RESUMO
Considering the background of morbidity and mortality caused by human rotavirus, detection methods that use rotavirus group antigen (VP6) in either enzyme immunoassay (EIA) or latex agglutination test (LAT) has been employed routinely in clinical diagnostic and epidemiological studies. In order to develop a rapid and sensitive rotavirus group A LAT, part of segment 6 corresponding to conserved N-terminal portion of the VP6 (1-245 aa) was cloned in Escherichia coli expression pGEX vector (glutathione S-transferase-GST gene fusion system) that has been modified previously containing a histidine tail at C-terminus. The immunological propriety of the recombinant VP6 having a total molecular weight of 52 kDa was evaluated by Western blot and by the ability of inducing anti-recombinant VP6 polyclonal antibodies in rabbit. The polyclonal serum produced was conjugated to a latex support to detect rotavirus in stool specimens. The percentage values for sensitivity and specificity of the rotavirus group A LAT were 98.5% and 100%, respectively.
Assuntos
Anticorpos Antivirais , Testes de Fixação do Látex/métodos , Infecções por Rotavirus/diagnóstico , Rotavirus/isolamento & purificação , Animais , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Criança , Clonagem Molecular , Escherichia coli/genética , Fezes/virologia , Expressão Gênica , Humanos , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infecções por Rotavirus/virologia , Sensibilidade e EspecificidadeRESUMO
Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry.