Assaying for hLH or hCG with immobilized hormone receptors

Claim 2

A process for preparing an hLH-enzyme complex or an hCG-enzyme complex which comprises a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 3

The product produced by the process of claim 1.

Claim 4

The process of claim 1, wherein said compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 5

The product produced by the process of claim 3.

Claim 6

A process for preparing an hLH-dye complex or an hCG-dye complex which comprises a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 7

A process for preparing an hLH-dye complex or an hCG-dye complex which comprises a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 8

The product produced by the process of claim 5.

Claim 9

The process of claim 5, wherein the compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 10

The product produced by the process of claim 7.

Claim 11

A solid phase for an hLH or hCG-receptor assay comprising a solid, inert support carrying a gonadotropin receptor material containing a common receptor for hLH and hCG, wherein said material is prepared by the process comprising: (A) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (B) separating and concentrating the liquid fraction; (C) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (D) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (E) separating the aqueous phase from the remainder of the product of step (D); (F) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (G) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (H) recovering the resultant purified receptor.

Claim 12

A solid phase for an hLH or hCG-receptor assay comprising a solid, inert support carrying a gonadotropin receptor material containing a common receptor for hLH and hCG, wherein said material is prepared by the process comprising: (A) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (B) separating and concentrating the liquid fraction; (C) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (D) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (E) separating the aqueous phase from the remainder of the product of step (D); (F) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (G) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (H) recovering the resultant purified receptor.

Claim 13

The solid phase for an hLH or hCG receptor assay of claim 9, wherein said process additionally comprises, after step (H), the following step (I): (I) further purifying the receptor by subjecting it to immunoaffinity chromatography.

Claim 14

The solid phase of claim 9, wherein in said process: (i) the receptor source material is bovine corpora lutea; (ii) the concentration in step (B) is carried out by ultrafiltration to remove material below about 50,000 molecular weight; (iii) the fractionation of step (C) is carried out by preparative sucrose linear density gradient ultracentrifugation, and before re-dispersion step (D), the active fraction is concentrated by ultrafiltration to remove sucrose and then centrifuged; (iv) a detergent is used as a re-dispersion aid in step (D); (v) in step (F), multi-stage chromatography is used to separate the adenyl cyclase and 5'-nucleotidase activity fractions and, wherein prior to step (G), the reactive fractions are ultrafiltered and gel-filtered to remove excess detergent; and (vi) in step (G), vertical zone electrophoresis is carried out on a cellulose column and the active fractions are re-chromatographed.

Claim 15

The solid phase of claim 9, wherein in step (G) said electrophoresis is vertical-zone electrophoresis.

Claim 16

The solid phase of claim 9, wherein said gonadotropin receptor material has a binding capacity of at least 2,000 pM hCG/mg protein.

Claim 17

The solid phase of claim 9, wherein said support is selected from the group consisting of a glass bead, a test-tube, a strip and a rod.

Claim 18

A reagent kit comprising: (A) a hormone-enzyme complex or a hormone-dye complex, wherein said hormone is selected from the group consisting of hLH and hCG; (B) a solid phase comprising a solid, inert support carrying a gonadotropin receptor material containing a common receptor for hLH and hCG, wherein said receptor material is prepared by the process comprising: (1) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (2) separating and concentrating the liquid fraction; (3) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (4) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (5) separating the aqueous phase from the remainder of the product of step (4); (6) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (7) subjecting the hLH and hCG specific binding fraction of the receptor in electrophoresis to separate said fraction from other remaining protein fractions; and (8) recovering the resultant purified receptor; and (C) when a hormone-enzyme complex is employed, a material which forms a color when reacted with the enzyme of said hormone-enzyme complex.

Claim 19

A reagent kit comprising: (A) a hormone-enzyme complex or a hormone-dye complex, wherein said hormone is selected from the group consisting of hLH and hCG; (B) a solid phase comprising a solid, inert support carrying a gonadotropin receptor material containing a common receptor for hLH and hCG, wherein said receptor material is prepared by the process comprising: (1) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (2) separating and concentrating the liquid fraction; (3) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (4) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (5) separating the aqueous phase from the remainder of the product of step (4); (6) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (7) subjecting the hLH and hCG specific binding fraction of the receptor in electrophoresis to separate said fraction from other remaining protein fractions; and (8) recovering the resultant purified receptor; and (C) when a hormone-enzyme complex is employed, a material which forms a color when reacted with the enzyme of said hormone-enzyme complex.

