Claims for Patent: 4,341,755

Summary for Patent: 4,341,755

Title:	Parathyroid radioimmunoassay
Abstract:	A method for the radioimmunoassay of parathyroid hormone in mammalian serum. The method is an improvement on previous double antibody radioimmunoassays in that spurious assay results caused by the nonspecific interaction of serum proteins with the labeled peptide is eliminated by the labeling of a specific portion of either human or bovine parathyroid molecule. The 65-84 portion of human or bovine parathyroid hormone, as radioactively labeled, is incorporated in the assay as the labeled peptide. A chicken antibody with a high affinity for the 65-84 portion of human parathyroid hormone is incorporated in the assay as the first antibody. The invention further pertains to compounds useful in practicing the method, namely X.sup.64 -hPTH.sup.65-84 and Y-X.sup.64 -hPTH.sup.65-84 wherein X is either histidyl, tyramyl, histamyl, or tyrosyl, and wherein Y is either .sup.125 I or .sup.131 I.
Inventor(s):	Lindall; Arnold W. (Marine on St. Croix, MN)
Assignee:	Immuno Nuclear Corporation (Stillwater, MN)
Application Number:	06/169,492
Patent Claims:	1. A method of double antibody radioimmunoassay measurement of the concentration in biological or other fluids of mammalian parathyroid hormone and/or C-terminal fragments thereof which comprises incorporating in the assay as the radioactively labeled peptide a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH. 2. The method of claim 1 wherein the mammalian parathyroid hormone and/or C-terminal fragments thereof are respective members of the group consisting of intact rat PTH and C-terminal rat PTH fragments, intact bovine PTH and C-terminal bovine PTH fragments, and intact hPTH and C-terminal hPTH fragments. 3. The method of claim 2 wherein said respective members are intact hPTH and C-terminal hPTH fragments. 4. The method of claim 3 wherein said double antibody radioimmunoassay comprises: A. Adding together: 1. a measured quantity of the biological or other fluid to be assayed; 2. a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH, in sufficient quantity and of sufficient radioactivity to give a measurable counting rate of later formed precipitated second antibody bound first antibody bound radioactively labeled fragment; 3. a first antibody, which has a substantially high affinity for intact hPTH and the radioactively labeled fragment, in sufficient quantity to bind a significant quantity of the intact hPTH, the C-terminal hPTH fragments, and the radioactively labeled fragment; and 4. a first buffering agent to buffer the mixture to a pH suitable for reaction of the first antibody with intact hPTH, C-terminal hPTH fragments, and the radioactively labeled fragment; B. Allowing reaction of the first antibody with the intact hPTH, the C-terminal hPTH fragments, and the radioactively labeled fragment to proceed substantially to equilibrium to thereby produce first antibody bound intact hPTH, first antibody bound C-terminal hPTH fragments, and first antibody bound radioactively labeled fragment; C. Adding: 1. a second antibody-precipitating complex, wherein the second antibody has a substantially high affinity for the first antibody, as bound to the radioactively labeled fragment, in a sufficient quantity to give a measurable counting rate of precipitated second antibody bound first antibody bound radioactively labeled fragment; and 2. a second buffering agent to buffer the mixture to a pH suitable for reaction of the second antibody with first antibody bound radioactively labeled fragment; D. Allowing reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment to proceed substantially to equilibrium to thereby produce precipitated second antibody bound first antibody bound radioactively labeled fragment; E. Separating the precipitated from non-precipitated radioactively labeled fragment; F. Measuring the radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment; G. Correlating the respective measured radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment for the biological or other fluid with that for known standards. 5. The method of claim 4 wherein the biological or other fluid is human serum or plasma. 6. The method of claim 4 wherein precipitated is separated from non-precipitated radioactively labeled fragment by centrifuging until the supernatant is substantially clear of the precipitate, and decanting the supernatant. 7. The method of claim 4 wherein the reaction of the first antibody with intact hPTH, C-terminal hPTH fragments, and radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 8. The method of claim 4 wherein the reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 9. The method of claim 4 wherein said correlation comprises: A. Preparing a standard curve for which known standards are plotted respectively against the measured radioactivity of their precipitated and/or non-precipitated radioactively labeled fragment; and B. Correlating via the standard curve the quantity of intact hPTH and C-terminal hPTH fragments in the biological or other fluid with the measured radioactivity of its precipitated and/or non-precipitated radioactively labeled fragment. 