Russell G A Jones - Academia.edu (original) (raw)

Papers by Russell G A Jones

Biologicals, Sep 30, 2006

Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes curren... more Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes currently defined (AeG). 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 AeF. 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.

Journal of Immunological Methods, May 1, 2002

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should c... more 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 (V 13 kDa) fragments while leaving the f 100-kDa F(abV) 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(abV) 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(abV) 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(abV) 2 /l serum. This novel, high yield method for processing serum to highly pure F(abV) 2 avoids salt precipitation and centrifugation and should be suitable for large-scale production.

Journal of pharmaceutical and biomedical analysis, Jan 29, 2015

Highly purified specific Fab antibody fragments derived from sheep have a long history of therape... more Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab')2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab')2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the mole...

Journal of Pharmaceutical and Biomedical Analysis, Jan 5, 2016

Highly purified specific Fab antibody fragments derived from sheep have a long history of therape... more Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab′)2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab′)2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the molecules and mapped onto the individual models. A theoretical order of digestion was subsequently constructed, which appeared to agree with the experimental data, suggesting an accurate prediction model for ovine IgG1 and IgG2 subclasses. These findings lay the foundations for a more detailed analysis of pepsin cleavage fragments in the future. Additionally, the F(ab′)2 generated following pepsin digestion were predicted to contain subclass unique C-terminal octapeptide neoepitopes, despite the high 89% sequence identity of the intact gamma-1 and gamma-2 chain constant regions. These neoepitopes have the potential to be utilised for identification purposes once confirmed experimentally.

Critical Reviews in Biotechnology, Jan 1, 2016

Therapeutic antibodies provide important tools in the "medicine chest" of today's clinician for t... more Therapeutic antibodies provide important tools in the "medicine chest" of today's clinician for the treatment of a range of disorders. Typically monoclonal or polyclonal antibodies are administered in large doses, either directly or indirectly into the circulation, via a systemic route which is well suited for disseminated ailments. Diseases confined within a specific localized tissue, however, may be treated more effectively and at reduced cost by a delivery system which targets directly the affected area. To explore the advantages of the local administration of antibodies, we reviewed current alternative, non-systemic delivery approaches which are in clinical use, being trialed or developed. These less conventional approaches comprise: (a) local injections, (b) topical and (c) peroral administration routes. Local delivery includes intra-ocular injections into the vitreal humor (i.e. Ranibizumab for age-related macular degeneration), subconjunctival injections (e.g. Bevacizumab for corneal neovascularization), intra-articular joint injections (i.e. anti-TNF alpha antibody for persistent inflammatory monoarthritis) and intratumoral or peritumoral injections (e.g. Ipilimumab for cancer). A range of other strategies, such as the local use of antibacterial antibodies, are also presented. Local injections of antibodies utilize doses which range from 1/10th to 1/100th of the required systemic dose therefore reducing both side-effects and treatment costs. In addition, any therapeutic antibody escaping from the local site of disease into the systemic circulation is immediately diluted within the large blood volume, further lowering the potential for unwanted effects. Needle-free topical application routes become an option when the condition is restricted locally to an external surface. The topical route may potentially be utilized in the form of eye drops for infections or corneal neovascularization or be applied to diseased skin for psoriasis, dermatitis, pyoderma gangrenosum, antibiotic resistant bacterial infections or ulcerated wounds. Diseases confined to the gastrointestinal tract can be targeted directly by applying antibody via the injection-free peroral route. The gastrointestinal tract is unusual in that its natural immuno-tolerant nature ensures the long-term safety of repeatedly ingesting heterologous antiserum or antibody materials. Without the stringent regulatory, purity and clean room requirements of manufacturing parenteral (injectable) antibodies, production costs are minimal, with the potential for more direct low-cost targeting of gastrointestinal diseases, especially with those caused by problematic antibiotic resistant or toxigenic bacteria (e.g. Clostridium difficile, Helicobacter pylori), viruses (e.g. rotavirus, norovirus) or inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease). Use of the oral route has previously been hindered by excessive antibody digestion within the gastrointestinal tract; however, this limitation may be overcome by intelligently applying one or more strategies (i.e. decoy proteins, masking therapeutic antibody cleavage sites, pH modulation, enzyme inhibition or encapsulation). These aspects are additionally discussed in this review and novel insights also provided. With the development of new applications via local injections, topical and peroral routes, it is envisaged that an extended range of ailments will increasingly fall within the clinical scope of therapeutic antibodies further expanding this market.

Journal of Medical Microbiology, Jun 1, 2013

Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks o... more Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. The active dichain neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the Carboxy-terminal end consisting of a receptor binding (HC) domain, while the Amino-terminal (HN) domain is linked by a critical disulfide bond to a light chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally used to confirm the presence of toxin in serum or food, its sensitivity is insufficient to detect low toxin levels in approximately 30 to 60% of botulism patients. A novel FDC (Functional Dual Coating) microtitre plate immuno-biochemical assay, which quantitates botulinum toxicity by measuring the HC (receptor binding) domain linked with L-chain endopeptidase activity, was modified to allow human serum (lysed or unlysed) to be tested without interference from the matrix, with toxin detection down to 0.03 mouse LD50 ml-1 serum or 0.13 pg ml-1 using just 100 µl of clinical samples. The assay was specific for type A toxin and could additionally be applied to whole blood and food samples. Low levels of 1 to 2 mouse LD50 ml-1 serum of type A toxin were quantitated for the first time using the modified FDC assay in two severely intoxicated UK patients that required mechanical ventilation and antitoxin. Toxin levels in recovered food sample extracts were also detected and one MBA negative sample found to contain 0.32 LD50 ml-1 extract. The FDC assay provides a real alternative for Public Health Laboratories to unambiguously confirm all cases of type A botulism and, due to its sensitivity, a promising new tool in toxin pharmacokinetic studies.

