Inhibin isolated from ovarian follicular fluid

• US 5,102,807 A
• Filed: 04/11/1989
• Issued: 04/07/1992
• Est. Priority Date: 06/08/1984
• Status: Expired due to Term

• Current Assignee: Biotechnology Australia Pty., Limited (Danaher Corporation), Monash Medical Centre, Monash University, St. Vincent'S Institute Of Medical Research
• Original Assignee: Biotechnology Australia Pty., Limited (Danaher Corporation), Monash Medical Centre, Monash University, St. Vincent'S Institute Of Medical Research
• Inventors: Forage, Robert G., Weitenhall, Richard E. H., Milne-Robertson, David, Hearn, Milton T. W., Burger, Henry G., de Kretser, David M., Findlay, John K., Morgan, Francis J.
• Primary Examiner(s): Saunders, David A.
• Application Number:
  US07/336,052
• Time in Patent Office: 1,092 Days
• Field of Search: 436/518, 436/536, 436/543-545, 436/547, 436/548, 436/817, 530/387, 530/397, 530/399, 530/806, 530/808, 530/850
• US Class Current: 436/518
• 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 applications in vitro and in vivo of inhibin and antibody to inhibin, are described.

There is provided a purified protein, inhibin, characterised in that

a. the apparent molecular weight as determined by SDS-PAGE is 56,000±1,000

b. the isoelectric point is in the range 6.9-7.3

c. the protein can bind specifically to Concanavalin A-Sepharose

d the protein consists of two sub-units, characterized in that

i. their apparent molecular weights as determined by SDS-PAGE are 44,000±3,000 and 14,000±2,000 respectively.

ii. the isoelectric point of the 44,000 molecular weight sub-unit is in the range 6.0-7.0

iii. the N-terminal amino acid sequences of the two sub-units are as described herein

e. the protein can suppress follicle stimulating hormone (FSH) but not luteinising hormone (LH), thyroid stimulating hormone or prolactin in an in vitro bioassay system

f. the protein can be labeled with radioactive iodine.

There is also provided a method for isolating and purifying inhibin from mammalian ovarian follicular fluid, characterised by

a. one or more gel permeation chromatography steps;

b. one or more reversed-phase high performance liquid chromatography steps;

c. one or more preparative polyacrylamide gel electrophoresis steps;

d. Electrophoretic elution of the purified inhibin.

Claims

1. A composition of matter comprising purified inhibin of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein said purified inhibin suppresses production of follicle stimulating hormone without suppressing production of luteinizing hormone.

7. The composition of matter as defined by any one of claims 1-4, wherein said band is visualized with silver staining.

8. The composition of matter as defined by any one of claims 1-4, wherein said band is visualized with Coomassie blue staining.

9. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter comprises at least a first subunit and a second subunit, and further wherein said first subunit and said second subunit are joined by at least one disulfide bridge.

10. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter has an apparent molecular weight of 56,000±

3,000.

11. The composition of matter as defined by claim 10, wherein said composition of matter has an apparent molecular weight of 56,000±

1,000.

12. The composition of matter as defined by claim 11, wherein said composition of matter has an apparent molecular weight of about 56,000.

13. The composition of matter as defined by any one of claims 1-3 or 5, wherein said suppression of follicle stimulating hormone occurs in rat anterior pituitary cells in culture.

14. The composition of matter as defined by any one of claims 1-5, wherein said suppression of follicle stimulating hormone is dose-dependent.

15. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter does not suppress thyroid stimulating hormone.

16. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter does not suppress prolactin.

17. The composition of matter as defined by claim 16, wherein said composition of matter does not suppress thyroid stimulating hormone.

18. The composition of matter as defined by claim 17, further wherein said composition of matter does not produce a non-specific, toxic suppression of hormone production.

19. The composition of matter as defined by any one of claims 1-5 wherein said composition of matter is a glycoprotein.

20. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter comprises at least two non-identical subunits.

21. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter is obtained from follicular fluid.