10. The method of claim 4 wherein the radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH comprises the fragment bound at the end opposite the carboxyl group by a member of the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl, and radioiodinated tyrosyl. 11. The method of claim 10 wherein the radioiodine is .sup.125 I. 12. The method of claim 10 wherein the fragment bound by said member is hPTH.sup.65-84. 13. The method of claim 12 wherein the radioactively labeled fragment is .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. 14. The method of claim 12 wherein the first antibody is chicken anti-hPTH.sup.65-84 and the first buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 15. The method of claim 14 wherein the first buffering agent comprises a borate buffer with a pH of about 8.4. 16. The method of claim 14 wherein the first buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 17. The method of claim 14 wherein the reaction of chicken anti-hPTH.sup.65-84 with intact hPTH, C-terminal hPTH fragments, and said radioactively labeled hPTH.sup.65-84 is allowed to proceed in the aforesaid manner by vortexing gently and incubating for 16 hours at 2.degree.-8.degree. C. 18. The method of claim 14 wherein the second antibody-precipitating complex is rabbit anti-chicken precipitating (RAC-PPT) complex and the second buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 19. The method of claim 18 wherein the second buffering agent comprises a borate buffer with a pH of about 8.4. 20. The method of claim 18 wherein the second buffering agent comprises BSA-borate buffer with a pH of about 8.4. 21. The method of claim 18 wherein the reaction of RAC-PPT complex with the chicken anti-hPTH.sup.65-84 bound radioactively labeled hPTH.sup.65-84 is allowed to proceed in the aforesaid manner by vortexing and incubating for a minimum of 2 hours at room temperature or greater than 16 hours at 2.degree.-8.degree. C. 22. The method of claim 3 wherein said double antibody radioimmunoassay comprises: A. Adding, to a measured quantity of the biological or other fluid to be assayed: 1. a first antibody, which has a substantially high affinity for intact hPTH, C-terminal hPTH fragments, and later to be added radioactively labeled fragment, in a sufficient quantity to bind a significant quantity of the intact hPTH and C-terminal hPTH fragments in the biological or other fluid; and 2. a first buffering agent to buffer the mixture to a pH suitable for reaction of the first antibody with intact hPTH, C-terminal hPTH fragments, and later to be added radioactively labeled fragment; B. Allowing reaction of the intact hPTH and C-terminal hPTH fragments with the first antibody to proceed substantially to equilibrium to thereby produce first antibody bound intact hPTH and first antibody bound C-terminal hPTH fragments; C. Adding a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH, in sufficient quantity to give a measurable counting rate of later formed precipitated second antibody bound first antibody bound radioactively labeled fragment; D. Allowing reaction of the radioactively labeled fragment with the first antibody bound intact hPTH and first antibody bound C-terminal hPTH fragments to proceed substantially to equilibrium to thereby bind a portion of the radioactively labeled fragment while freeing a portion of the bound intact hPTH and C-terminal hPTH fragments; E. Adding: 1. a second antibody-precipitating complex, wherein the second antibody has a substantially high affinity for the first antibody, as bound to the radioactively labeled fragment, in a sufficient quantity to give a measurable counting rate of precipitated second antibody bound first antibody bound radioactively labeled fragment; and 2. a second buffering agent to buffer the mixture to a pH suitable for reaction of the second antibody with first antibody bound radioactively labeled fragment; F. Allowing reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment to proceed substantially to equilibrium to thereby produce precipitated second antibody bound first antibody bound radioactively labeled fragment; G. Separating the precipitated from non-precipitated radioactively labeled fragment; H. Measuring the radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment; I. Correlating the respective measured radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment for the biological or other fluid with that for known standards. 23. The method of claim 22 wherein the biological or other fluid is human serum or plasma. 24. The method of claim 22 wherein precipitated is separated from non-precipitated radioactively labeled fragment by centrifuging until the supernatant is substantially clear of the precipitate, and decanting the supernatant. 