US4822747A
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Polyacrylamide gel particles having hapten moieties bound thereto as immunoassay reagent
Assignee: Bayer Corp
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Filing: 1986-12-09
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Granted: 1989-04-18
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Expires: 2006-12-09
Abstract
Abstract An immobilized hapten reagent for use in a specific binding assay for the determination of a hapten or binding analog thereof in a liquid test sample and methods for preparing and using the immobilized hapten reagent. The immobilized hapten reagent comprises a hapten moiety covalently linked substantially only to the external surface of a polyacrylamide gel particle. The immobilized hapten reagent is prepared by forming a reaction mixture comprising the hapten moiety and the carrier material in a nonswelling solvent wherein the gel particle is substantially impervious to the hapten moiety and wherein an analytically insignificant amount of the hapten moiety is nonspecifically bound to the gel particle. The immobilized hapten reagent is characterized by being substantially stable in aqueous solutions and exhibits an insignificant amount of leakage of the hapten moiety into a liquid test medium.
Inventors
- 👤 Richard D. Johnson
- 👤 H. Volker Runzheimer
- 👤 Ronald G. Sommer
- 👤 Kin F. Yip
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 21:27
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/544; G01N33/548; G01N33/54313; G01N33/72; G01N33/721; G01N33/723; G01N33/96
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
169 words
A method of preparing a stable immobilized hapten reagent for use in the specific binding assay determination of a hapten or binding analog thereof in a liquid test sample, the method comprising the steps of: (a) reacting a hapten moiety with a polyacrylamide gel particle comprising a plurality of external and internal chemically active functional groups wherein the reaction is performed in a solvent in which the gel particle is substantially nonswollen and under conditions to form a covalent bond between the hapten moiety and said external active functional groups which is substantially stable in aqueous solutions; (b) washing the gel particle resulting from step(a) with a nonswelling solvent; (c) washing the gel particle resulting from step(b) with an aqueous solution; and (d) isolating the immobilized hapten reagent resulting from step(c) comprising said gel particle and said hapten moieties bound thereto wherein substantially all of said bound hapten moieties are covalently linked to the external surface functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 2
Independent Claim
169 words
A method of preparing a stable immobilized hapten reagent for use in the specific binding assay determination of a hapten or binding analog thereof in a liquid test sample, the method comprising the steps of: (a) reacting a hapten moiety with a polyacrylamide gel particle comprising a plurality of external and internal chemically active functional groups wherein the reaction is performed in a solvent in which the gel particle is substantially nonswollen and under conditions to form a covalent bond between the hapten moiety and said external active functional groups which is substantially stable in aqueous solutions; (b) washing the gel particle resulting from step(a) with a nonswelling solvent; (c) washing the gel particle resulting from step(b) with an aqueous solution; and (d) isolating the immobilized hapten reagent resulting from step(c) comprising said gel particle and said hapten moieties bound thereto wherein substantially all of said bound hapten moieties are covalently linked to the external surface functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein the nonswelling solvent is an organic solvent.
Claim 4
Dependent Claim
26 words
The method of claim 2 wherein said organic solvent is selected from the group consisting of dimethylformamide, dimethylsulfoxide, acetone, chlorinated hydrocarbons, and cyclic and acyclic alkylethers.
Claim 5
Dependent Claim
22 words
The method of claim 1 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.
Claim 6
Dependent Claim
29 words
The method of claim 4 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and bovine serum albumin.
Claim 7
Dependent Claim
22 words
The method of claim 4 wherein the linking group is a bifunctional residue of 6-aminocaproic acid and the hapten moiety is digitoxigenin.
Claim 8
Dependent Claim
14 words
The method of claim 1 wherein the hapten moiety is a glycosylated peptide sequence.
Claim 9
Dependent Claim
24 words
The method of claim 7 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylenedi-4,1-phenylene]bis-maleimide, bismaleimido-hexane and bismaleimido-hexethylene glycol.
Claim 10
Dependent Claim
30 words
The method of claim 7 wherein said glycosylated peptide sequence corresponds with the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.
Claim 11
Dependent Claim
35 words
The method of claim 1 wherein less than from about 1×10 -12 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 12
Dependent Claim
35 words
The method of claim 10 wherein less than from about 1×10 -13 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 13
Dependent Claim
21 words
The method of claim 1 wherein the polyacrylamide gel particle in its nonswollen state is substantially impervious to the hapten moiety.
Claim 14
Dependent Claim
88 words
A substantially stable immobilized hapten reagent for use in the specific binding assay determination of a hapten or binding analog thereof in a liquid test sample, the immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal functional groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 15
Dependent Claim
88 words
A substantially stable immobilized hapten reagent for use in the specific binding assay determination of a hapten or binding analog thereof in a liquid test sample, the immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal functional groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 16
Dependent Claim
24 words
The immobilized hapten reagent of claim 13 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.
Claim 17
Dependent Claim
31 words
The immobilized hapten reagent of claim 14 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane,, 6-aminocaproic acid, and bovine serum albumin.
