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Equine influenza is a highly contagious viral disease, specially among 1-5 years old naive horses. Vaccination is considered the best way to control the disease spread and outbreaks. Although foals are the main animal used for evaluation of equine influenza vaccines, guinea pigs were chosen as an alternative model in the present work, as they have a negligible antibody titer against equine influenza virus and are cheaper and easier to handle than foals. Five equine influenza vaccine batches were evaluated in two animal models, foals and guinea pigs, by injection of two doses/animal with 4 weeks apart using 2 mL/animal/dose and evaluation of immune responses by hemagglutination inhibition test and enzyme-linked immunosorbent assay. On the 7th week post vaccination, equine influenza antibodies titers reached maximum values of 9-10.2 and 8.7-10 hemagglutination inhibition units for foals and guinea pigs, respectively; sample/negative ratios were 0.126-0.464 and 0.128-0.445 for both animals, respectively. The use of guinea pigs as an animal model for the evaluation of equine influenza vaccines could be recommended instead of foals.
La gripe equina es una enfermedad viral muy contagiosa, especialmente entre los caballos jóvenes de 1 a 5 años de edad. La vacunación se considera la mejor forma de controlar la propagación y los brotes de la enfermedad. Aunque los potros son el principal animal utilizado para la evaluación de vacunas contra la gripe equina, en el presente trabajo se eligieron cobayos como modelo alternativo, ya que tienen un título insignificante de anticuerpos contra el virus de la gripe equina y son más baratos y fáciles de manejar que los potros. Se evaluaron cinco lotes de vacunas contra la gripe equina en dos modelos animales, potros y cobayos, mediante la inyección de dos dosis/animal con 4 semanas de intervalo utilizando 2 mL/animal/dosis y la evaluación de las respuestas inmunitarias mediante la prueba de inhibición de la hemaglutinación y el ensayo inmunoenzimático. En la 7ª semana posvacunación, los títulos de anticuerpos contra la gripe equina alcanzaron valores máximos de 9-10,2 y 8,7-10 unidades de inhibición de la hemaglutinación para potros y cobayos, respectivamente; las relaciones muestras/negativos fueron de 0,126-0,464 y 0,128-0,445 para ambos animales, respectivamente. Podría recomendarse el uso de cobayos como modelo animal para la evaluación de vacunas contra la gripe equina, en lugar de potros.
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Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties.
O germoplasma local e exótico do tomate continua sendo uma importante fonte de melhoramento genético. A avaliação de linhagens para estresses bióticos, particularmente as doenças virais, é o critério mais importantes para seleção no Paquistão, onde o vírus da folha amarela do tomate (TYLCV) e o vírus do mosaico do tomateiro (ToMV) são as principais doenças/vírus. Um conjunto de 40 acessos (incluindo linhagens indígenas do Paquistão e germoplasma exótico da Europa, dos Estados Unidos e da Ásia) foi avaliado quanto à resistência/resposta à infecção ao ToMV com inoculação artificial em casa de vegetação. A resposta à infecção foi quantificada por meio de pontuação da doença e de teste DAS-ELISA (para ToMV). Um subconjunto de 24 linhas foi posteriormente rastreado para TYLCV usando pontuação de doença e TAS-ELISA. As linhas testadas apresentaram variabilidade significativa para resistência ao ToMV. Apenas um acesso (Acc-17878) foi resistente ao ToMV, enquanto sete acessos (Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352 e CLN-362) expressaram resistência ao TYLCV. A correlação entre a avaliação fenotípica foi confirmada pelos resultados do ELISA nas duas doenças, embora ambas as ferramentas tenham se complementado para avaliar o estado da infecção viral. No futuro, os programas de melhoramento de tomate devem considerar aperfeiçoamentos para resistência ao ToMV e TYLCV (usando germoplasma identificado em nosso estudo) de modo a fornecer variedades de tomate resistentes a vírus.
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Solanum lycopersicum , Mejoramiento Genético , Virus del MosaicoRESUMEN
@#Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein). One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen. Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and antimouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established. The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined. Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL. No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80% — 120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than15%. For the residual protein in GLP preparation,about 62% of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than0. 01% — 0. 1% in biological preparations.