The Botulinum J, 2013

Aims: The mouse bioassay (MBA) is the biologically relevant gold standard method for assessment o... more Aims: The mouse bioassay (MBA) is the biologically relevant gold standard method for assessment of BoNTs. However, the aspiration for replacement, reduction, and refinement – the 3Rs principles – of animal use in potency assays entail development of alternative methods. Pluripotent embryonic stem cells provide a continuous source of appropriate cells that allow evaluation of receptor binding, internalisation, translocation, and catalytic activity of BoNTs. We aim to develop a cell-based assay that captures all biological functions of BoNTs and is equally sensitive as the MBA.
Methods: Directed differentiation of murine ES cells, strain E14TG2a, into motoneurons was achieved in two steps: 1) embryoid bodies (EBs) were generated and treated with RA (1 μM), SHH (100 ng/ml) and purmorphamine (1 μM); 2) dissociated EBs were differentiated on laminin coated plates in neurobasal medium. Mixed population of mature cholinergic and adrenergic motoneurons were identified by immunolabeling against HB9, MAP-2, tau, synaptophysin, PSD-95, choline-acetyltransferase, and noradrenaline. We demonstrate that they express BoNT/A receptor SV2A and its intracellular target SNAP25. Motoneurons were intoxicated with a range of LD50 values of purified (Metabiologics) and formulated (NIBSC reference) BoNT/A1 samples for 12 to 72 hours. Time and dose dependent changes in the level of cleaved intracellular SNAP25 were evaluated using in-house developed sandwich-ELISA assay: 96 well ELISA plates were coated overnight with affinity purified capture Ab (-amino acids 190-197) specific to epitope exposed upon BoNT/A cleavage of intact SNAP25. Total cell lysates were prepared and added to plates for 90 minutes. Binding of cleaved SNAP25 to coated Ab was detected by addition of a mixture of sheep anti-SNAP25 Abs directed to SNAP25 amino acids 1–57 and 111–157, and reaction was visualised with commercially sourced rabbit anti-sheep HRP conjugated Ab.
Results: Differentiation procedure was optimised to enhance sensitivity of cells to BoNT/A. Optimal sensitivity was achieved when 50,000 cells/well in 96-well plates were cultured for nine days and intoxicated for 48 hours. The preliminary results indicate that intoxication with purified (Metabiologics) BoNT/A1 results in dose dependent proteolysis of SNAP25 respectively within 12 h, 24 h, 48 h and 72 h with EC50s of 10, 3.5, 1, and 0.7 LD50 mL, respectively.
Keywords: botulinum neurotoxins; bioassay; SNA; proteolysis.

Analytical Biochemistry, Mar 6, 2012

Conventional capture (“Sandwich”) ELISAs equally detect denatured inactive and native active botu... more Conventional capture (“Sandwich”) ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtitre plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin’s Hc domain, it was possible to develop a highly sensitive (130 Attomolar LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g. 1.2M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favourable buffer containing zinc and DTT reduced the inter-chain disulphide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1–206) substrate. A neoepitope antibody specific for the newly exposed Q197 epitope was used to quantify the cleaved SNAP25(1–197). The assay’s requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments and stressed samples containing partially or fully denatured material. This is the first known immuno-biochemical assay that correlates with in vivo potency and provides a realistic alternative.

Toxicon, Jun 2008

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Tr... more Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylating with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude, to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600 times higher than those produced with formaldehyde toxoid, despite producing equivalent ELISA anti-toxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxin's structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins.

PhD thesis, 2001

Large volumes of antisera were generated against Apis mellifera venom with which to develop a nov... more Large volumes of antisera were generated against Apis mellifera venom with which to develop a novel, platform technology for the inexpensive production of antivenoms. The ovine sera contained high levels of specific antibodies which neutralised the myotoxic, phospholipase A2 and in vivo activities of the venom.
Methods of processing the antisera to provide Fab or F(ab')2 were investigated. F(ab')2 was thought to be clinically advantageous and, by determining the conditions necessary for the preferential breakdown of Fc and serum components other than F(ab')2, it was possible to avoid salt precipitation. Diafiltration was then used to remove most of the unwanted small fragments and anion-exchange chromatography to remove any remaining acidic impurities such as pepsin and large aggregates. The F(ab')2 was 97% pure and the yield 19g per L of serum. This is the first specific therapy for mass envenoming by European or Africanised bees.
Spiders of the genus Latrodectus (black widows) are distributed widely and about 2,500 bites are reported annually in the USA. The neurotoxic effects of the venom were studied on the isolated phrenic nerve diaphragm preparation. Low venom concentrations (1mg/L) were stimulatory while high concentrations (10mg/L) caused nerve blockade which was potentiated by increased calcium levels.
Although effective, the Merck antivenom, which is unprocessed horse serum, causes unacceptable risks. The second purpose of this project was to prepare an improved Latrodectus spider antivenom using the new platform technology.
Different immunisation schedules were studied to optimise the humoral immune response. Sheep immunised with 2mg La. hesperus venom produced the highest levels of specific antibodies as assessed by ELISA, using the isolated nerve diaphragm preparation or in vivo in mice. The new process provided a pure F(ab')2 antivenom retaining 78% of the original antisera ED50 neutralising power and was twice as effective as the Merck antivenom.

