Analyte detection

Patent metadata

Field	Value
Publication number	US-10330684-B1
Application number	US-201815946498-A
Country	US
Kind code	B1
Filing date	Apr 5, 2018
Priority date	Apr 5, 2018
Publication date	Jun 25, 2019
Grant date	Jun 25, 2019

Abstract

This disclosure relates generally to detection and, in particular, to detecting objects within a sample or fraction thereof.

First claim

What is claimed is:

1. A method of detecting an analyte, comprising:
providing a sample or a fraction thereof;
adding, to the sample or fraction thereof, a first plurality of stains directed to a white blood cell marker, a granulocyte marker, and an endothelial cell marker after having performed a first antigen retrieval;
adding, to the sample or fraction thereof, a stain directed to a mesenchymal cell marker after having performed a second antigen retrieval; and
detecting at least one target analyte, wherein the at least one target analyte expresses expressing the mesenchymal cell marker, and wherein the at least one target analyte does not express any one of the white blood cell marker, the granulocyte marker, and the endothelial cell marker.

2. The method of claim 1 , wherein the detection moiety of the mesenchymal cell marker stain provides a signal having a different color than the detection moieties of the first plurality of stains.

3. The method of claim 2 , wherein the detecting step also detects at least one non-target analyte expressing at least one biomarker to which at least one stain of the first plurality of stains is directed.

4. The method of claim 3 , wherein the detection moieties of the first plurality of stains provide a signal having the same color.

5. The method of claim 3 , wherein the detection moieties of the first plurality of stains have the same emission wavelength.

6. The method of claim 2 , wherein the first adding step further comprises amplifying a signal generated by at least one of the stains.

7. The method of claim 6 , wherein the amplifying step uses horseradish peroxidase.

8. The method of claim 1 , wherein each affinity molecule of the first plurality of stains is derived from the same species.

9. A method of detecting an analyte, comprising:
providing a sample or a fraction thereof;
performing a first antigen retrieval on the sample or fraction thereof at 50-90° C.;
adding, to the sample or fraction thereof, a first plurality of stains directed to a white blood cell marker, a granulocyte marker, and an endothelial cell marker;
adding, to the sample or fraction thereof, a stain directed to a mesenchymal cell marker after having performed a second antigen retrieval; and
detecting at least one target analyte expressing the mesenchymal cell marker and not expressing the white blood cell marker, the granulocyte marker, or the endothelial cell marker.

10. The method of claim 9 , further comprising performing a second antigen retrieval on the sample or fraction thereof at a temperature greater than the temperature at which the first antigen retrieval step is performed,
wherein the second antigen retrieval step is performed after the first adding step and before the second adding and detecting steps.

11. The method of claim 1 , wherein the target analyte is a mesenchymal cell.

12. The method of claim 11 , wherein the non-target analyte is a white blood cell or an endothelial cell.

13. A method of detecting an analyte, comprising:
providing a sample or a fraction thereof;
performing a first antigen retrieval on the sample or fraction thereof at 50-85° C.;
adding, to the sample or fraction thereof, a first plurality of stains directed to a white blood cell marker, a granulocyte marker, and an endothelial cell marker;
adding, to the sample or fraction thereof, a stain directed to a mesenchymal cell marker after having performed a second antigen retrieval; and

detecting at least one target analyte expressing the mesenchymal cell marker and not expressing the white blood cell marker, the granulocyte marker, or the endothelial cell marker.

14. The method of claim 13 , further comprising performing a first antigen retrieval on the sample or fraction thereof at 50-80° C.,
wherein the first antigen retrieval step is performed before the first adding step.

15. The method of claim 14 , further comprising performing a first antigen retrieval on the sample or fraction thereof at 70° C.,
wherein the first antigen retrieval step is performed before the first adding step.

16. The method of claim 1 , wherein the granulocyte marker is CD11b, CD66b, or CD15.

17. The method of claim 16 , wherein the endothelial cell marker is CD105 or CD144.

18. The method of claim 16 , wherein the endothelial cell marker is CD31.

19. The method of claim 1 , wherein the mesenchymal cell marker is vimentin, fibronectin, or N-cadherin.

20. The method of claim 1 , wherein the second adding step further comprises amplifying a signal generated by at least one of the stains.

21. The method of claim 20 , wherein the amplifying step uses a hapten and an anti-hapten.

22. The method of claim 1 , wherein the detecting step further detects at least one non-target analyte expressing:
at least one of the white blood cell marker, the granulocyte marker, or the endothelial cell marker, and
the mesenchymal cell marker.

Assignees

• Rarecyte Inc

Inventors

• Campton Daniel E
• Ramirez Arturo B
• Duplessis Melinda R

Classifications

• G01N33/5759 Primary

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/57492 Primary

  Physics · mapped topic

• G01N33/56966 Primary

  Animal cells · CPC title