25. The method of claim 22 wherein the reaction of the first antibody with intact hPTH and C-terminal hPTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 26. The method of claim 22 wherein said reaction of the radioactively labeled fragment with the first antibody bound intact hPTH and first antibody bound C-terminal hPTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 27. The method of claim 22 wherein the reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 28. The method of claim 22 wherein said correlation comprises: A. Preparing a standard curve for which known standards are plotted respectively against the measured radioactivity of their precipitated and/or non precipitated radioactively labeled fragment; and B. Correlating via the standard curve the quantity of intact hPTH and C-terminal hPTH fragments in the biological or other fluid with the measured radioactivity of its precipitated and/or non-precipitated radioactively labeled fragment. 29. The method of claim 22 wherein the radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH comprises the fragment bound at the end opposite the carboxyl group by a member of the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl, and radioiodinated tyrosyl. 30. The method of claim 29 wherein the radioiodine is .sup.125 I. 31. The method of claim 29 wherein the fragment bound by said member is hPTH.sup.65-84. 32. The method of claim 31 wherein the radioactively labeled fragment is .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. 33. The method of claim 31 wherein the first antibody is chicken anti-hPTH.sup.65-84 and the first buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 34. The method of claim 33 wherein the first buffering agent comprises a borate buffer with a pH of about 8.4. 35. The method of claim 33 wherein the first buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 36. The method of claim 33 wherein the reaction of chicken anti-hPTH.sup.65-84 with intact hPTH and C-terminal hPTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating for a minimum of 4 hours at 2.degree.-8.degree. C. 37. The method of claim 33 wherein the reaction of the radioactively labeled fragment with the first antibody bound intact hPTH and first antibody bound C-terminal hPTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating for at least 20 hours at 2.degree.-8.degree. C. 38. The method of claim 33 wherein the second antibody-precipitating complex is rabbit anti-chicken precipitating (RAC-PPT) complex and the second buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 39. The method of claim 38 wherein the second buffering agent comprises a borate buffer with a pH of about 8.4. 40. The method of claim 38 wherein the second buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 41. The method of claim 38 wherein the reaction of RAC-PPT complex with the chicken anti-hPTH.sup.65-84 bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing and incubating for a minimum of 2 hours at room temperature or greater than 16 hours at 2.degree.-8.degree. C. 42. The method of claim 2 wherein said respective members are intact rat PTH and C-terminal rat PTH fragments. 43. The method of claim 42 wherein said double antibody radioimmunoassay comprises: A. Adding together: 1. a measured quantity of the biological or other fluid to be assayed; 2. a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH, in sufficient quantity and of sufficient radioactivity to give a measurable counting rate of later formed precipitated second antibody bound first antibody bound radioactively labeled fragment; 3. a first antibody, which has a substantially high affinity for intact rat PTH and the radioactively labeled fragment, in sufficient quantity to bind a significant quantity of the intact rat PTH, the C-terminal rat PTH fragments, and the radioactively labeled fragment; and 4. a first buffering agent to buffer the mixture to a pH suitable for reaction of the first antibody with intact rat PTH, C-terminal rat PTH fragments, and the radioactively labeled fragment; B. Allowing reaction of the first antibody with the intact rat PTH, the C-terminal rat PTH fragments, and the radioactively labeled fragment to proceed substantially to equilibrium to thereby produce first antibody bound intact rat PTH, first antibody bound C-terminal rat PTH fragments, and first antibody bound radioactively labeled fragment; C. Adding: 1. a second antibody-precipitating complex, wherein the second antibody has a substantially high affinity for the first antibody, as bound to the radioactively labeled fragment, in a sufficient quantity to give a measurable counting rate of precipitated second antibody bound first antibody bound radioactively labeled fragment; and 2. a second buffering agent to buffer the mixture to a pH suitable for reaction of the second antibody with first antibody bound radioactively labeled fragment; D. Allowing reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment to proceed substantially to equilibrium to thereby produce precipitated second antibody bound first antibody bound radioactively labeled fragment; E. Separating the precipitated from non-precipitated radioactively labeled fragment; F. Measuring the radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment; p1 G. Correlating the respective measured radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment for the biological or other fluid with that for known standards. 44. The method of claim 43 wherein the biological or other fluid is rat serum or plasma. 