Patent US4550075A successfully refreshed from Google Patents.
US4550075A
Expired / Lapsed
US
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Method for ligand determination utilizing an immunoassay monitorable by biotin-containing enzymes, and compositions therefor
Assignee: SANOFI K Co
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Filing: 1983-06-22
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Granted: 1985-10-29
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Expires: 2003-06-22
Abstract
Abstract Methods and compositions are disclosed for the qualitative and quantitative identification of specific ligand in a fluid medium suspected of containing the ligand. The method of the present invention involves the formation of a liquid reaction mixture comprising an enzyme, a ligand-specific binding component, a conjugate comprising ligand coupled to a modulator label, and sample fluid medium suspected of containing ligand. Once the reaction mixture is formed, enzyme activity is monitored and the presence and amount of ligand in the sample fluid medium is determined by changes in enzyme activity. Novel compositions useful in practicing the disclosed methods are provided. These include a biotin binding protein enzyme modulator, novel ligand-enzyme modulator conjugates, and biotin containing enzymes. The methods and compositions of the present invention are particularly useful in the qualitative and quantitative identification of pharmaceuticals and any such antigenic units or sites, including haptens and fragments of large molecules present in such biologic fluids as blood, urine, spinal fluid, amniotic fluid, lymphatic fluid and the like.
Inventors
- 👤 Cathy A. Bacquet
- 👤 Daniel Y. Twumasi
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 14:33
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/535; G01N33/536; G01N33/542; G01N33/58; G01N33/581; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/975
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
122 words
A method for quantitating a first ligand in fluid medium suspected of containing said first ligand comprising intermixing with said fluid medium a biotin-containing enzyme, a labeled conjugate comprising avidin coupled to a second ligand said second ligand comprising a substance identical to said first ligand or an analog of said first ligand, appropriate substrate and cofactors for said biotin-containing enzyme, and a ligand-specific binding component, the activity of said enzyme capable of being modulated by said labeled conjugate, said labeled conjugate having the characteristics that when bound by said ligand-specific binding component it is unable to modulate said enzyme; and thereafter determining the amount of said first ligand in said fluid medium by measuring the change in activity of said enzyme.
Claim 2
Independent Claim
122 words
A method for quantitating a first ligand in fluid medium suspected of containing said first ligand comprising intermixing with said fluid medium a biotin-containing enzyme, a labeled conjugate comprising avidin coupled to a second ligand said second ligand comprising a substance identical to said first ligand or an analog of said first ligand, appropriate substrate and cofactors for said biotin-containing enzyme, and a ligand-specific binding component, the activity of said enzyme capable of being modulated by said labeled conjugate, said labeled conjugate having the characteristics that when bound by said ligand-specific binding component it is unable to modulate said enzyme; and thereafter determining the amount of said first ligand in said fluid medium by measuring the change in activity of said enzyme.
Claim 3
Dependent Claim
12 words
A method according to claim 1 wherein said first ligand is dilantin.
Claim 4
Dependent Claim
12 words
A method according to claim 1 wherein said first ligand is theophylline.
Claim 5
Dependent Claim
12 words
A method according to claim 1 wherein said first ligand is lidocaine.
Claim 6
Dependent Claim
13 words
A method according to claim 1 wherein said fluid medium is human serum.
Claim 7
Dependent Claim
12 words
A method according to claim 1 wherein said enzyme is pyruvate carboxylase.
Claim 8
Dependent Claim
13 words
A method according to claim 1, wherein said enzyme activity is measured spectrophotometrically.
Claim 9
Dependent Claim
14 words
A method according to claim 1 wherein said ligand-specific binding component is ligand-specific antibody.
Claim 10
Dependent Claim
12 words
A method according to claim 8 wherein said antibody is polyclonal antibody.
Claim 11
Dependent Claim
12 words
A method according to claim 8 wherein said antibody is monoclonal antibody.
