Methods for diagnosis of low grade astrocytoma
View Patent ↗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.
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.