Toxicon, Jul 1996

Brown snakes (Pseudonaja ssp.) are amongst the most deadly found in Australia, due in part, to th... more Brown snakes (Pseudonaja ssp.) are amongst the most deadly found in Australia, due in part, to the pre- (textilotoxin) and post-synaptic (pseudonajatoxin a and b) neurotoxins in their venom. Studies using the murine phrenic nerve hemidiaphragm preparation have shown the venom to produce rapid neurotoxic effects with no myotoxicity. We have developed a new ovine Fab based antivenom which, when pre-mixed with the venom neutralises, completely, all neurotoxins under all test conditions. We have also demonstrated, possibly for the first time, that an antivenom is capable of achieving a full and rapid ( < 1 hr), reversal of the dominant post-synaptic neurotoxic effects in vitro. Only under conditions favouring pre-synaptic phospholipase type neurotoxins, with increased temperature and frequency of nerve stimulation, was it possible to demonstrate the minor but essentially irreversible presynaptic effects of textilotoxin. From these results we concluded that venom neurotoxicity was due, predominantly, to a high affinity post-synaptic component which can be reversed rapidly by a high affinity specific antivenom.

Toxicon, Feb 1996

Brown snakes (Pseudonaja sp.) are among the most deadly found in Australia, the venom containing ... more Brown snakes (Pseudonaja sp.) are among the most deadly found in Australia, the venom containing both presynaptic and postsynaptic neurotoxins. The presynaptic component, textilotoxin, is thought to be the most potent and complex toxin isolated from a snake venom, while the predominant postsynaptic neurotoxin, pseudonajatoxin-a, binds irreversibly to acetylcholine receptors and is unusually large for a venom component of this type (12,280 mol. wt). A murine hemidiaphragm preparation has been used to show, for the first time, the rapid (<1 hr) in vitro reversal of the neurotoxicity produced by brown snake venom. Presynaptic and postsynaptic components were distinguished by increasing the temperature and frequency of nerve stimulation, and it was concluded that venom toxicity was predominantly of a postsynaptic nature, and was devoid of any myotoxic activity.

Toxicon, Feb 1996

Brown snakes (Pseudonaja sp.) are responsible for most snakebites and snakebite deaths in Austral... more Brown snakes (Pseudonaja sp.) are responsible for most snakebites and snakebite deaths in Australia. Such envenomation results in neurotoxicity and, usually, a defibrination coagulopathy caused by a potent prothrombin activator. Respiratory support usually alleviates the sequelae of neurotoxicity, so that coagulopathy and associated cerebral haemorrhage is the major cause of death. There are four clinically important brown snakes (P. affinis, P. textilis, P. nuchalis and P. inframacula), whose venoms exhibit wide variation in bioactivity. For example, P. inframacula is 80 times more potent at coagulating citrated human plasma than P. textilis and P. affinis, which are both much more potent than P. nuchalis. Such variations highlight the need for a broad spectrum antivenom. A new Fab-based polyvalent antivenom has been raised by immunizing sheep with a pool of the four clinically important venoms. Specific antibody levels were assessed by ELISA and small-scale affinity chromatography and 1 mg of venom per month was optimal. The antivenom is significantly more effective than a current commercial equine F(ab)΄2 equivalent, and murine ED50 i.v. studies demonstrated that it provides considerable protection. Clinical trials are planned.

Toxicon, May 1995

There are currently five distinct subspecies of Russell’s viper found erratically distributed fro... more There are currently five distinct subspecies of Russell’s viper found erratically distributed from Pakistan (Daboia russelli russelli) in the west to China (D. russelli siamensis) and Taiwan (D. russelli formosensis) in the east. These snakes are clinically best known for their action of producing coagulation abnormalities (via the activation of factors V and X). However, there are some major differences between the symptoms produced by the different subspecies, such as acute renal failure, which is most frequently found in Burma and Sri Lanka. In Sri Lanka, however, there is also a neurotoxic action associated with bites by Daboia russelli pulchella. In Sri Lanka this snake kills more people than does any other species, with symptoms attributed to neurotoxicity being the most common sign of systemic envenoming. The two antivenoms used in Sri Lanka are imported from India and as a result raised against the native snakes of India (e.g. D. russelli russelli). This has resulted in the relative ineffectiveness of antivenoms such as those produced by the Haffkine Biopharmaceutical Corporation which is the major antivenom used in Sri Lanka. At Therapeutic Antibodies Inc. (TAb) we have developed a low-cost ovine monospecific Fab antivenom raised against D. russelli pulchella venom that is papain free, using a new solid-phase papain digestion system. In order to assess this new antivenom, an in vitro assay demonstrating the neurotoxic action of this venom was required. The isolated left mouse phrenic nerve-hemidiaphragm preparation was chosen, and by the application of 12.5 mg/litre of D. russelli pulchella venom, it was possible to distinguish clearly between the neurotoxic and myotoxic actions of this venom over the course of a 3 hr period. The venom produced a definite neurotoxic action at lower doses (12.5−25 mg/litre), but at higher doses (50 mg/litre) the neurotoxic action became almost indistinguishable from the rapid myotoxic action. By premixing either the Haffkine (2000 mg/litre) or TAb (500 mg/litre) antivenoms with 25 mg/litre of D. r. pulchella venom it was possible to show that the TAb antivenom was considerably better at neutralizing neurotoxicity using a dose of 1/4 that of the Haffkine antivenom. In vivo neutralization using the standard mouse ED50 test showed that the TAb antivenom produced an ED50 of 1.5 mg/mouse (against 5 x LD50 = 1 mg/mouse); however, the Haffkine product proved totally ineffective at the maximum dose of 16 mg/mouse. Owing to the inherently limited nature of bioassays, in the near future the new TAb antivenom will be compared clinically with the existing antivenoms found in Sri Lanka.

