612 J Neurol Neurosurg Psychiatry 1999;66:612–616 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.66.5.612 on 1 May 1999. Downloaded from Comparison of mouse bioassay and immunoprecipitation assay for antibodies

Philip A Hanna, Joseph Jankovic, Angela Vincent

Abstract botulinum toxin type A (BTX-A).1–10 In Objective—To compare a recently devel- addition, BTX-A is also being used for various oped immunoprecipitation assay (IPA) to non-neurological (for example, cosmetic) indi- the mouse protection bioassay (MPB), cations. As the range of uses for BTX-A currently considered the “gold standard”, continues to expand, there is a growing for detecting antibodies against botuli- concern regarding the development of immu- num toxin A (BTX-A) and to correlate noresistance secondary to blocking antibodies these assay results with clinical responses (Ab).211 The reported frequency of such to BTX-A injections. antibodies has ranged from 3% to 57% 12 13 Methods—MPB and IPA assays were per- depending on the assay method used. The formed on serum samples from 83 pa- standard assay for detecting BTX Ab is the in tients (38 non-responders, 45 responders) vivo mouse protection bioassay (MPB), which who received BTX-A injections. Six non- evaluates the ability of increasing dilutions of a responders had serum tested on two sepa- patient’s serum to protect mice from lethal rate occasions. Some patients also doses of BTX-A.14 In vitro assays, including the received a “test” injection into either the sphere linked immunodiagnostic assay 13 right eyebrow (n=29) or right frontalis (SLIDA), enzyme linked immunosorbent 15 16 (n=19). assay (ELISA), a monoclonal antibody 17 —All patients antibody positive based immunoassay, and western blot Results 18 (Ab+) by MPB were also Ab+ by IPA, technique have also been reported to detect whereas an additional 19 patients (17 with such antibodies. These assays, however, do not reduced or no clinical response) who were correlate well with clinical responses because MPB Ab− were Ab+, with low titres, by they do not detect specific blocking Ab. IPA. Two of these 19 patients (non- The MPB has been shown to have high spe- 18 responders) were initially MPB Ab− but cificity, but its sensitivity is relatively low. The later became MPB Ab+. Similar to previ- primary aim of this study was to compare the MPB with a more recent immunoprecipitation ous studies, the sensitivity for the MPB 19 was low; 50% for clinical, 38% for eyebrow, assay (IPA) developed by Palace et al and to correlate the presence of antibodies detected and 30% for frontalis responses whereas http://jnnp.bmj.com/ the IPA sensitivity was much higher at by these two assays to the patients’ clinical Parkinson’s Disease 84% for clinical (p<0.001), 77% for eye- response to BTX-A injections. The results Center and Movement described by Palace et al19 needed to be Disorders Clinic, brow (p=0.111, NS) and 90% for frontalis responses (p<0.02). The IPA specificity confirmed using a larger number of patients, as Department of well as incorporating more clinical details Neurology, Baylor was 89% for clinical, 81% for eyebrow, and College of Medicine, 89% for frontalis responses, whereas the including correlation with facial (eyebrow and Houston, Texas, USA MPB specificity was 100% for all three frontalis) “test” injections. Additionally, we P A Hanna evaluated the utility of eyebrow or frontalis response types, which were all non- on September 28, 2021 by guest. Protected copyright. J Jankovic 18 significant diVerences. injections as clinical “tests” for immunoresist- ance. Neurosciences Group, Conclusions—Both assays had high spe- Department of Clinical cificity although the sensitivity of the IPA Neurology, Institute of was higher than the MPB. In addition, the Methods Molecular Medicine, IPA seems to display positivity earlier Eighty three patients (17 men and 66 women) John RadcliVe than the MPB, and as such, it may Hospit