Generation of a mouse monoclonal TSH receptor antibody with stimulating activity.
A Balb/c mouse was subjected to genetic immunization with a cDNA construct encoding the human thyrotropin receptor (TSHr). The immune response of the mouse resulted in the production of immunoglobulins recognizing the TSHr in three different assays: (1) flow immunocytometry (FACS) with CHO cells expressing the receptor; (2) receptor-dependent stimulation of cAMP production in the same cell line; and (3) competition with labeled TSH for binding to the receptor. One thousand hybridomas were generated from the spleen of the mouse and their supernatants were screened. A single monoclonal, IRI-SAb1, scored positive in all three assays and was studied further. It stimulated 13-fold cAMP production in TSHr-expressing CHO cells, with an EC50 in the low nanomolar range. When compared with bovine TSH, IRI-SAb1 behaved as a partial agonist. Contrary to the expectation from the characteristic of autoantibodies of Graves' patients, IRI-SAb1 recognized a linear epitope, which was localized in a segment encompassing the first 281 residues of the receptor.
Costagliola
S
S
I.R.I.B.H.M., ULB, 808 Lennik street, B-1070 Brussels, Belgium.
Franssen
J D F
JD
Bonomi
M
M
Urizar
E
E
Willnich
M
M
Bergmann
A
A
Vassart
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000276:immunology*; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP