Patent US4378428A successfully refreshed from Google Patents.
US4378428A
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Method for carrying out non-isotopic immunoassays, labeled analytes and kits for use in such assays
Assignee: Roche Diagnostics Corp
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Filing: 1981-03-30
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Granted: 1983-03-29
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Expires: 2001-03-30
Abstract
Abstract A highly sensitive, immunoassay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration is provided. Sample; a polypeptide-labeled analog of the analyte, an antibody specific for said analyte, a polypeptide partner capable of non-covalently binding with the polypeptide-labeled analyte to form a complex having catalytic activity, and a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex are brought together in a medium. The polypeptide-labeled analyte analog is capable of competitively binding to the antibody and the polypeptide partner, the antibody inhibiting the formation of a catalytically active complex in the absence of analyte, and the concentrations of the antibody, polypeptide partner and polypeptide-labeled analyte are such as to cause varying amounts of analyte to be directly related to the conversion of the substrate to the reporter molecule. Conversion of the substrate to the reporter molecule is then determined, and compared to conversions of substrate to reporter molecule obtained with known concentrations of the analyte.
Inventors
- 👤 Peter R. Farina
- 👤 James R. Golke
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 09:35
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/542; G01N33/531; G01N33/532; G01N33/535; G01N33/58; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/967; Y10S435/968; Y10S435/971; Y10S435/975
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
222 words
A non-isotopic competitive binding assay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration, which comprises the steps of: (a) forming a reaction mixture by bringing together in a medium, (1) said sample, (2) polypeptide-labeled analyte, (3) an antibody specific for said analyte, (4) a polypeptide partner capable of non-covalently binding with the polypeptides of the polypeptide-labeled analyte to form a complex having catalytic activity, and (5) a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, said polypeptide-labeled analyte being capable of competitively binding to said antibody and to said polypeptide partner, said antibody inhibiting the formation of a catalytically active complex in the absence of analyte, the concentrations of said antibody, polypeptide partner and polypeptide-labeled analyte being such as to cause varying amounts of analyte in the sample to be directly related to the conversion of said substrate to said reporter molecule as a function of the catalytic activity of the complex; (b) measuring the conversion of said substrate to said reporter molecule in said reaction mixture, and (c) determining the amount of analyte in said sample by comparing the conversion of said substrate to said reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte.
Claim 2
Dependent Claim
222 words
A non-isotopic competitive binding assay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration, which comprises the steps of: (a) forming a reaction mixture by bringing together in a medium, (1) said sample, (2) polypeptide-labeled analyte, (3) an antibody specific for said analyte, (4) a polypeptide partner capable of non-covalently binding with the polypeptides of the polypeptide-labeled analyte to form a complex having catalytic activity, and (5) a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, said polypeptide-labeled analyte being capable of competitively binding to said antibody and to said polypeptide partner, said antibody inhibiting the formation of a catalytically active complex in the absence of analyte, the concentrations of said antibody, polypeptide partner and polypeptide-labeled analyte being such as to cause varying amounts of analyte in the sample to be directly related to the conversion of said substrate to said reporter molecule as a function of the catalytic activity of the complex; (b) measuring the conversion of said substrate to said reporter molecule in said reaction mixture, and (c) determining the amount of analyte in said sample by comparing the conversion of said substrate to said reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte.
Claim 3
Dependent Claim
28 words
The method of claim 1 wherein the complex formed by the non-covalent binding of the polypeptide partner with the polypeptide-labeled analyte exhibits catalytic activity characteristic of a ribonuclease.
Claim 4
Dependent Claim
15 words
The method of claim 2 wherein said complex exhibits catalytic activity characteristic of ribonuclease A.
Claim 5
Dependent Claim
20 words
The method of claim 2 wherein the polypeptide label for the analyte is S-peptide and the polypeptide partner is S-protein.
Claim 6
Dependent Claim
20 words
The method of claim 2 wherein the polypeptide label for the analyte is S-protein and the polypeptide partner is S-peptide.
Claim 7
Dependent Claim
23 words
The method of claim 1 wherein at least one of the polypeptide label and the polypeptide partner were obtained by cleaving an enzyme.
