Method for determining follicle stimulating hormone

• US 4,921,808 A
• Filed: 06/25/1986
• Issued: 05/01/1990
• Est. Priority Date: 06/25/1986
• Status: Expired due to Term

• Current Assignee: Albany Medical College (Albany Medical Center)
• Original Assignee: Albany Medical College of Union University
• Inventors: Schneyer, Alan L., Sluss, Patrick M., Dattatreyamurty, Bosukonda, Reichert, Leo E. Jr.
• Primary Examiner(s): Draper, Garrette D.
• Application Number:
  US06/878,159
• Time in Patent Office: 1,406 Days
• Field of Search: 436/803, 436/501, 436/504, 436/543, 436/544, 436/545, 436/804, 436/817
• US Class Current: 436/503
• CPC Class Codes: G01N 2333/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

The present disclosure relates to an improved method based on hormone-receptor binding for the determination of follicle stimulating hormone and to improved reagents useful for the determination of follicle stimulating hormone in a hormone-receptor binding assay.

Claims

1. In a method for the determination of follicle stimulating hormone in a sample, comprising the steps of:

(a) contacting said sample with a purified follicle stimulating hormone receptor preparation capable of selectively binding said hormone;

(b) providing a signalling entity for signalling whether said binding has taken place; and

(c) observing said signalling entity to determine the presence of said hormone in said sample,

the improvement comprising said signalling entity being a substantially pure follicle stimulating hormone labeled with a detectable label, in which labeled hormone the ratio of specific binding to non-specific binding is at least about 5 when measured using a partially purified calf testes plasma membrane preparation containing from about 5 to about 25 fmol of receptors per milligram of membrane wet weight, and said purified follicle stimulating hormone receptor preparation having at least about 600 fmol/mg protein of high affinity receptors for follicle stimulating hormone.

3. The method of claim 1 or 2, wherein said contact between said sample and said purified follicle stimulating hormone receptor preparation recited in step (a) comprises an incubation in a buffer comprising about 10 nM CaCl₂, about 5 mM MgCl₂, about 2 mM ethylendediamine-tetraacetic acid, about 0.1merthiolate, and about 0.15M sucrose for about 20-24 hours at about 20°

C.

4. The method of claim 1 or 2, wherein said labeled follicle stimulating hormone is labeled with a radioactive label.

2. In a method for the determination of follicle stimulating hormone in a sample, comprising the steps of:

(a) contacting said sample with a purified follicle stimulating hormone receptor preparation capable of selectively binding said hormone;

(b) providing a signalling entity for signalling whether said binding has taken place; and

(c) observing said signalling entity to determine the presence of said hormone in said sample,

the improvement comprising said signalling entity being a substantially pure follicle stimulating hormone labeled with a detectable label, prepared from an unpurified labeled follicle stimulating hormone and characterized as follows;

(i) the substantially pure labeled follicle stimulating hormone co-migrates as a single band with unlabeled follicle stimulating hormone when electrophoresed on 7.5% polyacrylamide gel with an acrylamide to BIS ratio of about 19;

1 in an electrophoresis buffer containing 9.3 g of tris(hydroxymethyl)tetraacetic acid (disodium salt), 0.7 g of boric acid and 5 mg of merthiolate per liter of water at a pH of from 8.9 to 10; and

(ii) when the unpurified, labeled follicle stimulating hormone is chromatographed on a G-25 column and a portion of the labeled follicle stimulating hormone of any fraction from the column having the same amount of detectable label as used in part (i) is electrophoresed as described in part (i), the amount of detectable label found in the single band of part (i) is at least about three times the amount of detectable label in the co-migrating band of material from the fraction of the column;

and said purified follicle stimulating hormone receptor preparation having at least about 600 fmol-/mg protein of high affinity receptors for follicle stimulating hormone.

5. The method of claim wherein said radioactive label is ¹²⁵ I or ¹³¹ I.

6. The method of claim 5, wherein said sample is an aqueous sample.

7. The method of claim 6, wherein said aqueous sample is a biological fluid.

8. The method of claim 7, wherein the biological fluid is from a human, sheep, rat, pig, cow, dolphin or porpoise.

9. The method of claim 8, wherein said biological fluid is serum or plasma.