Competitive immunoassay using complexed analyte derivatives
View Patent ↗The invention relates to a method for the detection of an analyte in a competitive immunoassay in the presence of an analyte derivative and first and second receptor molecules, and to a kit for carrying out this detection method.
1. A method for determining the presence or amount of an analyte in a sample in a competitive immunoassay, which comprises the following steps:
(a) incubating an analyte derivative with the sample, a first receptor molecule which is specific for the analyte and the analyte derivative, and a second receptor molecule which is specific for the analyte derivative and the analyte, wherein the analyte derivative is immobilized on a solid phase;
(b) binding the analyte derivative with the first receptor molecule, wherein the first receptor molecule is labeled with a label, and wherein the second receptor molecule masks the analyte derivative;
(c) separating the first receptor molecule not bound to the analyte derivative; and
(d) detecting the first receptor molecule bound to the analyte derivative and determining the presence or amount of the analyte in the sample.
2. The method of claim 1 , wherein prior to step (a) the analyte derivative is incubated with the second receptor molecule which is specific for the analyte derivative and the analyte to form a pre-incubated analyte derivative and wherein step (a) comprises incubating the sample, a first receptor molecule which is specific for the analyte and the analyte derivative, and the pre-incubated analyte derivative, wherein the pre-incubated analyte derivative is immobilized on a solid phase.
3. The method of claim 1 , wherein the label is selected from the group consisting of a radioactive label, chemiluminescent label, bioluminescent label, fluorescent label, phosphorescent label, electroluminescent label, an enzyme, biotin, and a group capable of absorption.
4. The method of claim 1 , wherein step (a) comprises three steps, step a1, step a2, and step a3 and wherein
step a1 comprises immobilizing the analyte derivative on a solid phase;
step a2 comprises incubating the immobilized analyte derivative with the second receptor molecule to form a pre-incubated analyte derivative; and
step a3 comprises incubating the sample, the first receptor molecule which is specific for the analyte and the analyte derivative, and the pre-incubated analyte derivative.
5. The method of claim 1 , wherein the first and the second receptor molecule are identical with the exception of a label.
6. The method of claim 1 , wherein the first and the second receptor molecule are different.
7. The method of claim 1 , wherein the first or the second receptor molecule, or both, are an antibody or an antibody fragment.
8. The method of claim 1 , wherein the analyte is a ligand.
9. The method of claim 1 , wherein the ligand is thyroxine, triiodothyronine or a steroid.
10. The method of claim 1 , wherein prior to step (a) the sample is incubated with the first receptor molecule.
11. A method for determining the presence or amount of an analyte in a sample in a competitive immunoassay, which comprises the following steps:
(a) incubating an analyte derivative with the sample, a first receptor molecule which is specific for the analyte and the analyte derivative, and a second receptor molecule which is specific for the analyte derivative and the analyte, wherein the first receptor molecule is immobilized on a solid phase;
(b) binding the analyte derivative with the first receptor molecule, wherein the analyte derivative is labeled with a label, and wherein the second receptor molecule masks the analyte derivative;
(c) separating the analyte derivative not bound to the first receptor molecule; and
(d) detecting the analyte derivative bound to the first receptor molecule and determining the presence or amount of the analyte in the sample.
12. The method of claim 11 , wherein prior to step (a) the analyte derivative is incubated with the second receptor molecule which is specific for the analyte derivative and the analyte to form a pre-incubated analyte derivative and wherein step (a) comprises incubating the sample, the first receptor molecule which is specific for the analyte and the analyte derivative, and the pre-incubated analyte derivative, wherein the pre-incubated analyte derivative is immobilized on a solid phase.
13. The method of claim 11 , wherein the label is selected from the group consisting of a radioactive label, chemiluminescent label, bioluminescent label, fluorescent label, phosphorescent label, electroluminescent label, an enzyme, biotin, and a group capable of absorption.
14. The method of claim 11 , wherein step (a) comprises three steps, step a1, step a2, and step a3 and wherein
step a1 comprises immobilizing the first receptor molecule on a solid phase;
step a2 comprises incubating the analyte derivative with the second receptor molecule to form a pre-incubated analyte derivative; and
step a3 comprises incubating the sample, the immobilized first receptor molecule which is specific for the analyte and the analyte derivative, and the pre-incubated analyte derivative.
15. The method of claim 11 , wherein the first and the second receptor molecule are identical with the exception of a label.
16. The method of claim 11 , wherein the first and the second receptor molecule are different.
17. The method of claim 11 , wherein the first or the second receptor molecule, or both, are an antibody or an antibody fragment.
18. The method of claim 11 , wherein the analyte is a ligand.
19. The method of claim 18 , wherein the ligand is thyroxine, triiodothyronine or a steroid.
20. The method of claim 12 , wherein prior to step (a) the sample is incubated with the first receptor molecule.
21. A kit for performing the method of claim 1 , said kit comprising:
(a) an analyte derivative, a labeled analyte derivative, or both;
(b) a first receptor molecule specific for an analyte and the analyte derivative, a first labeled receptor molecule specific for an analyte, or the analyte derivative, or both; and
(c) a second receptor molecule specific for the analyte derivative and the analyte.
22. A kit for performing the method of claim 11 , said kit comprising:
(a) an analyte derivative, a labeled analyte derivative, or both;
(b) a first receptor molecule specific for an analyte and the analyte derivative, a first labeled receptor molecule specific for an analyte, or the analyte derivative, or both; and
(c) a second receptor molecule specific for the analyte derivative and the analyte.