This research was undertaken in order to design techniques and processes that enable the manufacture of effective antivenoms. To prepare a broad specificity anti venom for European vipers from chicken yolk it was first necessary to develop a simple effective method to extract avian immunoglobulin (lgY). A specific fluoroimmunoassay was developed to monitor IgY recovery and serum IgY levels in immunised hens. The most promising extraction methods from the literature were compared using a triglyceride kit to monitor lipoprotein removed and SDS-PAGE and ELISA to monitor purity and activity respectively. Caprylic acid followed by ammonium sulphate proved the best method. Unfortunately only low levels of specific IgY were achieved and it was necessary to include an affinity purification step to demonstrate their effectiveness in an EDso test. Pepsin, papain and trypsin all produced Fab' fragments from IgY but only pepsin digested the resultant Fc fragments. Pepsin could also digest other proteins in egg yolk, thereby avoiding the need to salt fractionate IgY prior to its digestion with a consequent improvement in the recovery of Fab'. A small scale affinity purification (SSAP) assay was developed, characterised and used to determine specific antibody levels in ovine antisera. Small doses (l5IAg) of venom produced significant specific levels but larger doses produced a better response and were used to produce anti venom. Binding studies with SSAP demonstrated a high concentration of specific antibodies in V.latastei antisera that bind to components in the venoms of other European vipers. A specific ovine F(ab')2-based V.latastei antivenom approximately twice as potent as the anti venom used currently in Spain was prepared from the ovine antisera. Evidence is presented that SSAP should supersede manual ELISA for assessing specific antibody levels in antisera. No major gain in recovery and purity resulted from processing whole blood rather than serum for preparing antivenom.

Large volumes of antisera were generated against Apis melli/era venom with which to develop a novel, platform technology for the inexpensive production of anti venoms. 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 - 199 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 (ImgIL) were stimulatory while high concentrations (IOmg/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 ED so neutralising power and was - twice as effective as the Merck antivenom.

O escorpião amarelo é uma das principais espécies de interesse médico no Brasil e o tratamento recomendado em caso de acidentes é o uso do antiveneno. Pouco se sabe sobre os componentes proteolíticos de seu veneno e seus efeitos no envenenamento. O objetivo deste trabalho foi isolar peptidases do veneno de T. serrulatus e as caracterizar bioquimicamente, além de avaliar o potencial neutralizante de antivenenos. O veneno total foi caracterizado, buscando atividades proteolíticas relevantes. Após isso, foram isoladas duas metalloserrulases (ms3 e ms4) e uma ACE-like do veneno. Estudos bioquímicos como determinação de temperatura e pHs ótimos, influência de sais, determinação de constantes catalíticas e pontos de hidrólise dos substratos foram determinados para as proteases purificadas. Conclui-se que ativação/inativação de peptídeos bioativos in vitro pelas proteases são informações importantes e que deverão continuar sendo estudadas no futuro. / The yellow scorpion is one of the main species of medical interest in Brazil and the recommended treatment in case of accidents is the use of antivenom. Little is known about the proteolytic components of its venom and its effects on envenomation. The objective of this work was to isolate peptidases from T. serrulatus venom and to characterize them biochemically, besides to evaluating the neutralizing potential of antivenoms. The total venom was characterized, in searching for relevant proteolytic activities. After that, two metalloserrulases (ms3 and ms4) and one ACE-like venom were isolated. Biochemical studies such as determination of temperature and optimum pHs, influence of salts, determination of catalytic constants and hydrolysis points of the substrates were determined for the purified proteases. It was concluded that the activation / inactivation of bioactive peptides in vitro by proteases are important information and should be further studied in the future.

O escorpião amarelo é uma das principais espécies de interesse médico no Brasil e o tratamento recomendado em caso de acidentes é o uso do antiveneno. Pouco se sabe sobre os componentes proteolíticos de seu veneno e seus efeitos no envenenamento. O objetivo deste trabalho foi isolar peptidases do veneno de T. serrulatus e as caracterizar bioquimicamente, além de avaliar o potencial neutralizante de antivenenos. O veneno total foi caracterizado, buscando atividades proteolíticas relevantes. Após isso, foram isoladas duas metalloserrulases (ms3 e ms4) e uma ACE-like do veneno. Estudos bioquímicos como determinação de temperatura e pHs ótimos, influência de sais, determinação de constantes catalíticas e pontos de hidrólise dos substratos foram determinados para as proteases purificadas. Conclui-se que ativação/inativação de peptídeos bioativos in vitro pelas proteases são informações importantes e que deverão continuar sendo estudadas no futuro. / The yellow scorpion is one of the main species of medical interest in Brazil and the recommended treatment in case of accidents is the use of antivenom. Little is known about the proteolytic components of its venom and its effects on envenomation. The objective of this work was to isolate peptidases from T. serrulatus venom and to characterize them biochemically, besides to evaluating the neutralizing potential of antivenoms. The total venom was characterized, in searching for relevant proteolytic activities. After that, two metalloserrulases (ms3 and ms4) and one ACE-like venom were isolated. Biochemical studies such as determination of temperature and optimum pHs, influence of salts, determination of catalytic constants and hydrolysis points of the substrates were determined for the purified proteases. It was concluded that the activation / inactivation of bioactive peptides in vitro by proteases are important information and should be further studied in the future.