45. The method of claim 43 wherein precipitated is separated from non-precipitated radioactively labeled fragment by centrifuging until the supernatant is substantially clear of the precipitate, and decanting the supernatant. 46. The method of claim 43 wherein the reaction of the first antibody with intact rat PTH, C-terminal rat PTH fragments, and radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 47. The method of claim 43 wherein the reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 48. The method of claim 43 wherein said correlation comprises: A. Preparing a standard curve for which known standards are plotted respectively against the measured radioactivity of this precipitated and/or non-precipitated radioactively labeled fragment; and B. Correlating via the standard curve the quantity of intact rat PTH and C-terminal rat PTH fragments in the biological or other fluid with the measured radioactivity of its precipitated and/or non-precipitated radioactively labeled fragment. 49. The method of claim 43 wherein the radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH comprises the fragment bound at the end opposite the carboxyl group by a member of the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl, and radioiodinated tyrosyl. 50. The method of claim 49 wherein the radioiodine is .sup.125 I. 51. The method of claim 49 wherein the fragment bound by said member is hPTH.sup.65-84. 52. The method of claim 51 wherein the radioactively labeled fragment is .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. 53. The method of claim 51 wherein the first antibody is chicken anti-hPTH.sup.65-84 and the first buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 54. The method of claim 53 wherein the first buffering agent comprises a borate buffer with a pH of about 8.4. 55. The method of claim 53 wherein the first buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 56. The method of claim 53 wherein the reaction of chicken anti-hPTH.sup.65-84 with intact rat PTH, C-terminal rat PTH fragments, and said radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating for 16 hours at 2.degree.-8.degree. C. 57. The method of claim 53 wherein the second antibody-precipitating complex is rabbit anti-chicken precipitating (RAC-PPT) complex and the second buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 58. The method of claim 57 wherein the second buffering agent comprises a borate buffer with a pH of about 8.4. 59. The method of claim 57 wherein the second buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 60. The method of claim 57 wherein the reaction of RAC-PPT complex with the chicken anti-hPTH.sup.65-84 bound radioactively labeled hPTH.sup.65-84 is allowed to proceed in the aforesaid manner by vortexing and incubating for a minimum of 2 hours at room temperature or greater than 16 hours at 2.degree.-8.degree. C. 61. The method of claim 42 wherein said double antibody radioimmunoassay comprises: A. Adding, to a measured quantity of the biological or other fluid to be assayed: 1. a first antibody, which has a substantially high affinity for intact rat PTH, C-terminal rat PTH fragments, and later to be added radioactively labeled fragment, in a sufficient quantity to bind a significant quantity of the intact rat PTH and C-terminal rat PTH fragments in the biological or other fluid; and 2. a first buffering agent to buffer the mixture to a pH suitable for reaction of the first antibody with intact rat PTH, C-terminal rat PTH fragments, and later to be added radioactively labeled fragment; B. Allowing reaction of the intact rat PTH and C-terminal rat PTH fragments with the first antibody to proceed substantially to equilibrium to thereby produce first antibody bound intact rat PTH and first antibody bound C-terminal rat PTH fragments; C. Adding a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH, in sufficient quantity to give a measurable counting rate of later formed precipitated second antibody bound first antibody bound radioactively labeled fragment; D. Allowing reaction of the radioactively labeled fragment with the first antibody bound intact rat PTH and first antibody bound C-terminal rat PTH fragments to proceed substantially to equilibrium to thereby bind a portion of the radioactively labeled fragment while freeing a portion of the bound intact rat PTH and C-terminal rat PTH fragments; E. Adding: 1. a second antibody-precipitating complex, wherein the second antibody has a substantially high affinity for the first antibody, as bound to the radioactively labeled fragment, in a sufficient quantity to give a measurable counting rate of precipitated second antibody bound first antibody bound radioactively labeled fragment; and 2. a second buffering agent to buffer the mixture to a pH suitable for reaction of the second antibody with first antibody bound radioactively labeled fragment; F. Allowing reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment to proceed substantially to equilibrium to thereby produce precipitated second antibody bound first antibody bound radioactively labeled fragment; G. Separating the precipitated from non-precipitated radioactively labeled fragment; H. Measuring the radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment; I. Correlating the respective measured radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment for the biological or other fluid with that for known standards. 