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@#Objective To prepare rabbit polyclonal antibodies against pertussis toxin(PT) and develop a double antibody sandwich ELISA for quantitative determination of PT antigen,identify and apply the method.Methods The rabbit polyclonal antibody against PT was prepared by immunizing Chinchilla rabbit with PT using traditional method.The reaction conditions of ELISA system were optimized,the double antibody sandwich ELISA method for quantitative determi-nation of PT was developed,and the specificity,linearity,accuracy,precision and sensitivity were verified.The developed method was used to detect PT antigen content in fimbriae proteins(FIM) stock solution of samples during detoxification and other purification process of pertussis antigen.Results The working condition of double antibody sandwich ELISA for detection of PT antigen content was the coating concentration of PT rabbit polyclonal antibody of 1 μg/mL,and the enzyme-labeled antibody dilution of 1:8 000.This detection system showed specific reaction with PT purified protein,but had no cross reaction with filamentous hemagglutinin,diphtheria toxoid and tetanus toxoid;the linear detection range of the developed double antibody sandwich ELISA was within 25—400 ng/mL;the recovery rates of PT at high,moderate and low concentrations were 103.27%,91.48% and 103.52%,respectively;both the intra-and inter-coefficients of variation(CVs)were less than 10%;the sensitivity of the method was 20.719 ng/mL,and the detection limit was 41.438 ng/mL.Thirty-five batches of samples were detected under five different detoxification process conditions and at different sampling time points,and the changes of antigen content were all consistent with the trend of detoxification reaction.Conclusion The PT rabbit polyclonal antibody was successfully prepared,and a double antibody sandwich ELISA with high precision and accuracy was developed for the quantitative determination of PT antigen content,which can be used for the antigen content detection of chemically detoxified samples in the production process of component DPT vaccines
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@#Objective To establish and verify a universal and stable potency test method in vitro for SARS-CoV-2 mRNA vaccine,so as to use it for the quality control of SARS-CoV-2 mRNA vaccine.Methods ELISA kits that could bind well to S protein of SARS-CoV-2 variants,as well as transfected cells,cell plating concentrations and doses for transfection were screened,and then a potency test method for SARS-CoV-2 mRNA vaccine in vitro was established and verified.Results An ELISA kit was found with good binding ability to S protein of each variant,and HEK293T cells were determined as the transfection cells,with the plating concentration of 2.5 × 10~5 cells/mL and the transfection dose of 4 μg/well in the 6-well plate.An universal and stable potency test method for SARS-CoV-2 mRNA vaccine in vitro was established.The verification results showed that the method met the quality control needs.Conclusion The established potency test method in vitro for SARS-CoV-2 mRNA vaccine has good relative accuracy,linearity,intermediate precision and range,and can be applied to the quality control of SARS-CoV-2 mRNA vaccines.
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Abstract Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties.
RESUMO O germoplasma local e exótico do tomate continua sendo uma importante fonte de melhoramento genético. A avaliação de linhagens para estresses bióticos, particularmente as doenças virais, é o critério mais importantes para seleção no Paquistão, onde o vírus da folha amarela do tomate (TYLCV) e o vírus do mosaico do tomateiro (ToMV) são as principais doenças/vírus. Um conjunto de 40 acessos (incluindo linhagens indígenas do Paquistão e germoplasma exótico da Europa, dos Estados Unidos e da Ásia) foi avaliado quanto à resistência/resposta à infecção ao ToMV com inoculação artificial em casa de vegetação. A resposta à infecção foi quantificada por meio de pontuação da doença e de teste DAS-ELISA (para ToMV). Um subconjunto de 24 linhas foi posteriormente rastreado para TYLCV usando pontuação de doença e TAS-ELISA. As linhas testadas apresentaram variabilidade significativa para resistência ao ToMV. Apenas um acesso (Acc-17878) foi resistente ao ToMV, enquanto sete acessos (Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352 e CLN-362) expressaram resistência ao TYLCV. A correlação entre a avaliação fenotípica foi confirmada pelos resultados do ELISA nas duas doenças, embora ambas as ferramentas tenham se complementado para avaliar o estado da infecção viral. No futuro, os programas de melhoramento de tomate devem considerar aperfeiçoamentos para resistência ao ToMV e TYLCV (usando germoplasma identificado em nosso estudo) de modo a fornecer variedades de tomate resistentes a vírus.
