THREE-STEP ACID DISSOCIATION ENZYME LINKED IMMUNOSORBENT (TADELIS) ASSAY

• US 20190011440A1
• Filed: 06/15/2018
• Published: 01/10/2019
• Est. Priority Date: 12/16/2015
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: JORDAN, Gregor, STAACK, Roland
• Granted Patent: US 10,458,981 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Abstract

Herein is reported a method for determining in a sample the (total, i.e. binding competent) amount of a ligand of a ligand-binding protein (therapeutic) comprising the following steps in the following order: subjecting the sample to an acid treatment, forming in solution a non-covalent complex comprising i) an anti-ligand antibody, ii) the ligand, and iii) labelled ligand-binding protein, by adding anti-ligand antibody and labelled ligand-binding protein to the sample, and determining the amount of the complex and thereby determining the amount of the ligand of the ligand-binding protein.

Claims

1. A method for determining in a sample the (total) amount of a ligand of a ligand-binding protein (therapeutic) comprising the following steps in the following order:

subjecting the sample to an acid treatment,

forming in solution a ternary complex comprising

i) an anti-ligand antibody,

ii) the ligand, and

iii) labelled ligand-binding protein,

by adding first the labelled ligand-binding protein to the sample to form a binary labelled ligand-binding protein-ligand complex, and by adding after the formation of the binary complex the anti-ligand antibody to the sample to form a ternary labelled ligand-binding protein-ligand-anti-ligand antibody complex, and

determining the amount of the ternary complex,

and

thereby determining the amount of the ligand of the ligand-binding protein.

2. The method according to claim 1, wherein the method is for determining the amount of a ligand that can (specifically) be bound by the ligand-binding protein.

3. The method according to claim 1, wherein the sample is a plasma sample or a serum sample.

4. The method according to claim 1, wherein the sample is not diluted prior to the acid treatment.

5. The method according to claim 1, wherein the forming of the complex is by incubating the acid-treated sample with an excess of labelled ligand-binding protein.

6. The method according to claim 1, wherein the forming of the complex is by incubating the acid-treated sample firstly with an excess of labelled ligand-binding protein and secondly with the anti-ligand antibody.

7. The method according to claim 1, wherein in the forming of the complex the labelled ligand-binding protein is conjugated to a detectable label.

8. The method according to claim 1, wherein the anti-ligand antibody is conjugated to a first member of a binding pair.

9. The method according to claim 1, wherein the method comprises after the forming of the ternary complex the step of immobilizing the formed ternary complex on a solid support.

10. The method according to claim 9, wherein the solid support is conjugated to the second member of the binding pair (that can form a complex with the first member of the binding pair conjugated to the anti-ligand antibody).

11. The method according to claim 1, wherein the anti-ligand antibody is a polyclonal antibody.

12. The method according to claim 1,

wherein the method comprises the following steps in the following order:

subjecting the sample to an acid treatment,

incubating the sample with labelled ligand-binding protein, which is conjugated to a detectable label,

incubating the sample with a polyclonal anti-ligand antibody, which is conjugated to a first member of a binding pair,

applying the sample to a solid support conjugated to a second member of the binding pair that forms with the first member of the binding pair a non-covalent complex, and

determining the amount of a ligand of a ligand-binding protein by determining the amount of solid phase immobilized labelled ligand binding protein.

13. The method according to claim 1, wherein the ligand-binding protein is an antibody.

14. The method according to claim 8, wherein the first member of the binding pair is biotin.

15. The method according to claim 1, wherein the ratio of the added labelled ligand binding protein and the added anti-ligand antibody is about 8-9:

1 based on molecular weight.

16. The method according to claim 1, wherein the ternary complex is a non-covalent complex.

17. The method according to claim 10, wherein the second member of the binding pair is streptavidin.