US4243749A
Expired / Lapsed
US
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Immunoassay employing an enzyme label
Assignee: Hypolab SA
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Filing: 1978-03-16
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Granted: 1981-01-06
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Expires: 1998-03-16
Abstract
Abstract According to the present invention there is provided a sensitive quantitative assay for the determination of a hapten "X" which comprises: preparing a conjugate of the hapten X which conjugate is adapted to ellicit antibody formation, and injecting a mammal with said conjugate, resulting in a specific antibody against the said hapten, "anti-X"; preparing a conjugate of the hapten X and of another entity Y, which is either another hapten or a larger molecule, injecting a mammal with the entity Y if it is a larger molecule, or with a conjugate thereof if it is a small molecule which does not by itself ellicit antibody formation, so as to form anti-Y antibodies, preparing a conjugate of a suitable enzyme and the antibody against Y, i.e. an (enzyme)-(anti-Y)-conjugate; adsorbing the antibodies against X, i.e. anti-X on a solid support, contacting said solid support with a mixture containing the unknown quantity of hapten X and a known quantity of the X-Y conjugate; removing unreacted X and X-Y, leaving all anti-X sites occupied, adding enzyme-labelled anti-Y antibody; adding a suitable substrate resulting in a color reacting and measuring the intensity of color indicating the quantity of bound enzyme and deducing from calibration curves the quantity of the hapten X; and to a kit for carrying out such assay.
Inventors
- 👤 Dora Sadeh
- 👤 Charles S. Hexter
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 02:24
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54306; G01N33/74; G01N33/88; Y; Y10; Y10S; Y10S435/00; Y10S435/964; Y10S435/969; Y10S435/975; Y10S436/00; Y10S436/805; Y10S436/807; Y10S436/81; Y10S436/811; Y10S436/814; Y10S436/815; Y10S436/817; Y10S436/826
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
255 words
A quantitative assay method for the determination of an unknown quantity of a hapten, hereinafter designated hapten X, which method comprises: a. preparing a conjugate of the hapten X which conjugate is adapted to ellicit antibody formation, and injecting a mammal with said conjugate so as to result in a specific antibody against said hapten, anti-X; b. preparing a conjugate of the hapten X and of another entity Y, which is either another hapten or a larger molecule; c. injecting a mammal with the entity Y if it is a larger molecule, or with a conjugate thereof if it is a small molecule which does not by itself ellicit antibody formation, so as to form anti-Y antibodies; d. preparing a conjugate of an enzyme and the antibody against Y to form enzyme-labelled anti-Y antibody; e. adsorbing the anti-X antibodies on a solid support; f. contacting said solid support with a mixture containing the unknown quantity of hapten X and a known quantity of the X-Y conjugate so as to cause all anti-X sites to be occupied; g. removing unreacted X and X-Y, leaving all anti-X sites occupied; h. contacting the support carrying occupied anti-X sites with enzyme-labelled anti-Y antibody; i. contacting the resulting support carrying occupied anti-X-sites and enzyme-labelled anti-Y antibody with a substrate for the enzyme which when acted upon by the enzyme produces a color reaction and measuring the resulting intensity of color so as to determine the quantity of bound enzyme and deducing from calibration curve the quantity of the hapten X.
Claim 2
Dependent Claim
255 words
A quantitative assay method for the determination of an unknown quantity of a hapten, hereinafter designated hapten X, which method comprises: a. preparing a conjugate of the hapten X which conjugate is adapted to ellicit antibody formation, and injecting a mammal with said conjugate so as to result in a specific antibody against said hapten, anti-X; b. preparing a conjugate of the hapten X and of another entity Y, which is either another hapten or a larger molecule; c. injecting a mammal with the entity Y if it is a larger molecule, or with a conjugate thereof if it is a small molecule which does not by itself ellicit antibody formation, so as to form anti-Y antibodies; d. preparing a conjugate of an enzyme and the antibody against Y to form enzyme-labelled anti-Y antibody; e. adsorbing the anti-X antibodies on a solid support; f. contacting said solid support with a mixture containing the unknown quantity of hapten X and a known quantity of the X-Y conjugate so as to cause all anti-X sites to be occupied; g. removing unreacted X and X-Y, leaving all anti-X sites occupied; h. contacting the support carrying occupied anti-X sites with enzyme-labelled anti-Y antibody; i. contacting the resulting support carrying occupied anti-X-sites and enzyme-labelled anti-Y antibody with a substrate for the enzyme which when acted upon by the enzyme produces a color reaction and measuring the resulting intensity of color so as to determine the quantity of bound enzyme and deducing from calibration curve the quantity of the hapten X.
Claim 3
Dependent Claim
22 words
An assay method according to claim 1, wherein the hapten which is being determined is a hormone, vitamin, cardiac glycoside, or polypeptide.
Claim 4
Dependent Claim
23 words
An assay method according to claim 2, wherein the hapten is selected from the group consisting of estradiol, progesterone, prostaglandin, thyroxine, and triiodothyronine.
Claim 5
Dependent Claim
13 words
An assay method according to claim 1 wherein said hapten is a drug.
Claim 6
Dependent Claim
39 words
An assay method according to claim 1, wherein the entity Y is selected from the group consisting of trinitrophenyl-lysine, sulfanilic acid, p-amino-benzoic acid, p-azophenyl-β-lactoside, hippuric acid, iodoacetylaminobenzene-azo hippuric acid, 6-carboxypurine, 5-acetyl uracil, thymidin, uridin, dinitrophenyl lysine and arsanil tyrosine.
Claim 7
Dependent Claim
13 words
An assay method according to claim 1, wherein the enzyme is a peroxidase.
Claim 8
Dependent Claim
13 words
An assay method according to claim 6, wherein the peroxidase is horseradish peroxidase.
Claim 9
Dependent Claim
15 words
An assay method according to claim 1, wherein the solid support is a polymeric substance.
Claim 10
Dependent Claim
12 words
An assay method according to claim 8, wherein the support is polystyrene.
Claim 11
Dependent Claim
33 words
An assay method according to claim 1, wherein the support is in the form of a test tube and step e comprises coating the inside of the test tubes with the anti-X antibodies.
Claim 12
Dependent Claim
15 words
An assay method according to claim 10, wherein the support is a polystyrene test tube.
References Cited (Backward Citations)
US3998943A
Cited
US4017597A
Cited
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1984-01-06 | 1984-07-07 | 1985-01-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-01-06 | 1988-07-07 | 1989-01-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-01-06 | 1992-07-07 | 1993-01-06 | $7700.00 | Window Closed / Lapsed |