A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies
Gd. Chazenbalk et al., A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies, J CLIN END, 84(2), 1999, pp. 702-710
We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (trunc
ated at amino acid residue 289 with a B-histidine tail) to investigate prop
erties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A
and Ni-chelate chromatography extracted milligram quantities of TSHR-289 (
similar to 20 -40% purity) from the culture medium. Nanogram quantities of
this material neutralized the TSH binding inhibitory activity in all 15 Gra
ves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, t
o partially purified TSHR-289. Screening of a TSHR complementary DNA fragme
nt expression library localized the 3BD10 epitope to 27 amino acids at the
N-terminus of the TSHR, a cysteine-rich segment predicted to be highly conf
ormational. 3BD10 preferentially recognized native, as opposed to reduced a
nd denatured, TSHR-289, but did not interact with the TSH holoreceptor on t
he cell surface, Moreover, mAb 3BD10 could extract from culture medium TSHR
-289 nonreactive with autoantibodies, but not the lesser amount (similar to
25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Althoug
h the active form of TSHR-289 in culture medium was stable at ambient tempe
rature, stability was reduced at 37 C, explaining the mixture of active and
inactive molecules in medium harvested from cell cultures.
In conclusion, studies involving a TSHR ectodomain variant indicate the exq
uisite conformational requirements of TSHR autoantibodies. Even under "nati
ve" conditions, only a minority of molecules in highly potent TSHR-289 prep
arations neutralize patients' autoantibodies. Therefore, Graves' disease is
likely to be caused by even lower concentrations of autoantibodies than pr
eviously thought. Finally, reciprocally exclusive binding to TSHR-289 by hu
man autoantibodies and a mouse mAb with a defined epitope suggests that the
extreme N-terminus of the TSHR is important for autoantibody recognition.