Method for improving cell permeability to foreign particles

• US 8,632,963 B2
• Filed: 07/27/2006
• Issued: 01/21/2014
• Est. Priority Date: 07/28/2005
• Status: Active Grant

• Current Assignee: ID-Fish Technology, Inc.
• Original Assignee: ID-Fish Technology, Inc.
• Inventors: Shah, Jyotsna, Weltman, Helena
• Primary Examiner(s): HIBBERT, CATHERINE S
• Application Number:
  US11/494,430
• Publication Number:
  US 20070042358A1
• Time in Patent Office: 2,735 Days
• Field of Search: None
• US Class Current: 435/4
• 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 and cell membranes of Mycobacterium sp., said composition comprising:

GuSCN (guanidine thiocyanate) at a concentration of approximately 2.0 to 3.3 M,

Tris-HCL at a concentration of approximately 10.0 to 100.0 mM and at a pH of approximately 7.0 to 9.0,

EDTA at a concentration of approximately 5 to 50 mM,

IGEPAL (octylphenoxy poly(ethyleneoxy)ethanol) at a concentration of approximately 0.1 to 2.0 percent (v/v),

acetic acid at a concentration of approximately 1.0 to 10 percent (v/v),

methanol at a concentration of approximately 20 to 50 percent (v/v),

sodium cholate at a concentration of approximately 0.02 to 2.5 percent (w/v) and,

sodium deoxycholate at a concentration of approximately 0.02 to 2.5 percent (w/v),

whererin said composition is effective in permeabilizing the cell walls and cell membranes of Mycobacterium sp. but not effective in lysing Mycobacterium sp.

2. A method for detecting a target in a cell, comprising:

a) Contacting the cell with the composition of claim 1 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).

3. The method of claim 2, wherein said target is selected from a group consisting of nucleic acids, peptides, glycoproteins, lipids, lipoproteins, viruses and prions.

4. The method of claim 2, wherein said binding agent is selected from a group consisting of nucleic acids, peptide nucleic acids, peptides, lipoproteins, glycoproteins and lipids.

5. The method of claim 2, wherein said binding agent additionally comprises a detection moiety.

6. The method of claim 5, wherein the detection moiety is selected from a group consisting of fluorescent markers, radioactive markers, dyes, colloidal metals, biotin/avidin and horseradish peroxidase.

7. The method of claim 2, wherein said detection is via a labeled antibody with affinity for said binding agent.

8. The method of claim 2, wherein said target is a nucleic acid from the microorganism Mycobacterium tuberculosis.

9. The method of claim 2, wherein said binding agent is an oligonucleotide complementary to a nucleic acid of the microorganism from Mycobacterium tuberculosis.

10. The method of claim 2, wherein said method additionally comprises background staining.

11. The method of claim 2, wherein said binding agent is an oligonucleotide complementary to a nucleic acid of the microorganism from Mycobacterium sp.

12. The method of claim 2, wherein said binding agent is an oligonucleotide complementary to a nucleic acid of fungi.