Reactivity of Thyrotropin Receptor Autoantibodies with the Thyrotropin Receptor on Western Blots

Abstract

Affinity purified recombinant human thyrotropin receptor (TSHR) was run on sodium dodecyl sulfate (SDS) gels and subjected to a renaturing and blotting procedure. Twenty sera from thyrotropin receptor autoantibodies (TRAb)-positive patients with a history of hyperthyroidism and 20 sera with high levels of TSH blocking activity were analyzed. Four of 20 sera with blocking-type of TRAb (i.e., TSH antagonist activity) were able to recognize the mature, fully glycosylated 120-kd form of the receptor on blots of gels run under reducing conditions. No sera recognized the 100-kd high mannose precursor form of the TSHR. Three of the four recognized a 74-kd band and 2 of the 4 recognized a 50-kd band. These bands are probably proteolytic cleavage fragments of the mature 120-kd TSHR. In the absence of reducing agent the same 4 of 20 sera described above together with a further serum sample (i.e., 5/20 in total) reacted with the 120-kd form of the receptor. No specific reaction with the TSHR was observed on Western blots with the remaining 15 sera with TSH blocking activity, nor with 20 sera from patients with a history of hyperthyroidism, nor with sera from 10 healthy blood donors, 10 Hashimoto sera (negative for TRAb) and 10 systemic lupus erythematosus sera. No clear differences were observed in the TRAb positive sera that were reactive and nonreactive on Western blots in terms of their ability to inhibit TSH binding or to immunoprecipitate 125I-labeled TSHR. Overall, our results indicate that the mature 120-kd form of the TSHR that is principally responsible for binding TSH is also responsible for binding TRAb (when this binding can be detected). These observations together with immunoprecipitation and TSH binding inhibition studies, emphasize the close relationship between the receptor's binding sites for TSH and TRAb.

Characteristics of a Monoclonal Antibody to the Thyrotropin Receptor that Acts as a Powerful Thyroid-Stimulating Autoantibody Antagonist

“…Consequently, none of these mAbs resembled patient sera TSHR autoantibodies with TSH antagonist activity. Also these seven mAbs recognised linear epitopes on the TSHR (amino acid 378-387 or amino acid 381-415) and as such differed further from patient serum TSHR autoantibodies with blocking activity which almost always recognize conformational epitopes (1)(2)(3)36).…”

Thyroid-Stimulating Monoclonal Antibodies

“…Sera A-D were diluted in a pool of healthy blood donor sera in all experiments. TSH blocking activity was determined as described previously (43). FIG.…”

“…In particular, no reactivity could be demonstrated between TSmAbs 1-3 and the TSHR by Western blotting, TSHR peptide ELISA or using 35 S-labeled TSHR (produced in vitro) and similar lack of reactivity is observed with TSH, and with almost all TSHR autoantibodies (16,42,43).…”

Free-flow isoelectric focusing of proteins remaining in cell fragments following sonication of thyroid carcinoma cells

“…After blocking, membranes were incubated overnight at 47C with primary antibodies raised against a peptide comprising amino acids 236-249 of human 5'DI [12] (Antiserum 1068 was a kind gift of G. G. J. M. Kuiper and T. J. Visser, Rotterdam) or against human TSH-R [13] (purchased from Acris, Hiddenhausen, Germany). Details of the procedure as well as the detection of the antibodies binding specifically to their antigens have been described by Menth et al [14].…”