In the ELISA kit, TSH-R autoantibodies in patient sera, calibrators and controls are allowed to interact with TSH receptors coated onto ELISA plate wells.

After a 2 hour incubation, the samples are discarded leaving TRAb bound to the immobilised receptor. A human monoclonal autoantibody to the TSHR labelled with biotin (M22-biotin) is added in a second incubation step, where it interacts with immobilised TSH receptors which have not been blocked by bound TRAb.

The amount of M22-biotin bound to the plate is then determined in a third incubation step by addition of streptavidin peroxidase, which binds specifically to biotin.

Excess unbound streptavidin peroxidase is then washed away and addition of 3,3’,5,5’-tetramethylbenzidine (TMB) resulting in the formation of a blue colour.

This reaction is stopped by addition of stop solution causing the well contents to turn from blue to yellow. The absorbance of the yellow reaction mixture at 450nm is then read using an ELISA plate reader. A lower absorbance indicates the presence of TRAb in a test sample (as TRAb inhibit the binding of M22-biotin to TSHR coated plate wells).

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