Clinical determination and/or quantification of thyrotropin and a variety of thyroid stimulatory or inhibitory factors performed in vitro with an improved thyroid cell line, FRTL-5

Claim 2

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibiting factors to cause or prevent, respectively, incorporation of thymidine in a continuous strain of FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said FRTL-5 thyroid cells in a nutrient medium comprising calf serum and thyrotropin; (b) removing thyrotropin from said plated FRTL-5 thyroid cells; (c) introducing labeled thymidine, a nutrient medium and at least one of a thyroid stimulator and inhibitory factor, to said plated FRTL-5 thyroid cells; (d) incubating the mixture of said FRTL-5 thyroid cells, said labeld thymidine, said nutrient medium, and said at least one of a thyroid stimulatory and inhibitory factor; (e) terminating growth of said FRTL-5 thyroid cells; (f) recovering said labeled thymidine; and (g) detecting said labeled thymidine.

Claim 3

The assay method according to claim 1 wherein said nutrient medium further includes Coon's modified F-12 medium.

Claim 4

The assay method according to claim 1 wherein said nutrient medium contains about 2.5 to about 15 percent, by volume, calf serum.

Claim 5

The assay method according to claim 3 wherein said nutrient medium contains about 5 percent, by volume, calf serum.

Claim 6

The assay method according to claim 1 wherein the medium in which said FRTL-5 ATCC CRL 8305 thyroid cells are plated or incubated additionally includes a mixture of hormones comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate, and somatostatin.

Claim 7

The assay method according to claim 1 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are subjected to a growth period after plating of between 1 to 10 days.

Claim 8

The assay method according to claim 6 wherein during said growth period a mixture of hormones is added, said hormone mixture comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate, and somatostatin.

Claim 9

The assay method according to claim 1 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are washed with isotonic buffer, nutrient medium, or one of the foregoing with calf serum, to remove said thyrotropin.

Claim 10

The assay method according to claim 1 wherein said FRTL-5 ATCC CRL 8305 cells are maintained in a thyrotropin-free nutrient medium for a period of from about 1 to about 10 days between steps (b) and (c).

Claim 11

The assay method according to claim 9 wherein said maintenance period is about 5 days.

Claim 12

The assay method according to claim 9 wherein said FRTL-5 ATCC CRL 8305 cells are maintained in a mixture of hormones comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate and somatostatin.

Claim 13

The assay method according to claim 9 wherein said thyrotropin-free medium is changed at least once during said maintenance period.

Claim 14

The assay method according to claim 1 wherein said thymidine is labeled with 3 H or 14 C.

Claim 15

The assay method according to claim 1 wherein the incubation is continued for a period of approximately 72 hours.

Claim 16

The assay method according to claim 1 wherein said growth is terminated with a phosphate buffered saline solution or Hanks' Balanced Salt Solution.

Claim 17

The assay method according to claim 1 wherein said growth termination step is maintained at a temperature within the range of about 0 to about 4 degrees C.

Claim 18

The assay method according to claim 1 wherein protein-containing material is precipitated and separated from supernatant liquid containing said labeled thymidine after growth of said FRTL-5 ATCC CRL 8305 thyroid cells is terminated.

Claim 19

The assay method according to claim 17 wherein trichloroacetic acid of ethanol is employed to precipitate said protein-containing material.

Claim 20

The assay method according to claim 17 wherein said protein-containing material is analyzed for protein.

Claim 21

The assay method according to claim 17 wherein said protein-containing material is analyzed for DNA.

Claim 22

The assay method according to claim 17 wherein said labeled thymidine in said supernatant liquid is quantified.

Claim 23

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibitory factors to cause or prevent, respectively, cAMP elevation in a continuous strain of functional FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said FRTL-5 thyroid cells in a nutrient medium comprising calf serum and thyrotropin; (b) removing thyrotropin from said plated FRTL-5 thyroid cells; (c) introducing a buffered medium and at least one of a thyroid stimulatory and inhibitory factor, to said plated FRTL-5 thyroid cells; (d) incubating the mixture of said FRTL-5 thyroid cells, said at least one of a thyroid stimulatory and inhibitory factor, and said buffered medium; (e) terminating the reaction occurring during incubation; (f) recovering cAMP; and (g) detecting cAMP.

Claim 24

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibitory factors to cause or prevent, respectively, cAMP elevation in a continuous strain of functional FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said FRTL-5 thyroid cells in a nutrient medium comprising calf serum and thyrotropin; (b) removing thyrotropin from said plated FRTL-5 thyroid cells; (c) introducing a buffered medium and at least one of a thyroid stimulatory and inhibitory factor, to said plated FRTL-5 thyroid cells; (d) incubating the mixture of said FRTL-5 thyroid cells, said at least one of a thyroid stimulatory and inhibitory factor, and said buffered medium; (e) terminating the reaction occurring during incubation; (f) recovering cAMP; and (g) detecting cAMP.