British Journal of Pharmacology, Feb 1999

Brown snake (Pseudonaja) venom has been reported to produce 'irreversible' post synaptic neurotox... more Brown snake (Pseudonaja) venom has been reported to produce 'irreversible' post synaptic neurotoxicity (Harris & Maltin, 1981; Barnett et al., 1980). A murine phrenic nerve/diaphragm preparation was used to study the neurotoxic effects of this venom and pre- and post-synaptic components were distinguished by varying the temperature and frequency of nerve stimulation. There were no myotoxic effects and the neurotoxicity proved irreversible by washing alone. The effects of a new Fab based ovine antivenom have been investigated and proved able to produce a complete, rapid (< 1 h) reversal of the neurotoxicity induced by Brown snake venom. A reversal was also possible when the antivenom addition was delayed for a further 60 min. We believe that this is the first time such a reversal has been shown.

The American Journal of Tropical Medicine and Hygiene, Sep 1999

The frequency of mass bee attacks has dramatically increased in the Americas following the introd... more The frequency of mass bee attacks has dramatically increased in the Americas following the introduction and spread of the aggressive Africanized 'killer' bee (Apis mellifera scutellata). As yet no specific therapy is available, which led us to develop an ovine Fab-based antivenom as a potential new treatment. Sera from sheep immunized against the venom contained high levels of specific antibodies, as demonstrated by ELISA and by small-scale affinity chromatography, against both whole (A. m. mellifera) venom and purified melittin. A nerve muscle preparation was used to show the myotoxic effects of the venom and neutralization by the antivenom. Antivenom neutralizing ability was also demonstrated using assays for venom phospholipase A2 and in vivo activities. Venom from both European and Africanized bees appeared identical when analyzed by acid-urea gel electrophoresis. This antivenom may therefore provide the first specific therapy for the treatment of mass envenomation by either European or Africanized 'killer' bees.

Food and Bioproducts Processing, Jun 2002

A simple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for th... more A simple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for the facile preparation of antivenoms is described. The production of polyclonal F(ab′)2 fragments from serum commonly involves the initial purification of IgG's prior to their proteolytic cleavage and further purification. To reduce the number of processing steps the authors have compared the digestion of whole serum by free and immobilized pepsin with that of pure IgG. It was observed that with equal units of pepsin activity, caprylic acid pre-purifi ed IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was signifi cantly greater in the latter case. The effi ciencies of whole serum digestion by the same total units of free and immobilized pepsin was also compared. IgG digestion was complete after 12 hours, as was serum digestion. About 80% of the antigen binding activity was retained in either case after 24 hours though the immobilized pepsin lost about 40% of its proteolytic activity after first use. The purifi cation of F(ab′)2 on two ion exchange adsorbents (StreamlineTMSP and paramagnetic CM beads) on paramagnetic thiophilic beads and on a hydrophobic custom assembled guanidine agarose was studied. The binding capacity on CM beads was too low to be useful for F(ab′)2 preparation. Most of the F(ab′)2 bound to the Streamline SP at low pH (pH 4.0 and 3.0), as did significant amounts of low molecular weight digestion products. The F(ab′)2 fragments were eluted completely with 0.3M NaCl whilst the low molecular weight digestion products required 1M NaCl to elute them. F(ab′)2 also bound to the thiophilic beads, free from the low molecular weight digestion products, and could be eluted with low ionic strength solutions. The guanidine agarose beads showed a lower capacity than the thiophilic beads but their selectivity was comparable. Overall the thiophilic and hydrophobic matrices showed better selectivity than the cation exchange matrices.

Movement disorders : official journal of the Movement Disorder Society, 2004

After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only som... more After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only some of these will have the capacity to neutralise the toxin activity. In fact,
the ability of toxoid vaccine to induce toxin neutralising antibodies has provided the basis for the use of therapeutic antitoxins and immunoglobulins for the prophylaxis and treatment of diseases caused by bacterial toxins. Increasing indications for the chronic use of botulinum toxin for therapy have inevitably resulted in concern for patients becoming unresponsive because of the presence of circulating toxin-specific antibodies. Highly sensitive and relevant assays to detect only clinically relevant toxin neutralising antibodies are essential. Although immunoassays often provide the sensitivity, their relevance and specificity is often questioned. The mouse protection LD50 bioassay is considered most relevant but can often only detect 10 mIU/ml of antitoxin. This sensitivity, although sufficient for confirming protective immunity, is inadequate for patients undergoing toxin therapy. An intramuscular paralysis assay improves the sensitivity to ca. 1 mIU/ml, and a mouse ex vivo diaphragm assay, with sensitivity of
0.5 mIU/ml, is the most sensitive functional assay to date for this purpose. Alternative approaches for the detection of antibodies to botulinum toxin have included in vitro endopeptidase activity neutralisation. Unlike any other functional assay, this approach is not reliant on serotype-specific antibodies for specificity. Most recent promising developments are focused on cellular assays utilising
primary rat embryonic cord cells or more conveniently in vitro differentiated established cell lines such as human neuroblastoma cells.

