title: Mouse diaphragm assay for detection of antibodies against botulinum toxin type B.
abstract: 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.
authors.affiliation: Department of Neurology, Rostock University, Rostock, Germany. dirk.dressler@med.uni-rostock.de
authors.forename: Dirk
authors.initials: D
authors.lastname: Dressler
authors.forename: M
authors.initials: M
authors.lastname: Lange
authors.forename: Hans
authors.initials: H
authors.lastname: Bigalke
mesh_terms: D000818:Animals; D018726:Anti-Dyskinesia Agents / Q000276:immunology* / Q000627:therapeutic use; D000906:Antibodies / Q000032:analysis*; D001681:Biological Assay / Q000379:methods; D001905:Botulinum Toxins / Q000276:immunology* / Q000627:therapeutic use; D019274:Botulinum Toxins, Type A; D003964:Diaphragm / Q000187:drug effects / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D013997:Time Factors; D014103:Torticollis / Q000097:blood / Q000188:drug therapy / Q000276:immunology*; D017211:Treatment Failure
chemical_list: D018726:Anti-Dyskinesia Agents; D000906:Antibodies; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A