Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies.

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. Potency testing of toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay which is associated with extreme suffering of large numbers of animals to ensure high precision. The mouse phrenic nerve-diaphragm assay is an ex vivo assay that closely mimics in vivo respiratory paralysis offering substantial refinement and reduction in the number of animals used. A range of botulinum antitoxin standards, one licenced product and experimental antitoxins were tested for neutralising potency using ex vivo hemidiaphragm assay and compared with in vivo determined activities. Overall, there was an excellent agreement between neutralising activity detected by the two assay systems and for each toxin serotype using only 4-7 replicates for each product (almost perfect concordance for type A antitoxins: ρ = 0.997, and substantial concordance for type B antitoxins: ρ = 0.991 and type E antitoxins: ρ = 0.964, respectively). These findings confirm that the mouse nerve-diaphragm preparation can provide a functional ex vivo replacement assay for specific, sensitive and precise assessment of toxin and antitoxin activity.

Measurement of botulinum types A, B and E neurotoxicity using the phrenic nerve-hemidiaphragm: improved precision with in-bred mice.

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. The current method for assessing the potency of botulinum toxin and antitoxins is the mouse LD50 assay. The mouse phrenic nerve-diaphragm assay is an in vitro assay that closely mimics in vivo respiratory paralysis. In this study, we have further improved the assay by using gelatin as a non-frothing alternative to albumin and investigated the effects of botulinum toxin serotypes A, B and E on phrenic nerve-hemidiaphragms from out-bred MF1 and in-bred Balb/c mice. Improved reproducibility was found with in-bred mice. Balb/c mice were also found to be much less sensitive to type B toxin perhaps indicating differences in the expression of receptor components. Hemidiaphragm preparations from Balb/c mice were approximately 7 times more sensitive to type A toxin and 7-12 times more sensitive to type E toxin relative to type B toxin. These findings indicate that when fully optimised the mouse nerve-diaphragm preparation can provide a functional in vitro model for accurate and reproducible assessment of toxin activity.

Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.

Botulinum type A toxin neutralisation by specific IgG and its fragments: a comparison of mouse systemic toxicity and local flaccid paralysis assays.

In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin neutralising activity of specific immunoglobulin G (IgG) and its fragments (F(ab')(2), Fab', Fab) purified from pentavalent botulinum antisera raised in goats. Each assay method was repeated on three separate occasions in mice and relative potencies calculated with respect to a type A equine reference antitoxin. The conventional assay, which measures the number of mice surviving typically after 72 or 96 h following the intraperitoneal administration of a mixture of toxin and antitoxin, gave the following order of potency IgG>F(ab')(2)>Fab'>Fab (6.8>4.7>3.5>2.6 IU/mg). Differences in potency are likely to be due to differences in the pharmacokinetics of the antitoxins, which are related to their molecular weight. The alternative local flaccid paralysis assay, where toxin and antitoxin are injected subcutaneously into the left inguinocrural region, gave results with a narrower range of activities: IgG>Fab'>F(ab')(2)>Fab (6.0>5.9>5.5>4.6 IU/mg). Comparison of the two assay methods showed no significant differences for IgG, F(ab')(2) or Fab', although the Fab fragment was significantly more potent in the non-lethal assay probably because of the reduced influence of antitoxin pharmacokinetics in this localised assay. These findings show that a local flaccid paralysis assay provides a less time consuming and more humane alternative to the lethal assay for the potency testing of botulinum IgG and F(ab')(2) antitoxins.

A review of WHO International Standards for botulinum antitoxins.

Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes currently defined (A-G). These toxins can cause a life threatening systemic toxicity whether through natural causes such as food poisoning, infant botulism, wound botulism, or through use as bio-terror agents (e.g. inhalational botulism). It was realised early on that standard reference botulinum antitoxins were required to reduce the variation between assays and ensure a consistent potency of therapeutic antitoxins and vaccines, and to define the serotype. This led to the International Unit being defined by the World Health Organisation (WHO) in the 1960s with the establishment of the first International Standards (IS) for serotypes A-F. Since then botulinum antitoxin ISs have been used world wide as the 'yard stick' to measure the neutralising potency of antitoxins. These primary WHO ISs are used to calibrate in house working reagents that are more extensively utilised. A definition of the International Unit for serotype G antitoxin has yet to be defined or accepted by the WHO and urgently needs addressing. However, before September 11th 2001 there was very little interest in botulinum antitoxin IS and as a result stocks of most of the original preparations are now completely exhausted or depleted and replacements long overdue. We have reviewed the extensive history and availability of the primary WHO ISs and interim materials. All type A and B antitoxin materials were recently assayed and their relative activities confirmed against the original IS preparations. The recent increase in demand for these materials has further exacerbated the shortage. We describe here the production and characterization of stable freeze dried potential candidate replacements along with a new prospective first IS for type G antitoxin. Available toxin A reference preparations are also briefly reviewed.

A protocol for 'enhanced pepsin digestion': a step by step method for obtaining pure antibody fragments in high yield from serum.

The digestion of ovine antiserum under acidic conditions (pH 3.5) by pepsin is highly effective at reducing all unwanted serum components to low molecular weight (< or =13 kDa) fragments while leaving the approximately 100-kDa F(ab')(2) intact. The pH is then raised to 6 to stop further digestion and the reaction mixture centrifuged or filtered to remove any insoluble contaminants. Next, unwanted low molecular weight fragments are removed by diafiltration with a 30-kDa nominal molecular weight cut-off membrane leaving an F(ab')(2) solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. Material of this purity is suitable for many applications but, since all the contaminants are highly acidic, they can be easily removed by passage down an anion-exchange column to yield F(ab')(2) that is essentially free from pepsin and aggregates with a typical purity of over 96% and yields of 16-19 g/l serum. When an antivenom was processed, approximately 78% of the original serum's toxin neutralising capacity was recovered. This simple, high yield protocol for processing serum to highly purified F(ab')(2) avoids the need for an initial or any subsequent salt precipitation step and can be utilised for either bench or large scale production. If required, a mild reducing agent may be used finally to create Fab fragments.

Enhanced pepsin digestion: a novel process for purifying antibody F(ab')(2) fragments in high yield from serum.

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only 'highly pure' immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (< or =13 kDa) fragments while leaving the approximately 100-kDa F(ab')(2) intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab')(2) solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab')(2) produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab')(2)/l serum. This novel, high yield method for processing serum to highly pure F(ab')(2) avoids salt precipitation and centrifugation and should be suitable for large-scale production.