22. The composition of matter as defined by claim 21, wherein said composition of matter is obtained from mammalian follicular fluid.

23. The composition of matter as defined by claim 22, wherein said composition of matter is obtained from ovarian follicular fluid.

24. The composition of matter as defined by claim 22, wherein said composition of matter is obtained from a bovine.

25. The composition of matter as defined by claim 22, wherein said composition of matter is obtained from an ovine.

26. The composition of matter as defined by claim 22, wherein said composition of matter is obtained from a human.

27. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter has a pI of between about 6.9 and 7.3.

28. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter can bind specifically to a gel permeation matrix comprising a bead formed gel prepared from agarose.

29. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter can bind with horseradish peroxidase-labelled wheat germ lectin following fractionation of said composition of matter into fractions on SDS-PAGE and electrotransfer of said fractions to nitrocellulose.

30. A method for producing monoclonal antibodies specific to inhibin, comprising producing said monoclonal antibodies using the composition of matter as defined by any one of claims 1-5 as an immunogen.

31. A composition of matter comprising monoclonal antibodies obtained according to the method as defined in claim 30.

32. A method for assaying for inhibin in a sample, comprising:

providing the purified inhibin as defined by claim 1 in radiolabelled form;

adding the radiolabelled purified inhibin and antibodies specific to inhibin to the sample;

allowing the antibodies to bind to inhibin present in the sample; and

separating the antibody-bound from the unbound radiolabelled inhibin.

33. A method for detecting and diagnosing infertility in a mammal, comprising assaying a sample from said mammal by the radioimmunoassay procedure as defined by claim 32.

112. A method for producing antibodies specific to inhibin, comprising immunizing a mammal with the preparation of purified inhibin as defined by claim 1 in combination with an adjuvant.

113. A composition of matter, comprising antibodies specific to inhibin produced according to the method as defined by claim 112.

114. The method as defined in claim 112, further comprising the steps of:

a) dissolving said purified inhibin in buffer;

b) emulsifying said purified inhibin dissolved in said buffer to yield an emulsion;

c) injecting said emulsion into said mammal.

115. The method as defined in claim 112, wherein said buffer is a phosphate buffer.

116. The method as defined in claim 115, wherein said phosphate buffer has a pH of about 7.4.

117. The method as defined in claim 115, further comprising emulsifying said purified inhibin dissolved in said phosphate buffer with an approximately equal volume of oil-based adjuvant.

118. The method as defined in claim 114, further comprising injecting said mammal at least a first time and a second time.

119. The method as defined in claim 114, further comprising injecting said mammal at at least one intramuscular site.

120. The method as defined in claim 119, further comprising also injecting said mammal subcutaneously.

121. The method as defined in claim 112, further comprising isolating antisera containing said antibodies from said mammal.

122. A composition of matter comprising antisera containing antibodies specific to inhibin, said antisera being produced according to the method as defined by claim 121.

123. The composition of matter as defined by claim 122, wherein said antisera neutralizes inhibin activity in vitro.

124. The method as defined in claim 121, further comprising isolating said antibodies from said antisera.

132. The composition of matter as defined by any one of claims 1-5, wherein said composition of matter is labelled.

2. A composition of matter comprising purified proteinaceous material comprising at least one subunit, said subunit having an apparent molecular weight of 14,000±

2,000, wherein said purified proteinaceous material is of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, and wherein said purified proteinaceous material suppresses production of follicle stimulating hormone without suppressing production of luteinizing hormone.

3. A composition of matter comprising purified proteinaceous material of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein said purified proteinaceous material suppresses cellular content of follicle stimulating hormone without suppressing cellular content of luteinizing hormone.

4. A composition of matter comprising purified proteinaceous material of a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein said purified proteinaceous material suppresses cellular content of follicle stimulating hormone in rate anterior pituitary cells in culture without suppressing cellular content of luteinizing hormone.

5. A composition of matter comprising purified proteinaceous material of at least 75% purity, wherein said purified proteinaceous material suppresses production of follicle stimulating hormone without suppressing production of luteinizing hormone.

6. The composition of matter as defined by claim 5, wherein said purified proteinaceous material yields a single major band on non-reducing SDS-PAGE.

34. A purified proteinaceous material having a purity sufficient to yield a single major band on non-reducing SDS-PAGE, wherein said purified proteinaceous material suppresses production of follicle stimulating hormone without suppressing production of luteinizing hormone, and which, when administered as an antigen in mammals, raises antibodies that neutralize the follicle stimulating hormone suppressing activity of said purified proteinaceous material.

35. A composition of matter comprising a purified inhibin of a purity sufficient to yield a single major band on non-reducing SDS-PAGE having an apparent molecular weight of between about 54,000 and 57,000, said purified inhibin being capable of suppressing production of follicle stimulating hormone by rat anterior pituitary cells in culture without inhibiting the production of luteinizing hormone by said cells.

36. The composition of matter as defined by claim 35, wherein said apparent molecular weight is between about 55,000 and 57,000.

37. The composition of matter as defined by claim 36, wherein said apparent molecular weight is about 56,000.

38. The composition of matter as defined by claim 35, wherein said purified inhibin comprises at least a first subunit and a second subunit.

39. A composition of matter comprising a first subunit of the composition of matter as defined by claim 38.

40. A composition of matter comprising a second subunit of the composition of matter as defined by claim 38.

41. The composition of matter as defined by claim 38, wherein said second subunit is a glycoprotein.

42. The composition of matter as defined by claim 38, wherein said first subunit has an apparent molecular weight of between about 12,000 and 16,000.

43. The composition of matter as defined by claim 42, wherein said first subunit has an apparent molecular weight of between about 13,000 and 15,000.

44. The composition of matter as defined by claim 43, wherein said first subunit has an apparent molecular weight of about 14,000.

45. The composition of matter as defined by claim 38, wherein said second subunit has an apparent molecular weight of between about 41,000 and 47,000.

46. The composition of matter as defined by claim 45, wherein said second subunit has an apparent molecular weight of between about 43,000 and 46,000.

47. The composition of matter as defined by claim 46, wherein said second subunit has an apparent molecular weight of about 44,000.

48. The composition of matter as defined by claim 38, wherein said purified inhibin comprises a first subunit having an apparent molecular weight of about 14,000 and a second subunit having an apparent molecular weight of about 44,000.

49. A method for purifying inhibin from a starting material containing inhibin comprising:

a) subjecting said starting material to a first gel permeation chromatography to yield a first eluent;

b) subjecting said first eluent to a second gel permeation chromatography to yield a second eluent;

c) subjecting said second eluent to reverse phase high performance liquid chromatography to yield a third eluent; and

d) subjecting said third eluent to preparative polyacrylamide gel electrophoresis.

50. A composition of matter comprising purified inhibin obtained according to the method as defined by claim 49.

51. The composition of matter as defined by claim 50, wherein said purified inhibin has a specific activity of at least about 3 times greater than the specific activity of said starting material.

52. The composition of matter as defined by claim 51, wherein said purified inhibin has a specific activity of at least about 10 times greater than the specific activity of said starting material.

53. The composition of matter as defined by claim 52, wherein said purified inhibin has a specific activity of at least about 20 times greater than the specific activity of said starting material.

54. The composition of matter as defined by claim 53, wherein said purified inhibin has a specific activity of at least about 50 times greater than the specific activity of said starting material.

55. The composition of matter as defined by claim 54, wherein said purified inhibin has a specific activity of at least about 160 times greater than the specific activity of said starting material.

56. The method as defined in claim 49, wherein said starting material is follicular fluid.

57. The method as defined in claim 56, wherein said starting material is mammalian follicular fluid.

58. The method as defined in claim 57, wherein said starting material is bovine follicular fluid.

59. The method as defined in claim 57, wherein said starting material is ovine follicular fluid.

60. The method as defined in claim 57, wherein said starting material is human follicular fluid.

61. The method as defined in claim 49, further comprising diluting said starting material with ammonium acetate prior to said first gel permeation chromatography.

