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Hannover, Germany

Dressler D.,Hannover Medical School | Bigalke H.,Toxogen Gmbh
Journal of Neural Transmission | Year: 2016

Most botulinum toxin (BT) drugs are stored as powders which need to be reconstituted with normal saline before clinical use. As botulinum neurotoxin (BNT), the therapeutically active ingredient, is a large double-stranded protein the process of reconstitution should be performed with special attention to mechanical stress applied. We wanted to test the mechanical stability of BNT during the reconstitution process. For this, 100 MU onabotulinumtoxinA (Botox®, Irvine, CA, USA) was reconstituted with 2.0 ml of NaCl/H2O. Gentle reconstitution (GR) was performed with a 5 ml syringe, a 0.90 × 70 mm injection needle, one cycle of injection-aspiration-injection and two gentle shakes of the vial. Aggressive reconstitution (AR) was performed with a 5 ml syringe, a 0.40 × 40 mm injection needle, ten injection-aspiration-injection cycles and 30 s of continuous shaking of the vial. AR increased the time to paralysis in the mouse hemidiaphragm assay (HDA) from 72.0 ± 4.6 to 106.0 ± 16.0 min (*p = 0.002, two-tailed t test after Kolmogorov-Smirnova test with Lilliefors correction for normal distribution). Construction of a calibration curve revealed that the increase in the time to paralysis was correlated with a loss of potency of from 100 to 58 MU (−42 %). BT users should use large diameter injection needles for reconstitution, apply two or three injection-aspiration-injection cycles and, maybe, shake the vials a few times to rinse the entire glass wall. Aggressive reconstitution with small diameter needles, prolonged injection-aspiration-injection and violent shaking should be avoided. © 2016, Springer-Verlag Wien. Source

Worbs S.,Robert Koch Institute | Fiebig U.,Robert Koch Institute | Zeleny R.,European Commission | Schimmel H.,European Commission | And 3 more authors.
Toxins | Year: 2015

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. © 2015 by the authors; licensee MDPI, Basel, Switzerland. Source

Toxogen Gmbh and Miprolab Gmbh | Date: 2012-07-18

This invention relates to a method of determining presence, amount and/or activity of a clostridial neurotoxin in a sample, the method comprising or consisting of the following steps: (a) bringing said sample into contact with a liposome, said liposome comprising (aa) at least one receptor on its outer surface, said receptor being capable of binding said neurotoxin and comprising or consisting of (i) a glycolipid and (ii) a peptide or protein; and (ab) a substrate in its interior, said substrate (i) being cleavable by the peptidase comprised in said neurotoxin and (ii) generating a detectable signal upon cleavage, said detectable signal preferably being generated by (1) the donor of a FRET pair, said donor exhibiting increased fluorescence upon cleavage by said peptidase, (2) a luminescent compound formed upon said cleavage, or (3) an enzyme formed upon said cleavage; and (b) determining whether an increase in signal occurs as compared to the absence of said sample, wherein such increase is indicative of the presence of said neurotoxin and/or the degree of such increase is indicative of the amount and/or activity of said neurotoxin in said sample.

The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively. © 2015 by the authors; licensee MDPI, Basel, Switzerland. Source

The invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by

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