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


METHODS
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.
Jordan
N J
NJ
Department of Medical Biochemistry, UWCM and University Hospital of Wales NHS Trust, Cardiff, UK.
Rinderle
C
C
Ashfield
J
J
Morgenthaler
N G
NG
Lazarus
J
J
Ludgate
M
M
Evans
C
C
D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D001681:Biological Assay / Q000379:methods; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000032:analysis; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013961:Thyroid Gland / Q000276:immunology*; D013972:Thyrotropin; D014162:Transfection
D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D008156:Luciferases