Methods for diagnosis of low grade astrocytoma

• US 7,279,292 B2
• Filed: 08/17/2006
• Issued: 10/09/2007
• Est. Priority Date: 10/11/2001
• Status: Expired due to Fees

• Current Assignee: Huntington Medical Research Institutes, The Governors of the University of Alberta (University of Alberta)
• Original Assignee: Huntington Medical Research Institutes, The Governors of the University of Alberta (University of Alberta)
• Inventors: Malhotra, Sudarshan, Imam, S. Ashraf
• Primary Examiner(s): Yaen; Christopher H.
• Assistant Examiner(s): Sang; Hong
• Application Number:
  US11/506,688
• Publication Number:
  US 20060281138A1
• Time in Patent Office: 418 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/57407 Specifically defined cancers

Abstract

A method for identifying low grade astrocytoma cells in a sample is provided, wherein the method distinguishes between low grade astrocytoma cells and normal astrocytes, thus permitting early diagnosis of astrocytoma. The method uses antibody directed against J1-31 to test astrocytes in the sample for the presence or absence of J1-31 polypeptide, the low grade astrocytoma cells being characterized by the absence of J1-31 while normal astrocytes are characterized by the presence of J1-31.

Claims

1. A method for identifying low grade astrocytoma cells in a biological sample, comprising the steps of:

preparing slides from a biological sample;

serially incubating said biological sample in at least ethanol and hydrogen peroxide;

heating said slides in an unmasking solution including a mixture of at least sodium citrate and citric acid;

incubating the biological sample with anti-J1-31 antibody under conditions in which the antibody binds to J1-31 antigen in the sample, said anti-J1-31 antibody being produced by the hybridoma cell line J1-31, ATCC designation number CRL-2253;

contacting the biological sample with a secondary antibody selected so as to form an immune complex with the anti-J1-31 antibody bound to J1-31 antigen;

detecting cells expressing the J1-31 antigen by detecting the immune complex; and

detecting astrocytes in the sample in which the immune complex is not present, to thereby identify low-grade astrocytoma cells.

2. The method of claim 1, wherein the secondary antibody is biotinylated.

3. The method of claim 2, wherein the immune complex is detected by contacting the sample with an avidin-peroxidase conjugate, followed by a chromogen suitable for use as a peroxidase substrate.

4. The method of claim 1, wherein the secondary antibody is labeled with a fluorescent dye.

5. The method of claim 1, wherein the secondary antibody is labeled with a metal particle.

6. The method of claim 1, wherein after incubating the biological sample with anti-J1-31 antibody, the slides are washed at least four times with a wash solution, wherein the wash solution includes at least an 0.5% solution of Tween-20.

7. The method of claim 1, wherein the biological sample is fixed in one of either paraformaldehyde or methanol.

8. The method of claim 1, wherein the sample is further stained with a non-cell specific stain.

9. The method of claim 1, further comprising the step of contacting the sample with anti-GFAP antibody under conditions in which the anti-GFAP antibody binds to GFAP antigen in the sample.

10. The method of claim 9, wherein the anti-GFAP antibody is labeled with fluorescent dye selected to fluoresce at a distinct wavelength.

11. The method of claim 1 further comprising, before the step of detecting:

incubating the immune complex with a conjugate; and

exposing the conjugated immune complex with a chromogen.