Fluorescent Antibody Staining of Street and Fixed Rabies Virus Antigens

Goldwasser, Robert A.; Kissling, R. E.

doi:10.3181/00379727-98-23996

Cited by 223 publications

(63 citation statements)

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“…7 The fluorescent antibody (FA) test for rabies diagnosis is a fast and reliable laboratory test. 6 However, one disadvantage is that ideally the diagnostic procedure requires unfixed (fresh or frozen) brain tissue.…”

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confidence: 99%

Immunoperoxidase Test for Rabies: Utility as a Diagnostic Test

Hamir

Moser

1994

J VET Diagn Invest

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Abstract. Although a fast and reliable fluorescent antibody (FA) test for rabies diagnosis is available, ideally the diagnostic procedure requires fresh or frozen brain tissues. In some instances (and particularly for retrospective studies), fresh or frozen tissues may not be available. In such cases, immunohistochemical tests may be utilized. Because such tests have been used only in a limited number of cases, their diagnostic reliability has not been thoroughly evaluated. This study documents the results of a streptavidin-biotin complex (ABC) immunoperoxidase test on formalin-fixed, paraffin-embedded brain tissues of domestic and wild animals that were positive on FA test for rabies prior to fixation. Thirty-nine of 40 rabies cases were positive with the ABC technique. Based on these preliminary results, the ABC technique appears reliable. However, more corroborative test results are needed before the ABC method can be used as a routine diagnostic procedure, especially with field samples and negative controls.The eastern USA is enzootic for raccoon (Procyon lotor) rabies. 1 Within this area, the disease is compartmentalized in wildlife and on many occasions has spilled over into the domestic animal population. 7 The fluorescent antibody (FA) test for rabies diagnosis is a fast and reliable laboratory test. 6 However, one disadvantage is that ideally the diagnostic procedure requires unfixed (fresh or frozen) brain tissue.the ABC technique for rabies antigen detection has not been used routinely, its reliability as a diagnostic test has not been documented. Therefore, the following study was conducted to correct this deficiency. The validity of the ABC technique for diagnosis of rabies was tested on a variety of domestic and wild animals that were previously found to be positive for rabies by the FA test. Recently, the Laboratory of Large Animal Pathology (University of Pennsylvania) was confronted with 2 instances where rabies was suspected in animals, but unfixed tissue samples were unavailable. In the first case, a puppy had bitten a child; because the dog was unvaccinated and was from a rabies-enzootic area, it was euthanized. The referring veterinarian removed the brain from the carcass and placed it in formalin. In the second case, a neurologically impaired cat from a nonenzootic area was euthanized and necropsied, and tissues were fixed in formalin for histopathology. Microscopic examination revealed an extensive nonsuppurative meningoencephalitis, and rabies was considered as a differential diagnosis. In both cases, only formalin-fixed tissues were available. Materials and methodsSeveral methods have been previously described for the detection of rabies antigen in formalin-fixed, paraffin-embedded tissue sections. 2-5,9,12 An immunoperoxidase method utilizing the streptavidin-biotin complex (ABC) has been used for a retrospective survey of rabies in equine necropsy cases 7 and for documenting the distribution of lesions and rabies antigen in tissues of raccoons that had contracted the disease. 8 Because...

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Immunoperoxidase Test for Rabies: Utility as a Diagnostic Test

Hamir

Moser

1994

J VET Diagn Invest

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“…El sistema implica una fuerte amplificación de la señal del antigeno buscado por el uso de los anticuerpos primario y secundario y por la incubación con el complejo enzima-avidina (3-5), de tal manera que, cantidades de antigeno indetectables por otros medios, se hacen evidentes. por inmunofluorescencia directa, usando un anticuerpo primario acoplado a isotiocianato de fluoresceina (6)(7)(8). Con esta técnica se obtienen resultados reproducibles, aunque adolece de varios problemas, como el ser una preparación cuya vida útil e s muy corta, utilizar una concentración elevada de anticuerpo primario y requerir de un microscopio de fluorescencia, costoso y no siempre disponible.…”

