The potential misuse of botulinum neurotoxins (BoNTs) as biological weapons, the accidental intoxication through food and their increasing applications as therapeutic drugs for treatment of many neurological and non-neurological disorders require meaningful methods for detection and quantification. Because of their high potencies, the concentrations of deadly or clinically effective doses are far below the detection limit of most standard chemical detection methods. Furthermore, pure chemical methods do not differentiate between active and inactive BoNT or peptidic fragments thereof [
1]. Therefore, the method of first choice is a reliable bioassay that can detect BoNT concentrations in the low nanogram or upper picogram range. For decades, the mouse bioassay (MBA) determining the median lethal dose (MLD) of BoNT in mice represented the gold standard among various biological, chemical or immunological detection systems for BoNT [
1,
2]. The MLD can easily be calculated from the numbers of deceased
versus surviving mice after treatment with increasing doses of toxin [
3,
4,
5,
6] and has been accepted by pharmaceutical regulatory agencies worldwide as well as being implemented in official standard operating procedures like German DIN10102 [
7] and US Association of Official Analytical Chemists (AOAC Official Method of Analysis 977.26) [
8] for detection of BoNT in food. It comprises, however, many disadvantages. The MBA is costly, it lasts for a long period (up to 5 days) and, most important, many mice suffer from botulism and die painfully by respiratory failure due to flaccid paralysis of the diaphragm muscles which represents its major end point [
9]. The death of mice, however, could also be caused by a wide range of other systemic effects,
i.e., pneumonia or heart failure causing limited specificity and precision of the MBA. A shortened version of the MBA is the mouse time-to-death method employing high concentrations of BoNT which reduces the experimental time to a single day [
10]. Local paralysis assays like the digital abductions score (DAS) are sublethal and reflect the pharmacokinetics of intra muscularly injected BoNT, but still constitute an animal experiment and lack precision due to subjective read out. In contrast, the isolated mouse phrenic nerve (MPN) hemidiaphragm assay, an
ex vivo method examining the full physiological pharmacodynamic of BoNT by closely reproducing
in vivo respiratory failure, replaces the MBA since sacrificing animals, e.g., for scientific purposes, is by definition not an animal experiment. A precursor of the MPN test performed with rat organs was first published by Bülbring in 1946 [
11] and later adapted to tissue of mice [
12,
13]. The change of the species increased the sensitivity of the test dramatically. However, no difference in the paralytic half-time was observed, e.g., when phrenic nerve hemidiaphragm preparations from mice of outbred strain Naval Medical Research Institute (NMRI) and inbred strain C57BL/6 were compared [
14]. Furthermore, the MPN assay not only replaces animal experiments, it also reduces consumption of mice. Whereas LD
50 determination of a single BoNT by MBA requires at least 100 mice (10 BoNT dilutions for groups of 10 mice) [
4], the MPN assay requires less than 15 hemidiaphragm preparations. In addition, although only the left phrenic nerve is nicely exposed after opening the chest an experienced operator can also successfully dissect the right phrenic nerve despite being located behind vital organs and closely attached to main blood vessels. Hereby, the use of left and right hemidiaphragms further halves the consumption of animals. After the application of BoNT to an organ bath in which the MPN preparation has been mounted, the contraction amplitude of the indirectly stimulated muscle declined after a short lag time continuously in a characteristic sigmoidale pattern until complete paralysis occurs (
Figure 1). The contractions of the hemidiaphragm are recorded via a force transducer and appropriate hard- and software for analysis over time. The time period between application of BoNT into the organ bath and the time point when the contraction amplitude is halved to its original value (paralysis time or
t1/2) is the read out to determine the presence of BoNT, its efficacy and potency as well as its concentration in comparison to BoNT standard material. It has been demonstrated that the paralysis time correlates with the toxicity (MLD, LD
50, Units) determined by the MBA [
15]. Thus, the MPN is precise, reduces consumption of animals, replaces animal experiments, and hence represents a superior substitute for the mouse bioassay [
16]. Therefore, it is listed, e.g., as an alternative method for assaying pharmaceutical preparations of injectable BoNT/A in the European Pharmacopoeia [
17]. In addition, the MPN assay has been used in numerous scientific publications to decipher the mechanism of action of botulinum neurotoxins, identify its cellular receptors and screen for inhibitors of BoNT [
13,
14,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36]. Furthermore, the MPN assay is suitable to identify BoNT serotypes employing neutralizing antitoxins, characterize neutralizing antibodies as pharmaceutical countermeasure against botulism and measure the occurrence of neutralizing anti-BoNT antibodies in patients’ sera treated with BoNT pharmaceuticals [
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
49,
50,
51,
52]. The MPN has also been proven in the real world. The detection of BoNT/B in ham has helped physicians to find the right diagnosis (BoNT/B poisoning) and appropriate treatment in an unclear human case marked by neurological symptoms [
53].
Figure 1.
Time course of hemidiaphragm paralysis caused by BoNT/A. The amplitude of muscle contraction in mN represents the difference between recorded basal and maximal tensions. All samples were administered at
t0 = 300 s. Addition of 0.1% BSA/PBS lead to spontaneous reduction of only ~35% over a period of 4 h. Upon administration of 500 pg/mL BoNT/A the amplitude remained unchanged for a dose dependent latent period and then decayed in a steep sigmoidale curve down to zero. The time period between application
t0 and the inflection point when the contraction amplitude halved, the so called paralysis time
t1/2, was used for construction of the calibration curves (
Figure 2). Whereas 500 pg/mL BoNT/A yielded
t1/2 = 51 min, thermal denaturation or neutralization by serotype-specific antiserum of BoNT/A did not cause paralysis, but only led to spontaneous partial reduction of the contraction amplitude similar to the negative control sample.