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Background: Dexamethasone is a synthetic corticosteroid similar to cortisol produced naturally by the adrenal glands. As an anti- inflammatory and immunosuppressive agent, it is used in many diseases such as rheumatoid arthritis and allergic anaphylactic shock, and its suppression test to diagnose Cushing's syndrome. Its further use includes its administration before antibiotics in bacterial meningitis, antitumor treatment, for treatment of glucocorticoid resistance, Addison抯 disease, and congenital adrenal hyperplasia. The drug is abused by using it in animal husbandry as a growth promoter and in horse sports to enhance their performance. Methods: In this study, the development of homologous ELISA using Dexamethasone-21-hemisuccinate (DEX-21-HS)-Bovine serum albumin antiserum and Dexamethasone-21-hemisuccinate (DEX-21-HS)-Horseradish peroxidase enzyme conjugate has been done. The n-hydroxysuccinimide ester method was used to prepare the immunogen and enzyme conjugate. Results: The sensitivity 0.25 ng/mL, affinity 2.8x10-8 L/mol and ED50 4.98 ng/mL of the assay were found. The cross-reactivity of the assay was checked and found with three steroids (Corticosterone- 1.13%, Progesterone- 2.25% and Prednisolone- 6.3%) out of 48 structurally related steroids. Then, analytical variables of the developed assay were studied, such as recovery (98.55% to 105.08%), precision (Inter and Intra- assay coefficient of variation <9.28%), correlation (R2= 0.98) by utilizing a commercially available Dexamethasone kit for comparison. Conclusion: This study concluded that low-cost indigenous ELISA for Dexamethasone had been developed, which can give results within 75-80 minutes.
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Background: Membranous nephropathy (MN) is a pattern of glomerular injury. Exact categorization into primary membranous nephropathy (PMN) or secondary membranous nephropathy (SMN) is essential for treatment. An endogenous podocyte antigen, M-type phospholipase A2 receptor (PLA2R) has been discovered to be involved in the pathogenesis of PMN. Aims and Objectives: In this article, we aimed to analyze renal tissue PLA2R and serum anti-PLA2R antibodies in MN cases and determined the diagnostic utility. Materials and Methods: The study was of prospective type carried out from March 2019 to August 2020. Analysis of cases of MN was performed with PLA2R paraffin immunoflourescence and serum anti-PLA2R antibody ELISA. Results: Overall sensitivity, specificity, PPV, and NPV of serum anti-PLA2R ELISA for PMN was 91.3%, 80%, 75%, and 93.3%, respectively, and of tissue PLA2R staining for PMN was 91.67%, 81.08%, 75.86%, and 93.75%, respectively. There was strong concordance between two methods. In the patients that were followed up, we found baseline serum anti-PLA2R antibody was less in complete remission group than that in non-remission group and the reduction in serum anti-PLA2R antibody was more in complete remission group than that in non-remission group. Conclusion: Routine light and immunofluorescence examination are incapable of giving exact categorical opinion regarding PMN and SMN. Serum anti-PLA2R antibody detection and renal tissue PLA2R analysis are sensitive and specific in detecting PMN. Baseline serum anti-PLA2R antibody and anti-PLA2R antibody quantification trends are related to prognosis of PMN. So they can be incorporated as additional biomarker.
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RESUMEN Objetivos. Determinar la seropositividad a anticuerpos anti-IgG por infección de Echinococcus granulosus, Fasciola hepatica y cisticerco de Taenia solium y describir las características de los infectados en 13 regiones de la sierra peruana entre 2016 y 2019. Materiales y métodos. Estudio observacional transversal, que analizó 7811 fichas epidemiológicas de la vigilancia basada en laboratorio de las zoonosis parasitarias del periodo 2016-2019. El diagnóstico se realizó mediante la detección de anticuerpos tipo IgG anti E. granulosus, F. hepatica y cisticerco de T. solium utilizando antígenos nativos mediante el ensayo inmunoabsorbente ligado a enzimas (ELISA) e Inmunoblot. La diferencia en la frecuencia de casos de estas zoonosis según características identificadas se realizó mediante la prueba chi-cuadrado de Pearson y prueba exacta de Fisher. Resultados. Se determinó una seropositividad de 7,9% para fascioliasis, 4,9% para equinococosis quística, y 2,3% para cisticerco de T. solium. Estas frecuencias fueron mayores en Cerro de Pasco para equinococosis quística (24,5%), en Ayacucho para cisticerco de T. solium (4,5%) y en Puno para fascioliasis (40,6%). Entre las características sociodemográficas, se encontró una diferencia estadísticamente significativa en la frecuencia de casos para todas las zoonosis según grupo etario, ocupación, y región de residencia. Además, se encontró diferencia con el consumo de verduras en emolientes, y entre las características clínico-epidemiológicas con tener antecedentes familiares de las zoonosis parasitarias. Conclusiones. A partir de las 7811 muestras evaluadas, se encontró que estas zoonosis parasitarias están distribuidas en 13 regiones de la sierra del Perú, ocasionando un problema de salud importante, con frecuencias que varían según diversas características.
