Recombinant Polypeptides and Methods for Detecting and/or Quantifying Autoantibodies Against Tsh Receptor
AssigneesDr. Fenning Biomed GmbH, Micromol Corporation
InventorsSteve Fenning, Xiaoping Yang, Andreas Dreusch, Wolfgang Rudy
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Abstract
The present invention relates to unglycosylated isolated and purified recombinant polypeptides comprising a fusion protein able to bind to autoantibodies produced in response to an autoimmune disease associated with an immune reaction to a TSH-receptor (TSHR). Also disclosed are methods of detecting and/or quantifying such autoantibodies using the isolated and purified recombinant polypeptides and respective kits.
Claims46
An unglycosylated isolated and purified recombinant polypeptide comprising a fusion protein able to bind to autoantibodies produced in response to an autoimmune disease associated with an immune reaction to a TSH-receptor obtainable by the method comprising,
- a) transforming a prokaryotic host with no mutation allowing formation of disulfide bonds in the cytoplasm of the prokaryotic host cells with a vector comprising an isolated and purified nucleic acid sequence encoding said fusion protein, wherein one or more TSH receptor partial sequences is fused to a polypeptide and wherein said TSH receptor partial sequences consist essentially of the extracellular domain of the TSH receptor, fragments thereof or variants thereof;
- b) culturing said prokaryotic host cells whereby said fusion protein is expressed and accumulated in said prokaryotic host cells;
- c) isolating said prokaryotic host cells;
- d) lysing said prokaryotic host cells in the presence of a detergent;
- e) separating soluble components of the lysate from insoluble components;
- and
- f) purifying said fusion protein from said soluble components of said lysate.
An unglycosylated isolated and purified recombinant polypeptide expressed in a prokaryotic host with no mutation allowing formation of disulfide bonds in the cytoplasm of the prokaryotic host cells, said unglycosylated isolated and purified recombinant polypeptide comprising a fusion protein able to bind to autoantibodies produced in response to an autoimmune disease associated with an immune reaction to a TSH-receptor, wherein one or more TSH receptor partial sequences is fused to a polypeptide consisting essentially of the glutathione S transferase (GST), fragments thereof or variants thereof, and wherein said TSH receptor partial sequence consists essentially of the extracellular domain of the TSH receptor, fragments thereof or variants thereof.
An unglycosylated TSH receptor partial sequence able to bind to autoantibodies produced in response to an autoimmune disease associated with an immune reaction to a TSH-receptor, said unglycosylated TSH receptor partial sequence consisting essentially of the extracellular domain of the TSH receptor, fragments thereof or variants thereof obtainable by the method comprising,
- a) transforming a prokaryotic host with no mutation allowing formation of disulfide bonds in the cytoplasm of the prokaryotic host cells with a vector comprising an isolated and purified nucleic acid sequence encoding a fusion protein able to bind to autoantibodies produced in response to an autoimmune disease associated with an immune reaction to a TSH-receptor, wherein one or more TSH receptor partial sequences is fused to a polypeptide and wherein said TSH receptor partial sequences consist essentially of the extracellular domain of the TSH receptor, fragments thereof or variants thereof;
- b) culturing said prokaryotic host cells whereby said fusion protein is expressed and accumulated in said prokaryotic host cells;
- c) isolating said prokaryotic host cells;
- d) lysing said prokaryotic host cells in the presence of a detergent;
- e) separating soluble components of the lysate from insoluble components;
- f) purifying said fusion protein from said soluble components of said lysate;
- g) digesting or cleaving said polypeptide fused to said TSH receptor partial sequence;
- and
- h) recovering said TSH receptor partial sequence, fragments thereof or variants thereof.
An epitope of an unglycosylated TSH receptor partial sequence selected from the group consisting of
- a) an epitope comprising at least 5 consecutive amino acids of the amino acid sequence selected from SEQ ID NOS:
- 10-27;
- b) an epitope comprising an amino acid sequence selected from SEQ ID NOS:
- 10-27;
- c) an epitope which is at least 60% identical to the amino acid sequences as defined in a) or b).
Dependencies are inferred from the claim text.