Claim 18
Dependent Claim
20 words
The immobilized hapten reagent of claim 14 wherein the linking group is 6-aminocaproic acid and the hapten moiety is digitoxigenin.
Claim 19
Dependent Claim
16 words
The immobilized hapten reagent of claim 13 wherein the hapten moiety is a glycosylated peptide sequence.
Claim 20
Dependent Claim
26 words
The immobilized hapten reagent of claim 17 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and bismaleimido-hexaethylene glycol.
Claim 21
Dependent Claim
32 words
The immobilized hapten reagent of claim 17 wherein said glycosylated peptide sequence corresponds with the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.
Claim 22
Dependent Claim
37 words
The immobilized hapten reagent of claim 13 wherein less than from about 1×10 12 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 23
Dependent Claim
37 words
The immobilized hapten reagent of claim 20 wherein less than from about 1×10 -13 moles hapten/g gel particles dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 24
Dependent Claim
172 words
In an immunometric assay method for determining a hapten or analog thereof in a liquid test sample, wherein the test sample is contacted with a labeled antibody reagent capable of binding with said hapten or analog thereof and with an immobilized form of the hapten capable of binding with said antibody reagent, and wherein the amount of labeled antibody reagent which becomes bound to said hapten or analog thereof in the sample compared to that which becomes bound to the immobilized reagent is determined and related to the presence of the hapten or analog thereof in the test sample, the improvement which comprises employing as the immobilized reagent a substantially stable immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal function groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 25
Dependent Claim
172 words
In an immunometric assay method for determining a hapten or analog thereof in a liquid test sample, wherein the test sample is contacted with a labeled antibody reagent capable of binding with said hapten or analog thereof and with an immobilized form of the hapten capable of binding with said antibody reagent, and wherein the amount of labeled antibody reagent which becomes bound to said hapten or analog thereof in the sample compared to that which becomes bound to the immobilized reagent is determined and related to the presence of the hapten or analog thereof in the test sample, the improvement which comprises employing as the immobilized reagent a substantially stable immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal function groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 26
Dependent Claim
23 words
The assay method of claim 22 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.
Claim 27
Dependent Claim
30 words
The assay method of claim 22 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and bovine serum albumin.
Claim 28
Dependent Claim
19 words
The assay method of claim 23 wherein the linking group is 6-aminocaproic acid and the hapten moiety is digitoxigenin.
Claim 29
Dependent Claim
15 words
The assay method of claim 22 wherein the hapten moiety is a glycosylated peptide sequence.
Claim 30
Dependent Claim
25 words
The assay method of claim 26 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and bismaleimido-hexaethylene glycol.
Claim 31
Dependent Claim
31 words
The assay method of claim 26 wherein said glycosylated peptide sequence corresponds with the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.
Claim 32
Dependent Claim
36 words
The assay method of claim 22 wherein less than from about 1×10 -12 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 33
Dependent Claim
36 words
The assay method of claim 29 wherein less than from about 1×10 -13 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 34
Dependent Claim
125 words
In a reagent system for determining a hapten or analog thereof in a liquid test sample, which reagent system comprises (1) a labeled reagent capable of binding with said hapten or analog thereof and (2) an immobilized reagent comprising an immobilized form of the hapten capable of binding with said labeled reagent, the improvement which comprises employing as the immobilized reagent a substantially stable immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal functional groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 35
Dependent Claim
125 words
In a reagent system for determining a hapten or analog thereof in a liquid test sample, which reagent system comprises (1) a labeled reagent capable of binding with said hapten or analog thereof and (2) an immobilized reagent comprising an immobilized form of the hapten capable of binding with said labeled reagent, the improvement which comprises employing as the immobilized reagent a substantially stable immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal functional groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.
Claim 36
Dependent Claim
23 words
The reagent system of claim 31 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.
Claim 37
Dependent Claim
30 words
The reagent system of claim 32 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and bovine serum albumin.
Claim 38
Dependent Claim
19 words
The reagent system of claim 32 wherein the linking group is 6-aminocaproic acid and the hapten moiety is digitoxigenin.
Claim 39
Dependent Claim
15 words
The reagent system of claim 31 wherein the hapten moiety is a glycosylated peptide sequence.
Claim 40
Dependent Claim
25 words
The reagent system of claim 35 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and bismaleimido-hexaethylene glycol.
Claim 41
Dependent Claim
31 words
The reagent system of claim 35 wherein said glycosylated peptide sequence corresponds with the glucosylatjed N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.
Claim 42
Dependent Claim
36 words
The reagent system reagent of claim 31 wherein less than from about 1×10 -12 moles hapten/g of particle dissociates from the particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
Claim 43
Dependent Claim
36 words
The reagent system reagent of claim 38 wherein less than from about 1×10 -13 moles hapten/g gel particle dissociates from the particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
References Cited (Backward Citations)
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) | 1992-04-17 | 1992-10-17 | 1993-04-18 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-04-17 | 1996-10-17 | 1997-04-18 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-04-17 | 2000-10-17 | 2001-04-18 | $7700.00 | Window Closed / Lapsed |