Site-specific conjugation of immunoglobulins and detectable labels

• US 5,191,066 A
• Filed: 12/07/1990
• Issued: 03/02/1993
• Est. Priority Date: 12/07/1990
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Bond, Howard E., Bieniarz, Christopher, Husain, Mazhar
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US07/624,169
• Time in Patent Office: 816 Days
• Field of Search: 530/390, 530/391.1, 530/391.3, 530/391.5, 530/391.7, 530/391.9, 424/85.91
• US Class Current: 530/391.1
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 16/065 Purification, fragmentation

Abstract

A process for producing a derivatized immunoglobulin and conjugates prepared therewith are provided. The derivatized immunoglobulin is produced by introducing sulfhydryl groups in the Fc region of an immunoglobulin wherein the Fab portion of the immunoglobulin is not altered. The derivatized immunoglobulin is used to prepare conjugates with various detectable moieties wherein a controlled distance exists between the Fc region of the immunoglobulin and the detectable moiety. The conjugates are particularly useful for performing immunoassays, and exhibit improved longevity, specificity and sensitivity.

Claims

1. A process for producing a derivatized immunoglobulin, said process comprising the steps of:

(a) site-specifically oxidizing the glycosylated zone of the Fc region of an immunoglobulin with an oxidant to convert at least two hydroxyl groups in said zone of said immunoglobulin to an aldehyde group;

(b) reacting the oxidized immunoglobulin of step (a) with a disulfide compound wherein at least one of said aldehyde groups is reductively aminated to introduce a disulfide moiety onto the Fc region of said immunoglobulin; and

(c) selectively reducing the disulfide bonds of the disulfide moiety of the reacted immunoglobulin of step (b) with an amount of a reductant sufficient to convert at least one of said disulfide moieties of said immunoglobulin into two sulfhydryl groups but insufficient to reduce the disulfide bonds in the complementarity defined region and the hinge region of said immunoglobulin thereby leaving the disulfide bonds in said complementarity defined region and said hinge region essentially unaffected.

2. The process of claim 1 wherein said disulfide compound in step (b) is of the formula:

space="preserve" listing-type="equation">X-R.sub.1 -S-S-R.sub.1 -X

wherein

X is an aminoalkyl group or a semihydrazone group;

R₁ is a group containing from 1 through 20 carbon atoms; and

R₁ and X together can form with the disulfide atoms between them a macrocyclic, heterocyclic ring or interimmunoglobulin disulfide containing chain to convert at least one aldehyde group per immunoglobulin molecule to a group which contains a disulfide moiety of the formula;

space="preserve" listing-type="equation">-X-R.sub.1 -S-S-

wherein;

R₁ and X are as defined above.

3. The process of claim 1 wherein the oxidant in step (a) is a chemical oxidant.

4. The process of claim 2 wherein X is selected from the group consisting of CH₂ --NH₂, ##STR7## a fragment of glutathione, a fragment of reduced hydrazidolipoamide, alkyl chain modified-length extended glutathione, and alkyl chain modified-length extended hydrazidolipoamide.

5. The process of claim 3 wherein the chemical oxidant is selected from the group consisting of alkali metal periodate, iodine and bromine.

6. The process of claim 3 wherein the chemical oxidant is sodium periodate.

7. The process of claim 1 wherein the oxidant in step (a) is an enzymatic oxidant.

8. The process of claim 7 wherein said enzymatic oxidant comprises neuraminidase and galactose oxidase and the oxidation step comprises sequentially contacting said immunoglobulin with neuraminidase and galactose oxidase.

9. The process of claim 1 wherein the disulfide compound in step (b) is selected from the group consisting of cystamine, oxidized glutathione, oxidized hydrazidolipoic acid, alkyl chain modified-length extended oxidized glutathione, and alkyl chain modified-length extended oxidized hydrazidolipoic acid.

10. The process of claim 1 wherein the reductant in step (c) is a chemical reductant selected from the group consisting of mercaptoethanol, dithiothreitol, sodium borohydride and sodium dithionite.

11. The process of claim 1 wherein the reductant in step (c) is an enzymatic reductant selected from the group consisting of glutathione reductase and lipoamide dehydrogenase.

12. The process of claim 1 wherein the sulfhydryl group-containing immunoglobulin of step (c) is contacted with a detectable moiety containing a maleimide group to produce a conjugate.

13. The process of claim 12 wherein said detectable moiety is selected from the group consisting of enzymes, chromophores, fluorescent molecules, chemiluminescent molecules, phosphorescent molecules, colored particles, and luminescent molecules.

14. The process of claim 12 wherein said detectable moiety is an enzyme.

15. The process of claim 12 wherein said maleimide group is part of a linking group.

16. The process of claim 15 wherein said linking group is a heterobifunctional linking group.

17. The process of claim 1 wherein the reductant in claim 1 is present in an amount whereby essentially no disulfide bonds of the native immunoglobulin in step (a) are reduced.

18. The process of claim 17 wherein the reductant comprises form between about 1 mM and about 5 mM dithiothreitol.

19. The process of claim 14 wherein the reductant comprises about 2 mM dithiotreitol.