Damon C. Smith

An affinity purified ovine antivenom for the treatment of Vipera berus envenoming

A novel antivenom for treating patients with Vipera berus bite has been developed. Sheep are immunised monthly with relatively small amounts of Vipera berus (common adder) venom and the resultant antisera pooled. The immunoglobulin fraction is precipitated with sodium sulphate then cleaved with papain to produce Fab fragments. Finally, those Fab fragments that are directed specifically against components in the venom are purified by affinity chromatography on columns comprising V. berus venom coupled to cyanogen bromide activated Sepharose 4B. The resultant product is some three times more effective than the non-purified Fab in protecting mice against the lethal venom effects.

First clinical experiences with specific sheep Fab fragments in snake bite. Report of a multicentre study of Vipera berus envenoming.

Objectives. To evaluate the efficacy and safety of specific, ovine Fab fragments in the treatment of envenoming by the common adder, Vipera berus.

A comparison of ovine and equine antivenoms

Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venoms pharmacological effects (haemolysis, platelet aggregation and coagulation), and in neutralizing phospholipase A2 activity. Comparison of one ovine and three equine F(ab)2 products raised against V. a. ammodytes venom showed that all were at least 95% pure; that all protected mice; and that all contained antibody populations directed against most components of V. a. ammodytes and V. b. berus venoms. The ovine antivenoms generally contained a higher concentration of specific antibodies than the equine products. Finally, the ovine antivenoms raised against E. ocellatus, V. lebetina deserti, V. b. berus, M. f. fulvius and N. naja venoms provided better in vivo protection to mice than the equine antivenoms, but the equine antivenoms to N. kaouthia and C. atrox were more protective than the ovine product.

Experimental assessment of a new, low-cost antivenom for treatment of carpet viper (Echis ocellatus) envenoming

Morbidity and mortality due to envenoming by the carpet viper (Echis ocellatus) in northern Nigeria remains unacceptably high and constitutes a severe economic and public health problem to the local farming community in particular. The only effective treatment of systemic envenoming is antivenom, but supplies are very limited as the little that is available is either too expensive, ineffective or both. Here, we describe a new ovine antivenom, designed both to be effective and to be available at low cost. The antivenom, a polyclonal ovine Fab preparation, provides superior protection, both in vivo and in vitro, to the best alternatives, the monospecific South African Institute of Medical Research antivenom and the polyspecific Pasteur Isper Africa antivenom. Fab fragments, which have the advantages of large volumes of distribution and, theoretically, low immuno-reactivity, are produced by a reusable solid-phase papain matrix which eliminates enzyme contamination of the product and reduces cost. The antivenom is lyophilised for increased stability and extended shelf-life in tropical climates where it is often impossible to keep such products cool.

A new antivenom to treat eastern coral snake (Micrurus fulvius fulvius) envenoming

An Fab based ovine antivenom has been prepared and compared both in vitro and in vivo with two commercial preparations. The product was found to be at least four times more effective on a weight basis. The increased potency, combined with the low incidence of side-effects associated with ovine Fab, should result in a safer, more effective antivenom.

Development and clinical application of immunoassays for European adder (Vipera berus berus) venom and antivenom

An ovine affinity purified Fab antivenom was used in a clinical trial in Sweden to treat European adder (Vipera berus berus) envenoming. Immunoassays were developed to measure V. b. berus venom and antivenom concentrations in clinical samples to help assess the efficacy of treatment. A radioimmunoassay (RIA) was developed, optimized and validated to measure plasma levels of V. b. berus venom and compared with a conventional ELISA. Both showed a similar variation of zero binding in biological samples and the results obtained correlated closely. However, the ELISA was quicker and more sensitive (0.8 compared with 2 micrograms/litre). Before administration of antivenom, V. b. berus venom concentrations in plasma ranged from 10 to 53 micrograms/litre; 12 hr after the Fab infusion, no patient had measurable levels. However, two patients had low venom levels 24 hr after treatment. ELISA and RIA were also developed, optimized and used to measure concentrations of free Fab in plasma. There was a biexponential fall of Fab concentration with a fast distribution phase (t 1/2 = 0.9 hr) and a slower elimination phase (t 1/2 = 18 hr). The amount of Fab excreted in urine was low.

Assessment of an ovine antivenom raised against venom from the desert black cobra (Walterinnesia aegyptia).

The desert black cobra (Walterinnesia aegyptia) is an elapid widely distributed throughout the deserts of Saudi Arabia and currently available antivenoms are ineffective in the treatment of its envenoming. Walterinnesia aegyptia venom was assessed for several of its physicochemical, enzymatic and biological characteristics. An antivenom was raised in sheep using a low-dose immunization schedule and digested with papain to provide Fab fragments. The antivenom neutralized all of the above enzymatic and biological activities and provided good protection in mice (ED50 0.25 g/kg), whereas the commercial polyspecific products showed only partial neutralization and did not protect mice.

Experimental evaluation of ovine antisera to Thai cobra (Naja kaouthia) venom and its α-neurotoxin

Conventional treatment of Naja kaouthia (Thai cobra) envenoming requires large volumes (up to 600 ml) of equine antivenom, which results in a high incidence of serum reactions. The inefficiency of the antivenom is assumed to be related to the high percentage (approx. 20%) of alpha-neurotoxin, a relatively weak and highly toxic immunogen, present in the native venom. First, antibodies to N. kaouthia venom were raised in sheep, which protected mice against challenge with whole venom. Second, ovine antibodies to the purified neurotoxin and to three different neurotoxin conjugates were developed and their neutralising abilities against either whole venom or neurotoxin were compared using murine ED50 tests. High titre antibodies, assessed by enzyme immunoassay and Western blot, were obtained from all four neurotoxin immunisation regimens. Neurotoxin conjugated to rabbit anti-sheep IgG produced the highest titres against both neurotoxin and whole venom. This antiserum provided protection against neurotoxin challenge but failed to protect against whole venom. Furthermore, the addition of neurotoxin antibodies to whole venom antiserum did not enhance the neutralisation efficacy of the latter. These findings raise the possibility that in mice other toxins apart from the neurotoxin may significantly contribute to the lethal effect of N. kaouthia venom.