Produção de anticorpos policlonais para a pesquisa de produção de toxina diftérica por cepas de Corynebacterium diphtheriae

• Autor(a) principal: Brito, Wagner Oliveira
• Data de Publicação: 2016
• Outros Autores: [e-mail]
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/19965
• topic: Diphtheria; rabbit antibodies diphtheria antitoxin; SAD; Diphtheria toxin; Immunofluorescence; Corynebacterium diphtheriae; Difteria; Anticorpos de coelho antitoxina diftérica; SAD; Toxina diftérica; Imunofluorescência; Corynebacterium diphtheriae; CIENCIAS BIOLOGICAS::MICROBIOLOGIA

Despite the success of mass immunization in many countries, including in Brazil, diphtheria continues to be a potentially lethal resurgent infectious disease, which requires the use of diphtheria antitoxin (produced in horse) for treatment and diagnosis of toxin production. In this investigation the antiserum against diphtheria toxin was produced in rabbits and, after fractionation, immunoglobulin preparation was tested in parallel with horse antitoxin (SAD). Rabbits (n=2) were immunized with dimerized (non-toxic) diphtheria toxin. The experimental immunization was performed with consent of the University Experimental Animal Board (CEUA/033/2015). Clinical and reference strains were tested for the confirmation of species and toxin production by multiplex PCR. Reactivity against toxin produced by the strains was performed by phenotypic methods (Radial immunodiffusion and modified Elek tests). The capability of toxin neutralization was evaluated by VERO cell toxicity test. The detection of toxin in the surface of a toxin producer (ATCC 27012), and a non-toxin producer (ATCC 27010) strains was evaluated after biotinilation of Igs fraction, and revealed by fluorescence with streptavidin coupled to fluorescein. Horse antitoxin (SAD) presented many precipitation lines that can be mislead to toxin precipitin under modified Elek test, and many non-toxigenic strains gave positive results under radial immunodiffusion reaction analysis. An 80% discordance of toxin production result was observed by RID test amongst tox– strains, and 40% by Elek test, using SAD. The results with rabbit antibody fraction presented variable results for only one tox– strain (HC03). In general, a concordance of 90% was obtained by Elek test using rabbit antibody. The rabbit antibody fraction was capable to neutralize the toxin in a dilution of 1:1200 correlated to 0,1 IU of SAD. Preimmune sera of both rabbits were incapable to neutralize or react by Elek test. Biotinilated antibodies were capable to bind the tox+ ATCC 27012 strain, by fluorescence method. The rabbit antiserum rendered a more accurate reagent to identify tox+ C. diphtheriae strains, with a good neutralization titer. Biotinilated antibodies can be used for diagnosis of toxigenic C. diphtheriae strains