Method for specific identification of target biomolecules

Patent metadata

Field	Value
Publication number	US-10732176-B2
Application number	US-201214128379-A
Country	US
Kind code	B2
Filing date	Jun 27, 2012
Priority date	Jun 29, 2011
Publication date	Aug 4, 2020
Grant date	Aug 4, 2020

Abstract

The present invention relates to a method for identification of specific target proteins in a protein sample following a detection procedure, such as a Western blotting procedure, wherein the membrane is probed with at least two primary antibodies directed against the same and/or different epitopes of the same target protein, and wherein specific binding to the target protein in a sample is differentiated from unspecific binding to the target protein by comparing the resulting sample patterns, such as bands or spot patterns, with each other.
In a further step signals from the true target proteins are enhanced while signals resulting from unspecific binding are diminished.

First claim

The invention claimed is:

1. A method for identification of specific target biomolecules in a sample in a detection procedure, comprising:
probing a gel or membrane with at least two probes that are directed against and specifically bind to a same target biomolecule to form bands or spot patterns on the gel or membrane;
obtaining image sample patterns corresponding to the bands or spot patterns;
overlaying the image sample patterns with each other;
generating image data by applying an algorithm that uses pixel-wise multiplication to enhance overlapping features and diminish non-overlapping features of the bands or spot patterns in the overlayed image sample patterns;
scaling the image data using a root function; and
differentiating the bands or spot patterns corresponding to specific binding of the probes to the target biomolecule in the sample from non-specific binding of the probes to other biomolecules in the sample using the scaled image data.

2. The method of claim 1 , wherein the target biomolecule is a protein, a peptide, an aggregated biomolecule, a fusion protein, a modified protein, a phosphorylated protein, a glycosylated protein, an ubiquitinated protein, a SUMOylated protein, or an acetylated protein.

3. The method of claim 1 , wherein the scaling of the image data uses a function involving image data from original images.

4. The method of claim 1 , wherein the resulting sample pattern is quantitatively analyzed.

5. The method of claim 1 , further comprising:
pre-processing the sample; and
separating the sample by electrophoresis after the pre-processing step.

6. The method of claim 5 , wherein the pre-processing of the sample comprises affinity separation by using antibodies against the target biomolecule.

7. The method of claim 5 , wherein at least two images represent aliquots of a sample that have been covalently labeled with different dyes and mixed together during the pre-processing of the sample.

8. The method of claim 1 , wherein the detection procedure is a Western blotting procedure.

9. The method of claim 8 , wherein the image data is in a form of one or more intensity profiles or 3D surfaces.

10. The method of claim 8 , wherein the image data is in a form of one or more 3D surfaces.

11. The method of claim 1 , wherein a final result is the image data presented in a form of one or more images where color coding has been used to highlight the overlapping and/or non-overlapping features of the bands or spot patterns.

12. The method of claim 11 , wherein the color coding represents a result of applying the algorithm to the image data.

13. The method of claim 1 , wherein at least two primary antibodies are used as probes.

14. The method of claim 13 , wherein the at least two primary antibodies are from different species.

15. The method of claim 13 , wherein the at least two primary antibodies are from the same species.

16. The method of claim 13 , wherein the at least two primary antibodies are differentially labelled.

17. The method of claim 13 , wherein one or more of the antibodies are detected using Enhanced chemiluminescence (ECL).

18. The method of claim 13 , wherein the primary antibodies are labeled with fluorescent dyes.

19. The method of claim 13 , wherein differentially labelled secondary antibodies are used against the at least two primary antibodies.

20. The method of claim 19 , wherein the secondary antibodies are labeled with fluorescent dyes.

21. The method of claim 20 , wherein the fluorescent dyes are cyanine dyes.

Assignees

• Bjorkesten Lennart
• Edlund Sofia
• Hagner Mcwhirter Asa
• Ge Healthcare Bio Sciences Ab

Inventors

• Bjorkesten Lennart
• Edlund Sofia
• Hagner-Mcwhirter Asa

Classifications

• G01N33/561 Primary

  Immunoelectrophoresis · CPC title

• G01N27/44726

  using specific dyes, markers or binding molecules · CPC title

• G01N33/543 Primary

  with an insoluble carrier for immobilising immunochemicals · CPC title