Method of obtaining antibodies

• US 4,478,744 A
• Filed: 01/25/1982
• Issued: 10/23/1984
• Est. Priority Date: 01/25/1982
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Sherwood Medical Company (Medtronic Plc)
• Inventors: Huang, John J., Mezei, Louis M., Lovins, Robert E., Chen, Jung S.
• Primary Examiner(s): Phillips, Delbert R.
• Application Number:
  US06/342,418
• Time in Patent Office: 1,002 Days
• Field of Search: 260/112.5 R, 260/112 R, 424/177, 424/88, 424/85
• US Class Current: 530/322
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins

Abstract

Antibodies specific to glycosylated hemoglobin and substantially free of cross-reactivity with nonglycosylated forms of hemoglobin are obtained by administering a synthetic antigen to a host animal. The antigen comprises a glycosylated synthetic peptide having an amino acid sequence corresponding to that of the NH₂ -terminus of the β-chain of hemoglobin. The peptide comprises a sequence of between about four and ten, and preferably seven, amino acids, and is conjugated with glucose and a protein. The protein preferably comprises an immunoglobulin which is foreign to the host animal.

Claims

1. A method of preparing an antigen useful in obtaining antibodies against HbAlc, which antibodies are substantially free of cross-reactivity to nonglycosylated hemoglobin, said method comprising the steps of:

(a) synthesizing a hapten consisting of a peptide having the formula Val--His--Leu--Y wherein Y is chosen from the group consisting of Thr--, Thr--Pro--, Thr--Pro--Glu--, Thr--Pro--Glu--Glu--, Thr--Pro--Glu--Glu--Lys--, Thr--Pro--Glu--Glu--Lys--Ser--, and Thr--Pro--Glu--Glu--Lys--Ser--Ala--, said hapten thereby having an amino acid sequence corresponding to the NH₂ terminus of the hemoglobin β

-chain;

(b) reacting the peptide of step (a) with glucose to obtain a glycosylated peptide having the formula

space="preserve" listing-type="equation">glucose--Val--His--Leu--Y;

and

(c) conjugating the glycosylated peptide of step (b) with a protein to form an antigen suitable for injection into a host animal whose metabolism does not naturally produce HbAlC for production of antibodies thereto.

2. The method of claim 1 wherein said protein is an immunoglobulin.

3. The method of claim 2 wherein said protein comprises IgG.

4. The method of claim 1 wherein said peptide is covalently linked to said protein.

5. The method of claim 4 wherein said peptide is conjugated to said protein through a carbodiimide linkage.

6. The method of claim 2 wherein said peptide comprises d-glucose--Val--His--Leu--Thr--Pro--Glu--Glu--, said protein is bovine IgG, and said peptide is covalently linked to said bovine IgG through a carbodiimide linkage.