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.

Made available in DSpace on 2014-06-11T19:32:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08Bitstream added on 2014-06-13T19:22:54Z : No. of bitstreams: 1 arauco_emr_dr_botfmvz.pdf: 810943 bytes, checksum: 6acc5913059689bd4615fef648271508 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O néctar é coletado pelas operárias coletoras e transportado em sua vesícula nectarífera para a colméia. Durante a sua transformação em mel, o néctar recebe enzimas provenientes das glândulas hipofaringeanas, as quais atuam em seu processamento. A invertase é uma das enzimas adicionadas ao néctar e promove a inversão da sacarose em frutose e glicose. Com o objetivo de se conhecer o pH ideal dessa enzima nas abelhas operárias foram extraídas as glândulas hipofaringeanas de abelhas com oito dias de idade. O método utilizado para a dosagem da invertase foi o do ácido 3-5þ dinitrosalicílico (DNS) para determinação de açúcares redutores, utilizando-se medida de leitura espectrofotométrica em absorbância da glicose em comprimento de onda (?) igual a 540nm. De acordo com os resultados, verificou-se que a atividade catalítica da enzima foi mais eficiente no pH 5,0 e em 40 minutos de reação em comparação com outros valores de pH e tempos de reação. Não foi possível a reutilização das glândulas. Desta forma, concluiu-se que a enzima invertase aumentou sua eficiência em função do tempo de reação de forma linear e significativa em pH 5,0. / The nectar is collected by the collecting workers that carry it in its nectar gallbladder to the beehive. During its transformation in honey, the nectar receives enzymes proceeding from the hypopharyngeal glands, which act in its processing. Invertase is one of enzymes added to the nectar and promotes the inversion of sacarose in fructose and glucose. With the aim of knowing the ideal pH, in the worker bees, the bee's hypopharyngeal glands with eight days of age had been extracted. The used method, for the dosage of invertase, had been the 3-5 ' dinitrosalisilic acid for determination of reducing sugars, using absorbance spectrophotometry data from the glucose in ? equal to 540nm. According to the results, it had been verified that the enzyme catalytic activity had been more efficient in pH 5,0 and in 40 minutes of reaction, comparing to other pH values and times of reaction. The glands reuse had not been possible. At this way, it was concluded that the invertase enzyme had increased its efficiency by the reaction time at linear and significant way in pH 5,0.

Resumo: O néctar é coletado pelas operárias coletoras e transportado em sua vesícula nectarífera para a colméia. Durante a sua transformação em mel, o néctar recebe enzimas provenientes das glândulas hipofaringeanas, as quais atuam em seu processamento. A invertase é uma das enzimas adicionadas ao néctar e promove a inversão da sacarose em frutose e glicose. Com o objetivo de se conhecer o pH ideal dessa enzima nas abelhas operárias foram extraídas as glândulas hipofaringeanas de abelhas com oito dias de idade. O método utilizado para a dosagem da invertase foi o do ácido 3-5þ dinitrosalicílico (DNS) para determinação de açúcares redutores, utilizando-se medida de leitura espectrofotométrica em absorbância da glicose em comprimento de onda (?) igual a 540nm. De acordo com os resultados, verificou-se que a atividade catalítica da enzima foi mais eficiente no pH 5,0 e em 40 minutos de reação em comparação com outros valores de pH e tempos de reação. Não foi possível a reutilização das glândulas. Desta forma, concluiu-se que a enzima invertase aumentou sua eficiência em função do tempo de reação de forma linear e significativa em pH 5,0. / Abstract: The nectar is collected by the collecting workers that carry it in its nectar gallbladder to the beehive. During its transformation in honey, the nectar receives enzymes proceeding from the hypopharyngeal glands, which act in its processing. Invertase is one of enzymes added to the nectar and promotes the inversion of sacarose in fructose and glucose. With the aim of knowing the ideal pH, in the worker bees, the bee's hypopharyngeal glands with eight days of age had been extracted. The used method, for the dosage of invertase, had been the 3-5 ' dinitrosalisilic acid for determination of reducing sugars, using absorbance spectrophotometry data from the glucose in ? equal to 540nm. According to the results, it had been verified that the enzyme catalytic activity had been more efficient in pH 5,0 and in 40 minutes of reaction, comparing to other pH values and times of reaction. The glands reuse had not been possible. At this way, it was concluded that the invertase enzyme had increased its efficiency by the reaction time at linear and significant way in pH 5,0. / Orientador: Sílvia Regina Cunha Funari / Coorientador: Carlos Ducatti / Doutor