Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample.
OBJECTIVE
Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes.


METHODS
Two heterohybridoma cell lines secreting TSHR autoantibodies were isolated using standard techniques from the lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies (160 units/l by inhibition of TSH binding). The ability of the two new monoclonal antibodies (MAbs; K1-18 and K1-70) to bind to the TSHR and compete with TSH or TSHR antibody binding was analysed. Furthermore, the effects of K1-18 and K1-70 on cyclic AMP production in Chinese hamster ovary cells (CHO) cells expressing the TSHR were investigated.


RESULTS
One MAb (K1-18) was a strong stimulator of cyclic AMP production in TSHR-transfected CHO cells and the other (K1-70) blocked stimulation of the TSHR by TSH, K1-18, other thyroid-stimulating MAbs and patient serum stimulating type TSHR autoantibodies. Both K1-18 (IgG1 kappa) and K1-70 (IgG1 lambda) bound to the TSHR with high affinity (0.7 x 10(10) l/mol and 4 x 10(10) l/mol, respectively), and this binding was inhibited by unlabelled K1-18 and K1-70, other thyroid-stimulating MAbs and patient serum TSHR autoantibodies with stimulating or blocking activities. V region gene analysis indicated that K1-18 and K1-70 heavy chains used the same V region germline gene but different D and J germline genes as well as having different light chains. Consequently, the two antibodies have evolved separately from different B cell clones.


CONCLUSIONS
This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.
Evans
Michele
M
FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK.
Sanders
Jane
J
Tagami
Tetsuya
T
Sanders
Paul
P
Young
Stuart
S
Roberts
Emma
E
Wilmot
Jane
J
Hu
Xiaoling
X
Kabelis
Katarzyna
K
Clark
Jill
J
Holl
Sabrina
S
Richards
Tonya
T
Collyer
Alastair
A
Furmaniak
Jadwiga
J
Smith
Bernard Rees
BR
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001323:Autoantibodies / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects / Q000276:immunology; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D006825:Hybridomas; D007037:Hypothyroidism / Q000097:blood / Q000276:immunology*; D007133:Immunoglobulin Joining Region / Q000276:immunology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism; D007457:Iodine Radioisotopes; D008875:Middle Aged; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007133:Immunoglobulin Joining Region; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D000249:Adenosine Monophosphate; D013972:Thyrotropin