Method of modifying serum hormone levels using purified inhibin materials

• US 5,364,837 A
• Filed: 08/03/1989
• Issued: 11/15/1994
• Est. Priority Date: 06/08/1984
• Status: Expired due to Term

• Current Assignee: Biotechnology Australia Pty., Limited, Monash Medical Centre, Monash University, St. Vincent'S Institute Of Medical Research
• Original Assignee: Biotechnology Australia Pty., Limited, Monash Medical Centre, Monash University, St. Vincent'S Institute Of Medical Research
• Inventors: Forage, Robert G., de Kretser, David M., Findlay, John K., Morgan, Francis J., Hearn, Milton T. W., Milne-Robertson, David M., Burger, Henry G.
• Primary Examiner(s): Hill, Jr., Robert J.
• Assistant Examiner(s): Fitzgerald, David L.
• Application Number:
  US07/389,328
• Time in Patent Office: 1,930 Days
• Field of Search: 514/2, 514/8, 530/350, 530/387, 530/397, 530/399
• US Class Current: 514/9.9
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: G01N 33/689 related to pregnancy or the gonads
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins
• CPC Class Codes: Y10S 530/85 Reproductive organs or embryos

Abstract

A protein which satisfies all the biological criteria which are characteristic of inhibin has been isolated from a gonadal source. The purification and characterization of inhibin and the use of the purified material to raise antibodies, the use of inhibin and said antisera in a quantative radioimmunoassay, and application in vitro and in vivo of inhibin and antibody to inhibin, are described. There is provided a purified protein, inhibin characterized in that the apparent molecular weight as determined by SDS-PAGE is 56,000±1,000, the isoelectric point is in the range 6.9-7.3, and the protein can bind specifically to Concanavalin A-Sepharose. Moreover, the protein includes two subunits, characterized in that their apparent molecular weights as determined by SDS-PAGE are 44,000±3,000 and 14,000±2,000, respectively. Furthermore, the isoelectric point of the 44,000 molecular weight sub-unit is in the range of 6.0-7.0. In addition, the N-terminal amino acid sequence of the two subunits are described. With regard to the purified protein, inhibin, the protein can suppress follicle stimulating hormone (FSH) but not luteinizing hormone (EACH), thyroid stimulating hormone or prolactin in an in vitro bioassay system, and the protein can be labeled with radioactive iodine. In addition to the forgoing, there is also provided a method for isolating and purifying inhibin from mammalian ovarian follicular fluid, characterized by one or more gel permeation chromatography steps; reversed-phase high performance liquid chromatography steps; preparative polyacrylamide gel electrophoresis steps; and electrophoretic elution of the purified inhibin.

Claims

1. A method of modifying mammal serum levels of follicle stimulating hormone by producing antibodies to inhibin in a mammal comprising administering into a mammal a composition of matter consisting essentially of inhibin of a purity sufficient to yield a single band on non-reducing SDS-PAGE, wherein said purified inhibin has an apparent molecular weight of 56,000±

1000 as determined by non-reducing SDS-PAGE, consists of two subunits having apparent molecular weights of 44,000±

3,000 and 14,000±

2,000 as determined by reducing SDS-PAGE, and which suppresses production of the follicle stimulating hormone without suppressing production of luteinizing hormone in said mammal.

4. The method of claim 1, 2, or 3 wherein said mammal is bovine.

2. A method of modifying mammal serum levels of follicle stimulating hormone by producing antibodies to inhibin in a mammal comprising administering into a mammal a composition of matter consisting essentially of purified proteinaceous material of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein said purified proteinaceous material has an apparent molecular weight of 56,000±

1,000 as determined by non-reducing SDS-PAGE, consisting of two subunits having apparent molecular weight of 44,000±

3,000 and 14,000±

2,000 as determined by reducing SDS-PAGE, and which suppresses cellular content of follicle stimulating hormone without suppressing cellular content of luteinizing hormone in said mammal.

3. A method of modifying mammal serum levels of follicle stimulating hormone by producing antibodies to inhibin in a mammal comprising administering into a mammal a composition of matter consisting essentially of purified proteinaceous material of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein said purified proteinaceous material has an apparent molecular weight of 56,000±

1,000 as determined by non-reducing SDS-PAGE, consists of two subunits having apparent molecular weight of 44,000±

3,000 and 14,000±

2,000 as determined by reducing SDS-PAGE, and capable of suppressing cellular content of follicle stimulating hormone in rat anterior pituitary cells in culture without suppressing cellular content of luteinizing hormone.