ABSTRACT Objectives. To determine seropositivity to anti-IgG antibodies against Echinococcus granulosus, Fasciola hepatica and Taenia solium cysticercus infection and to describe the characteristics of the infected patients in 13 regions of the Peruvian highlands between 2016 and 2019. Materials and methods. Cross-sectional, observational study, in which we analyzed 7811 epidemiological records of laboratory-based surveillance of parasitic zoonoses from 2016 to 2019. Diagnosis was established by detecting IgG type anti-E. granulosus, F. hepatica and T. solium cysticercus antibodies using native antigens by enzyme-linked immunosorbent assay (ELISA) and Immunoblot. We evaluated the difference in the frequency of the cases according to identified characteristics using Pearson's chi-square test and Fisher's exact test. Results. Seropositivity was 7.9% for fascioliasis, 4.9% for cystic echinococcosis, and 2.3% for T. solium cysticercus. These rates were higher in Cerro de Pasco for cystic echinococcosis (24.5%), in Ayacucho for T. solium cysticercus (4.5%) and in Puno for fascioliasis (40.6%). Regarding the sociodemographic characteristics, we found a statistically significant difference in the frequency of cases for all zoonoses according to age group, occupation, and region of residence. We also found a difference with the consumption of vegetables in emollients, and between clinical-epidemiological characteristics and having a family history of parasitic zoonoses. Conclusions. From the 7811 samples, we found that these parasitic zoonoses are distributed in 13 regions of the Peruvian highlands, and represent a major health problem, with frequencies that change according to different characteristics.
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Humanos , Masculino , Femenino , Niño , Adolescente , Adulto , Persona de Mediana Edad , Sistema Único de SaludRESUMEN
Purpose: Ocular surface discomfort and dry eye disease are caused by a dysfunctional tear film. The efficacy of lubricating eye drops on the human eye is known, but the compositions may show differential effects on rescuing the tear film. Mucins form a critical layer of the tear film, a reduction of which may be causative for ocular surface conditions. Therefore, it is essential to develop relevant human?derived models to test mucin production. Methods: Human corneoscleral rims were obtained from a healthy donor (n = 8) post?corneal keratoplasty and cultured in DMEM/F12 media. Hyperosmolar stress mimicking dry eye disease was induced by exposing the corneoscleral rim tissues to +200 mOsml NaCl?containing media. The corneoscleral rims were treated with polyethylene glycol–propylene glycol (PEG–PG)?based topical formulation. Gene expression analysis was performed for NFAT5, MUC5AC, and MUC16. Secreted mucins were measured by enzyme?linked immunosorbent assay (ELISA) (Elabscience, Houston, TX, USA) for MUC5AC and MUC16. Results: The corneoscleral rims responded to hyperosmolar stress by upregulating NFAT5, a marker for increased osmolarity, as observed in the case of dry eye disease. The expression of MUC5AC and MUC16 was reduced upon an increase in hyperosmotic stress. The corneoscleral rim tissues showed induction of MUC5AC and MUC16 expression upon treatment with PEG–PG topical formulation but did not show significant changes in the presence of hyperosmolar treatments. Conclusion: Our findings showed that PEG–PG?based topical formulation slightly alleviated hyperosmolar stress?induced decrease in MUC5AC and MUC16 gene expression that is encountered in DED
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Introdução: O conhecimento da aerobiologia local é fundamental para o alergista. Os aeroalérgenos são capazes de sensibilizar e levar ao desenvolvimento de doenças respiratórias alérgicas, portanto devem ser monitorados rotineiramente, tendo em vista possíveis mudanças locais conforme alterações climáticas, poluição e atividades agroindustriais. Objetivo: Verificar a presença e concentração do alérgeno principal da poeira da casca da soja (Gly m 1) na atmosfera da cidade de Maringá-PR e possíveis associações aos fatores climáticos. A escolha da soja deve-se a alta prevalência desta cultura no Brasil e nesta região do país. Até o presente momento, há apenas um