Genetically engineered enzymes and their conjugates for diagnostic assays

• US 5,843,634 A
• Filed: 06/04/1996
• Issued: 12/01/1998
• Est. Priority Date: 03/09/1993
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Russell, John C., March, Steven C., Mandecki, Wlodzimierz, Brate, Elaine M, Yue, Vincent T., Bridon, Dominique P., Brennan, Catherine A., Krafft, Grant A., Jaffe, Keeve D.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/657,392
• Time in Patent Office: 910 Days
• Field of Search: 435/4, 435/7.1, 435/7.6, 435/7.9, 435/195, 435/196, 530/402, 536/23.2
• US Class Current: 435/4
• CPC Class Codes: C07K 14/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12Q 1/42 involving phosphatase

Abstract

This invention relates to genetically engineered enzymes, their ligand conjugates, their manufacture, and their use in qualitative or quantitative assays. A hybrid enzyme, such as an AP-epitope, has a foreign amino acid moiety (an epitope) inserted near the active site of the starting AP enzyme. The foreign amino acid moiety binds with an analyte, and, as a consequence of this binding, the enzymatic activity of the hybrid enzyme, AP-epitope, is modified. The changes in the enzymatic activity are dependent upon the presence, or the amount, of the analyte. In another embodiment, the hybrid enzyme consists of a cysteine introduced near the active site of an AP to give a hybrid enzyme. The cysteine on the hybrid enzyme serves as a point of conjugation of a ligand, such as theophylline, ferritin, thyroxine, or digoxigenin, to form the hybrid enzyme-ligand conjugate. The ligand binds with an antibody, an analyte or a binding molecule to an analyte and as a result of this binding, the enzymatic activity of the hybrid enzyme-ligand conjugate is modified or modulated.

Claims

1. A hybrid enzyme-ligand conjugate, wherein amino acid Asparagine at position 263 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

11. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 1.

2. A hybrid enzyme-ligand conjugate, wherein amino acid Lysine at position 177 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

12. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 2.

3. A hybrid enzyme-ligand conjugate, wherein amino acid Lysine at position 209 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

13. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 3.

4. A hybrid enzyme-ligand conjugate, wherein amino acid Lysine at position 328 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

14. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 4.

5. A hybrid enzyme-ligand conjugate, wherein amino acid Glutamine at position 291 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

15. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 5.

6. A hybrid enzyme-ligand conjugate, wherein amino acid Aspartic Acid at position 294 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

16. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 6.

7. A hybrid enzyme-ligand conjugate, wherein amino acid Glutamic Acid at position 407 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

17. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 7.

8. A hybrid enzyme-ligand conjugate, wherein amino acid Aspartic Acid at position 408 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

18. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 8.

9. A hybrid enzyme-ligand conjugate, wherein amino acid Aspartic Acid at position 380 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

19. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 9.

10. A hybrid enzyme-ligand conjugate, wherein amino acid Asparagine at position 117 of a starting alkaline phosphatase, numbered in accordance with SEQ ID NO:

2, is replaced by Cysteine, and the hybrid enzyme-ligand conjugate exhibits an enzymatic activity of the starting alkaline phosphatase.

20. A recombinant DNA molecule encoding an alkaline phosphate wherein the recombinant DNA molecule comprises a synthetic DNA fragment encoding the Cysteine as recited in claim 10.