US4043872A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Polyiodothyronine immunoassay
Assignee: Syva Co
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Filing: 1975-12-29
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Granted: 1977-08-23
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Expires: 1995-12-29
Abstract
Abstract Method and enzyme conjugates are provided for determining physiological amounts of polyiodothyronine in physiological fluid, particularly serum. A homogeneous enzyme immunoassay is provided employing an enzyme-bound-polyiodothyronine having less than about 50 percent of the original enzyme activity, whose activity increases upon binding with a receptor. The enzyme-bound-polyiodothyronine, the unknown suspected to containing a polyiodothyronine, and receptor for the polyiodothyronine are combined in an aqueous medium containing substrate for said enzyme and the enzymatic activity of the solution determined. By comparing the result obtained with results obtained from standards having known amounts of polyiodothyronine, the amount of polyiodothyroninein the unknown can be determined. The enzyme-bound-polyiodothyronine employs an enzyme which is reversibly deactivated by polyiodothyronine, so as to be reactived by receptor binding to the polyiodothyronine. Particular enzymes include malate dehydrogenase and triose phosphate isomerase.
Inventors
- 👤 Judith I. Blakemore
- 👤 Richard K. Leute
- 👤 Roberta D. Ernst
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 14:43
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/74; G01N33/78; G01N33/53; G01N33/536; G01N33/542; Y; Y10; Y10S; Y10S435/00; Y10S435/964
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
112 words
A method for determining the presence of thyroxine in a medium suspected of containing said thyroxine, which comprises: bringing together in an aqueous liquid zone: (1) said medium; (2) soluble ENZ-bound-thyroxine; and (3) antithyroxine, said antithyroxine having sites common to and capable of binding to said thyroxine and said ENZ-bound-thyroxine, wherein ENZ is an enzyme selected from malate dehydrogenase or tricore phosphate isomerase and both of which are reversibly inhibited by said thyroxine and said antithyroxine is present at a concentration resulting in substantial enhancement of said enzyme activity in the absence of thyroxine; and analyzing in said zone for the effect of said medium on the enzymatic activity of said ENZ-bound-thyroxine.
Claim 2
Independent Claim
112 words
A method for determining the presence of thyroxine in a medium suspected of containing said thyroxine, which comprises: bringing together in an aqueous liquid zone: (1) said medium; (2) soluble ENZ-bound-thyroxine; and (3) antithyroxine, said antithyroxine having sites common to and capable of binding to said thyroxine and said ENZ-bound-thyroxine, wherein ENZ is an enzyme selected from malate dehydrogenase or tricore phosphate isomerase and both of which are reversibly inhibited by said thyroxine and said antithyroxine is present at a concentration resulting in substantial enhancement of said enzyme activity in the absence of thyroxine; and analyzing in said zone for the effect of said medium on the enzymatic activity of said ENZ-bound-thyroxine.
Claim 3
Dependent Claim
14 words
A method according to claim 1, wherein ENZ is malate dehydrogenase to form MDH-bound-thyroxine.
Claim 4
Dependent Claim
12 words
A method according to claim 1, where ENZ is triose phosphate isomerase.
Claim 5
Dependent Claim
63 words
A method according to claim 1, wherein said aqueous liquid zone is buffered at a pH in the range of about 7 to 10.5, and said ENZ-bound-thyroxine has less than about 50 percent of the enzyme activity of the ENZ prior to conjugation to form ENZ-bound-thyroxine, but not less than about 0.5 percent of the enzyme activity of said ENZ prior to conjugation.
Claim 6
Dependent Claim
39 words
A method according to claim 4, wherein ENZ is malate dehydrogenase and said enzymatic activity is determined by the addition of NAD and malate to said aqueous liquid zone at a pH in the range of 8.5 to 10.5.
Claim 7
Dependent Claim
41 words
A method according to claim 4, wherein ENZ is triose phosphate isomerase and said enzymatic acitivty is determined by the addition of glyceraldehyde-3-phosphate,α-glycerophosphate dehydrogenase and NADH to said aqueous liquid zone at a pH in the range of 7 to 9.
Claim 8
Dependent Claim
21 words
A method according to claim 2, wherein said medium and said antithydroxine are combined prior to the addition of said MDH-bound-thyroxine.
Claim 9
Dependent Claim
49 words
A method according to claim 2, wherein said MDH-bound-thyroxine has from about 1 to 10 thyroxine per MDH, said MDH is mitochrondial MDH and is deactivated to the extent of from about 65 to 99.5 percent of the enzyme activity of the MDH prior to conjugation to form MDH-bound-thyroxine.
Claim 10
Dependent Claim
17 words
A method according to claim 2, wherein the MDH in said MDH-bound-thyroxine is derived from mitochrondial MDH.
Claim 11
Independent Claim
110 words
A method for determining the presence of thyroxine in a medium suspected of containing said thyroxine, which comprises: bringing together in an aqueous buffered liquid zone at a pH in the range of 6-10.5: (1) said medium; (2) ENZ-bound-thyroxine; (3) antithydroxine, said antithyyroxine having sites common to and capable of binding to said thyroxine and said ENZ-bound-thyroxine, wherein ENZ is malate dehydrogense reversibly inhibited by said thyroxine, and said antithydroxine is present at a concentration resulting in substantial enhancement of said enzyme activity in the absence of thyroxine; and analyzing in said zone and NAD or NADH for the effect of said medium on the enzymatic activity of said ENZ-bound-thyroxine.
Claim 12
Independent Claim
110 words
A method for determining the presence of thyroxine in a medium suspected of containing said thyroxine, which comprises: bringing together in an aqueous buffered liquid zone at a pH in the range of 6-10.5: (1) said medium; (2) ENZ-bound-thyroxine; (3) antithydroxine, said antithyyroxine having sites common to and capable of binding to said thyroxine and said ENZ-bound-thyroxine, wherein ENZ is malate dehydrogense reversibly inhibited by said thyroxine, and said antithydroxine is present at a concentration resulting in substantial enhancement of said enzyme activity in the absence of thyroxine; and analyzing in said zone and NAD or NADH for the effect of said medium on the enzymatic activity of said ENZ-bound-thyroxine.
Claim 13
Dependent Claim
14 words
A method according to claim 10, wherein said analyzing is with NAD and malate.
Claim 14
Dependent Claim
19 words
A method according to claim 11 wherein said medium and said antithyroxine are brought together, followed by said ENZ-bound-thyroxine.
References Cited (Backward Citations)
US3850752A
Cited
US3817837A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1980-08-22 | 1981-02-21 | 1981-08-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-08-22 | 1985-02-21 | 1985-08-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-08-22 | 1989-02-21 | 1989-08-23 | $7700.00 | Window Closed / Lapsed |