estudo piloto feito por este mesmo grupo avaliando a presença deste alérgeno no Brasil. Métodos: Foram realizadas coletas de material atmosférico, durante o período de março de 2017 a março de 2018, durante 24 ou 48 horas distribuídas no decorrer do período, totalizando 70 amostras, das quais 10 foram excluídas por problemas técnicos de coleta. As amostras foram avaliadas pelo método ELISA (Enzyme linked immunosorbent assay ) para Gly m 1, sendo que todas as amostras apresentaram níveis detectáveis do alérgeno. Resultados: A mediana de concentração de Gly m 1 foi de 4,89 ng/m3. Os valores encontrados variaram de 0,66 ng/ m3 a 1826,1 ng/m3. Das 60 amostras analisadas, 23% delas apresentaram valores superiores a 90 ng/m3, sendo os meses de junho/2017 e março/2018 com concentrações mais elevadas. Houve correlação positiva das concentrações de Gly m 1 com as temperaturas máxima, média e mínima, umidade relativa, vento e insolação. Conclusão: Os dados evidenciam exposições constantes da população ao alérgeno do Gly m 1, por vezes em níveis elevados possivelmente capazes de gerar sensibilização e sintomas.
Introduction: Knowledge of local aerobiology is essential for allergists. Because airborne allergens can sensitize the population and lead to allergic respiratory diseases, they must be routinely monitored for the effects of climate change, pollution, and agroindustry. Objective: To verify the airborne presence and concentration of the main soy hull dust allergen (Gly m 1) in Maringá, PR, Brazil and possible associations with climatic factors. Soybeans were selected due to the high prevalence of this crop in this region. To date, only 1 pilot study (conducted by our group) has evaluated this allergen's presence in Brazil. Methods: Atmospheric material was collected between March 2017 and March 2018 in 24- or 48-hour intervals, totaling 70 samples, of which 10 were excluded due to technical problems. The samples were tested for Gly m 1 using enzyme-linked immunosorbent assay, and all samples showed detectable levels of the allergen. Results: The median concentration of Gly m 1 was 4.89 ng/m3, with values ranging from 0.66 ng/m3 to 1826.1 ng/m3. Of the 60 samples, 23% showed values > 90 ng/m3, with June 2017 and March 2018 having the highest concentrations. There was a positive correlation between Gly m 1 concentration and maximum, mean, and minimum temperatures, relative humidity, wind, and insolation. Conclusion: The data show that the population is constantly exposed to the Gly m 1 allergen, sometimes at high levels, which may lead to sensitization and symptoms.
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HumanosRESUMEN
Background & objectives: Chikungunya is a reemerging arbovirus infection. Laboratory diagnosis can be done by Classical test involving Rapid Immunochromatography, Enzyme-Linked Immunosorbent assay and Molecular methods. The present study was undertaken to know the genotype of the Chikungunya virus (CHICKV) among patients suspected of CHICKV and investigated by virus culture, partial sequencing, Rapid Immunochromatography, and Enzyme-linked Immunosorbent assay (ELISA). To understand different techniques used in Chikungunya diagnosis viz., virus culture, partial sequencing along with Immunochromatography and ELISA. Methods: This is a prospective, laboratory-based study at a tertiary care center. Lateral flow chromatography and ELISA was carried out on serum samples. All 50 samples were cultured and indirect Immunofluorescence was performed on positive samples at Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth Medical College Pune, Maharashtra, India. Virus isolates were subjected to partial sequencing for identification of genotype after confirmation by PCR. Statistical Package of Social Science (SPSS) version 22.0 software was used to calculate the Receiver operating curve (ROC) for different tests. Results: Out of 50 samples, 20 were positive by Immunochromatography, 23 by ELISA, and 3 by culture, PCR confirmed CHIKV isolates and sequencing identified genotypes as East Central South African type. Interpretation & conclusion: CHIKV culture isolates of East Central South African type lineage were predominantly found in the present study. These are also common genotypes present in Asia including India.