Claim 20

The reagent kit of claim 15, wherein said process additionally comprises, after step (8), the following step (9): (9) further purifying the receptor by subjecting it to an immunoaffinity chromatography.

Claim 21

The reagent kit of claim 15, wherein in said process: (i) the receptor source material is bovine corpora lutea; (ii) the concentration in step (2) is carried out by ultrafiltration to remove material below about 50,000 molecular weight; (iii) the fractionation of step (3) is carried out by preparative sucrose linear density gradient ultracentrifugation, and before re-dispersion step (4), the active fraction is concentrated by ultrafiltration to remove sucrose and then centrifuged; (iv) a detergent is used as a re-dispersion aid in step (4); (v) in step (6), multi-stage chromatography is used to separate the adenyl cyclase and 5'-nucleotidase activity fractions and, wherein prior to step (7), the reactive fractions are ultrafiltered and gel-filtered to remove excess detergent; and (vi) in step (7), vertical zone electrophoresis is carried out on a cellulose column and the active fractions are re-chromatographed.

Claim 22

The reagent kit of claim 15, wherein in step (7) said electrophoresis is vertical-zone electrophoresis.

Claim 23

The reagent kit of claim 5, wherein said gonadotropin receptor material has a binding capacity of at least 2,000 pM hCG/mg protein.

Claim 24

The reagent kit of claim 5, wherein the hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 25

The reagent kit of claim 15, wherein the hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein said compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 26

The reagent kit of claim 15, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 27

The reagent kit of claim 15, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein the compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 28

The reagent kit of claim 15, wherein said support is selected from the group consisting of a glass bead, a test-tube, a strip and a rod.

Claim 29

The reagent kit of claim 15, wherein the quantities of reagents are predetermined to provide a qualitative response to the presence or absence of sufficient hLH to indicate ovulation.

Claim 30

The reagent kit of claim 15, wherein the quantities of reagents are predetermined to provide a qualitative response to the presence or absence of sufficient hCG to indicate pregnancy.

Claim 31

The reagent kit of claim 15, wherein the hormone of said hormone-enzyme complex is prepared by the process comprising: (1) contacting an impure hLH or hCG hormone with said a gonadotropin receptor material containing a common receptor for hLH and hCG under conditions to allow the hLH or hCG hormone to complex with said receptor; (2) separating the receptor phase from the remainder; and (3) releasing the hLH or hCG from said receptor.

Claim 32

The reagent kit of claim 27, wherein complexation is carried out by admixing in an aqueous solution.

Claim 33

The reagent kit of claim 27, wherein said complexation is carried out by immunoaffinity chromatography.

Claim 34

A reagant kit comprising: (A) a hormone-enzyme complex or a hormone-dye complex, wherein said hormone is selected from the group consisting of hLH or hCG; (B) a solid phase comprisng a solid, inert support carrying an antibody to hLH or an antibody to hCG; (C) when a hormone-enzyme complex is employed, a material which forms a color when reacted with the enzyme of said hormone-enzyme complex, wherein said hLH or hCG is prepared by the process comprising: (1) contacting an impure hLH or hCG hormone with a gonadotropin receptor material containing a common receptor for hLH and hCG under conditions to allow the hLH or hCG hormone to complex with said receptor; (2) separating the receptor phase from the remainder; and (3) releasing the hLH or hCG from said receptor, wherein said receptor material is prepared by the process comprising: (a) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (b) separating and concentrating the liquid fraction; (c) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (d) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (e) separating the aqueous phase from the remainder of the product of step (d); (f) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (g) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (h) recovering the resultant purified receptor.

Claim 35

A reagant kit comprising: (A) a hormone-enzyme complex or a hormone-dye complex, wherein said hormone is selected from the group consisting of hLH or hCG; (B) a solid phase comprisng a solid, inert support carrying an antibody to hLH or an antibody to hCG; (C) when a hormone-enzyme complex is employed, a material which forms a color when reacted with the enzyme of said hormone-enzyme complex, wherein said hLH or hCG is prepared by the process comprising: (1) contacting an impure hLH or hCG hormone with a gonadotropin receptor material containing a common receptor for hLH and hCG under conditions to allow the hLH or hCG hormone to complex with said receptor; (2) separating the receptor phase from the remainder; and (3) releasing the hLH or hCG from said receptor, wherein said receptor material is prepared by the process comprising: (a) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (b) separating and concentrating the liquid fraction; (c) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (d) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (e) separating the aqueous phase from the remainder of the product of step (d); (f) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (g) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (h) recovering the resultant purified receptor.

