Determination of glycated (glycosylated) hemoglobin in blood

• US 5,206,144 A
• Filed: 10/30/1990
• Issued: 04/27/1993
• Est. Priority Date: 03/29/1985
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novo Industri A/S (Novo Nordisk A/S)
• Inventors: Zeuthen, Jesper, Kruse, Viggo, Prento, Annette
• Primary Examiner(s): Saunders, David
• Application Number:
  US07/607,766
• Time in Patent Office: 910 Days
• Field of Search: 435/7.5, 435/7.92, 435/172.2, 435/240.27, 435/948, 435/7.25, 436/501, 436/518, 436/531, 436/548, 436/809, 935/110
• US Class Current: 435/7.25
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

The concentration of glycated hemoglobin, HbA_1c, in hemoglobin from diabetic patients is regarded as a useful means of assessing the adequacy of diabetes control. This invention comprises a monoclonal antibody which, due to its specific binding of HbA_1c, can be used for the determination of HbA_1c in the absence of prior denaturation, in blood samples by such analytical methods as radioimmuno assay or enzyme-linked immunosorbent assay. The invention also comprises hybridoma cell lines which produce monoclonal antibodies of the invention.

Claims

1. A method for the determination of glycated hemoglobin (HbA_1c) in a sample comprising a mixture of HbA_1c and non-glycated hemoglobin (HbA_o) in the absence of prior denaturation, which comprises:

(A) insolubilizing hemoglobin in said sample on a solid phase;

(B) contacting said insolubilized hemoglobin with a murine monoclonal antibody which has binding selectivity for HbA_1c relative to HbA_o when said HbA_1c is insolubilized on a solid phase;

(C) measuring the amount of bound monoclonal antibody; and

(D) relating said measured amount of bound monoclonal antibody to the amount of glycated hemoglobin in said sample.

3. The method of claim 1 or 2 wherein said antibody is produced by the hybridoma cell line having the ECACC designation 85020101.

4. The method of claim 1 or 2 wherein said hemoglobin is insolubilized on a multiwell plate.

5. The method of claim 1 or 2 wherein said monoclonal antibody is detectably labeled.

6. The method of claim 5, wherein said detectable label is an enzyme or a radiolabel.

7. The method of claim 1 or 2 wherein said bound monoclonal antibody is measured by adding thereto a labeled second antibody with specificity therefor.

8. The method of claim 1 or 2 wherein said monoclonal antibody is attached to biotin.

9. The method of claim 8, wherein said attached monoclonal antibody is detected by adding thereto horseradish peroxidase conjugated to avidin.

2. A method for the determination of glycated hemoglobin (HbA_1c) in a sample comprising a mixture of HbA_1c and non-glycated hemoglobin (HbA_o), consisting essentially of:

(A) insolubilizing hemoglobin in said sample on a solid phase;

(B) contacting said insolubilized hemoglobin with a murine monoclonal antibody which has binding selectivity for HbA_1c relative to HbA_o when said HbA_1c is insolubilized on a solid phase;

(C) measuring the amount of bound monoclonal antibody; and

(C) relating said measured amount of bound monoclonal antibody to the amount of glycated hemoglobin in said sample.

10. A kit for the determination of glycated human hemoglobin (HbA_1c), which kit comprises:

(A) a carrier being compartmentalized to receive in close confinement therein two or more containers;

(B) a first container containing a monoclonal antibody produced by the hybridoma cell line having the designation ECACC 85020101 and having binding selectivity of HbA_1c relative to non-glycated hemoglobin (HbA_o), when said HbA1c is insolubilized on a solid phase; and

(C) at least a second container containing HbA_1c at a predetermined concentration.

11. The kit of claim 10, wherein said monoclonal antibody is detectably labeled.

12. The kit of claim 11, wherein said detectable label is an enzyme or a radiolabel.

13. The kit of claim 10, which contains a third container containing HbA_1c at a predetermined concentration which is different from that of HbA_1c in said second container.