Epitope mapping of a recombinant human TSH receptor extracellular domain: identification of a predominant epitope using animal sera.
The extracellular domain of the TSH receptor (TSHR-561, amino acids #78-389) was expressed as a hexa-histidine fusion protein in bacteria. The recombinant protein was purified to homogeneity and used to immunize porcine and ovine species. High titre antibodies were obtained from both species that recognized the recombinant protein in Western blot analysis but failed to interfere with the TSH radio receptor assay. An epitope library was constructed and screened with affinity purified ovine and porcine antisera and detected a number of positive clones. Sequence analysis revealed that all of the epitopes contained sequences derived from the carboxyl terminus of the recombinant immunogen. One clone defined an epitope covering 16 amino acids from the carboxyl terminus and was the common epitope found in all of the other clones. Western blot screening of a large panel of Graves' sera with recombinant TSH receptor protein identified one patient sera that also recognized linear epitopes in the TSHR-561 protein. Experimentation demonstrated that the linear epitope recognized by this human sera was identical to the sequence recognised by the animal antisera. This sequence is unique to the TSH receptor and will be useful in further studies to analyze the TSH receptor protein.
Hunt
N
N
Institute for Hormone and Fertility Research, University of Hamburg, Germany.
Willey
K P
KP
Abend
N
N
Northemann
W
W
Leidenberger
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000235:genetics*; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods; D006111:Graves Disease / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D012756:Sheep / Q000276:immunology; D013552:Swine / Q000276:immunology
D000939:Epitopes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins