Expression of recombinant human thyroid peroxidase by the baculovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease.
The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection of Spodoptera frugiperda (Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by conventional methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n = 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.
Haubruck
H
H
Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
Mauch
L
L
Cook
N J
NJ
Steffens
U
U
Hunt
N
N
Berthold
H
H
Niemann
H
H
Wirbelauer
C
C
Northemann
W
W
D000818:Animals; D001323:Autoantibodies / Q000097:blood; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006111:Graves Disease / Q000175:diagnosis*; D006801:Humans; D007453:Iodide Peroxidase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D009036:Moths; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013967:Thyroiditis, Autoimmune / Q000175:diagnosis*
D001323:Autoantibodies; D011993:Recombinant Fusion Proteins; D007453:Iodide Peroxidase