Compositions and methods for detecting autoantibodies
View Patent ↗The invention provides compositions and methods for detecting thyroid hormone blocking immunoglobulin (TBI). The invention's methods are sensitive and specific for TBI, and may be used for the dual detection of both TBI and TSI. The invention's compositions and methods are useful for the diagnosis of diseases that are associated with the presence of TBI and/or TSI, for monitoring the progress of disease and/or treatment regimens, therapeutics, vaccines, etc., and for assisting clinicians in making treatment decisions.
1. A method for detecting thyroid hormone blocking immunoglobulin (TBI) in a sample, comprising
a) combining
i) transgenic cells stably transfected with one or more expression vector comprising a
1) a first nucleic acid sequence that encodes a reporter, wherein said first nucleotide sequence is operably linked to a cAMP-inducible promoter, and
2) the nucleotide sequence of SEQ ID NO: 2 that encodes a chimeric TSH receptor (TSHR), wherein said SEQ ID NO: 2 is operably linked to a constitutive promoter,
wherein said cells express said chimeric TSHR on the cell membrane,
ii) a thyroid stimulating polypeptide that stimulates said chimeric TSHR upon binding to said TSHR, wherein said thyroid stimulating polypeptide is selected from the group consisting of thyroid stimulating hormone (TSH), a thyroid stimulating monoclonal antibody, and thyroid stimulating polyclonal antibody, and
iii) a sample, wherein said sample is a control sample or is a test sample,
wherein said combining said transgenic cells and said TSH with
A) said control sample produces a first sample, and
B) said test sample produces a second sample, and
is under conditions for binding of said TSH to said chimeric TSHR, and
b) measuring the level of expression of said reporter in said first sample and in said second sample, wherein a reduced level of expression of said reporter in said second sample compared to said first sample indicates the presence of TBI in said test sample.
2. The method of claim 1 , wherein the IC 50 for TBI is from 5 fold to 15 fold smaller than the IC 50 for TBI when the method is performed with said transgenic cells in which chimeric TSHR is substituted with wild type TSHR.
3. The method of claim 1 , wherein the IC 50 for TBI is from 10 fold to 30 fold smaller when the thyroid stimulating polypeptide is TSH than when the thyroid stimulating polypeptide is an anti-TBI monoclonal antibody.
4. The method of claim 1 , wherein said method further comprises detecting a reduced level of expression of said reporter in said second sample compared to said first sample.
5. The method of claim 1 , wherein said method further comprises determining the level of TBI in said test sample by comparing
a) the level of expression of said reporter after said contacting with said test sample, with
b) the level of expression of said reporter after contacting said transgenic cells with one or more standard samples, each containing a known concentration of TSH.
6. The method of claim 1 , wherein said method is TBI specific.
7. The method of claim 1 , wherein said thyroid stimulating polypeptide is TSH and wherein said TSH is present in at least one of said first sample and said second sample at a concentration of 0.2 mIU/ml to 100 mIU/ml.
8. The method of claim 1 , wherein said reporter comprises a bioluminescence protein.
9. The method of claim 8 , wherein said bioluminescence protein comprises Renilla luciferase amino acid sequence SEQ ID NO:3.
10. The method of claim 1 , wherein said transgenic cells are Chinese hamster ovary (CHO) cells or human Rhabdomyosarcoma (RD) cells.
11. The method of claim 1 , wherein said thyroid stimulating polypeptide is a thyroid stimulating monoclonal antibody.
12. The method of claim 1 , wherein said thyroid stimulating polypeptide is a thyroid stimulating polyclonal antibody.
13. A method for detecting thyroid hormone blocking immunoglobulin (TBI) and thyroid hormone stimulating immunoglobulin (TSI) in a sample, comprising
a) combining
i) transgenic cells stably transfected with one or more expression vector comprising a
1) a first nucleic acid sequence that encodes a reporter, wherein said first nucleic acid sequence is operably linked to a cAMP-inducible promoter, and
2) the nucleotide sequence of SEQ ID NO: 2 that encodes a chimeric TSH receptor (TSHR), wherein said SEQ ED NO: 2 is operably linked to a constitutive promoter,
wherein said cells express said chimeric TSHR on the cell membrane,
ii) a thyroid stimulating hormone (TSH),
iii) a control sample or a test sample,
wherein combining said transgenic cells and said TSH with
A)said control sample produces a first sample, and
B) said test sample produces a second sample,
combining is under conditions for binding of said TSH to said chimeric TSHR, and
b) measuring the level of expression of said reporter in said transgenic cells before said combining and after said combining, wherein
i) a reduced level of expression of said reporter in said second sample compared to said first sample indicates the presence of TBI in said test sample, and
ii) an increased level of expression of said reporter in said second sample compared to said first sample indicates the presence of TSI in said test sample.
14. The method of claim 13 , wherein said method further comprises detecting a reduced level of expression of said reporter in said second sample compared to said first sample, wherein said detecting of a reduced level of expression of said reporter indicates the presence of TBI in said test sample.
15. The method of claim 13 , wherein said transgenic cells are Chinese hamster ovary (CHO) cells or human Rhabdomyosarcoma (RD) cells.