Section: Discussionunclassified

Inmunodetección por peroxidasa de células de ganglio sensorial infectadas por virus de rabia

Castellanos

Castañeda²,

Hurtado³

et al. 1996

biomedica

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ResumenCon el objetivo de establecer una técnica de inmunocitoquimica por avidina-peroxidasa biotinilada para la detección de células infectadas in vitro por el virus rábico, se usaron cultivos de ganglio de la raíz dorsal de ratón. En este trabajo se presentan los resultados del proceso de estandarización de la inmunoperoxidasa, en la que también se usa el conjugado antirrábico producido en el lnstituto Nacional de Salud. Las diluciones de anticuerpo, que normalmente se usan en la inmunofluorescencia, muestran un excesivo ruido de fondo en la inmunoperoxidasa. Se discuten las posibles razones de este artefacto y se presenta un protocolo para la detección por inmunoperoxidasa de células de ganglio sensorial infectadas in vitro por virus de rabia. SummaryAn immunoperoxidase ABC technique was developed to detect rabies virus in dorsal root ganglion cells. This paper presents the standarization of the immunoperoxidase ABC technique which uses the antirabies conjugate produced by the Colombian lnstituto Nacional de Salud. Antibody dilution normally used in the fluorescence routine technique shows an excesive background in the ABC assay. We discuss the possible reasons and presenta protocol that minimizes the high background in the ABC immunoassay.El sistema de inmunodetección que utiliza un anticuerpo y una enzima biotinilados unidos por medio de una molécula de avidina, fue desarrollado en la década de los 80 por Hsu (1, 2) y, desde entonces, ha demostrado ser una metodologia fácil, reproducible y económica para la inmunocitoquímica. El sistema implica una fuerte amplificación de la señal del antigeno buscado por el uso de los anticuerpos primario y secundario y por la incubación con el complejo enzima-avidina (3-5), de tal manera que, cantidades de antigeno indetectables por otros medios, se hacen evidentes. por inmunofluorescencia directa, usando un anticuerpo primario acoplado a isotiocianato de fluoresceina (6-8). Con esta técnica se obtienen resultados reproducibles, aunque adolece de varios problemas, como el ser una preparación cuya vida útil e s muy corta, utilizar una concentración elevada de anticuerpo primario y requerir de un microscopio de fluorescencia, costoso y no siempre disponible.El Laboratorio de Neurobiologia del lnstituto Nacional de Salud está trabajando en un modelo M vitro para estudiar la interacción entre el virus rábico v las células del sistema nervioso. Con el La inmunodetección de las células infectadas in fin de obviar las limitantes señaladas en el vitro por el virus rábico se hace, generalmente, trabajo con fluorescencia, se presentó la

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“…The antigen was prepared by mixing 200 mg RNP in 1 mL PBS with 0.5 mL complete Freund adjuvant for the first dose, and incomplete for the following injections, all given subcutaneously at several sites. Ten days after the last booster, blood samples were collected from the auricular vein and used for titration by indirect immunofluorescence (IIF) (18).…”

Section: Immunization Schedulementioning

confidence: 99%

First production of fluorescent anti‐ribonucleoproteins conjugate for diagnostic of rabies in Brazil

Caporale¹,

Silva²,

Peixoto³

et al. 2009

Clinical Laboratory Analysis

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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.

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Fluorescent Antibody Staining of Street and Fixed Rabies Virus Antigens

Cited by 223 publications

References 0 publications

Immunoperoxidase Test for Rabies: Utility as a Diagnostic Test

Immunoperoxidase Test for Rabies: Utility as a Diagnostic Test

Inmunodetección por peroxidasa de células de ganglio sensorial infectadas por virus de rabia

First production of fluorescent anti‐ribonucleoproteins conjugate for diagnostic of rabies in Brazil

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