Claim 36

The reagent kit of claim 30, wherein said process additionally comprises, following after step (h), the step (i): (i) further purifying the receptor by subjecting it to immunoaffinity chromatography.

Claim 37

The reagent kit of claim 30, wherein in said process: (i') the receptor source material is bovine corpora lutea; (ii') the concentration in step (b) is carried out by ultrafiltration to remove material below about 50,000 molecular weight; (iii') the fractionation of step (c) is carried out by preparative sucrose linear density gradient ultracentrifugation, and before re-disperson step (d), the active fraction is concentration by ultrafiltration to remove sucrose and then centrifuged; (iv') a detergent is used as a re-dispersion aid in step (d); (v') in step (f), multi-stage chromatography is used to separate the adenyl cyclase and 5'-nucleotidase activity fractions and, wherein prior to step (g), the reactive fractions are ultrafiltered and gel-filtered to remove excess detergent; and (vi') in step (g), vertical zone electrophoresis is carried out on a cellulose column and the active fractions are re-chromatographed.

Claim 38

The reagent kit of claim 30, wherein in step (g) said electrophoresis is vertical-zone electrophoresis.

Claim 39

The reagent kit of claim 30, wherein complexation is carried out by admixing in an aqueous solution.

Claim 40

The reagent kit of claim 30, wherein said complexation is carried out by immunoaffinity chromatography.

Claim 41

The reagent kit of claim 30, wherein said gonadotropin receptor material has a binding capacity of at least 2,000 pM hCG/mg protein.

Claim 42

The reagent kit of claim 30, wherein the hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) Separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 43

The reagent kit of claim 30. wherein the hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein said compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 44

The reagent kit of claim 30, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 45

The reagent kit of claim 30, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein the compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 46

The reagent kit of claim 30, wherein said support is selected from the group consisting of a glass bead, a test-tube, a strip and a rod.

Claim 47

The reagent kit of claim 30, wherein the quantities of reagents are predetermined to provide a qualitative response to the presence or absence of sufficient hLH to indicate ovulation.

Claim 48

The reagent kit of claim 30, wherein the quantities of reagents are predetermined to provide a qualitative response to the presence or absence of sufficient hCG to indicate pregnancy.

Claim 49

A process for qualitatively or quantitatively determining the hLH or hCG level in an unknown sample comprising: (A) admixing an inert, solid support carrying hLH or hCG receptor with an unknown sample, and an hLH-enzyme complex or hCG-enzyme complex, or hLH-dye complex or hCG-dye complex; (B) incubating the resulting admixture of step (A) under conditions to allow the hLH or hCG to react with said receptor; (C) separating the resulting solid phase from the resulting liquid phase; (D) when an enzyme complex is used, adding a material which forms a colored compound in the presence of the enzyme to either the liquid phase or the solid phase; (E) qualitatively or quantitatively measuring the amount of colored material produced, wherein when a quantitative measurement is made, comparing the amount of colored material produced with a standard curve to determine the quantitative result, wherein the receptor is a product produced by the process comprising: (1) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (2) separating and concentrating the liquid fraction; (3) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (4) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (5) separating the aqueous phase from the remainder of the product of step (4); (6) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (7) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (8) recovering the resultant purified receptor.

Claim 50

A process for qualitatively or quantitatively determining the hLH or hCG level in an unknown sample comprising: (A) admixing an inert, solid support carrying hLH or hCG receptor with an unknown sample, and an hLH-enzyme complex or hCG-enzyme complex, or hLH-dye complex or hCG-dye complex; (B) incubating the resulting admixture of step (A) under conditions to allow the hLH or hCG to react with said receptor; (C) separating the resulting solid phase from the resulting liquid phase; (D) when an enzyme complex is used, adding a material which forms a colored compound in the presence of the enzyme to either the liquid phase or the solid phase; (E) qualitatively or quantitatively measuring the amount of colored material produced, wherein when a quantitative measurement is made, comparing the amount of colored material produced with a standard curve to determine the quantitative result, wherein the receptor is a product produced by the process comprising: (1) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (2) separating and concentrating the liquid fraction; (3) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (4) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (5) separating the aqueous phase from the remainder of the product of step (4); (6) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (7) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (8) recovering the resultant purified receptor.