Claim 8
Dependent Claim
20 words
The method of claim 6 wherein the polypeptide label and the polypeptide partner are cleaved fragments of a parent enzyme.
Claim 9
Dependent Claim
33 words
The method of claim 7 wherein the complex formed by the non-covalent binding of the polypeptide partner with the polypeptide-labeled analyte recovers at least 5% of the catalytic activity of said parent enzyme.
Claim 10
Dependent Claim
28 words
The method of claim 1 wherein the equilibrium constant for said complex is a value of about 10 5 M -1 to about 10 11 M -1 .
Claim 11
Dependent Claim
22 words
The method of claim 1 wherein said analyte has a molecular weight of at least about 100 to about 10 6 .
Claim 12
Dependent Claim
52 words
The method of claim 1 wherein said analyte is a member selected from the group consisting of a drug or its metabolite, an opiate, narcotic, hormone, steroid, vitamin, polypeptide hormone, tumor associated antigen, immunoglobin, enzyme, industrial pollutant, pesticide or its metabolite, food additive, herbicide or its metabolite, flavoring agent, and food poison.
Claim 13
Dependent Claim
69 words
The method of claim 1 wherein said analyte is a member selected from the group consisting of dilantin, ethosuximide, penobarbital, primidone, lidocaine, theophylline, morphine, codeine, heroin, marijuana, gentamicin, tobramycin, methotrexate, digitoxin, thyroxine, testosterone, cortisol, immunoglobulin, triiodothyronine, digoxin, folic acid, angiotensin II, progesterone, prostaglandin F2, estrogens, vitamin B 12 , growth hormone, thyroid stimulating hormone, calcitonin, gastrin, luteinizing hormone, follicle stimulating hormone, glucagon, human chorionic gonadotropin, aldosterone and carcinoembryonic antigen.
Claim 14
Dependent Claim
10 words
The method of claim 12 wherein said analyte is dilantin.
Claim 15
Dependent Claim
10 words
The method of claim 12 wherein said analyte is thyroxine.
Claim 16
Dependent Claim
10 words
The method of claim 12 wherein said analyte is cortisol.
Claim 17
Dependent Claim
23 words
The method of claim 1 wherein said analyte is dilantin, the polypeptide label for dilantin is S-peptide and the polypeptide partner is S-protein.
Claim 18
Dependent Claim
23 words
The method of claim 1 wherein said analyte is thyroxine, the polypeptide label for thyroxine is S-peptide and the polypeptide partner is S-protein.
Claim 19
Dependent Claim
23 words
The method of claim 1 wherein said analyte is cortisol, the polypeptide label for cortisol is S-peptide and the polypeptide partner is S-protein.
Claim 20
Dependent Claim
23 words
The method of claim 1 wherein the antibody is present in a concentration sufficient to provide an inhibition of at least about 10%.
Claim 21
Dependent Claim
23 words
The method of claim 19 wherein the antibody is present in a concentration sufficient to provide an inhibition of at least about 50%.
Claim 22
Dependent Claim
25 words
The method of claim 1 wherein the substrate is present in a concentration such as to provide essentially linear rates over the rate measurement period.
Claim 23
Dependent Claim
22 words
The method of claim 1 wherein the substrate is present in a concentration of about 10 -4 to about 10 -2 molar.
Claim 24
Dependent Claim
101 words
The method of claim 1 wherein the substrate has the following structural formula: ##STR35## wherein B is a nucleotide base capable of assisting in hydrolysis of the phosphate ester at the 3'-position; R is a moiety selected from the group consisting of umbelliferonyl, 4-methyl umbelliferonyl, 3-flavonyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, dinitrophenyl, cyanophenyl, acylphenyl, carboxyphenyl, phenylsulfonate, phenylsulfonyl and phenylsulfoxide; R' is a moiety selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, aryl, araalkyl, acyl, oxaalkyl, thioalkyl, oxacycloalkyl and thiocycloalkyl; and R" is hydrogen or a cation selected from the group consisting of calcium, barium, lithium, sodium, ammonium, substituted ammonium and pyridinium.
Claim 25
Dependent Claim
30 words
The method of claim 23 wherein B is uracil, R is 4-methylumbelliferonyl, R' is acetyl and R" is a member selected from the group consisting of calcium, pyridinium and sodium.