Claim 12
Independent Claim
144 words
A method for quantitating a first ligand in a fluid medium suspected of containing said first ligand, comprising the steps of: (a) intermixing said fluid medium with reaction components comprising a labeled conjugate comprising avidin conjugated to a second ligand, said second ligand comprising a substance identical to said first ligand or an analog of said first ligand, a ligand-specific binding component capable of specifically binding to said first ligand or to said labeled conjugate, the biotin-containing enzyme pyruvate carboxylase, and an appropriate substrate and cofactors for said enzyme; (b) allowing said reaction mixture to incubate at about 20° C. to about 40° C. for a period of about 0 to about 15 minutes; (c) thereafter measuring any change in activity of said enzyme; and (d) thereafter correlating said change in enzyme activity to the concentration of said first ligand in said fluid medium.
Claim 13
Independent Claim
144 words
A method for quantitating a first ligand in a fluid medium suspected of containing said first ligand, comprising the steps of: (a) intermixing said fluid medium with reaction components comprising a labeled conjugate comprising avidin conjugated to a second ligand, said second ligand comprising a substance identical to said first ligand or an analog of said first ligand, a ligand-specific binding component capable of specifically binding to said first ligand or to said labeled conjugate, the biotin-containing enzyme pyruvate carboxylase, and an appropriate substrate and cofactors for said enzyme; (b) allowing said reaction mixture to incubate at about 20° C. to about 40° C. for a period of about 0 to about 15 minutes; (c) thereafter measuring any change in activity of said enzyme; and (d) thereafter correlating said change in enzyme activity to the concentration of said first ligand in said fluid medium.
Claim 14
Dependent Claim
17 words
A method according to claim 11 wherein the change in activity of said enzyme is measured spectrophotometrically.
Claim 15
Dependent Claim
13 words
A method according to claim 11 wherein said fluid medium is human serum.
Claim 16
Dependent Claim
12 words
A method according to claim 11 wherein said first ligand is dilantin.
Claim 17
Dependent Claim
12 words
A method according to claim 11 wherein said first ligand is theophylline.
Claim 18
Dependent Claim
12 words
A method according to claim 11 wherein said first ligand is lidocaine.
Claim 19
Dependent Claim
15 words
A method according to claim 11 wherein said ligand-specific binding component is a ligand-specific antibody.
Claim 20
Dependent Claim
13 words
A method according to claim 17 wherein said antibody is a polyclonal antibody.
Claim 21
Dependent Claim
13 words
A method according to claim 17 wherein said antibody is a monoclonal antibody.
Claim 22
Dependent Claim
61 words
A composition useful in the method for quantitating a first ligand in a fluid medium employing a competitive immunoassay technique, a ligand specific binding component capable of specifically binding to said first ligand, enzyme modulators, and monitoring enzymes, comprising a substance identical to said first ligand or an analog of said first ligand conjugated to avidin to form a labeled conjugate.
Claim 23
Dependent Claim
61 words
A composition useful in the method for quantitating a first ligand in a fluid medium employing a competitive immunoassay technique, a ligand specific binding component capable of specifically binding to said first ligand, enzyme modulators, and monitoring enzymes, comprising a substance identical to said first ligand or an analog of said first ligand conjugated to avidin to form a labeled conjugate.
Claim 24
Dependent Claim
11 words
A composition according to claim 20 wherein said ligand is dilantin.
Claim 25
Dependent Claim
11 words
A composition according to claim 20 wherein said ligand is theophylline.
Claim 26
Dependent Claim
11 words
A composition according to claim 20 wherein said ligand is lidocaine.
Claim 27
Dependent Claim
18 words
A composition according to claim 20 wherein said labeled conjugate can inhibit the activity of a biotin-containing enzyme.
Claim 28
Dependent Claim
12 words
A composition according to claim 20 wherein said enzyme is pyruvate carboxylase.
Claim 29
Dependent Claim
32 words
A composition according to claim 20 wherein the ratio of said substance to said enzyme modulator in said labeled conjugate is no less than about 1 and no more than about 10.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1988-10-28 | 1989-04-29 | 1989-10-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-10-28 | 1993-04-29 | 1993-10-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-10-28 | 1997-04-29 | 1997-10-29 | $7700.00 | Window Closed / Lapsed |