62. The method as defined in claim 61, wherein said ammonium acetate is used at a concentration of about 0.05M.

63. The method as defined in claim 61, wherein said ammonium acetate has a pH of about 7.0.

64. The method as defined in claim 61, further comprising centrifuging said starting material after said ammonium acetate is added thereto.

65. The method as defined in claim 64, further comprising centrifuging said starting material at about 12,000 g.

66. The method as defined in claim 49, wherein a gel permeation matrix comprising covalently cross-linked allyl dextran and N, N¹ -methylene bisacrylamide is used to perform said first gel permeation chromatography.

67. The method as defined in claim 49, wherein said first eluent comprises a void volume fraction from said gel permeation chromatography.

68. The method as defined by claim 49, further comprising acidifying said first eluent with acid prior to subjecting said first eluent to said second gel permeation chromatography.

69. The method as defined in claim 68, wherein said acid is acetic acid.

70. The method as defined in claim 69, wherein said first eluent is acidified with acetic acid to a final concentration of about 4.0M.

71. The method as defined in claim 49, wherein a gel permeation matrix comprising a bead formed gel of cross-linked dextran and epichlorohydrin is used to perform said second gel permeation chromatography.

72. The method as defined in claim 49, wherein said second gel permeation chromatography is performed on an analytical column.

73. The method as defined in claim 72, further comprising eluting said second eluent with acetic acid.

74. The method as defined in claim 73, wherein said acetic acid is used at a concentration of about 4.0M.

75. The method as defined in claim 49, further comprising performing said reverse phase high performance liquid chromatography using a chemically-bonded N-alkylsilica column material.

76. The method as defined in claim 75, wherein an Ultrapore RPSC is used for said column.

77. The method as defined in claim 76, wherein said N-alkylsilica column material has a particle size distribution of between about 5 and 10 microns.

78. The method as defined by claim 75, further comprising using a water-miscible organic solvent to produce said third eluent.

79. The method as defined in claim 78, wherein said water-miscible organic solvent is selected from the group consisting of methanol, acetonitrile, and isopropanol.

80. The method as defined in claim 79, further comprising using a gradient of about 0-50% acetonitrile to produce said third eluent.

81. The method as defined in claim 80, wherein said acetonitrile is present in a solution of about 0.1% trifluoroacetic acid.

82. The method as defined in claim 80, wherein said third eluent comprises a portion of said gradient elution corresponding to about 30.0% acetonitrile.

83. The method as defined in claim 78, further comprising modifying said water-miscible organic solvent with an ionic modifier.

84. The method as defined in claim 83, wherein said ionic modifier is selected from the group consisting of trifluoroacetic acid, ammonium bicarbonate, ammonium acetate, and sodium phosphate.

85. The method as defined in claim 49, further comprising dissolving said third eluent in non-reducing sample buffer prior to fractionating said third eluent on a polyacrylamide gel electrophoresis apparatus.

86. The method as defined in claim 85, further comprising incubating said third eluent at about room temperature prior to fractionating.

87. The method as defined in claim 86, wherein said third eluent is incubated at about room temperature overnight prior to fractionating.

88. The method as defined in claim 86, wherein said third eluent is incubated at about room temperature for about one hour prior to fractionating.

89. The method as defined in claim 49, further comprising using, for said preparative polyacrylamide gel electrophoresis, a separation gel;

a stacking gel;

a non-reducing sample buffer; and

an electrophoretic buffer.

90. The method as defined in claim 89, wherein said separating gel comprises Tris-HCl, SDS acrylamide, bis acrylamide, and ammonium persulphate.

91. The method as defined in claim 90, wherein said separating gel comprises about 0.38M Tris-HCl at a pH of about 8.8, 0.1% w/v SDS, 7.5% acrylamide, 0.2% bis acrylamide, and 0.03% ammonium persulphate.

