Original Assignee: Signet Laboratories Incorporated (Revvity, Inc.)
• Inventors: Bukusoglu, Cuneyt
• Application Number:
  US10/484,220
• Publication Number:
  US 20040248325A1
• Time in Patent Office: Days
• US Class Current: 436/548
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/5088 of vertebrates
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

Monoclonal antibodies that are specific for a physiological disorder of interest are used to conduct an immunometric assay for antigens correlated with that specific disorder. The invention provides a method which comprises a process having one probe and one target detection. Multiple probes are applied tissue samples without destroying the integrity of the tissue sample. The inventive method utilizes a specific reagent system that is a Probe-Tagging system. This system utilizes a cleavable linker and a specific tagging molecule that upon dissociation may be analyzed using micro-array-based detection systems or other detection methods known within the art.

Claims

1. A method of forming a probe with a cleavable tag for use in an immunometric assay for determining the presence or concentration of a target in a sample comprising the steps of:

providing a compound capable of reacting with said target to be assayed;

modifying said compound with a linking agent forming a compound with linkage;

reducing said linkage with a reducing agent forming a compound with a reactive group;

providing a detectable tag;

modifying said tag with a linking agent forming a tag with a reactive group;

incubating said tag with a reactive group with said compound with said reactive group forming a compound having a cleavable tag specific for said target.

2. The method of claim 1 wherein said tag is a peptide.

3. The method of claim 1 wherein said compound is an antibody.

4. The method of claim 3 wherein said antibody is mouse anti-human BG-5 monoclonal specific antibody.

5. The method of claim 3 wherein said antibody is mouse anti-human AE1 monoclonal specific antibody.

6. The method of claim 1 where said linking agent is a heterobifunctional cross linking agent.

7. The method of claim 6 wherein said heterobifunctional cross linking agent is N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

8. The method of claim 1 wherein said reducing agent is Dithiothreitol.

9. The method of claim 1 wherein said tag is Lysozyme

10. The method of claim 1 wherein said tag is carbonic anhydrase.

11. The method of claim 1 wherein said tag is selected from the group consisting of peptides, proteins, carbohydrates, nucleic acids, DNA, RNA, PNA, oligonucleotides, beads, particles, vesicles, liposomes, nanobeads, nanobarcodes, nanocrystals.

12. The method of claim 1 wherein said compounds is selected from the group consisting of peptides, proteins, nucleic acid sequences, DNA, RNA, PNA carbohydrates, lipids, steroids.

13. The method of claim 1 wherein said compound having a cleavable tag specific for said target formed by the process is BG5-ss-Lysozyme.

14. The method of claim 1 wherein said a compound having a cleavable tag specific for said target formed by the process is AE1-ss-carbonic anhydrase.

15. A method for detecting antigens in tissue comprising the steps of:

providing an antibody specific for the antigen to be detected;

modifying said antibody wherein a cleavable tag is attached to said antibody forming an antibody with a cleavable tag;

incubating said tissue with said antibody with cleavable tag under conditions permitting an immunological reaction between said antibody with cleavable tag and said antigen to be detected thereby forming a complex;

cleaving said tag from said complex;

detecting said tag.

16. The method of claim 15 wherein said antibody is mouse anti-human BG-5 monoclonal specific antibody.

17. The method of claim 15 wherein said antibody is mouse anti-human AEI monoclonal specific antibody.

18. The method of claim 15 wherein said tag is Lysozyme

19. The method of claim 15 wherein said tag is carbonic anhydrase.

20. The method of claim 15 wherein said tag is selected from the group consisting of peptides, proteins, antigen complexes, nucleic acid complexes, DNA complexes, RNA complexes and enzyme complexes.

21. A method of forming a antibody with a cleavable tag for use in an immunometric assay for determining the presence or concentration of an antigenic substance in a sample comprising the steps of:

providing an antibody capable of immunologically reacting with said antigen to be assayed;

modifying said antibody with N-Succinimidyl 3-(2-pyridyldithio)propionate forming a reactive disulfide linkage;

reducing said disulfide linkage with Dithiothreitol forming an antibody with a free sulfhydryl group;

providing a detectable tag;

modifying said detectable tag with additional N-Succinimidyl 3-(2-puridyldithio)propionate to form a detectable tag with a reactive group;

incubating said detectable tag with a reactive group with said antibody with a free sulfhydryl group forming an antibody having a cleavable tag specific for said antigen.

