US5484735A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Immunoassay of glycosylated proteins employing antibody directed to reductively glycosylated N-terminal amino acids
Assignee: Northwestern University
•
Filing: 1993-11-12
•
Granted: 1996-01-16
•
Expires: 2013-11-12
Abstract
Abstract An immunoassay for a protein that is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid, the immunoassay comprising: providing a sample containing the glycosylated protein; reacting the glycosylated protein with a reducing agent so that the sugar residue on the N-terminal amino acid is reduced; contacting the reduced glycosylated protein with an antibody directed to Glc-ol-X which is prepared by immunizing an animal with an immunogen of the formula (Glc-ol-X-L) n -carrier; and detecting or quantitating the reduced glycosylated protein bound to the antibody. In the formula (Glc-ol-X-L) n -carrier: X is the N-terminal amino acid of the glycosylated protein, except that X cannot be lysine; L is a bond or a linker group; Glc-ol is the reduced form of the sugar attached to X on the glycosylated protein, and Glc-ol is attached to the alpha amino group of X; the carrier is an immunogenic compound other than the glycosylated protein; and n is from 1 to the number of available coupling sites on the carrier. Also, the antibody, the immunogen, and methods of making them. Finally, a kit for quantitating a protein glycosylated on its N-terminal amino acid.
Inventors
- 👤 Lyman E. Davis
- 👤 Byron E. Anderson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 12:22
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6803; G01N33/6842; G01N33/66; G01N33/72; G01N33/721; G01N33/723
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
163 words
A method of preparing an antibody which specifically binds a protein which is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid after the sugar on the N-terminal amino acid of the protein has been reduced, the method comprising immunizing an animal with a compound having the formula: (Glc-ol-X-L).sub.n -carrier wherein, X is the N-terminal amino acid of the non-enzymatically glycosylated protein, except that X cannot be lysine; L is a bond or a linker group, provided that if L is an amino acid or peptide, the combination of X and L does not correspond to the N-terminal sequence of the glycosylated protein to which it is desired to form an antibody; Glc-ol is the reduced form of the sugar that is attached to the alpha amino group of X on the glycosylated protein; the carrier is an immunogenic compound other than the glycosylated protein; and n is from 1 to the number of available coupling sites on the carrier.
Claim 2
Independent Claim
163 words
A method of preparing an antibody which specifically binds a protein which is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid after the sugar on the N-terminal amino acid of the protein has been reduced, the method comprising immunizing an animal with a compound having the formula: (Glc-ol-X-L).sub.n -carrier wherein, X is the N-terminal amino acid of the non-enzymatically glycosylated protein, except that X cannot be lysine; L is a bond or a linker group, provided that if L is an amino acid or peptide, the combination of X and L does not correspond to the N-terminal sequence of the glycosylated protein to which it is desired to form an antibody; Glc-ol is the reduced form of the sugar that is attached to the alpha amino group of X on the glycosylated protein; the carrier is an immunogenic compound other than the glycosylated protein; and n is from 1 to the number of available coupling sites on the carrier.
Claim 3
Dependent Claim
15 words
The method of claim 1 wherein the protein is glycosylated with glucose or a derivative.
Claim 4
Dependent Claim
11 words
The method of claim 2 wherein the glycosylated protein is hemoglobin.
Claim 5
Dependent Claim
20 words
The method of claim 3 wherein the glycosylated protein is HbA 1c , Glc-ol is glucitol and X is valine.
Claim 6
Dependent Claim
9 words
The method of claim 4 wherein L is glycine-glycine.
Claim 7
Dependent Claim
20 words
The method of claim 3 wherein the glycosylated protein is HbA 1c , Glc-ol is mannitol and X is valine.
Claim 8
Dependent Claim
9 words
The method of claim 6 wherein L is glycine-glycine.
Claim 9
Dependent Claim
106 words
An immunoassay for a protein that is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid, the immunoassay comprising: providing a sample containing the glycosylated protein; reacting the glycosylated protein with a reducing agent so that the sugar residue on the N-terminal amino acid is reduced; contacting the reduced glycosylated protein with an antibody which specifically binds Glc-ol-X wherein X is the N-terminal amino acid of the glycosylated protein, except that X cannot be lysine, and Glc-ol is the reduced form of the sugar attached to X on the glycosylated protein; and detecting or quantitating the reduced glycosylated protein bound to the antibody.
Claim 10
Dependent Claim
106 words
An immunoassay for a protein that is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid, the immunoassay comprising: providing a sample containing the glycosylated protein; reacting the glycosylated protein with a reducing agent so that the sugar residue on the N-terminal amino acid is reduced; contacting the reduced glycosylated protein with an antibody which specifically binds Glc-ol-X wherein X is the N-terminal amino acid of the glycosylated protein, except that X cannot be lysine, and Glc-ol is the reduced form of the sugar attached to X on the glycosylated protein; and detecting or quantitating the reduced glycosylated protein bound to the antibody.
Claim 11
Dependent Claim
15 words
The method of claim 8 wherein the protein is glycosylated with glucose or a derivative.
Claim 12
Dependent Claim
11 words
The method of claim 9 wherein the glycosylated protein is hemoglobin.