92. The method as defined in claim 89, wherein said stacking gel comprises Tris-HCl, SDS acrylamide, bis acrylamide, ammonium persulphate, and TEMED.

93. The method as defined in claim 92, wherein said stacking gel comprises about 0.125M Tris-HCl at a pH of about 6.8, about 0.1% w/v SDS, about 5% acrylamide, about 0.13% Bis acrylamide, and about 0.1% ammonium persulphate.

94. The method as defined in claim 89, wherein said non-reducing sample buffer comprises Tris-HCl, glycerol, SDS, and bromophenol blue.

95. The method as defined in claim 94, wherein said non-reducing sample buffer comprises about 0.06M Tris-HCl at a pH of about 6.8, about 12.5% glycerol, about 1.25% w/v SDS, and about 0.006% bromophenol blue.

96. The method as defined in claim 89, wherein said electrophoresis buffer comprises tris-glycine buffer containing SDS.

97. The method as defined in claim 96, wherein said electrophoresis buffer comprises about 2.5 mM Tris-glycine buffer containing about 0.05% (w/v) SDS.

98. The method as defined in claim 49, further comprising electrophoresing said third eluent at a current of about 20 mA until said third eluent migrates into said separating gel, and then increasing said current to about 30 mA until said electrophoresis is complete.

99. The method as defined in claim 49, further comprising isolating a gel portion from said preparative polyacrylamide gel electrophoresis containing inhibin and subjecting the inhibin contained in said gel portion to electrophoretic elution.

100. The method as defined in claim 99, wherein said electrophoretic elution comprises:

a) washing said gel portion in an elution buffer;

b) soaking said gel portion in a soaking buffer overlaid with an elution buffer;

c) initiating electrophoretic elution of said gel portion;

d) replacing said elution buffer with a dialysis buffer;

e) continuing electrophoretic elution of said gel portion until said inhibin has migrated out of said gel portion; and

f) collecting said inhibin.

101. The method as defined in claim 100, wherein said elution buffer comprises SDS and NH₄ HCO₃.

102. The method as defined in claim 101, further wherein said elution buffer comprises about 0.1% SDS in about 0.05M NH₄ HCO₃.

103. The method as defined in claim 100, wherein said soaking buffer comprises SDS and NH₄ HCO₃.

104. The method as defined in claim 103, further wherein said soaking buffer comprises about 2% SDS in about 0.4M NH₄ HCO₃.

105. The method as defined in claim 100, wherein said dialysis buffer comprises SDS and NH₄ HCO₃.

106. The method as defined in claim 105, further wherein said dialysis buffer comprises about 0.02% SDS in about 0.01M NH₄ HCO₃.

107. The method as defined in claim 49, wherein purified inhibin of a purity sufficient to yield a single major band on non-reducing SDS-PAGE is obtained.

108. The method as defined in claim 107, wherein said band is visualized with silver staining.

109. The method as defined in claim 107, wherein said band is visualized with Coosmassie blue staining.

110. The method as defined in claim 49, further comprising using, for said preparative polyacrylamide gel electrophoresis, a separation gel;

a stacking gel;

a reducing sample buffer; and

an electrophoretic buffer.

111. The method as defined in claim 110, further comprising preparing said reducing sample buffer by adding about 0.1% 2-mercaptoethanol to a non-reducing sample buffer.

125. A composition of matter comprising monoclonal antibodies that preferentially bind to an immunological determinant of the composition of matter as defined by anyone of claims 61-65.

126. The composition of matter as defined by claim 125, wherein said monoclonal antibodies are specific to inhibin.

127. The composition of matter as defined by claim 126, wherein said composition of matter is found in follicular fluid.

128. The composition of matter as defined by claim 127, wherein said follicular fluid is mammalian follicular fluid.

129. The composition of matter as defined by claim 128, wherein said follicular fluid is bovine follicular fluid.

130. The composition of matter as defined by claim 128, wherein said follicular fluid is ovine follicular fluid.

131. The composition of matter as defined by claim 128, wherein said follicular fluid is human follicular fluid.