Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovineleukemia virus (BLV) infection. However, commercially available kits test inSouth America detect only antibodies against the gp51 protein. With the aimto improve the sensitivity of the test, we developed here a two-step indirectdual ELISA test that included both proteins p24 and gp51, expressed andproduced in E. coli and baculovirus expression system respectively. Two hundredten BLV sera, stated as double positive or double negative by the combinationof commercial agar gel immunodiffusion (AGID) assay and a gp51-ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordancecorrelation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards.The statistical analysis demonstrated the value of sera correctly rankedhighest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.Fil: Larsen, Alejandra. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Corva, Santiago. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Panei, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Geisler, Christoph. University Of Wyoming; Estados UnidosFil: Mortola, Eduardo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentin

Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies

The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mousehorseradish peroxidase conjugate and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.Facultad de Ciencias Veterinaria

Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies

The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mousehorseradish peroxidase conjugate and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.Facultad de Ciencias Veterinaria

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51- ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.Facultad de Ciencias Veterinaria

ELISA indirecto (iELISA) para la detección de rutina de anticuerpos contra el virus diminuto del ratón (MVM) en colonias de ratones

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.Se desarrolló un ELISA indirecto para detectar anticuerpos contra el virus diminuto del ratón (Mice minute virus [MVM]), utilizando un antígeno producido a partir de células BHK-21 infectadas con la cepa prototipo del virus. Se establecieron las diluciones óptimas de antígeno y el suero a utilizar. Para analizar la variabilidad en el laboratorio, se determinaron la reproducibilidad y la repetibilidad dentro de una placa y entre placas. Luego se analizaron 460 sueros provenientes de bioterios convencionales y clasificados previamente como positivos o negativos por inmunofluorescencia indirecta. El valor de corte se determinó mediante una curva ROC. Los resultados se compararon con los obtenidos con la prueba de inmunofluorescencia indirecta. El ELISA mostró 100% de sensibilidad y un 99% de especificidad. Esta técnica demostró ser una herramienta útil para desarrollar en laboratorios de virología estándar y puede utilizarse como prueba tamiz para seleccionar animales de manera más rápida que con la tradicional prueba de inmunofluorescencia indirectaFil: Laborde, Juan Martin. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Animales de Laboratorio; ArgentinaFil: Sguazza, Guillermo Hernán. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Fuentealba, Nadia Analia. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Corva, Santiago Gerardo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas; ArgentinaFil: Carbone, Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Animales de Laboratorio; ArgentinaFil: Galosi, Cecilia Monica. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin

Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies

The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mousehorseradish peroxidase conjugate and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.Facultad de Ciencias Veterinaria

Infección experimental por herpesvirus bovino 1 en conejas gestantes

Natural infection with Bovine herpesvirus 1 (BoHV-1) produces several clinical manifestations including conjunctivitis, respiratory signs, genital diseases and abortion. The rabbit is a good model for studying of latency, pathogenicity of BoHV-5 and other bovine herpesviruses. This study was conducted in order to analyze the response to experimental infection with BoHV-1 in rabbits during different periods of pregnancy. The results obtained could be useful for better understanding of the infection in cattle. A new method of infection was used. The rabbits developed clinical signs. The virus was recovered from nasal swabs and histological lesions were found in the analyzed samples. The humoral response was demonstrated and viral DNA was detected from the placentas. This work showed for the first time the arrival of BoHV-1 to blood after intranasal infection with the virus. It also provides a useful tool that can be used to evaluate the pathogenicity of by BoHV-1 strains, the immune response and to study viral latency and reactivation.La infección natural por Herpesvirus bovino 1 (BoHV-1) se manifiesta por conjuntivitis, signos respiratorios (rinotraqueitis), lesiones genitales (vulvovaginitis pustular infecciosa o balanopostitis) y abortos. El conejo, hasta el momento, ha resultado ser el mejor modelo experimental para estudiar los diferentes aspectos de la infección por BoHV-1, el fenómeno de latencia, la neuropatogenicidad de BoHV-5 y el comportamiento de diferentes cepas virales. Este trabajo se desarrolló con el objetivo de estudiar la respuesta de conejas infectadas con BoHV-1 en diferentes períodos de la gestación para que los datos resultantes puedan ser utilizados para la mejor comprensión de la infección en el bovino. Se utilizó un nuevo método de infección intranasal. Los animales desarrollaron signos clínicos. Se recuperó virus a partir de hisopados nasales, se observaron lesiones histopatológicas en las muestras analizadas, se demostró la respuesta inmune humoral y se detectó ADN viral a partir de placentas de los animales gestantes infectados. En este trabajo se evidenció por primera vez en el modelo conejo la llegada del virus al torrente sanguíneo luego de la infección intranasal. Además se aporta una herramienta de utilidad que puede ser utilizada para evaluar la virulencia de diferentes cepas de BoHV-1 y la respuesta inmune a distintos inmunógenos como también para realizar estudios de latencia y reactivación

Development of a peptide ELISA for the diagnosis of equine arteritis virus

A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.Instituto de Genética VeterinariaFacultad de Ciencias Veterinaria

Development of a peptide ELISA for the diagnosis of Equine arteritis virus

A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.Fil: Metz, German Ernesto. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lorenzon, Esteban Nicolás. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Serena, Maria Soledad. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Corva, Santiago Gerardo. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Cátedra de Epidemiología; ArgentinaFil: Panei, Carlos Javier. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Diaz, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; ArgentinaFil: Maffud Cilli, Eduardo. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Echeverria, Maria Gabriela. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; Argentin

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51- ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.Facultad de Ciencias Veterinaria