Claim 25

The assay method according to claim 22 wherein said nutrient medium further includes Coon's modified F-12 medium.

Claim 26

The assay method according to claim 22 wherein said nutrient medium contains about 2.5 to about 15 percent, by volume, calf serum.

Claim 27

The assay method according to claim 22 wherein said nutrient medium contains about 5 percent, by volume, calf serum.

Claim 28

The assay method according to claim 22 wherein the medium in which said FRTL-5 ATCC CRL 8305 thyroid cells are plated or incubated additionally includes a mixture of hormones comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate, and somatostatin.

Claim 29

The assay method according to claim 22 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are subjected to a growth period after plating of between 1 and 10 days.

Claim 30

The assay method according to claim 27 wherein during said growth period a mixture of hormones is added, said hormone mixture comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate, and somatostatin.

Claim 31

The assay method according to claim 22 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are washed with isotonic buffer, nutrient medium, or one of the foregoing with calf serum to remove said thyrotropin.

Claim 32

The assay method according to claim 22 wherein said FRTL-5 ATCC CRL 8305 cells are maintained in a thyrotropin-free medium for a period of from about 1 to about 10 days between steps (b) and (c).

Claim 33

The assay method according to claim 30 wherein said maintenance period is about 7 to 10 days.

Claim 34

The assay method according to claim 30 wherein said FRTL-5 ATCC CRL 8305 cells are maintained in a mixture of hormones comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate and somatostatin.

Claim 35

The assay method according to claim 30 wherein said thyrotropin-free medium is changed at least once during said maintenance period.

Claim 36

The assay method according to claim 22 wherein the incubation is continued for a period of approximately 20 minutes to 3 hours.

Claim 37

The assay method according to claim 22, wherein an inhibitor of cAMP phosphodiesterase activity is added when said at least one of a thyroid stimulatory or inhibitory factor is introduced.

Claim 38

The assay method according to claim 35 wherein said inhibitor of cAMP phosphodiesterase is IMX.

Claim 39

The assay method according to claim 22, wherein a proteinaceous substance which prevents non-specific adsorption to an assay vessel of said at least one of a thyroid simulatory or inhibitory factor is added when said at least one of a thyroid stimulatory or inhibitory factor is introduced.

Claim 40

The assay method according to claim 37 wherein said non-specific adsorption preventing proteinaceous substance is bovine serum albumin, egg albumin or gelatin.

Claim 41

The assay method according to claim 30 wherein the thyrotropin-free medium in which said FRTL-5 ATCC CRL 8305 thyroid cells are maintained contains about 2.5 to 15 percent by volume of calf serum.

Claim 42

The assay method according to claim 30 wherein said thyrotropin-free medium contains approximately 5 percent, by volume, of serum.

Claim 43

The assay method according to claim 22 wherein said buffered medium in which said at least one of a thyroid stimulatory or inhibitory factor is introduced is a phosphate buffered saline solution or Hanks' Balanced Salt Solution.

Claim 44

The assay method according to claim 22 wherein the buffered medium in which said FRTL-5 ATCC CRL 8305 thyroid cells and said at least one of a thyroid stimulatory or inhibitory factor are incubated is a phosphate buffered saline solution or Hanks' Balanced Salt Solution.

Claim 45

The assay method according to claim 22 wherein the buffered medium in which said FRTL-5 ATCC CRL 8305 thyroid cells and said at least one of a thyroid stimulatory or inhibitory factor are incubated is reduced in salt content or is substantially free of NaCl.

Claim 46

The assay method according to claim 22 wherein said reaction is terminated by aspirating said buffered medium and said at least one of a thyroid stimulatory or inhibitory factor, said aspirated buffered medium being analyzed for cAMP.

Claim 47

The assay method according to claim 22 wherein protein containing material is precipitated and separated from cAMP after incubating the mixture of said FRTL-5 ATCC CRL 8305 thyroid cells and said at least one of a thyroid stimulatory or inhibitory factor.

Claim 48

The assay method according to claim 45 wherein ethanol is employed to precipitate and separate cAMP.

Claim 49

The assay method according to claim 45 wherein said protein-containing material is analyzed for protein.

Claim 50

The assay method according to claim 45 wherein said protein-containing material is analyzed for DNA.

Claim 51

The assay method according to claim 45 wherein said separated cAMP in the supernatant liquid is analyzed for cAMP.