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Background & objectives: West Nile virus (WNV) is transmitted by a mosquito-borne virus whose natural reservoir is birds. Humans and horses are considered accidental hosts. Even if the vast majority of WNV infections in humans have asymptomatic or mild disease settings, serious neurological disorders with lethal outcomes can also be observed in around 1% of the cases. We aimed to serologically investigate the presence of WNV in humans living in Black sea of Turkey, and to obtain epidemiological data that will contribute to the implementation of public health policies to control and prevent potentially other life-threatening arboviral infections. Methods: In the current study, a total of 416 human sera were collected from native patients of Samsun and its boroughs attending Samsun Training and Research Hospital; these sera were tested for WNV with pooling method, using anti-IgM and IgG ELISA commercial kits. All pools that were found positive for both IgM and IgG were individually retested for the detection of positive WNV sera. After that, all positive samples were tested using realtime PCR to detect the presence of WNV-RNA particles. Results: Total seropositivity rates of WNV in terms of IgM and IgG were found as 0.96% and 0.72%, respectively. No presence of WNV-RNA could be detected in positive samples. Interpretation & conclusion: According to the data, further studies should be conducted to better understand the epidemiological dynamics of WNV in Turkey. It is recommended that other antigenically related flaviviruses which can give cross-reaction with WNV should also be investigated.
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Introduction: Leptospirosis is a zoonotic disease caused by Leptospira interrogans and has been reported from various countries worldwide. As very few studies were conducted on leptospirosis from north India, this study was conducted to know the status of this disease in this region. This retrospective hospital Material & Methods: based study was conducted in the Department of Microbiology of a tertiary care super specialty teaching institute from north India for a period of two consecutive years. Blood specimens from acute febrile illness cases were tested for presence of IgM antibodies against Leptospira interrogans by rapid card (Leptocheck from TULIP) testing and ELISA (Leptospira IgM ELISA from PanBio). Out of total 216 samples Results: collected and included in this study, 40 were found to be positive for presence of IgM antibodies against Leptospira interrogans. Seropositivity for leptospirosis was observed to be 19%. Maximum number of patients were from economically productive age groups, 31-40 years of age group followed by 21-30 and 41-50 years of age groups. CONCLUSION: Leptospirosis was found to be a major cause of acute febrile illness from north India. It is neglected and under reported from most of the regions of India due to lack of clinician's suspicion. More studies with more samples are required on leptospirosis from this region to reach on final conclusion.
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Introducción: El Perú es endémico al virus linfotrópico T humano tipo 1 (HTLV-1), por esas razones es importante conocer la fiabilidad de las pruebas diagnósticas que se usan en el país, con la finalidad de continuar o no su uso. El objetivo fue evaluar el rendimiento de tres pruebas serológicas ELISA Murex, ELISA Wantai e IFI INS-Perú para la detección de anticuerpos anti HTLV-1 frente a muestras peruanas. El estudio. Las tres pruebas fueron evaluadas frente a 382 sueros: 215 positivos y 167 negativos a HTLV-1 (Gold Standar: inmunoblot). Hallazgos. IFI no presentó falsos positivos, Wantai tuvo más falsos negativos (siete) y Murex más falsos positivos (ocho). Las tres pruebas mostraron resultados superiores a 95% para los parámetros estimados de exactitud diagnóstica. Conclusiones. IFI INS-Perú y ELISA Murex tuvieron buen rendimiento diagnóstico para la detección de anticuerpos contra HTLV-1 y son buenos candidatos para continuar siendo usados en Perú.
Background: Peru is endemic to the human T-lymphotropic virus type 1 (HTLV-1), for these reasons it is important to know the reliability of the diagnostic tests used in the country, in order to continue their use or not. The objective was to evaluate the performance of three serological tests ELISA Murex, ELISA Wantai and IFI INS-Peru for the detection of anti-HTLV-1 antibodies against Peruvian samples. The study. The three tests were evaluated against 382 sera: 215 positive and 167 negative for HTLV-1 (Gold Standard: immunoblot). Findings. IFI had no false positives, Wantai had more false negatives (seven) and Murex more false positives (eight). The three tests showed results above 95% for the estimated parameters of diagnostic accuracy. Conclusions. IIF INS-Perú and ELISA Murex had good diagnostic performance for the detection of antibodies against HTLV-1 and are good candidates to continue being used in Peru.
