Demonstration of Thyroactive Smaller Components Released from TSAb-IgG by Protease Digestion
Thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) in fragments released from TSAb-IgG by protease digestion were examined. The unbound fraction (UF) and the bound fraction (BF) were separated using a prot...
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Veröffentlicht in: | Thyroid (New York, N.Y.) N.Y.), 1995-12, Vol.5 (6), p.449-454 |
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Zusammenfassung: | Thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) in fragments released from TSAb-IgG by protease digestion were examined. The unbound fraction (UF) and the bound fraction (BF) were separated using a protein A-Sepharose column after papain hydrolysis (more hydrolysis at pH 5.0 than pH 7.5) of TSAb-IgG. When both fractions were gel filtrated on a Sephadex G-100 column, the TS and TBI activity were found in both Fab fraction (M
r
50 kDa) and the retarded fraction (between M
r
50 and 20 kDa) in the UF, and also in the first fraction (undigested IgG, M
r
160 kDa), the second fraction (Fc with tracer amounts of Fab, M
r
50 kDa), and the retarded fraction (between M
r
50 and 20 kDa) of the BF. The biological activity in the second fraction was suggested as being derived from Fab, because the activity bound to the anti-F(ab')2 column but did not bind to the anti-Fc column. Anti-Tg and anti-TPO activities were found in Fab, but were not found in the retarded fraction that consisted of M
r
20–30 kDa. In pepsin hydrolysis the UF from the protein A column consisted of both F(ab')
2
(M
r
100 kDa) and pF'c (CH
3
) (M
r
25 kDa), and the BF consisted of only the undigested IgG. The biological activities were found in both the F(ab')2 fraction and the retarded fraction (between M
r
100 and 25 kDa). Anti-Tg and anti-TPO activities were found in F(ab')
2
, but no activity was observed in the Mr 25-kDa fraction. The present study showed that the biological activity of TSAb is distributed in not only the Fab or F(ab')
2
fragment, but also in thyroactive smaller components (TSC) (M
r
20–30 kDa) without antigen-binding activity such as anti-Tg and anti-TPO. We suggest that TSC may be released from the Fab fragment region of TSAb-IgG by protease hydrolysis. |
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ISSN: | 1050-7256 1557-9077 |
DOI: | 10.1089/thy.1995.5.449 |