Claim 51

The process of claim 44, wherein said process additionally comprises, after step (8), the following step (9): (9) further purifying the receptor by subjecting it to immunoaffinity chromatography.

Claim 52

The process of claim 44, wherein in said process: (i') the receptor source material is bovine corpora lutea; (ii') the concentration in step (2) is carried out by ultrafiltration to remove material below about 50,000 molecular weight; (iii') the fractionation of step (3) is carried out by preparative sucrose linear density gradient ultracentrifugation, and before re-dispersion step (4), the active fraction is concentrated by ultrafiltration to remove sucrose and then centrifuged; (iv') a detergent is used as a re-dispersion aid in step (4); (v') in step (6), multi-stage chromatography is used to separate the adenyl cyclase and 5'-nucleotidase activity fractions and, wherein prior to step (7), the reactive fractions are ultrafiltered and gel-filtered to remove excess detergent; and (vi') in step (7), vertical zone electrophoresis is carried out on a cellulose column and the active fractions are re-chromatographed.

Claim 53

The process of claim 44, wherein in step (7) said electrophoresis is vertical-zone electrophoresis.

Claim 54

The process of claim 44, wherein said gonadotropin receptor material has a binding capacity of at least 2,000 pM hCG/mg protein.

Claim 55

The process of claim 44, wherein said hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 56

The process of claim 44, wherein said hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein said compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 57

The process of claim 44, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 58

The process of claim 44, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein the compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 59

The process of claim 44, wherein said support is selected from the group consisting of a glass bead, a test-tube, a strip and a rod.

Claim 60

The process of claim 44, wherein said hLH or hCG is prepared by the process comprising: (1) contacting an impure hLH or hCG hormone with said a gonadotropin receptor material containing a common receptor for hLH or hCG under conditions to allow the hLH or hCG hormone to complex with said receptor; (2) separating the receptor phase from the remainder; and (3) releasing the hLH or hCG from said receptor.

Claim 61

The process of claim 54, wherein complexation is carried out by admixing in an aqueous solution.

Claim 62

The process of claim 54, wherein complexation is carried out by immunoaffinity chromatography.

Claim 63

A process for qualitatively or quantitatively determining the hLH or hCG level in a unknown sample comprising: (A) admixing an inert, solid support carrying hLH or hCG antibody with an unknown sample and an hLH-enzyme complex or hCG-enzyme complex or hLH-dye complex or hCG-dye complex; (B) incubating the resulting admixture of step (A) under conditions to allow hLH or hCG to react with said antibody; (C) separating the resulting solid phase from the liquid phase; (D) when an enzyme complex is used, adding a material which forms a colored compound in the presence of the enzyme to either the liquid phase or the solid phase; (E) qualitatively or quantitatively measuring the amount of colored material produced, wherein when a quantitative measurement is made, comparing the amount of colored material produced with a standard curve to determine the quantitative result, wherein said hLH or hCG is prepared by the process comprising: (1) contacting an impure hLH or hCG hormone with said a gonadotropin receptor material containing a common receptor for hLH or hCG under conditions to allow the hLH or hCG hormone to complex with said receptor; (2) separating the receptor phase from the remainder; and (3) releasing the hLH or hCG from said receptor, wherein said material is prepared by the process comprising: (a) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (b) separating and concentrating the liquid fraction; (c) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (d) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (e) separating the aqueous phase from the remainder of the product of step (d); (f) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (g) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (h) recovering the resultant purified receptor.