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@#Objective To develop and verify an indirect ELISA method for determination of specific IgG antibody of rhesus monkey serum against SARS-CoV-2 variant strain. Methods An indirect ELISA method for the determination of specific IgG antibody was developed using inactivated SARS-CoV-2 Beta variant strain inactivated vaccine as coating antigen,and optimized for the coating antigen concentration(1,2 and 4 μg/mL),dilution of serum(1∶800～1∶12 800),blocking solution(PBST containing 1% BSA,2% BSA,1% skim milk,2% skim milk and 1% BSA + 1% skim milk),blocking time(30,60 and 90 min),dilution of secondary antibody(1∶5 000,1∶10 000,1∶15 000 and 1∶20 000),incubation time of serum and secondary antibody(30,60 and 90 min),and TMB reaction time(5,10,15,20,25 and 30 min). 60 negative serum samples of rhesus monkeys were detected by the developed method,and the negative and positive critical values were determined. The sensitivity and precision of the methodology were verified. In addition,the specific IgG antibody and neutralizing antibody against SARS-CoV-2 Beta variant strain in 44 serum samples of rhesus monkey were detected by the developed method and microneutralization method,and the correlation and consistency between the two methods were compared. Results The optimum detection conditions were determined:the coating antigen concentration was 1 μg/mL;the blocking solution was PBST containing 1% skim milk,and the blocking time was 30 min;the serum samples to be tested were diluted to 1∶1 600 and incubated for 90 min,and the secondary antibody was diluted to 1∶10 000 and incubated for 30 min;the color development time of substrate was 25 min. The positive critical value and negative critical value of the method was 0. 093 and 0. 084 respectively,and the detection values between them were judged as suspicious. The dilution of5 positive serum samples that showed positive results was 1∶51 200;the coefficients of variation(CVs)of precision were all less than 15%. There was a strong correlation between IgG antibody titer and neutralizing antibody level in the 44 rhesus monkey serum samples(r = 0. 858 0,P < 0. 000 1);the total coincidence rate of the two methods was 90. 9%,the positive coincidence rate was 93. 6%,and the negative coincidence rate was 84. 6%;the consistency test Kappa value was 0. 783 8(95% CI:0. 586 5～0. 981 0). Conclusion The developed indirect ELISA method for eletermination of specific IgG antibody against SARS-CoV-2 Beta variant strain in rhesus monkey serum has good sensitivity and precision,and has strong consistency with microneutralization method,which can be used for the determination of IgG antibody in rhesus monkey serum.
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@#ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap2=0.995 6,P=0.000 1)。结论 建立的双抗体夹心ELISA法具有良好的准确性、精密性和特异性，可用于VZV疫苗中g E抗原含量的快速检测。 ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap(TM)Mabselect(TM)Mabselect(TM)Su Re and Hi Trap(TM)Su Re and Hi Trap(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1～14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of10(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1～14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of106～106～107with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95～1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)7with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95～1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)(1.49)]+3.99,and R(1.49)]+3.99,and R2value was 0.999.The recoveries of accuracy verification were 94.9%～114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2value was 0.999.The recoveries of accuracy verification were 94.9%～114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2=0.995 6,P=0.000 1).ConclusionThe developed double antibody sandwich ELISA had good accuracy,precision and specificity,which might be used for rapid detection of gE antigen content in VZV vaccine.