62. The method of claim 61 wherein the biological or other fluid is rat serum or plasma. 63. The method of claim 61 wherein precipitated is separated from non-precipitated radioactively labeled fragment by centrifuging until the supernatant is substantially clear of the precipitate, and decanting the supernatant. 64. The method of claim 61 wherein the reaction of the first antibody with intact rat PTH and C-terminal rat PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 65. The method of claim 61 wherein said reaction of the radioactively labeled fragment with the first antibody bound intact rat PTH and first antibody bound C-terminal rat PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 66. The method of claim 61 wherein the reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 67. The method of claim 64 wherein said correlation comprises: A. Preparing a standard curve for which known standards are plotted respectively against the measured radioactivity of their precipitated and/or non-precipitated radioactively labeled fragment; and B. Correlating via the standard curve the quantity of intact rat PTH and C-terminal rat PTH fragments in the biological or other fluid with the measured radioactivity of its precipitated and/or non-precipitated radioactively labeled fragment. 68. The method of claim 61 wherein the radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH comprises the fragment bound at the end opposite the carboxyl group by a member of the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl, and radioiodinated tyrosyl. 69. The method of claim 68 wherein the radioiodine is .sup.125 I. 70. The method of claim 68 wherein the fragment bound by said member is hPTH.sup.65-84. 71. The method of claim 70 wherein the radioactively labeled fragment is .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. 72. The method of claim 70 wherein the first antibody is chicken anti-hPTH.sup.65-84 and the first buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 73. The method of claim 72 wherein the first buffering agent comprises a borate buffer with a pH of about 8.4. 74. The method of claim 72 wherein the first buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 75. The method of claim 72 wherein the reaction of chicken anti-hPTH.sup.65-84 with intact rat PTH and C-terminal rat PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating for a minimum of 4 hours at 2.degree.-8.degree. C. 76. The method of claim 72 wherein the reaction of the radioactively labeled fragment with the first antibody bound intact rat PTH and first antibody bound C-terminal rat PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating for at least 20 hours at 2.degree.-8.degree. C. 77. The method of claim 72 wherein the second antibody-precipitating complex is rabbit anti-chicken precipitating (RAC-PPT) complex and the second buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 78. The method of claim 77 wherein the second buffering agent comprises a borate buffer with a pH of about 8.4. 79. The method of claim 77 wherein the second buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 80. The method of claim 77 wherein the reaction of RAC-PPT complex with the chicken anti-hPTH.sup.65-84 bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing and incubating for a minimum of 2 hours at room temperature or greater than 16 hours at 2.degree.-8.degree. C. 81. The method of claim 2 wherein said respective members are intact bovine PTH and C-terminal bovine PTH fragments. 82. The method of claim 81 wherein said double antibody radioimmunoassay comprises: A. Adding together: 1. a measured quantity of the biological or other fluid to be assayed; 2. a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH, in sufficient quantity and of sufficient radioactivity to give a measurable counting rate of later formed precipitated second antibody bound first antibody bound radioactively labeled fragment; 3. a first antibody, which has a substantially high affinity for intact bovine PTH and the radioactively labeled fragment, in sufficient quantity to bind a significant quantity of the intact bovine PTH, the C-terminal bovine PTH fragments, and the radioactively labeled fragment; and 4. a first buffering agent to buffer the mixture to a pH suitable for reaction of the first antibody with intact bovine PTH, C-terminal bovine PTH fragments, and the radioactively labeled fragment; B. Allowing reaction of the first antibody with the intact bovine PTH, the C-terminal bovine PTH fragments, and the radioactively labeled fragment to proceed substantially to equilibrium to thereby produce first antibody bound intact bovine PTH, first antibody bound C-terminal bovine PTH fragments, and first antibody bound radioactively labeled fragment; C. Adding: 1. a second antibody-precipitating complex, wherein the second antibody has a substantially high affinity for the first antibody, as bound to the radioactively labeled fragment, in