Claims for Patent: 4,711,856

Summary for Patent: 4,711,856

Title:	Nuclear binding assay for steroid receptor functionality in cancerous cells
Abstract:	A method for rapidly determining the presence of functional cellular steroid receptors by assaying a tissue sample for nuclear steroid binding is disclosed which comprises treating the tissue with collagenase, incubating the isolated cells with a labelled steroid capable of complexing said receptors and measuring the bound radioactivity and the DNA of the isolated cellular nuclei.
Inventor(s):	Spelsberg; Thomas C. (Rochester, MN)
Assignee:	Mayo Medical Resources (Rochester, MN)
Application Number:	06/652,295
Patent Claims:	1. A method for rapidly determining the presence of functional cellular steroid receptors by assaying a tissue sample for nuclear steroid binding comprising: (a) fragmenting said tissue sample; (b) digesting said fragmented tissue with collagenase; (c) isolating the cells from said digested tissue; (d) incubating said cells with an amount of a radiolabeled steroid capable of complexing with and saturating said receptors; (e) isolating the cellular nuclei; and (f) measuring the bound radioactivity and the total DNA of said nuclei. 2. The method of claim 1 wherein the tissue sample is carcinoma tissue. 3. The method of claim 2 wherein the carcinoma is selected from the group consisting of breast, endrometrium, testicular, lung, myeloma and prostate. 4. The method of claim 1 wherein about 1-1000 mg of tissue is fragmented. 5. The method of claim 4 wherein the tissue is contacted with about 0.5-15 units of collagenase per mg of tissue. 6. The method of claim 1 wherein the isolated cells are incubated in a medium which is about 10-50 nM in radiolabeled steroid. 7. The method of claim 6 wherein the radiolabeled steroid is [.sup.3 H]-progesterone or [.sup.3 H]-estradiol. 8. The method of claim 1 wherein the nuclei are isolated by layering a homogenate of said incubated cells onto a cold, neutral, aqueous medium comprising sucrose, glycerol, octoxynol-9, tris(hydroxymethyl) amino-methane hydrochloride and KCl and centrifuging said layered medium to obtain a pellet of purified nuclei. 9. The method of claim 8 further comprising dispersing said pellet in a neutral solution comprising tris(hydroxymethyl) amino-methane hydrochloride and glycerol, and collecting the dispersed nuclei by filtration. 10. The method of claim 1 wherein the DNA of said nuclei is measured by a diphenylamine assay. 11. The method of claim 1 wherein the DNA of said nuclei is measured by a method comprising treating said nuclei with a protease and ribonuclease, mixing the treated nuclei with ethidium bromide and measuring the fluorescence of said nuclei. 12. A method for rapidly determining the presence of functional steroid receptors in cancerous cells comprising assaying a tissue sample for nuclear steroid binding comprising: (a) obtaining a tissue sample by biopsy; (b) fragmenting said tissue sample; (c) disrupting said fragmented tissue sample in contact with about 1-5 units of collagenase per mg of tissue; (d) isolating the cells from said tissue sample; (e) incubating said cells with an amount of a radiolabeled steroid capable of complexing with and saturating said receptors; (f) homogenizing said incubated cells; (g) layering said homogenate onto a cold, neutral aqueous medium comprising sucrose, glycerol, octoxynol-9, tris(hydroxymethyl)aminomethane hydrochloride and KCl; (h) centrifuging said layered medium to obtain a pellet of purified nuclei; and (i) measuring the bound radioactivity and the total DNA of said nuclei. 13. The method of claim 1 wherein about 1-25 mg of tissue are obtained by biopsy. 14. The method of claim 1 wherein about 1-5 mg of tissue are obtained by biopsy. 15. The method of claim 1 wherein said cells are incubated in medium comprising about 20-30 nM [.sup.3 H]-progesterone or [.sup.3 H]-estradiol. 16. The method of claim 15 wherein the cells are incubated for about 0.5-1.5 hr at about 18.degree.-37.degree. C. 17. The method of claim 16 wherein the cells are incubated for about 1.0 hr at about 22.degree. C. 18. The method of claim 12 wherein step (h) further comprises dispersing the pellet of purified nuclei in a neutral aqueous solution comprising tris(hydroxymethyl) amino-methane hydrochloride and glycerol and collecting them by filtration.

Details for Patent 4,711,856

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-03-29