Mouse diaphragm assay for detection of antibodies against botulinum toxin type B

With the advent of a commercial preparation of botulinum toxin type B (BT‐B) for treatment of cervical dystonia detection of antibodies against BT‐B (BT‐B‐AB) becomes necessary. For this purpose, we carried out a mouse diaphragm assay (MDA) by continuous measurement of the twitch force of a mouse hemidiaphragm preparation elicited by electric stimulation of its phrenic nerve. After exposing the preparation to BT‐B 3 ng/ml the time to half‐maximal twitch force reduction (paralysis time [PT]) was 69 ± 4 min (n = 25). Addition of sera from patients with antibodies against BT‐A produced a PT of 68 ± 5 min (n = 24), whereas addition of sera from controls with antibodies against tetanus toxoid produced a PT of 67 ± 6 min (n = 30). When defined amounts of BT‐B‐AB were added to the MDA, PT was prolonged. This prolongation was correlated closely to the amount of BT‐B‐AB added, thus producing a calibration curve. The threshold for BT‐B‐AB detection was 0.4 mU/ml. When sera from 7 patients (4 women, 3 men; age 50.6 ± 14.2 years) with cervical dystonia (Toronto Western Spasmodic Torticollis Rating Scale score, 18.9 ± 2.9) and complete secondary failure of BT‐B therapy (NeuroBloc; Elan Pharmaceuticals, Shannon, Ireland; 12,229 ± 2,601 MU/injection series, 1.86 ± 0.69 injection series before complete secondary therapy failure; 100.4 ± 15.8 days between injection series with normal therapeutic effect) were tested, BT‐B‐AB titers of more than 10 mU/ml were found in all of them. The MDA can be used to measure neutralizing BT‐B‐AB titers quantitatively and with adequate sensitivity and specificity. Further studies are necessary to understand the role of intermediate BT‐B‐AB titers in partial BT‐B therapy failure. © 2005 Movement Disorder Society