Toxicon, 2006

In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin n... more 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.

Biologicals, Sep 30, 2006

Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes curren... more Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes currently defined (AeG). 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 AeF. 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.

Journal of Immunological Methods, May 1, 2002

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should c... more 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 (V 13 kDa) fragments while leaving the f 100-kDa F(abV) 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(abV) 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(abV) 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(abV) 2 /l serum. This novel, high yield method for processing serum to highly pure F(abV) 2 avoids salt precipitation and centrifugation and should be suitable for large-scale production.

Journal of pharmaceutical and biomedical analysis, Jan 29, 2015

Highly purified specific Fab antibody fragments derived from sheep have a long history of therape... more Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab')2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab')2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the mole...

Journal of Pharmaceutical and Biomedical Analysis, Jan 5, 2016

Highly purified specific Fab antibody fragments derived from sheep have a long history of therape... more Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab′)2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab′)2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the molecules and mapped onto the individual models. A theoretical order of digestion was subsequently constructed, which appeared to agree with the experimental data, suggesting an accurate prediction model for ovine IgG1 and IgG2 subclasses. These findings lay the foundations for a more detailed analysis of pepsin cleavage fragments in the future. Additionally, the F(ab′)2 generated following pepsin digestion were predicted to contain subclass unique C-terminal octapeptide neoepitopes, despite the high 89% sequence identity of the intact gamma-1 and gamma-2 chain constant regions. These neoepitopes have the potential to be utilised for identification purposes once confirmed experimentally.

Critical Reviews in Biotechnology, Jan 1, 2016

Therapeutic antibodies provide important tools in the "medicine chest" of today's clinician for t... more Therapeutic antibodies provide important tools in the "medicine chest" of today's clinician for the treatment of a range of disorders. Typically monoclonal or polyclonal antibodies are administered in large doses, either directly or indirectly into the circulation, via a systemic route which is well suited for disseminated ailments. Diseases confined within a specific localized tissue, however, may be treated more effectively and at reduced cost by a delivery system which targets directly the affected area. To explore the advantages of the local administration of antibodies, we reviewed current alternative, non-systemic delivery approaches which are in clinical use, being trialed or developed. These less conventional approaches comprise: (a) local injections, (b) topical and (c) peroral administration routes. Local delivery includes intra-ocular injections into the vitreal humor (i.e. Ranibizumab for age-related macular degeneration), subconjunctival injections (e.g. Bevacizumab for corneal neovascularization), intra-articular joint injections (i.e. anti-TNF alpha antibody for persistent inflammatory monoarthritis) and intratumoral or peritumoral injections (e.g. Ipilimumab for cancer). A range of other strategies, such as the local use of antibacterial antibodies, are also presented. Local injections of antibodies utilize doses which range from 1/10th to 1/100th of the required systemic dose therefore reducing both side-effects and treatment costs. In addition, any therapeutic antibody escaping from the local site of disease into the systemic circulation is immediately diluted within the large blood volume, further lowering the potential for unwanted effects. Needle-free topical application routes become an option when the condition is restricted locally to an external surface. The topical route may potentially be utilized in the form of eye drops for infections or corneal neovascularization or be applied to diseased skin for psoriasis, dermatitis, pyoderma gangrenosum, antibiotic resistant bacterial infections or ulcerated wounds. Diseases confined to the gastrointestinal tract can be targeted directly by applying antibody via the injection-free peroral route. The gastrointestinal tract is unusual in that its natural immuno-tolerant nature ensures the long-term safety of repeatedly ingesting heterologous antiserum or antibody materials. Without the stringent regulatory, purity and clean room requirements of manufacturing parenteral (injectable) antibodies, production costs are minimal, with the potential for more direct low-cost targeting of gastrointestinal diseases, especially with those caused by problematic antibiotic resistant or toxigenic bacteria (e.g. Clostridium difficile, Helicobacter pylori), viruses (e.g. rotavirus, norovirus) or inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease). Use of the oral route has previously been hindered by excessive antibody digestion within the gastrointestinal tract; however, this limitation may be overcome by intelligently applying one or more strategies (i.e. decoy proteins, masking therapeutic antibody cleavage sites, pH modulation, enzyme inhibition or encapsulation). These aspects are additionally discussed in this review and novel insights also provided. With the development of new applications via local injections, topical and peroral routes, it is envisaged that an extended range of ailments will increasingly fall within the clinical scope of therapeutic antibodies further expanding this market.

Journal of Medical Microbiology, Jun 1, 2013

Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks o... more Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. The active dichain neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the Carboxy-terminal end consisting of a receptor binding (HC) domain, while the Amino-terminal (HN) domain is linked by a critical disulfide bond to a light chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally used to confirm the presence of toxin in serum or food, its sensitivity is insufficient to detect low toxin levels in approximately 30 to 60% of botulism patients. A novel FDC (Functional Dual Coating) microtitre plate immuno-biochemical assay, which quantitates botulinum toxicity by measuring the HC (receptor binding) domain linked with L-chain endopeptidase activity, was modified to allow human serum (lysed or unlysed) to be tested without interference from the matrix, with toxin detection down to 0.03 mouse LD50 ml-1 serum or 0.13 pg ml-1 using just 100 µl of clinical samples. The assay was specific for type A toxin and could additionally be applied to whole blood and food samples. Low levels of 1 to 2 mouse LD50 ml-1 serum of type A toxin were quantitated for the first time using the modified FDC assay in two severely intoxicated UK patients that required mechanical ventilation and antitoxin. Toxin levels in recovered food sample extracts were also detected and one MBA negative sample found to contain 0.32 LD50 ml-1 extract. The FDC assay provides a real alternative for Public Health Laboratories to unambiguously confirm all cases of type A botulism and, due to its sensitivity, a promising new tool in toxin pharmacokinetic studies.

