Receptor binding assays and reagent kit for detecting TSH receptor autoantibodies
Improved receptor assays for the detection of thyroid stimulating hormone receptor (TSHR) autoantibodies are described which use immobilized, affinity-purified rTSHR preparations as specific binders. This format, as well as novel measures for neutralizing pathologically increased human TSH (hTSH) levels in the sera, e.g., by the addition of anti-hHSH antibody, and/or eliminating the influence of anti-bovine TSH antibody, result in increased assay reliability and open up the possibility of preparing the assay constituents in a ready to use and/or well-standardized form for automatic processing and/or convenient marketing.
1. A method for the determination of TSH receptor autoantibodies comprising:
(i) reacting a solid phase, comprising an affinity-purified immobilized functional recombinant human TSH receptor, with a liquid biological sample to be assayed for the presence of said autoantibodies;
(ii) separating a reacted solid phase from the liquid biological sample;
(iii) washing the reacted solid phase;
(iv) incubating the reacted solid phase with a buffer solution comprising an amount of labeled bovine TSH for a sufficient time to occupy all the TSH binding sites of the functional recombinant human TSH receptor not occupied by the autoantibodies; and
(v) determining the presence and/or amount of the autoantibodies on the basis of the amount of labeled bovine TSH bound to the solid phase;
wherein the functional recombinant human TSH receptor is immobilized to a solid support by a selective monoclonal antibody that recognizes only conformational epitopes of the human TSH receptor and is obtained by immunizing an animal with a DNA plasmid construct encoding the human TSH receptor and washed.
2. A method for the determination of TSH receptor autoantibodies comprising:
(i) reacting a solid phase comprising an affinity-purified immobilized functional recombinant human TSH receptor with a solution prepared from:
a) a serum-containing biological sample to be assayed for the presence of said autoantibodies, and
b) a buffer solution containing an amount of labeled bovine TSH for a sufficient time to occupy all the TSH binding sites of the functional recombinant human TSH receptor not occupied by the autoantibodies;
(ii) separating the solution from a reacted solid phase;
(iii) washing the reacted solid phase; and
(iv) determining the presence and/or amount of the autoantibodies on the basis of the amount of labeled bovine TSH bound to the solid phase;
wherein the functional recombinant human TSH receptor is immobilized to the solid support by a selective monoclonal antibody that recognizes only conformational epitopes of the human TSH receptor and is obtained by immunizing an animal with a DNA plasmid construct encoding the human TSH receptor and washed.
3. The method according to either claim 1 or claim 2 , wherein the solid phase is formed by the walls of test tubes, which are pre-coated with an animal-specific antibody for binding the selective monoclonal antibody against the human TSH receptor.
4. The method according to either claim 1 or claim 2 , carried out in an automated form, wherein the solid phase comprises suspended particles that are coated with said selective monoclonal antibody against the human TSH receptor, and wherein by adding the functional recombinant human TSH receptor and the sample a solution containing the suspended solid particles and the receptor is temporarily formed.
5. The method according to claim 1 , wherein the labeled bovine TSH is added in a serum-free buffer solution.
6. The method according to either claim 1 or claim 2 , wherein step (i) is carried out in the presence of at least one antibody against human TSH that does not cross-react with bovine TSH.
7. The method according to either claim 1 or claim 2 , wherein the autoantibodies are receptor-stimulating autoantibodies, whose occurrence in a human serum is characteristic of Graves' disease.
8. The method according to either claim 1 or claim 2 , wherein the affinity-purified immobilized functional recombinant human TSH receptor is in the presence of a buffer.
9. The method according to either claim 1 or claim 2 , wherein said sample is diluted with a buffer.
10. A method for the determination of TSH receptor autoantibodies comprising:
(i) reacting a solid phase, comprising an affinity-purified immobilized functional recombinant human TSH receptor, with a liquid biological sample to be assayed for the presence of said autoantibodies;
(ii) separating a reacted solid phase from the liquid biological sample;
(iii) washing the reacted solid phase;
(iv) incubating the reacted solid phase with a buffer solution comprising an amount of labeled bovine TSH for a sufficient time to occupy all the TSH binding sites of the functional recombinant human TSH receptor not occupied by the autoantibodies; and
(v) determining the presence and/or amount of the autoantibodies on the basis of the amount of labeled bovine TSH bound to the solid phase;
wherein the functional recombinant human TSH receptor is immobilized to a solid support by a selective monoclonal antibody that:
a) recognizes only conformational epitopes of the functional human TSH receptor and is obtained by immunizing an animal with a DNA plasmid construct encoding the human TSH receptor; and
b) does not bind to peptides representing short sequences of the human TSH receptor, but which shows strong binding to the complete functional recombinant human TSH receptor;
and then washed.