Claim 64

A process for qualitatively or quantitatively determining the hLH or hCG level in a unknown sample comprising: (A) admixing an inert, solid support carrying hLH or hCG antibody with an unknown sample and an hLH-enzyme complex or hCG-enzyme complex or hLH-dye complex or hCG-dye complex; (B) incubating the resulting admixture of step (A) under conditions to allow hLH or hCG to react with said antibody; (C) separating the resulting solid phase from the liquid phase; (D) when an enzyme complex is used, adding a material which forms a colored compound in the presence of the enzyme to either the liquid phase or the solid phase; (E) qualitatively or quantitatively measuring the amount of colored material produced, wherein when a quantitative measurement is made, comparing the amount of colored material produced with a standard curve to determine the quantitative result, wherein said hLH or hCG is prepared by the process comprising: (1) contacting an impure hLH or hCG hormone with said a gonadotropin receptor material containing a common receptor for hLH or hCG under conditions to allow the hLH or hCG hormone to complex with said receptor; (2) separating the receptor phase from the remainder; and (3) releasing the hLH or hCG from said receptor, wherein said material is prepared by the process comprising: (a) homogenizing a receptor source material in an aqueous medium to disperse the receptor in a liquid aqueous fraction; (b) separating and concentrating the liquid fraction; (c) fractioning the concentrate to separate at least one of inactive protein and lipid from the receptor; (d) re-dispersing the receptor in aqueous medium and extracting the aqueous medium with an organic solvent in which lipids are soluble to remove additional lipid from the aqueous phase; (e) separating the aqueous phase from the remainder of the product of step (d); (f) separating an hLH and hCG specific binding fraction of the receptor from adenyl cyclase and 5'-nucleotidase activity fractions of the receptor; (g) subjecting the hLH and hCG specific binding fraction of the receptor to electrophoresis to separate said fraction from other remaining protein fractions; and (h) recovering the resultant purified receptor.

Claim 65

The process of claim 57, wherein said process additionally comprises, after step (h), the following step (i): (i) further purifying the receptor by subjecting it to an immunoaffinity chromatography.

Claim 66

The process of claim 57, wherein in said process: (i') the receptor source material is bovine corpora lutea; (ii') the concentration in step (b) is carried out by ultrafiltration to remove material below about 50,000 molecular weight; (iii') the fractionation of step (c) is carried out by preparative sucrose linear density gradient ultracentrifugation, and before re-dispersion step (d), the active fraction is concentrated by ultrafiltration to remove sucrose and then centrifuged; (iv') a detergent is used as a re-dispersion aid in step (d); (v') in step (f), multi-stage chromatography is used to separate the adenyl cyclase and 5'-nucleotidase activity fractions and, wherein prior to step (g), the reactive fractions are ultrafiltered and gel-filtered to remove excess detergent; and (vi') in step (h), vertical zone electrophoresis is carried out on a cellulose column and the active fractions are re-chromatographed.

Claim 67

The process of claim 57, wherein in step (h) said electrophoresis is vertical-zone electrophoresis.

Claim 68

The process of claim 57, wherein complexation is carried out by admixing in an aqueous solution.

Claim 69

The process of claim 57, wherein said complexation is carried out by immunoaffinity chromatography.

Claim 70

The process of claim 57, wherein said gonadotropin receptor material has a binding capacity of at least 2,000 pM hCG/mg protein.

Claim 71

The process of claim 57, wherein said hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 72

The process of claim 57, wherein said hormone-enzyme complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with an enzyme containing an active thiol group to form a disulfide-linked hLH-enzyme complex or hCG-enzyme complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said enzyme-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated enzyme to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and an enzyme-disulfide complex, thiolating said enzyme-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein said compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 73

The process of claim 57, wherein said hormone-dye complex is the product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex.

Claim 74

The process of claim 57, wherein said hormone-dye complex is a product produced by a method selected from the group consisting of the following methods (A), (B) and (C): (A) complexing hLH or hCG with a compound providing a reactive disulfide moiety, and reacting the complex with a dye containing an active thiol group to form a disulfide-linked hLH-dye complex or hCG-dye complex through thiol-disulfide exchange; (B) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex and a dye-disulfide complex, thiolating the hLH-disulfide complex or hCG-disulfide complex and then reacting the thiolated complex with said dye-disulfide complex; and (C) separately complexing (1) hLH or hCG and (2) a non-thiolated dye to different portions of a compound providing a reactive disulfide moiety to form an hLH-disulfide complex or an hCG-disulfide complex, and a dye-disulfide complex, thiolating said dye-disulfide complex and then reacting the thiolated complex with said hLH-disulfide complex or said hCG-disulfide complex, wherein the compound providing a reactive disulfide moiety is N-succinimidyl 3-(2-pyridyldithio)propionate.

Claim 75

The process of claim 57, wherein said support is selected from the group consisting of a glass bead, a test-tube, a strip and rod.