Claim 13
Dependent Claim
20 words
The method of claim 10 wherein the glycosylated protein is HbA 1c , Glc-ol is glucitol and X is valine.
Claim 14
Dependent Claim
20 words
The method of claim 10 wherein the glycosylated protein is HbA 1c , Glc-ol is mannitol and X is valine.
Claim 15
Dependent Claim
34 words
The method of claim 10 wherein the sample is whole blood, and the blood is treated so as to lyse the red blood cells therein so as to release HbA 1c from the cells.
Claim 16
Dependent Claim
20 words
The method of claim 13 further comprising the step of coating a solid surface with the released HbA 1c .
Claim 17
Dependent Claim
14 words
The method of claim 14 wherein the lysing and reducing steps are performed simultaneously.
Claim 18
Dependent Claim
15 words
The method of claim 14 wherein the lysing, reducing and coating steps are performed simultaneously.
Claim 19
Dependent Claim
14 words
The method of claim 13 wherein the HbA 1c is detected or quantitated colorimetrically.
Claim 20
Dependent Claim
66 words
A kit for detecting or quantitating a protein that is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid comprising: a container of a first antibody which specifically binds Glc-ol-X, wherein X is the N-terminal amino acid of the glycosylated protein, except that X cannot be lysine, and Glc-ol is the reduced form of the sugar attached to X on the glycosylated protein.
Claim 21
Dependent Claim
66 words
A kit for detecting or quantitating a protein that is non-enzymatically glycosylated on the alpha amino group of its N-terminal amino acid comprising: a container of a first antibody which specifically binds Glc-ol-X, wherein X is the N-terminal amino acid of the glycosylated protein, except that X cannot be lysine, and Glc-ol is the reduced form of the sugar attached to X on the glycosylated protein.
Claim 22
Dependent Claim
21 words
The kit of claim 18 wherein the first antibody is labeled to allow for the detection or quantitation of the protein.
Claim 23
Dependent Claim
31 words
The kit of claim 18 further comprising a container of reducing agent for reducing the sugar residue on the alpha amino group of the N-terminal amino acid of the glycosylated protein.
Claim 24
Dependent Claim
26 words
The kit of claim 18 further comprising a container of a labeled component useful for detecting or quantitating reduced glycosylated protein bound to the first antibody.
Claim 25
Dependent Claim
18 words
The kit of claim 21 wherein the labeled component is a second antibody reactive with the first antibody.
Claim 26
Dependent Claim
34 words
The kit of claim 18 further comprising a container of a substance capable of lysing the red blood cells in a sample of whole blood so as to release the HbA 1c contained therein.
Claim 27
Dependent Claim
45 words
Antibody which specifically binds Glc-ol-X, wherein X is the N-terminal amino acid of a non-enzymatically glycosylated protein, except that X cannot be lysine, and Glc-ol is the reduced form of the sugar residue attached to the alpha amino group of X on the glycosylated protein.
Claim 28
Dependent Claim
45 words
Antibody which specifically binds Glc-ol-X, wherein X is the N-terminal amino acid of a non-enzymatically glycosylated protein, except that X cannot be lysine, and Glc-ol is the reduced form of the sugar residue attached to the alpha amino group of X on the glycosylated protein.
Claim 29
Dependent Claim
10 words
The antibody of claim 24 which is directed to glucitol-valine.
Claim 30
Dependent Claim
10 words
The antibody of claim 24 which is directed to mannitol-valine.
Claim 31
Dependent Claim
10 words
The antibody of claim 26 which is a monoclonal antibody.
Claim 32
Dependent Claim
10 words
The antibody of claim 27 which is an IgG1 antibody.
Claim 33
Dependent Claim
10 words
A hybridoma which produces monoclonal antibody which specifically binds mannitol-valine.
Claim 34
Dependent Claim
10 words
A hybridoma which produces monoclonal antibody which specifically binds mannitol-valine.
Claim 35
Dependent Claim
12 words
The hybridoma of claim 29 which produces antibody of the IgG1 class.
References Cited (Backward Citations)
US3941763A
Cited
US4247533A
Cited
US4269605A
Cited
US4200435A
Cited
US4268270A
Cited
US4255385A
Cited
US4376110A
Cited
US4349352A
Cited
US4649122A
Cited
US4423034A
Cited
US4371374A
Cited
US4399227A
Cited
US4372747A
Cited
US4438204A
Cited
US4478744A
Cited
US4629692A
Cited
EP0111211A1
Cited
US4778752A
Cited
US4636463A
Cited
US4629022A
Cited
JPS61172064A
Cited
EP0185870A2
Cited
US4647654A
Cited
DE3439610A1
Cited
US5206144A
Cited
EP0201187A1
Cited
US4727036A
Cited
US4658022A
Cited
US4868130A
Cited
US4837170A
Cited
US4879036A
Cited
US4797473A
Cited
US4835097A
Cited
US4876188A
Cited
US4806468A
Cited
US4861728A
Cited
US5149633A
Cited
US4810391A
Cited
JPH028743A
Cited
US5071767A
Cited
US5116762A
Cited
US4956301A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-01-15 | 1999-07-17 | 2000-01-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-01-15 | 2003-07-17 | 2004-01-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-01-15 | 2007-07-17 | 2008-01-16 | $7700.00 | Window Closed / Lapsed |