The Botulinum J, 2013

Aims: The mouse bioassay (MBA) is the biologically relevant gold standard method for assessment o... more Aims: The mouse bioassay (MBA) is the biologically relevant gold standard method for assessment of BoNTs. However, the aspiration for replacement, reduction, and refinement – the 3Rs principles – of animal use in potency assays entail development of alternative methods. Pluripotent embryonic stem cells provide a continuous source of appropriate cells that allow evaluation of receptor binding, internalisation, translocation, and catalytic activity of BoNTs. We aim to develop a cell-based assay that captures all biological functions of BoNTs and is equally sensitive as the MBA.
Methods: Directed differentiation of murine ES cells, strain E14TG2a, into motoneurons was achieved in two steps: 1) embryoid bodies (EBs) were generated and treated with RA (1 μM), SHH (100 ng/ml) and purmorphamine (1 μM); 2) dissociated EBs were differentiated on laminin coated plates in neurobasal medium. Mixed population of mature cholinergic and adrenergic motoneurons were identified by immunolabeling against HB9, MAP-2, tau, synaptophysin, PSD-95, choline-acetyltransferase, and noradrenaline. We demonstrate that they express BoNT/A receptor SV2A and its intracellular target SNAP25. Motoneurons were intoxicated with a range of LD50 values of purified (Metabiologics) and formulated (NIBSC reference) BoNT/A1 samples for 12 to 72 hours. Time and dose dependent changes in the level of cleaved intracellular SNAP25 were evaluated using in-house developed sandwich-ELISA assay: 96 well ELISA plates were coated overnight with affinity purified capture Ab (-amino acids 190-197) specific to epitope exposed upon BoNT/A cleavage of intact SNAP25. Total cell lysates were prepared and added to plates for 90 minutes. Binding of cleaved SNAP25 to coated Ab was detected by addition of a mixture of sheep anti-SNAP25 Abs directed to SNAP25 amino acids 1–57 and 111–157, and reaction was visualised with commercially sourced rabbit anti-sheep HRP conjugated Ab.
Results: Differentiation procedure was optimised to enhance sensitivity of cells to BoNT/A. Optimal sensitivity was achieved when 50,000 cells/well in 96-well plates were cultured for nine days and intoxicated for 48 hours. The preliminary results indicate that intoxication with purified (Metabiologics) BoNT/A1 results in dose dependent proteolysis of SNAP25 respectively within 12 h, 24 h, 48 h and 72 h with EC50s of 10, 3.5, 1, and 0.7 LD50 mL, respectively.
Keywords: botulinum neurotoxins; bioassay; SNA; proteolysis.

Analytical Biochemistry, Mar 6, 2012

Conventional capture (“Sandwich”) ELISAs equally detect denatured inactive and native active botu... more Conventional capture (“Sandwich”) ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtitre plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin’s Hc domain, it was possible to develop a highly sensitive (130 Attomolar LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g. 1.2M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favourable buffer containing zinc and DTT reduced the inter-chain disulphide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1–206) substrate. A neoepitope antibody specific for the newly exposed Q197 epitope was used to quantify the cleaved SNAP25(1–197). The assay’s requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments and stressed samples containing partially or fully denatured material. This is the first known immuno-biochemical assay that correlates with in vivo potency and provides a realistic alternative.

Toxicon, Jun 2008

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Tr... more Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylating with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude, to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600 times higher than those produced with formaldehyde toxoid, despite producing equivalent ELISA anti-toxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxin's structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins.

PhD thesis, 2001

Large volumes of antisera were generated against Apis mellifera venom with which to develop a nov... more Large volumes of antisera were generated against Apis mellifera venom with which to develop a novel, platform technology for the inexpensive production of antivenoms. The ovine sera contained high levels of specific antibodies which neutralised the myotoxic, phospholipase A2 and in vivo activities of the venom.
Methods of processing the antisera to provide Fab or F(ab')2 were investigated. F(ab')2 was thought to be clinically advantageous and, by determining the conditions necessary for the preferential breakdown of Fc and serum components other than F(ab')2, it was possible to avoid salt precipitation. Diafiltration was then used to remove most of the unwanted small fragments and anion-exchange chromatography to remove any remaining acidic impurities such as pepsin and large aggregates. The F(ab')2 was 97% pure and the yield 19g per L of serum. This is the first specific therapy for mass envenoming by European or Africanised bees.
Spiders of the genus Latrodectus (black widows) are distributed widely and about 2,500 bites are reported annually in the USA. The neurotoxic effects of the venom were studied on the isolated phrenic nerve diaphragm preparation. Low venom concentrations (1mg/L) were stimulatory while high concentrations (10mg/L) caused nerve blockade which was potentiated by increased calcium levels.
Although effective, the Merck antivenom, which is unprocessed horse serum, causes unacceptable risks. The second purpose of this project was to prepare an improved Latrodectus spider antivenom using the new platform technology.
Different immunisation schedules were studied to optimise the humoral immune response. Sheep immunised with 2mg La. hesperus venom produced the highest levels of specific antibodies as assessed by ELISA, using the isolated nerve diaphragm preparation or in vivo in mice. The new process provided a pure F(ab')2 antivenom retaining 78% of the original antisera ED50 neutralising power and was twice as effective as the Merck antivenom.

