Method for improving cell permeability to foreign particles

• US 20070042358A1
• Filed: 07/27/2006
• Published: 02/22/2007
• Est. Priority Date: 07/28/2005
• Status: Active Grant

• Current Assignee: ID-Fish Technology, Inc.
• Original Assignee: ID-Fish Technology, Inc.
• Inventors: Weltman, Helena, Shah, Jyotsna
• Granted Patent: US 8,632,963 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/183 Amino acids, e.g. glycine, EDTA or aspartame
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/567 utilising isolate of tissue or organ as binding agent
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/5695 Mycobacteria

Abstract

The present invention provides a method for allowing foreign particles to penetrate, very efficiently, the cell wall, cell membrane, organelle membrane and/or nuclear membrane of a cell and hybridizing or binding to the complimentary target in the cell. The cells may be from a culture or from specimens obtained from a patient. The foreign particle can be a probe consisting of, for example, either individually or in any combination of two or more of the following: DNA, RNA, peptide nucleic acids (PNA), glycopeptides, lipopeptides, glycolipids or prions. The target is a cell, a cell component or, preferably, a pathogen or pathogen component. The pathogen can be, for example, bacteria, fungi, yeast or viruses.

Claims

1. A composition for increasing the permeability of cell walls, cell membranes and nuclear membranes, said composition comprising:

GuSCN (guanidine thiocyanate), Tris-HCL, EDTA, IGEPAL (octylphenoxy poly(ethyleneoxy)ethanol), acetic acid, methanol, sodium chloride and sodium deoxycholate.

2. The composition of claim 1, wherein said GuSCN is at a concentration of approximately 2.0 to 3.3 M.

3. The composition of claim 1, wherein said Tris-HCL is at a concentration of approximately 10 to 100 mM.

4. The composition of claim 1, wherein said Tris-HCL is at a pH of approximately 7.0 to 9.0.

5. The composition of claim 1, wherein said EDTA is at a concentration of approximately 5 to 50 mM.

6. The composition of claim 1, wherein said IGEPAL is at a concentration of approximately 0.1 to 2.0 percent.

7. The composition of claim 1, wherein said Acetic acid is at a concentration of approximately 1.0 to 10 percent.

8. The composition of claim 1, wherein said methanol is at a concentration of approximately 20 to 50 percent.

9. The composition of claim 1, wherein said sodium cholate is at a concentration of approximately 0.02 to 2.5 percent.

10. The composition of claim 1, wherein said sodium deoxycholate is at a concentration of approximately 0.02 to 2.5 percent.

11. A method for staining a target in a cell, comprising:

a) contacting the cell with a composition comprising GuSCN (guanidine thiocyanate), Tris-HCL, EDTA, IGEPAL (octylphenoxy poly(ethyleneoxy)ethanol), acetic acid, methanol, sodium chloride and sodium deoxycholate to create a permeabilized cell;

b) contacting the permeabilized cell of step (a) with a binding agent specific for binding to said target, and;

c) detecting said binding agent of step (b).

12. The method of claim 11, wherein said target is selected from a group consisting of nucleic acids, peptide nucleic acids, peptides, glycoproteins, lipids, lipoproteins, viruses and prions.

13. The method of claim 11, wherein said binding agent is selected from a group consisting of nucleic acids, peptide nucleic acids, peptides, lipoproteins, glycoproteins and lipids.

14. The method of claim 11, wherein said binding agent additionally comprises a detection moiety.

15. The method of claim 14, wherein the detection moiety is selected from a group consisting of fluorescent markers, radioactive markers, dyes, colloidal metals, biotin/avidin and horseradish peroxidase.

16. The method of claim 11, wherein said detection is via a labeled antibody with affinity for said binding agent.

17. The composition of claim 11, wherein said GuSCN is at a concentration of approximately 2.0 to 3.3 M.

18. The composition of claim 11, wherein said Tris-HCL is at a concentration of approximately 10 to 100 mM.

19. The composition of claim 11, wherein said Tris-HCL is at a pH of approximately 7.0 to 9.0.

20. The composition of claim 11, wherein said EDTA is at a concentration of approximately 5 to 50 mM.

21. The composition of claim 11, wherein said IGEPAL is at a concentration of approximately 0.1 to 2.0 percent.

22. The composition of claim 11, wherein said Acetic acid is at a concentration of approximately 1.0 to 10 percent.

23. The composition of claim 11, wherein said methanol is at a concentration of approximately 20 to 50 percent.

24. The composition of claim 11, wherein said sodium cholate is at a concentration of approximately 0.02 to 2.5 percent.

25. The composition of claim 11, wherein said sodium deoxycholate is at a concentration of approximately 0.02 to 2.5 percent.

26. The method of claim 11, wherein said target is a nucleic acid from the microorganism Mycobacterium tuberculosis.

27. The method of claim 11, wherein said binding agent is an oligonucleotide complementary to a nucleic acid from the microorganism Mycobacterium tuberculosis.

28. The method of claim 11, wherein said method additionally comprises background staining.