First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil.
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.
Medeiros Caporale
Graciane Maria
GM
Rabies Diagnostic Laboratory, Instituto Pasteur de São Paulo, São Paulo, Brazil.
Rodrigues da Silva
Andréa de Cássia
Ade C
Peixoto
Zélia Maria Pinheiro
ZM
Chaves
Luciana Botelho
LB
Carrieri
Maria Luiza
ML
Vassão
Ruth Camargo
RC
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
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D012261:Ribonucleoproteins; D014764:Viral Proteins; D016650:Fluorescein-5-isothiocyanate