Toxicon, Jul 1996

Brown snakes (Pseudonaja ssp.) are amongst the most deadly found in Australia, due in part, to th... more Brown snakes (Pseudonaja ssp.) are amongst the most deadly found in Australia, due in part, to the pre- (textilotoxin) and post-synaptic (pseudonajatoxin a and b) neurotoxins in their venom. Studies using the murine phrenic nerve hemidiaphragm preparation have shown the venom to produce rapid neurotoxic effects with no myotoxicity. We have developed a new ovine Fab based antivenom which, when pre-mixed with the venom neutralises, completely, all neurotoxins under all test conditions. We have also demonstrated, possibly for the first time, that an antivenom is capable of achieving a full and rapid ( < 1 hr), reversal of the dominant post-synaptic neurotoxic effects in vitro. Only under conditions favouring pre-synaptic phospholipase type neurotoxins, with increased temperature and frequency of nerve stimulation, was it possible to demonstrate the minor but essentially irreversible presynaptic effects of textilotoxin. From these results we concluded that venom neurotoxicity was due, predominantly, to a high affinity post-synaptic component which can be reversed rapidly by a high affinity specific antivenom.

Toxicon, Feb 1996

Brown snakes (Pseudonaja sp.) are among the most deadly found in Australia, the venom containing ... more Brown snakes (Pseudonaja sp.) are among the most deadly found in Australia, the venom containing both presynaptic and postsynaptic neurotoxins. The presynaptic component, textilotoxin, is thought to be the most potent and complex toxin isolated from a snake venom, while the predominant postsynaptic neurotoxin, pseudonajatoxin-a, binds irreversibly to acetylcholine receptors and is unusually large for a venom component of this type (12,280 mol. wt). A murine hemidiaphragm preparation has been used to show, for the first time, the rapid (<1 hr) in vitro reversal of the neurotoxicity produced by brown snake venom. Presynaptic and postsynaptic components were distinguished by increasing the temperature and frequency of nerve stimulation, and it was concluded that venom toxicity was predominantly of a postsynaptic nature, and was devoid of any myotoxic activity.

Toxicon, Feb 1996

Brown snakes (Pseudonaja sp.) are responsible for most snakebites and snakebite deaths in Austral... more Brown snakes (Pseudonaja sp.) are responsible for most snakebites and snakebite deaths in Australia. Such envenomation results in neurotoxicity and, usually, a defibrination coagulopathy caused by a potent prothrombin activator. Respiratory support usually alleviates the sequelae of neurotoxicity, so that coagulopathy and associated cerebral haemorrhage is the major cause of death. There are four clinically important brown snakes (P. affinis, P. textilis, P. nuchalis and P. inframacula), whose venoms exhibit wide variation in bioactivity. For example, P. inframacula is 80 times more potent at coagulating citrated human plasma than P. textilis and P. affinis, which are both much more potent than P. nuchalis. Such variations highlight the need for a broad spectrum antivenom. A new Fab-based polyvalent antivenom has been raised by immunizing sheep with a pool of the four clinically important venoms. Specific antibody levels were assessed by ELISA and small-scale affinity chromatography and 1 mg of venom per month was optimal. The antivenom is significantly more effective than a current commercial equine F(ab)΄2 equivalent, and murine ED50 i.v. studies demonstrated that it provides considerable protection. Clinical trials are planned.

Toxicon, May 1995

There are currently five distinct subspecies of Russell’s viper found erratically distributed fro... more There are currently five distinct subspecies of Russell’s viper found erratically distributed from Pakistan (Daboia russelli russelli) in the west to China (D. russelli siamensis) and Taiwan (D. russelli formosensis) in the east. These snakes are clinically best known for their action of producing coagulation abnormalities (via the activation of factors V and X). However, there are some major differences between the symptoms produced by the different subspecies, such as acute renal failure, which is most frequently found in Burma and Sri Lanka. In Sri Lanka, however, there is also a neurotoxic action associated with bites by Daboia russelli pulchella. In Sri Lanka this snake kills more people than does any other species, with symptoms attributed to neurotoxicity being the most common sign of systemic envenoming. The two antivenoms used in Sri Lanka are imported from India and as a result raised against the native snakes of India (e.g. D. russelli russelli). This has resulted in the relative ineffectiveness of antivenoms such as those produced by the Haffkine Biopharmaceutical Corporation which is the major antivenom used in Sri Lanka. At Therapeutic Antibodies Inc. (TAb) we have developed a low-cost ovine monospecific Fab antivenom raised against D. russelli pulchella venom that is papain free, using a new solid-phase papain digestion system. In order to assess this new antivenom, an in vitro assay demonstrating the neurotoxic action of this venom was required. The isolated left mouse phrenic nerve-hemidiaphragm preparation was chosen, and by the application of 12.5 mg/litre of D. russelli pulchella venom, it was possible to distinguish clearly between the neurotoxic and myotoxic actions of this venom over the course of a 3 hr period. The venom produced a definite neurotoxic action at lower doses (12.5−25 mg/litre), but at higher doses (50 mg/litre) the neurotoxic action became almost indistinguishable from the rapid myotoxic action. By premixing either the Haffkine (2000 mg/litre) or TAb (500 mg/litre) antivenoms with 25 mg/litre of D. r. pulchella venom it was possible to show that the TAb antivenom was considerably better at neutralizing neurotoxicity using a dose of 1/4 that of the Haffkine antivenom. In vivo neutralization using the standard mouse ED50 test showed that the TAb antivenom produced an ED50 of 1.5 mg/mouse (against 5 x LD50 = 1 mg/mouse); however, the Haffkine product proved totally ineffective at the maximum dose of 16 mg/mouse. Owing to the inherently limited nature of bioassays, in the near future the new TAb antivenom will be compared clinically with the existing antivenoms found in Sri Lanka.

