Detection of autoantibodies to recombinant human thyroid peroxidase by sensitive enzyme immunoassay.
Autoantibodies to thyroid peroxidase (TPO), the thyroid 'microsomal' antigen, are widely utilized in the diagnosis of human autoimmune thyroid disease. Crude human thyroid preparations of TPO are of differing potency, contain residual thyroglobulin (Tg) and other human membrane antigens, and are available in only limited amounts. Hence, immunoassays for anti-TPO are unstandardized and of variable sensitivity and specificity. We co-transfected the Chinese hamster ovary (CHO) cell line with a full-length human TPO cDNA expression plasmid. We selected a high expressing recombinant TPO positive cell population (CHO-TPO) by Northern blot analysis, then fluorescence laser flow cytometry using both human polyclonal and murine monoclonal anti-TPO antibodies. Solubilized 100,000 g membrane preparations from both CHO-TPO and CHO cells were used as antigens in a specific ELISA with CHO antigen serving as background control. In a selected series of known anti-TPO positive (n = 46) and negative (n = 73) sera there was a high correlation between ELISAs utilizing recombinant or natural-TPO antigen (r = 0.93). There appeared to be no difference in the affinity of high titre human anti-TPO for recombinant and natural-TPO antigen with both ELISAs able to detect 0.05 U/ml of anti-TPO activity (reference preparation NIBSC 66/387). These data predict a new era in standardized thyroid autoantibody testing utilizing recombinant antigen preparations.
Kendler
D L
DL
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Martin
A
A
Magnusson
R P
RP
Davies
T F
TF
D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D001327:Autoimmune Diseases / Q000175:diagnosis*; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015342:DNA Probes; D004274:DNA, Recombinant; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007453:Iodide Peroxidase / Q000276:immunology*; D010053:Ovary; D013959:Thyroid Diseases / Q000175:diagnosis* / Q000276:immunology; D014162:Transfection
D001323:Autoantibodies; D015342:DNA Probes; D004274:DNA, Recombinant; D007453:Iodide Peroxidase