a sufficient quantity to give a measurable counting rate of precipitated second antibody bound first antibody bound radioactively labeled fragment; and 2. a second buffering agent to buffer the mixture to a pH suitable for reaction of the second antibody with first antibody bound radioactively labeled fragment; D. Allowing reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment to proceed substantially to equilibrium to thereby produce precipitated second antibody bound first antibody bound radioactively labeled fragment; E. Separating the precipitated from non-precipitated radioactively labeled fragment; F. Measuring the radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment; G. Correlating the respective measured radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment for the biological or other fluid with that for known standards. 83. The method of claim 82 wherein the biological or other fluid is bovine serum or plasma. 84. The method of claim 82 wherein precipitated is separated from non-precipitated radioactively labeled fragment by centrifuging until the supernatant is substantially clear of the precipitate, and decanting the supernatant. 85. The method of claim 82 wherein the reaction of the first antibody with intact bovine PTH, C-terminal bovine PTH fragments, and radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 86. The method of claim 82 wherein the reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 87. The method of claim 82 wherein said correlation comprises: A. Preparing a standard curve for which known standards are plotted respectively against the measured radioactivity of their precipitated and/or non-precipitated radioactively labeled fragment; and B. Correlating via the standard curve the quantity of intact bovine PTH and C-terminal bovine PTH fragments in the biological or other fluid with the measured radioactivity of its precipitated and/or non-precipitated radioactively labeled fragment. 88. The method of claim 82 wherein the radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH comprises the fragment bound at the end opposite the carboxyl group by a member of the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl, and radioiodinated tyrosyl. 89. The method of claim 88 wherein the radioiodine is .sup.125 I. 90. The method of claim 88 wherein the fragment bound by said member is hPTH.sup.65-84. 91. The method of claim 90 wherein the radioactively labeled fragment is .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. 92. The method of claim 90 wherein the first antibody is chicken anti-hPTH.sup.65-84 and the first buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 93. The method of claim 92 wherein the first buffering agent comprises a borate buffer with a pH of about 8.4. 94. The method of claim 92 wherein the first buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 95. The method of claim 92 wherein the reaction of chicken anti-hPTH.sup.65-84 with intact bovine PTH, C-terminal bovine PTH fragments, and said radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating for 16 hours at 2.degree.-8.degree. C. 96. The method of claim 92 wherein the second antibody-precipitating complex is rabbit anti-chicken precipitating (RAC-PPT) complex and the second buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 97. The method in claim 96 wherein the second buffering agent comprises a borate buffer with a pH of about 8.4. 98. The method of claim 96 wherein the second buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 99. The method of claim 96 wherein the reaction of RAC-PPT complex with the chicken anti-hPTH.sup.65-84 bound radioactively labeled hPTH.sup.65-84 is allowed to proceed in the aforesaid manner by vortexing and incubating for a minimum of 2 hours at room temperature or greater than than 16 hours at 2.degree.-8.degree. C. 100. The method of claim 81 wherein said double antibody radioimmunoassay comprises: A. Adding, to a measured quantity of the biological or other fluid to be assayed: 1. a first antibody, which has a substantially high affinity for intact bovine PTH, C-terminal bovine PTH fragments, and later to be added radioactively labeled fragment, in a sufficient quantity to bind a significant quantity of the intact bovine PTH and C-terminal bovine PTH fragments in the biological or other fluid; and 2. a first buffering agent to buffer the mixture to a pH suitable for reaction of the first antibody with intact bovine PTH, C-terminal bovine PTH fragments, and later to be added radioactively labeled fragment; B. Allowing reaction of the intact bovine PTH and C-terminal bovine PTH fragments with the first antibody to proceed substantially to equilibrium to thereby produce first antibody bound intact bovine PTH and first antibody bound C-terminal bovine PTH fragments; C. Adding a radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH, in sufficient quantity to give a measurable counting rate of later formed precipitated second antibody bound first antibody bound radioactively labeled fragment; D. Allowing reaction of the radioactively labeled fragment with the first antibody bound intact bovine PTH and first antibody bound