Claim 52

The assay method according to claim 22 wherein cAMP is detected by a radioimmunoassay. PG,66

Claim 53

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibitory factors to cause or prevent, respectively, cAMP mediated iodine uptake by a continuous strain of functional FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said FRTL-5 thyroid cells in a nutrient medium comprising calf serum and thyrotropin; (b) removing thyrotropin from said plated FRTL-5 thyroid cells; (c) introducing a nutrient medium and at least one of a thyroid stimulatory and inhibitory factor, to said plated FRTL-5 thyroid cells; (d) incubating the mixture of said FRTL-5 thyroid cells, said at least one of a thyroid stimulatory and inhibitory factor, and nutrient medium; (e) adding a source of radioactive iodide and performing a second incubation in a buffered medium; (f) terminating the reaction occurring during the second incubation of step (e); (g) recovering incorporated radioactive iodide; and (h) detecting incorporated radioactive iodide.

Claim 54

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibitory factors to cause or prevent, respectively, cAMP mediated iodine uptake by a continuous strain of functional FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said FRTL-5 thyroid cells in a nutrient medium comprising calf serum and thyrotropin; (b) removing thyrotropin from said plated FRTL-5 thyroid cells; (c) introducing a nutrient medium and at least one of a thyroid stimulatory and inhibitory factor, to said plated FRTL-5 thyroid cells; (d) incubating the mixture of said FRTL-5 thyroid cells, said at least one of a thyroid stimulatory and inhibitory factor, and nutrient medium; (e) adding a source of radioactive iodide and performing a second incubation in a buffered medium; (f) terminating the reaction occurring during the second incubation of step (e); (g) recovering incorporated radioactive iodide; and (h) detecting incorporated radioactive iodide.

Claim 55

The assay method according to claim 51 wherein said nutrient medium further includes Coon's modified F-12 medium.

Claim 56

The assay method according to claim 51 wherein said nutrient medium contains about 2.5 to about 15 percent by volume of calf serum.

Claim 57

The assay method according to claim 51 wherein said nutrient medium contains about 5 percent, by volume, calf serum.

Claim 58

The assay method according to claim 51 wherein the medium in which said FRTL-5 ATCC CRL 8305 thyroid cells are plated or incubated additionally includes a mixture of hormones comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate, and somatostatin.

Claim 59

The assay method according to claim 51 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are subjected to a growth period after plating of between 1 to 10 days.

Claim 60

The assay method according to claim 56 wherein during said growth period a mixture of hormones is added, said hormone mixture comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate, and somatostatin.

Claim 61

The assay method according to claim 51 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are washed with isotonic buffer, nutrient medium, or one of the foregoing with calf serum to remove said thyrotropin.

Claim 62

The assay method according to claim 51 wherein said FRTL-5 ATCC CRL 8305 thyroid cells are maintained in a thyrotropin-free nutrient medium for a period of from about 1 to about 10 days between steps (b) and (c).

Claim 63

The assay method according to claim 59 wherein said maintenance period is about 7 to 10 days.

Claim 64

The assay method according to claim 59 wherein said FRTL-5 cells are maintained in a mixture of hormones comprising insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lycine acetate and somatostatin.

Claim 65

The assay method according to claim 59 wherein said thyrotropin-free nutrient medium is changed at least once during said maintenance period.

Claim 66

The assay method according to claim 51 wherein the incubation is continued for a period of approximately 36 to about 48 hours.

Claim 67

The assay method according to claim 51 wherein the nutrient medium employed during the incubation of step (d) is removed by washing with an Na+-containing isotonic buffered medium.

Claim 68

The assay method according to claim 64 wherein said Na+-containing isotonic buffered medium comprises NaCl, KCl, CaCl 2 , MgSO 4 .7H 2 O. MgCl 2 , Na 2 HPO.7H 2 O, KH 2 PO 4 , glucose, and Hepes.

Claim 69

The assay method according to claim 51 wherein said second incubation is maintained for a period of about 20 minutes to about 60 minutes.

Claim 70

The assay method according to claim 51 wherein said second incubation is performed in Na+ and K+-containing isotonic buffered medium.

Claim 71

The assay method according to claim 51 wherein said second incubation is performed in a medium having a composition comprising NaCl, KCl, CaCl 2 , MgSO 4 .7H 2 O, MgCl 2 , Na 2 HPO 4 .7H 2 O, KH 2 PO 4 , glucose, and Hepes.

Claim 72

The assay method according to claim 51 wherein an inhibitor of cAMP phosphodiesterase activity is added when said at least one of a thyroid stimulatory or inhibitory factor is introduced.

Claim 73

The assay method according to claim 69 wherein said inhibitor of cAMP phosphodiesterase is IMX.

Claim 74

The assay method according to claim 51 wherein a proteinaceous substance which prevents non-specific adsorption to an assay vessel of said at least one of a thyroid stimulatory or inhibitory factor is added when said at least one of a thyroid stimulatory or inhibitory factor is introduced.

Claim 75

The assay method according to claim 71 wherein said non-specific adsorption preventing proteinaceous substance is bovine serum albumin, egg albumin or gelatin.

Claim 76

The assay method according to claim 51 wherein said reaction is terminated with a phosphate buffered saline solution or Hanks' Balanced Salt Solution.

Claim 77

The assay method according to claim 51 wherein the reaction terminating step is maintained at a temperature of about 0 to about 4 degrees C.

Claim 78

The assay method according to claim 51 wherein protein-containing material is precipitated and separated from supernatant liquid containing said radioactive iodide after said second incubation of said FRTL-5 ATCC CRL 8305 thyroid cells is terminated.

Claim 79

The assay method according to claim 75 wherein ethanol is employed to precipitate and separate radioactive iodide.

Claim 80

The assay method according to claim 75 wherein said protein-containing material is analyzed for protein.

Claim 81

The assay method according to claim 75 wherein said protein-containing material is analyzed for DNA.

Claim 82

The assay method according to claim 75 wherein said radioactive iodide in said supernatant liquid is quantified.

Claim 83

The assay method according to claim 51 wherein said source of radioactive iodide is a salt of 125 I or 131 I.

Claim 84

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibitory factors to cause changes in growth, function or concentration of cAMP present in a continuous strain of FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said rat thyroid cells in a culture medium; (b) removing said culture medium from said plated rat thyroid cells; (c) introducing an incubation medium containing at least one of a thyroid stimulatory and inhibitory factor, to the plated rat thyroid cells wherein, when the assay method measures the ability of said at least one of a thyroid stimulatory and inhibitory factor to cause changes in growth, then labeled thymidine is introduced to said medium containing said at least one of a thyroid stimulatory and inhibitory factor; (d) incubating the mixture of said rat thyroid cells, said incubation medium, said at least one of a thyroid stimulatory and inhibitory factor, and, when present, said labeled thymidine; (e) terminating incubation of said rat thyroid cells wherein, when said assay method measures the ability of said at least one of a thyroid stimulatory and inhibitory factor to cause or prevent, respectively, cAMP mediated incorporation of iodide, then after termination of incubation of said rat thyroid cells, a source of labeled iodide is added, a second incubation is performed, and said second incubation is terminated; (f) recovering, when present, at least one of said labeled thymidine, said labeled iodide and said cAMP; and (g) detecting, when present, at least one of said labeled thymidine, said labeled iodide and said cAMP.

Claim 85

An assay method for measuring thyroid stimulatory or inhibitory factors by determining the ability of said thyroid stimulatory or inhibitory factors to cause changes in growth, function or concentration of cAMP present in a continuous strain of FRTL-5 ATCC CRL 8305 rat thyroid cells capable of surviving thyrotropin withdrawal for a period of up to about 10 days comprising the sequential steps of: (a) plating said rat thyroid cells in a culture medium; (b) removing said culture medium from said plated rat thyroid cells; (c) introducing an incubation medium containing at least one of a thyroid stimulatory and inhibitory factor, to the plated rat thyroid cells wherein, when the assay method measures the ability of said at least one of a thyroid stimulatory and inhibitory factor to cause changes in growth, then labeled thymidine is introduced to said medium containing said at least one of a thyroid stimulatory and inhibitory factor; (d) incubating the mixture of said rat thyroid cells, said incubation medium, said at least one of a thyroid stimulatory and inhibitory factor, and, when present, said labeled thymidine; (e) terminating incubation of said rat thyroid cells wherein, when said assay method measures the ability of said at least one of a thyroid stimulatory and inhibitory factor to cause or prevent, respectively, cAMP mediated incorporation of iodide, then after termination of incubation of said rat thyroid cells, a source of labeled iodide is added, a second incubation is performed, and said second incubation is terminated; (f) recovering, when present, at least one of said labeled thymidine, said labeled iodide and said cAMP; and (g) detecting, when present, at least one of said labeled thymidine, said labeled iodide and said cAMP.

Claim 86

The assay method according to claim 81 wherein said nutrient medium further includes thyrotropin.

Claim 87

The assay method according to claim 82 wherein a period of about 1 to about 10 days is allowed between steps (b) and (c).

Claim 88

The assay method according to claim 81 wherein said incubation medium comprises a nutrient medium.

Claim 89

The assay method according to claim 81 wherein said culture medium comprises calf serum containing a nutrient medium.

Claim 90

The assay method according to claim 81 wherein said thyroid stimulatory or inhibitory factor is obtained from a human source.