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 hypothy roidism. Specific measurement of TBAb requires a bioassay, but current meth ods 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 transfecte d with human TSH-R together with G418 resistance and a cAMP responsive luci ferase construct. Stable pools of transfected cells were selected and clone s identified by limiting dilution. Clone lulu* gave the best response to st imulation by TSH and was used to develop a bioassay for TBAb. The luminesce nt bioassay conditions have been optimized and validated using 12 serum sam ples from patients found to be TBAb positive in a bioassay using an establi shed method quantifying cAMP by radioimmunoassay (RIA). The effect of thyro id stimulating antibodies (TSAb) on the calculation of Inhibition Index (In I) using two previously described formulae have been investigated and we ha ve used serum containing both TSAb and TBAb to investigate detection of TBA b in samples containing more than one type of activity. RESULTS Lulu* displays a dose dependent increase in luciferase expression i n response to stimulation with bovine (b) TSH which is more effective in se rum free medium than in salt free buffer. TSH stimulated luciferase express ion can be inhibited by TBAb in either serum or an immunoglobulin preparati on. Using optimized assay conditions, challenging 10% serum against 1 U/l b TSH in culture medium, we have tested 31 euthyroid sera to determine a refe rence range: InI values >23% were considered positive. Twelve samples previ ously shown to contain TBAb by an established method quantifying cAMP by RI A were positive by the luciferase-based assay. Of control sere, 20/20 syste mic lupus erythematosus, 13/14 rheumatoid arthritis, 12/12 multinodular goi tre were negative. We demonstrated that if more complex formulae are used to calculate InI, fa lse positive TBAb results can be obtained in samples containing only TSAb. Finally, when sera contain both TSAb and TBAb, the net activity of stimulat ing and blocking antibodies is detected in the bioassay. Where TSAb are als o 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 u se to develop a luminescent bioassay for TBAb suitable for clinical use. Co mparing 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.