Claim 26
Dependent Claim
11 words
The method of claim 1 wherein said rate is determined spectrophotometrically.
Claim 27
Dependent Claim
11 words
The method of claim 1 wherein said rate is determined fluorometrically.
Claim 28
Dependent Claim
90 words
A kit for use in carrying out an assay of an analyte, which comprises the following components: (1) a polypeptide-labeled analyte, (2) an antibody specific for the analyte, (3) a polypeptide partner capable of non-covalently binding with the polypeptide of the polypeptide-labeled analyte to form a complex having catalytic activity and (4) a substrate capable of being converted to a reporter molecule by the catalytic activity of the complex wherein the polypeptide-labeled analyte, antibody, polypeptide partner and substrate are present in relative amounts sufficient for the determination of the analyte.
Claim 29
Dependent Claim
90 words
A kit for use in carrying out an assay of an analyte, which comprises the following components: (1) a polypeptide-labeled analyte, (2) an antibody specific for the analyte, (3) a polypeptide partner capable of non-covalently binding with the polypeptide of the polypeptide-labeled analyte to form a complex having catalytic activity and (4) a substrate capable of being converted to a reporter molecule by the catalytic activity of the complex wherein the polypeptide-labeled analyte, antibody, polypeptide partner and substrate are present in relative amounts sufficient for the determination of the analyte.
Claim 30
Dependent Claim
22 words
The kit of claim 27 which includes a set of standard analyte solutions which covers the anticipated concentration range for the analyte.
Claim 31
Dependent Claim
15 words
The kit of claim 27 which includes a buffer for pH adjustment for the assay.
Claim 32
Dependent Claim
11 words
The kit of claim 27 wherein each component is separately packaged.
Claim 33
Dependent Claim
24 words
The kit of claim 27 wherein the polypeptide-labeled analyte and the substrate are packaged together and the antibody and polypeptide partner are packaged together.
Claim 34
Dependent Claim
14 words
The kit of claim 27 wherein the antibody and polypeptide partner are packaged together.
Claim 35
Dependent Claim
13 words
The kit of claim 27 wherein the antibody and substrate are packaged together.
Claim 36
Dependent Claim
169 words
A non-isotopic competitive binding assay method for carrying out a heterogeneous immunoassay of a sample containing a known analyte present in an unknown concentration which comprises: (a) forming a reaction mixture by bringing together in a medium (1) said sample, (2) a polypeptide-labeled analyte and (3) an antibody specific for said analyte; (b) separating the antibody-bound, polypeptide-labeled analyte from the free polypeptide-labeled analyte; (c) determining the amount of at least one of: (1) the antibody-bound, polypeptide-labeled analyte and (2) the free polypeptide-labeled analyte by adding a polypeptide partner capable of non-covalently binding with the polypeptide of the polypeptide-labeled analyte to form a complex having catalytic activity and a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, by measuring the conversion of said substrate to reporter molecule, and (d) determining the amount of analyte in said sample by comparing the conversion of said substrate to reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte.
Claim 37
Dependent Claim
169 words
A non-isotopic competitive binding assay method for carrying out a heterogeneous immunoassay of a sample containing a known analyte present in an unknown concentration which comprises: (a) forming a reaction mixture by bringing together in a medium (1) said sample, (2) a polypeptide-labeled analyte and (3) an antibody specific for said analyte; (b) separating the antibody-bound, polypeptide-labeled analyte from the free polypeptide-labeled analyte; (c) determining the amount of at least one of: (1) the antibody-bound, polypeptide-labeled analyte and (2) the free polypeptide-labeled analyte by adding a polypeptide partner capable of non-covalently binding with the polypeptide of the polypeptide-labeled analyte to form a complex having catalytic activity and a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, by measuring the conversion of said substrate to reporter molecule, and (d) determining the amount of analyte in said sample by comparing the conversion of said substrate to reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-03-28 | 1986-09-27 | 1987-03-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-03-28 | 1990-09-27 | 1991-03-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-03-28 | 1994-09-27 | 1995-03-29 | $7700.00 | Window Closed / Lapsed |