Enterotoxina, estafilococica tipo D : produção, purificação e obtenção de antissoro

AUTOR(ES)
DATA DE PUBLICAÇÃO

1994

RESUMO

In this work at was development a new method - modification of the method of Chang and Bergdoll (29) - for de purification of staphylococcal enterotoxin D (SED), and a new method of rabbit s immunization for the preparation of specific SED antisserum. The proposed immunization procedure is faster and uses considerably less toxin than the method used by the Food Research Institute (FRIJ. The enterotoxin was produced by Strain Staphylococcus aureus 1151m, from the FRI, incubated in Fernbach flasks of 2,8 liters of capacity, containing tryptone broth 3%, added of yeast extrat to a final concentration of 1%. The medium final pH was adjusted to 6,8. The enteroroxin purification was monitored by vertical gel eletroforesis in the presence of molecular weight standards. This procedure allowed to control each stage of the purification process. On the others side serological method of the precipitation simple (Oudin) and double (modified Ouchterlony - OSP) allowed to determine toxin concentration. The total protein was determined by the Lowry method. The first chromatography through Amberlite CG-50 had a yield of 72%. The modification introduced in this work was at the second chromatography, where CM-Celulose was substituted by CM-Sepharose CL-6B, and the toxin recovery in this step was 46%. The third chromatography through Sephadex G-75 had ayield of 77%. The total yield of SED purification was 25%. To obtain the specific antisserum, a lot of 5 rabbits was inoculated, each animal in two steps, with a total toxin concentration of 100 µg, for a total 35 days period. This procedure has the advantage of using less oxin and less time that the procedure recommended in the literature. The antiserum titers are lower than the titers obtained by the traditional methodology, but high enough to be used as reagents for the sorological quantification of the toxin. The toxin was inoculated in the rabbit tight by the subcutaneous route in two steps, in the first injection 50 µg toxin was inoculated with complete Freund adjuvant, and 21 days later, 50 µg of toxin with incomplete Freund adjuvante. The animals were bleeded to death by cardiac puncture on the second week after the second injection or after the level of antibodies was high enough (above 16) to follow the procedure. The highers titer obtained was 32. The serum titer was determined by the OSP method which allowed al so to determine the toxin purity, this one by the appearance of a single precipitation line - against antisserum produced with crude toxin

ASSUNTO(S)

soros enterotoxinas

ACESSO AO ARTIGO

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