title: First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil.
abstract: The laboratory tests recommended by the World Health Organization for detection of rabies virus and evaluation of specific antibodies are performed with fluorescent antibodies against the virus, the ribonucleoproteins (RNPs), or by monoclonal antibodies. In this study, we purified the rabies virus RNPs for the production of a conjugate presenting sensibility and specificity compatible with commercial reagents. The method employed for the purification of RNPs was ultracentrifugation in cesium chloride gradient, the obtained product being used for immunizing rabbits, from which the hyperimmune sera were collected. The serum used for conjugate production was the one presenting the highest titer (1/2,560) when tested by indirect immunofluorescence. The antibodies were purified by anion exchange chromatography (QAE-Sephadex A-50),conjugated to fluorescein isothiocyanate and separated by gel filtration (Sephadex G-50). The resulting conjugate presented titers of 1/400 and 1/500 when assayed by direct immunofluorescence (DIF) and simplified fluorescence inhibition microtest, respectively. Sensibility and specificity tests were performed by DIF in 100 central nervous system samples of different animal species, presenting 100% matches when compared with the commercial reagent used as standard, independent of the conservation state of the samples. The quality reached by our conjugate will enable the standardization of this reagent for use by the laboratories performing diagnosis of rabies in Brazil, contributing to the intensification of the epidemiological vigilance and research on this disease.
authors.affiliation: Rabies Diagnostic Laboratory, Instituto Pasteur de São Paulo, São Paulo, Brazil.
authors.forename: Graciane Maria
authors.initials: GM
authors.lastname: Medeiros Caporale
authors.forename: Andréa de Cássia
authors.initials: Ade C
authors.lastname: Rodrigues da Silva
authors.forename: Zélia Maria Pinheiro
authors.initials: ZM
authors.lastname: Peixoto
authors.forename: Luciana Botelho
authors.initials: LB
authors.lastname: Chaves
authors.forename: Maria Luiza
authors.initials: ML
authors.lastname: Carrieri
authors.forename: Ruth Camargo
authors.initials: RC
authors.lastname: Vassão
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D001938:Brazil; D002460:Cell Line; D006224:Cricetinae; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D008297:Male; D011817:Rabbits; D011818:Rabies / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D011820:Rabies virus / Q000276:immunology / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000276:immunology*; D014776:Virus Cultivation
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D012261:Ribonucleoproteins; D014764:Viral Proteins; D016650:Fluorescein-5-isothiocyanate