A luminescent bioassay for thyroid blocking antibodies.

OBJECTIVE: Thyroid blocking antibodies (TBAb) have a role in the development of hypothyroidism and in the neonate are responsible for transient hypothyroidism. Specific measurement of TBAb requires a bioassay, but current methods are lengthy and cumbersome. We describe a rapid luciferase-based method for the detection of TBAb using the lulu* cell line which is suitable for the provision of a clinical service PATIENTS AND MEASUREMENTS: Chinese hamster ovary (CHO) cells were transfected with human TSH-R together with G418 resistance and a cAMP responsive luciferase construct. Stable pools of transfected cells were selected and clones identified by limiting dilution. Clone lulu* gave the best response to stimulation by TSH and was used to develop a bioassay for TBAb. The luminescent bioassay conditions have been optimized and validated using 12 serum samples from patients found to be TBAb positive in a bioassay using an established method quantifying cAMP by radioimmunoassay (RIA). The effect of thyroid stimulating antibodies (TSAb) on the calculation of Inhibition Index (InI) using two previously described formulae have been investigated and we have used serum containing both TSAb and TBAb to investigate detection of TBAb in samples containing more than one type of activity. RESULTS: Lulu* displays a dose dependent increase in luciferase expression in response to stimulation with bovine (b) TSH which is more effective in serum free medium than in salt free buffer. TSH stimulated luciferase expression can be inhibited by TBAb in either serum or an immunoglobulin preparation. Using optimized assay conditions, challenging 10% serum against 1 U/l bTSH in culture medium, we have tested 31 euthyroid sera to determine a reference range: InI values >23% were considered positive. Twelve samples previously shown to contain TBAb by an established method quantifying cAMP by RIA were positive by the luciferase-based assay. Of control sera, 20/20 systemic lupus erythematosus, 13/14 rheumatoid arthritis, 12/12 multinodular goitre were negative. We demonstrated that if more complex formulae are used to calculate InI, false positive TBAb results can be obtained in samples containing only TSAb. Finally, when sera contain both TSAb and TBAb, the net activity of stimulating and blocking antibodies is detected in the bioassay. Where TSAb are also present, analysis of serum may be required at several dilutions to detect TBAb. CONCLUSIONS: We describe the production of a new cell line, lulu*, and its use to develop a luminescent bioassay for TBAb suitable for clinical use. Comparing two established methods of calculating TBAb, we found that they do not give identical results. In light of this, the high prevalence reported for TBAb in some studies has to be considered with caution.

AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: altered sub-cellular distribution of mutants lacking the PHD zinc fingers.

The gene responsible for autoimmune polyendocrino-pathy candidiasis ectodermal dystrophy (APECED) recently has been positionally cloned to 21q22.3. This novel gene, AIRE, encodes for a predicted 57.7 kDa protein featuring two PHD-type zinc fingers shared by other proteins involved in chromatin-mediated tran-scriptional regulation. APECED is an autosomal recessive condition characterized by multiple polyendocrinopathies, and the typical triad of APECED symptoms includes hypoparathyroidism, primary adrenocortical failure and chronic mucocutaneous candidiasis. The aetiology of APECED is linked directly to mutations within the coding region of AIRE. These mutations are predicted to lead to truncated forms of the protein lacking at least one of the PHD zinc fingers. In this study, we have investigated the sub-cellular localization of AIRE expressed transiently in COS cells and fibroblasts. We found that AIRE has a dual nuclear and cytoplasmic localization. The wild-type protein is directed to speckled domains in the nucleus and also shows co-localization with cytoskeletal filaments. N-terminal AIRE fragments deleted for the PHD domain show altered nuclear localization, suggesting that the APECED mutations may elicit their primary effects in the nucleus.

Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active.

We have expressed a cDNA for virus-induced chick interferon in Escherichia coli. The product, a 19-kDa protein lacking the signal peptide, was purified to homogeneity from the bacterial inclusion bodies. Proteins in the insoluble fraction of bacterial lysates were dissolved in guanidine hydrochloride and subjected to chromatography on Q-Sepharose and MonoS columns. Purified recombinant chick interferon has a specific antiviral activity of approximately 10(8) IU/mg and is a powerful inducer of the interferon-responsive promoter of the chicken Mx gene. Culture medium of transfected COS cells expressing full-length chick interferon cDNA contained up to 5 x 10(4) IU antiviral activity/ml that could be neutralized by antibodies to purified recombinant chick interferon. The antibodies precipitated proteins of 23-28 kDa from the supernatants of transfected COS cells. Treatment with endoglycosidase F reduced the size of the immunoprecipitated proteins to approximately 20 kDa, demonstrating that chick interferon is a glycoprotein.

Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity.

Crude preparations of chicken interferon (ChIFN) from various sources contain both antiviral and macrophage-activating factor (MAF) activity. Previous serological data indicated that unlike mammals, birds might express only a single type of IFN in response to viruses and mitogens that exhibits both activities. We have now expressed a complementary DNA for virus-induced ChIFN in transfected COS cells and in Escherichia coli. Purified recombinant ChIFN is a powerful antiviral agent and has high Mx promoter-inducing activity. However, as the sole agent, recombinant ChIFN lacks MAF activity: it does not induce the secretion of nitric oxide in primary monocyte-derived chicken macrophages. A neutralizing antiserum prepared against cloned ChIFN blocks most of the antiviral and Mx promoter-inducing activity present in preparations of natural ChIFN, but does not inhibit the MAF activity. These results demonstrate that chicken cells can be induced to secrete a novel cytokine which probably represents the avian homolog of mammalian IFN-gamma.