British Journal of Pharmacology, Feb 1999

Brown snake (Pseudonaja) venom has been reported to produce 'irreversible' post synaptic neurotox... more Brown snake (Pseudonaja) venom has been reported to produce 'irreversible' post synaptic neurotoxicity (Harris & Maltin, 1981; Barnett et al., 1980). A murine phrenic nerve/diaphragm preparation was used to study the neurotoxic effects of this venom and pre- and post-synaptic components were distinguished by varying the temperature and frequency of nerve stimulation. There were no myotoxic effects and the neurotoxicity proved irreversible by washing alone. The effects of a new Fab based ovine antivenom have been investigated and proved able to produce a complete, rapid (< 1 h) reversal of the neurotoxicity induced by Brown snake venom. A reversal was also possible when the antivenom addition was delayed for a further 60 min. We believe that this is the first time such a reversal has been shown.

The American Journal of Tropical Medicine and Hygiene, Sep 1999

The frequency of mass bee attacks has dramatically increased in the Americas following the introd... more The frequency of mass bee attacks has dramatically increased in the Americas following the introduction and spread of the aggressive Africanized 'killer' bee (Apis mellifera scutellata). As yet no specific therapy is available, which led us to develop an ovine Fab-based antivenom as a potential new treatment. Sera from sheep immunized against the venom contained high levels of specific antibodies, as demonstrated by ELISA and by small-scale affinity chromatography, against both whole (A. m. mellifera) venom and purified melittin. A nerve muscle preparation was used to show the myotoxic effects of the venom and neutralization by the antivenom. Antivenom neutralizing ability was also demonstrated using assays for venom phospholipase A2 and in vivo activities. Venom from both European and Africanized bees appeared identical when analyzed by acid-urea gel electrophoresis. This antivenom may therefore provide the first specific therapy for the treatment of mass envenomation by either European or Africanized 'killer' bees.

Food and Bioproducts Processing, Jun 2002

A simple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for th... more A simple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for the facile preparation of antivenoms is described. The production of polyclonal F(ab′)2 fragments from serum commonly involves the initial purification of IgG's prior to their proteolytic cleavage and further purification. To reduce the number of processing steps the authors have compared the digestion of whole serum by free and immobilized pepsin with that of pure IgG. It was observed that with equal units of pepsin activity, caprylic acid pre-purifi ed IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was signifi cantly greater in the latter case. The effi ciencies of whole serum digestion by the same total units of free and immobilized pepsin was also compared. IgG digestion was complete after 12 hours, as was serum digestion. About 80% of the antigen binding activity was retained in either case after 24 hours though the immobilized pepsin lost about 40% of its proteolytic activity after first use. The purifi cation of F(ab′)2 on two ion exchange adsorbents (StreamlineTMSP and paramagnetic CM beads) on paramagnetic thiophilic beads and on a hydrophobic custom assembled guanidine agarose was studied. The binding capacity on CM beads was too low to be useful for F(ab′)2 preparation. Most of the F(ab′)2 bound to the Streamline SP at low pH (pH 4.0 and 3.0), as did significant amounts of low molecular weight digestion products. The F(ab′)2 fragments were eluted completely with 0.3M NaCl whilst the low molecular weight digestion products required 1M NaCl to elute them. F(ab′)2 also bound to the thiophilic beads, free from the low molecular weight digestion products, and could be eluted with low ionic strength solutions. The guanidine agarose beads showed a lower capacity than the thiophilic beads but their selectivity was comparable. Overall the thiophilic and hydrophobic matrices showed better selectivity than the cation exchange matrices.

Movement disorders : official journal of the Movement Disorder Society, 2004

After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only som... more After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only some of these will have the capacity to neutralise the toxin activity. In fact,
the ability of toxoid vaccine to induce toxin neutralising antibodies has provided the basis for the use of therapeutic antitoxins and immunoglobulins for the prophylaxis and treatment of diseases caused by bacterial toxins. Increasing indications for the chronic use of botulinum toxin for therapy have inevitably resulted in concern for patients becoming unresponsive because of the presence of circulating toxin-specific antibodies. Highly sensitive and relevant assays to detect only clinically relevant toxin neutralising antibodies are essential. Although immunoassays often provide the sensitivity, their relevance and specificity is often questioned. The mouse protection LD50 bioassay is considered most relevant but can often only detect 10 mIU/ml of antitoxin. This sensitivity, although sufficient for confirming protective immunity, is inadequate for patients undergoing toxin therapy. An intramuscular paralysis assay improves the sensitivity to ca. 1 mIU/ml, and a mouse ex vivo diaphragm assay, with sensitivity of
0.5 mIU/ml, is the most sensitive functional assay to date for this purpose. Alternative approaches for the detection of antibodies to botulinum toxin have included in vitro endopeptidase activity neutralisation. Unlike any other functional assay, this approach is not reliant on serotype-specific antibodies for specificity. Most recent promising developments are focused on cellular assays utilising
primary rat embryonic cord cells or more conveniently in vitro differentiated established cell lines such as human neuroblastoma cells.

Toxicon, 2006

In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin n... more 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.