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Demonstration of fragments with thyroid stimulating activity from Thyroid stimulation blocking antibodies‐IgG molecules by papain digestion
Author(s) -
Kouki Tsuyoshi,
Inui Takehiro,
Yamashiro Kei,
Hachiya Takashi,
Ochi Yukio,
Kajita Yoshihiro,
Takasu Nobuyuki,
Sato Yasushi,
Nagata Atsuo
Publication year - 1997
Publication title -
clinical endocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.055
H-Index - 147
eISSN - 1365-2265
pISSN - 0300-0664
DOI - 10.1046/j.1365-2265.1997.3191139.x
Subject(s) - sephadex , papain , chemistry , antibody , thyroid , digestion (alchemy) , sepharose , medicine , endocrinology , stimulation , biological activity , enzyme , biochemistry , in vitro , chromatography , biology , immunology
OBJECTIVES Thyroid stimulation blocking antibodies (TSBAb) inhibit TSH action and may have a role in the pathogenesis of hypothyroidism. In order to study the relationship between blocking and stimulating activities we have examined the biologically active fragments in TSBAb‐IgG molecules after papain digestion. DESIGN Both thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibitory (TBI) activity (determined by TSH receptor assay) in sera from patients with primary hypothyroidism were examined after digestion with papain‐Sepharose in the presence of cysteine. The digested IgG was separated into unbound (UF) and bound (BF) fractions on a Protein A‐Sepharose column. Each fraction was then gel‐filtrated on a Sephadex G‐100 column. RESULTS TS activity was found within one hour after hydrolysis in 5 out of 7 antibodies, then gradually decreased after more prolonged incubation. Both TS and TBI activities in the UF and the BF from Protein A were found in Fab (Mr 50 kD) and the second protein peak (Fc with trace amounts of Fab), respectively. The biological activity in the second protein peak was suggested as being derived from Fab fraction, because the activity bound to the anti‐F(ab′)2 column. However, the first peak (undigested IgG) in the BF had neither TS nor TSB activity. The TS activity in the retarded fraction (less than Mr 20 kD) in the UF gradually increased with prolonged digestion. CONCLUSIONS The conversion of Thyroid stimulation blocking antibodies activity to thyroid stimulating activity by papain digestion suggests that the inherent thyroid stimulating activity located in the Fab portion of the IgG molecule is unmasked by papain cleavage. We also suggest that the thyroid stimulating activity in the retarded fraction in the unbound fraction may be released from hydrolysis of the Fab portion of the IgG molecule.