22. The method of claim 21 wherein said tag is selected from a group consisting of lysozymne and carbonic anhydrase

23. The method of claim 21 wherein said antibody is mouse anti-human BG-5 monoclonal specific antibody.

24. The method of claim 21 wherein said antibody is mouse anti-human AE1 monoclonal specific antibody.

25. A method of forming an antibody with a cleavable tag for use in an immunometric assay for determining the presence or concentration of an antigenic substance in a sample which comprises:

providing antigen detection means capable of immunologically reacting with said antigen to be assayed;

modifying said antigen detection means forming a compound reactive linkage means;

reducing said reactive linkage means to form a free reactive group;

providing detectable tag means;

modifying said detectable tag means with reactive group means;

incubating said modified detectable tag means with said reactive group means with said free reactive group of said antigen detection means forming antigen detection means specific for said antigen to be assayed and having a cleavable tag.

26. The method of claim 25 wherein said detectable tag means is selected from the group consisting of Lysozyme and Carbonic Anhydrase.

27. The method of claim 25 wherein said antigen detection means is a mouse anti-human AE1 antibody.

28. The method of claim 25 wherein said antigen detection means is a mouse anti human BG5 antibody.

29. The method of claim 25 further including the step of concentrating said detectable tag prior to said detecting step.

30. A method for detecting antigens in tissue comprising the steps of:

providing antigen detection means, specific for the antigen to be detected and having cleavable marking means;

contacting said tissue with said antigen detection means under conditions permitting the formation of antigen-antigen detection means complex to be formed;

cleaving said marking means from said complex;

detecting said marking means to determine its presence or absence; and

correlating said presence or absence with the presence or absence of said antigen to be detected.

31. A method of forming a compound having a probe anti-probe complex having distinct cleavable tags for use in an immunometric assay for determining the presence of targets in a sample comprising the steps of:

providing an anti-probe capable of binding with multiple probes wherein said probes react with said target to be assayed;

modifying said anti-probe with a linking agent forming a complex with linkage;

reducing said linkage with a reducing agent forming an anti-probe with a reactive group;

providing at least one detectable tag;

modifying said detectable tag with a linking agent forming a detectable tag with a reactive group;

incubating said detectable tag with a reactive group with said anti-probe with said reactive group forming at least one anti-probe having a cleavable detectable tag;

providing at least one probe; and

incubating said probe with said anti-probe having a cleavable detectable tag forming a probe anti-probe complex having a cleavable detectable tag.

32. The method of claim 31 wherein said anti-probe is goat anti-mouse antibody.

33. The method of claim 31 wherein said detectable tag is selected from the group consisting of peptides, proteins, carbohydrates, nucleic acids, DNA, RNA, PNA, oligonucleotides, beads, particles, vesicles, liposomes, nanobeads, nanobarcodes, nanocrystals.

34. The method of claim 31 wherein said probe is an antibody.

35. The method of claim 31 wherein said complex is selected from the group consisting of Goat anti mouse IgG, Rabbit anti goat IgG, Mouse anti Xpress, Mouse anti HiG, Mouse anti myc, Mouse anti Thio and Rabbit anti GFP.

36. The method of claim 31 wherein said anti-probe is selected from the group consisting of Protein A and Protein G, Lectins, Sugars, Nickel coupled Matrix and Heparin

37. A method for detecting antigens in tissue comprising the steps of:

providing an anti-probe;

modifying said anti-probe wherein a cleavable detectable tag is attached to said anti-probe forming an anti-probe with a cleavable detectable tag;

incubating said anti-probe with a cleavable detectable tag with a probe forming a probe anti probe complex having a cleavable detectable tag;

incubating said tissue with said probe anti probe complex having a cleavable detectable tag under conditions permitting an immunological reaction between said probe anti probe complex having a cleavable detectable tag said antigen to be detected thereby forning a complex;

cleaving said tag from said complex;

detecting said tag.

38. A method of using a probe with a cleavable tag and a connecting probe for use in proximity based assay system for determining the localization of bound-probe on a target tissue comprising the steps of:

providing two or more probes having cleavably linked tag molecules wherein said probes are capable of reacting with said target to be assayed;

providing a connecting probe capable of connecting said tag molecules and providing conditions necessary for said connecting probe to connect said tag molecules;

cleaving said linked tag molecules and releasing said connecting probe with connected tags

capturing said connecting probe with connected tags; and

detecting and quantifying said connecting probe with said connected tags.

39. The method of claim 38 wherein said probe is an antibody, peptide, protein, nucleic acid, DNA, RNA, carbohydrates, organic molecule and in-organic molecule.

40. The method of claim 38 wherein said tag molecule is selected from the group consisting of oligonucleotide, nucleic acid, DNA, RNA, PNA, peptide, protein.

41. The method of claim 38 wherein said connecting probe is selected from the group consisting of oligonucleotide, nucleic acid, DNA, RNA, PNA, protein, aptamer, antibody;

42. The method of claim 38 wherein said connecting probe is a polymer.

43. The method of claim 42 wherein said polymer is selected from the group consisting of nucleic acid, DNA, RNA, PNA, protein, antibody;

nanobarcodes, beads, nanocrystals.

44. The method of claim 38 wherein said connecting probe can connect specific tag molecules within proximity.