Mapping thyroid peroxidase epitopes using recombinant protein fragments.
The identification of autoantibody epitopes is important to the understanding of autoimmune thyroid diseases. In the case of thyroid peroxidase antibodies (TPO-ab), recent reports have disagreed about the number and type of autoantibody epitopes found in human TPO. In order to clarify the nature of these epitopes, we used an approach that provides recombinant human TPO produced by bacterial cells. The cDNA of four slightly overlapping fragments of human TPO-TPO 1(Glu 17-Ser 227), TPO 2(Tyr 226-Thr 476), TPO 3(Glu 471-Ser 720) and TPO 4(Phe 709-Leu 993)--were amplified by polymerase chain reaction and subcloned into the expression vector pMAL. In addition, a TPO 3 species for an alternatively spliced form of TPO of 876 amino acids was constructed (TPO 3M). Each of these constructs encodes a fusion protein, in which the amino terminal portion is maltose-binding protein, followed by the sequence of the fragment of human TPO. The plasmid constructs were transformed in Escherichia coli and, after growth, bacterial cells were harvested, lysed and the lysate was passed over an amylose affinity column and eluted with maltose. Western blots were performed using 33 sera from patients with autoimmune thyroid disease (group 1) and 17 sera from patients with nodular goiter and focal thyroiditis (group 2), all positive for TPO-ab measured by radio-immunoassay; sera from 10 healthy people with no clinical evidence of thyroiditis and positive for TPO-ab measured by radioimmunoassay (group 3) and sera from 30 patients with antigastric parietal cell antibodies without signs or symptoms of thyroiditis, 16 negative for TPO-ab (group 4a) and 14 positive for TPO-ab (group 4b), were included in the study.(ABSTRACT TRUNCATED AT 250 WORDS)
Tonacchera
M
M
Institut de Recherche Interdisciplinaire (IRIBHN), Brussels, Belgium.
Cetani
F
F
Costagliola
S
S
Alcalde
L
L
Uibo
R
R
Vassart
G
G
Ludgate
M
M
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D015153:Blotting, Western; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes; D005260:Female; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology*; D011994:Recombinant Proteins; D012016:Reference Values; D013959:Thyroid Diseases / Q000276:immunology
D000939:Epitopes; D015335:Molecular Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007453:Iodide Peroxidase