Pre-activated proteins for labelling oligonucleotide probes

• US 5,441,867 A
• Filed: 01/07/1992
• Issued: 08/15/1995
• Est. Priority Date: 12/04/1989
• Status: Expired due to Term

• Current Assignee: Avecia Biotechnology Inc (Nitto Denko Corporation)
• Original Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Inventors: Garman, Andrew J., Parker, John R.
• Primary Examiner(s): Kim, Kay K. A.
• Application Number:
  US07/815,881
• Time in Patent Office: 1,316 Days
• Field of Search: 530/591.1, 530/391.3, 530/391.5, 530/391.7, 530/341.9, 530/404, 530/405, 530/406, 530/408, 530/409, 530/410, 530/816, 435/177, 435/188, 435/174, 435/28, 435/21, 435/207, 435/192, 435/194, 435/964, 435/6, 435/7.9, 435/7.92, 436/544, 436/547, 422/61, 424/178.1, 424/179.1, 424/194.1
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2561/107 Enzyme complementation
• CPC Class Codes: C12Q 2563/119 the label being proteinic
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: Y10S 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

Abstract

Pre-activated stable protein reagents are provided. The reagents can be made by reaction of the protein and a heterobifunctional linker and have extended stability especially after lyophilisation. The reagents can be used in assay kits to form conjugates with proteins e.g. antibodies or polynucleotides e.g. oligonucleotides probes.

Claims

1. An antibody or an enzyme reagent, pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of a thiol containing protein, said reagent being lyophilized from a buffered carbohydrate solution having a pH of from 6 to 8 which buffered solution has concentration of non-volatile salts of not more than 10 mM, so as to provided said reagent in dry solid form and being stable for at least 3 months against hydrolysis, polymerization and loss of label activity and of capacity to form conjugate by said covalent coupling.

2. A reagent as claimed in claim 1 pre-activated by reacting said antibody or enzyme to a first reactive group of a heterobifunctional cross-linking agent having first and second reactive groups, which first reactive group is capable of reacting with protein functional groups, the second reactive group of the cross-linking agent being capable of reacting with thiol groups.

3. A reagent as claimed in claim 2 wherein the first reactive group is reactive towards protein amino groups.

4. A reagent as claimed in claim 3 wherein the first reactive group is a succinimido-N-oxy ester group.

5. A reagent as claimed in claim 2 wherein the second reactive group is a maleimido, haloacetyl or haloacetamidyl or an activated disulphide or thiol group.

6. A reagent as claimed in claim 1 wherein the enzyme is alkaline phosphatase or horseradish peroxidase.

7. A reagent as claimed in claim 1 wherein said carbohydrate is lactose.

13. A reagent according to claim 2 in which said antibody or enzyme carries 1 to 3 residues of cross linking agent per molecule of said antibody or enzyme.

8. A kit to enable the use of prepared conjugates comprising a pre-activated antibody or an enzyme reagent, pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of a thiol containing protein and instructions for use, said reagent being lyophilized from a buffered carbohydrate solution having a pH of from 6 to 8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM, so as to provide said reagent in dry solid form and being stable for at least 3 months against hydrolysis, polymerisation and loss of label activity and of capacity to form conjugate by said covalent coupling.

9. A kit as claimed in claim 8 also comprising one or more of the following components

a) solid thiolating agent;

b) a pre-packed column for removing excess thiolating reagent from the thiolated product;

c) buffer for equilibrating and/or running the column in 2;

d) buffer for reconstituting the thiolating agent;

e) a further column for the purification of conjugate;

f) buffer for equilibrating and/or running the column in 5;

g) stands for mounting the columns;

h) vials for collecting the product and intermediates;

i) an agent for quenching excess thiol reactive groups after conjugation; and

j) additives to stabilise the conjugate for prolonged storage.

11. A kit as claimed in claim 9 wherein the thiolating agent is 2-iminothiolane.

12. A kit as claimed in claim 8 comprising also solid thiolating agent.

14. A kit according to claim 8 in which the reagent is stored in the presence of a desiccant.

10. A method of making a conjugate which comprises reacting an antibody or an enzyme reagent, pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of a thiol containing protein with a thiolated polynucleotide or a thiol containing protein respectively, said reagent being lyophilized from a buffered carbohydrate solution having a pH of from 6 to 8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM, so as to provide said reagent in dry solid form and being stable for at least 3 months against hydrolysis, polymerisation and loss of label activity and capacity to form conjugate by said covalent coupling, and reconstituting said lyophilized reagent in buffer before use.

15. A reagent pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of a thiol containing protein, said reagent comprising a derivatized enzyme selected from the group consisting of alkaline phosphatase and horse radish peroxidase, said derivatization being by a reaction to give 1-3 substituents per enzyme molecule with a heterobifunctional cross-linking agent selected from the group consisting of:

##STR9## (SMCC) where R1 is H or SO₃ Na;

##STR10## (MBS) where R² is H or SO₃ Na;

##STR11## (SMPB) where R³ is H or SO₃ Na;

##STR12## (SIAB) where R⁴ is H or SO₃ Na; and

##STR13## and said reagent being in dry solid lyophilised form from a buffered carbohydrate solution having a pH of from 6 to 8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM, and being stable for at least 3 months against hydrolysis, polymerisation and loss of label activity and of capacity to form conjugate by said covalent coupling.

16. A storage stable freeze dried reagent pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of thiol containing protein, said reagent comprising:

alkaline phosphatase derivatised by 1-3 molecules per phosphatase molecule of the heterobifunctional cross linking agent;

##STR14## wherein said reagent has been lyophilized from a phosphate buffered solution containing lactose and having a pH of from pH 7.2 to pH 7.8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM, and being stable for at least 3 months against hydrolysis, polymerization and loss of label activity and of capacity to form conjugate by said covalent coupling.

17. A storage stable freeze dried reagent pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of a thiol containing protein, said reagent comprising:

alkaline phosphatase derivatised by 1-3 molecules per phosphatase molecule of the heterobifunctional cross linking agent;

##STR15## wherein said reagent has been lyophilized from a phosphate buffered solution containing lactose and having a pH of from pH 7.2 to pH 7.8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM, and being stable for at least 3 months against hydrolysis, polymerization and loss of label activity and of capacity to form conjugate by said covalent coupling.

18. A storage stable freeze dried reagent pre-activated for covalent coupling to a thiol group of a thiolated polynucleotide or to a thiol group of a thiol containing protein, said reagent comprising:

horseradish peroxidase derivatised by 1-3 molecules per peroxidase molecule of the heterobifunctional cross linking agent;

##STR16## wherein said reagent has been lyophilized from a phosphate buffered solution containing lactose and having a pH of from pH 7.2 to pH 7.8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM, and being stable for at least 3 months against hydrolysis, polymerization and loss of label activity and of capacity to form conjugate by said covalent coupling.

19. In a process for making a conjugate of an enzyme and a polynucleotide or protein by the steps of

(a) derivatising the polynucleotide or protein to apply to it one or more pendant thiol groups;

(b) derivatising the enzyme to apply to it one or more thiol-reactive groups; and

(c) reacting the derivatised polynucleotide or protein with the derivatised enzyme;

the improvement which comprises lyophilizing the derivatised enzyme in a buffered carbohydrate solution having a pH of from 6 to 8 which buffered solution has a concentration of non-volatile salts of not more than 10 mM so as to give a reagent stable for at least 3 months, storing it until required and reconstituting said reagent in buffer before use, whereby to decrease by one the number of chemical reaction stages at the time of making said conjugate.