C-terminal bovine PTH fragments to proceed substantially to equilibrium to thereby bind a portion of the radioactively labeled fragment while freeing a portion of the bound intact bovine PTH and C-terminal bovine PTH fragments; E. Adding: 1. a second antibody-precipitating complex, wherein the second antibody has a substantially high affinity for the first antibody, as bound to the radioactively labeled fragment, in a sufficient quantity to give a measurable counting rate of precipitated second antibody bound first antibody bound radioactively labeled fragment; and 2. a second buffering agent to buffer the mixture to a pH suitable for reaction of the second antibody with first antibody bound radioactively labeled fragment; F. Allowing reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment to proceed substantially to equilibrium to thereby produce precipitated second antibody bound first antibody bound radioactively labeled fragment; G. Separating the precipitated from non-precipitated radioactively labeled fragment; H. Measuring the radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment; I. Correlating the respective measured radioactivity of the precipitated and/or non-precipitated radioactively labeled fragment for the biological or other fluid with that for known standards. 101. The method of claim 100 wherein the biological or other fluid is bovine serum or plasma. 102. The method of claim 100 wherein precipitated is separated from non-precipitated radioactively labeled fragment by centrifuging until the supernatant is substantially clear of the precipitate, and decanting the supernatant. 103. The method of claim 100 wherein the reaction of the first antibody with intact bovine PTH and C-terminal bovine PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 104. The method of claim 100 wherein said reaction of the radioactively labeled fragment with the first antibody bound intact bovine PTH and first antibody bound C-terminal bovine PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 105. The method of claim 100 wherein the reaction of the second antibody-precipitating complex with the first antibody bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing gently and incubating the vortexed material until the reaction proceeds substantially to equilibrium. 106. The method of claim 100 wherein said correlation comprises: A. Preparing a standard curve for which known standards are plotted respectively against the measured radioactivity of their precipitated and/or non-precipitated radioactively labeled fragment; and B. Correlating via the standard curve the quantity of intact bovine PTH and C-terminal bovine PTH fragments in the biological or other fluid with the measured radioactivity of its precipitated and/or non-precipitated radioactively labeled fragment. 107. The method of claim 100 wherein the radioactively labeled fragment within the range of about 65-84 of bovine PTH or hPTH comprises the fragment bound at the end opposite the carboxyl group by a member of the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl, and radioiodinated tyrosyl. 108. The method of claim 107 wherein the radioiodine is .sup.125 I. 109. The method of claim 107 wherein the fragment bound by said member is hPTH.sup.65-84. 110. The method of claim 109 wherein the radioactively labeled fragment is .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. 111. The method of claim 109 wherein the first antibody is chicken anti-hPTH.sup.65-84 and the first buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 112. The method of claim 111 wherein the first buffering agent comprises a borate buffer with a pH of about 8.4. 113. The method of claim 111 wherein the first buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 114. The method of claim 111 wherein the reaction of chicken anti-hPTH.sup.65-84 with intact bovine PTH and C-terminal bovine PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating for a minimum of 4 hours at 2.degree.-8.degree. C. 115. The method of claim 111 wherein the reaction of the radioactively labeled fragment with the first antibody bound intact bovine PTH and first antibody bound C-terminal bovine PTH fragments is allowed to proceed in the aforesaid manner by vortexing gently and incubating for at least 20 hours at 2.degree.-8.degree. C. 116. The method of claim 111 wherein the second antibody-precipitating complex is rabbit anti-chicken precipitating (RAC-PPT) complex and the second buffering agent buffers the mixture to a pH in the range of about 7.5-8.6. 117. The method of claim 116 wherein the second buffering agent comprises a borate buffer with a pH of about 8.4. 118. The method of claim 116 wherein the second buffering agent comprises a BSA-borate buffer with a pH of about 8.4. 119. The method of claim 116 wherein the reaction of RAC-PPT complex with the chicken anti-hPTH.sup.65-84 bound radioactively labeled fragment is allowed to proceed in the aforesaid manner by vortexing and incubating for a minimum of 2 hours at room temperature or greater than 16 hours at 2.degree.-8.degree. C.

Details for Patent 4,341,755

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2039-03-29