Increased receptor binding by bovine (b) TSH bound to monoclonal antibody to bTSHbeta-subunit.
TSH receptor (R) binding and cAMP production by bovine (b) TSH-bound to a monoclonal antibody (MoAb) or polyclonal antibody (Ab) to bTSH were examined, using TSH receptor (R) coating tube and porcine thyroid cells. (125) I-bTSH bound-to MoAbs to bTSH(alpha) or discontinuous type MoAb showed TSHR binding (10%) similar to intact (125) I-bTSH. TSHR binding was completely decreased (<2%) when (125) I-bTSH was bound by polyclonal Abs to bTSH(alpha) in Graves' patient or rabbit polyclonal Abs to bTSH. When either of the two MoAb (No. 1 and 2) to bTSH(beta) was bound to (125) I-bTSH, TSHR binding was 4 times higher (40%) compared to intact (125) I-bTSH. Binding of another MoAb (No. 3) caused no increased binding. TSHR binding of intact (125) I-bTSH was decreased from 10% to 2% by excess amounts of bTSH. Binding of (125) I-bTSH bound to MoAb to bTSH(beta) (No. 1 and 2) decreased from 40% to 30% by excess amounts of bTSH. When (125) I-bTSH bound-Fab of MoAb was used, the binding was reduced from 30 to 10% (No. 1) and from 25 to 6% (No. 2), respectively. In contrast, cAMP production by bTSH was decreased by pre-binding of all MoAbs and polyclonal Abs. Binding of (125) I-MoAb to bTSH (beta) to a synthetic peptide array of bTSH (beta) sequence was examined by the radioautography. The epitope of MoAb to bTSH(beta) was suggested to be LPK (beta 42-44) for No. 1, KLF (beta 39-41) for No. 2 and PKYA (beta 43-46) for No. 3, respectively, although the existence of discontinuous epitope could not be ruled out. The increased TSHR binding and the decreased cAMP production by bTSH bound to MoAbs may be due to the conformational change of TSH molecule or TSHR by binding of both bTSH and MoAb.
Ochi
Yukio
Y
Research Institute for Production Development, Shimogamo, Kyoto, Japan.
Hamazu
Masanari
M
Kajita
Yoshihiro
Y
Hachiya
Takashi
T
Kunimatsu
Mitoshi
M
Sato
Takaaki
T
Nagata
Atsuo
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D006111:Graves Disease / Q000097:blood / Q000276:immunology; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D040081:Protein Array Analysis; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism*; D037322:Thyrotropin, beta Subunit / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D037322:Thyrotropin, beta Subunit; D013972:Thyrotropin; D000242:Cyclic AMP