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ObjectiveTo investigate the mechanism of Magnolia officinalis cortex for constipation-type irritable bowel syndrome（IBS-C） rats before and after sweating. MethodIBS-C rat model was established by gavage of ice water， and rats were randomly divided into the blank group， model group， mosapride group（1 mg·kg-1）， M. officinalis cortex group（10 g·kg-1） and sweated M. officinalis cortex group（10 g·kg-1）. The changes of body weight， fecal number and fecal water content of rats were observed， 16S rRNA sequencing was used to detect the structural changes of fecal intestinal flora in rats， the levels of 5-hydroxytryptamine（5-HT） and substance P（SP） in colonic tissues of rats were determined by enzyme-linked immunosorbent assay（ELISA）. ResultCompared with the model group， the fecal water content and fecal number of mosapride group， M. officinalis cortex group and sweated M. officinalis cortex group were significantly increased（P<0.05）. At the phylum level， the top four species of flora abundance were Firmicutes， Bacteroidetes， Spirochaetes and Proteobacteria. Compared with the blank group， the proportion of Firmicutes in the model group was significantly reduced（P<0.05）， while the proportion of Spirochaetes was significantly increased（P<0.05）. Compared with the model group， the proportion of Firmicutes and Spirochaetes in M. officinalis cortex group and sweated M. officinalis cortex group tended to be similar to that in the blank group， and the proportion of Spirochaetes in sweated M. officinalis cortex group was lower than that of M. officinalis cortex group. At the family level， the top four species of flora abundance were Lactobacillaceae， S24_7， Ruminococcaceae， Bacteroidaceae， compared with the blank group， the proportion of Lactobacillaceae in the model group decreased significantly（P<0.05）， and its proportion in the M. officinalis cortex group and sweated M. officinalis cortex group increased significantly after administration（P<0.05）， and the flora structure of the two groups tended to be similar to that of the blank group. At the genus level， the top four species of flora abundance were Lactobacillus， Unspecified_S24_7， Bacteroides and Treponema. Compared with the blank group， the proportion of Lactobacillus in the model group decreased significantly（P<0.05）， while the proportion of Treponema increased significantly（P<0.05）. Compared with the model group， ratio of bacterial structure of Lactobacillus and Treponema in the M. officinalis cortex group and sweated M. officinalis cortex group tended to be similar to those in the blank group， indicating that M. officinalis cortex could restore the intestinal microbial structure of IBS-C rats before and after sweating. Compared with the model group， the 5-HT content in mosapride group was significantly reduced（P<0.05）， the contents of 5-HT and SP in the M. officinalis cortex group and sweated M. officinalis cortex group were significantly increased（P<0.01）， and the sweated M. officinalis cortex group was higher than the M. officinalis cortex group. ConclusionM. officinalis cortex can play a therapeutic role on IBS-C rats by regulating 5-HT pathway and intestinal flora structure before and after sweating.
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@#Objective To develop and verify a double antibody sandwich ELISA method for quantitative detection of TrypLE.Methods The optimal concentration of capture antibody and detection antibody were determined by orthogonal experiments to develop TrypLE double antibody sandwich ELISA quantitative detection method,which was verified for linear range,specificity,limit of detection(LOD),limit of quantitation(LOQ),accuracy and reproducibility.A variety of biological products were detected by the developed method to verify the applicability.Results The TrypLE double antibody sandwich ELISA quantitative detection method was established by using 3 μg/mL capture antibody and 15 000 times dilution of detection antibody,with a linear range of 0.41 ～ 40.00 ng/mL,a LOD of 0.258 ng/mL,a LOQ of 0.5 ng/mL.The measurement deviation was less than 5% and the CV of reproducibility verification was less than 5% when detecting standards and samples.The recovery rates of different types of samples were within 80% ～ 120%.Conclusion The established TrypLE double antibody sandwich ELISA quantitative detection method accurately,effectively and quickly detected residual amount of TrypLE in various types of biological products with good specificity,accuracy and reproducibility.
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Bordetella pertussis es un patógeno exclusivo de humanos que causa la tos ferina, enfermedad respiratoria aguda que afecta principalmente a la población pediátrica. Existen dos tipos de vacunas comercializadas contra este patógeno: celulares y acelulares. Las vacunas celulares han sido extensamente utilizadas y siguen teniendo gran relevancia. El presente trabajo tuvo como objetivo la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra células enteras de Bordetella pertussis. Para ello se determinó la concentración de recubrimiento, el rango lineal de la curva, los parámetros de precisión intra e interensayo, la especificidad, el valor de corte y el límite de detección. Se determinó como concentración de recubrimiento 0,5 UO/mL de células enteras. La curva estándar utilizando un suero de referencia internacional presentó un buen ajuste a una función polinómica en un intervalo entre las diluciones 1/100 y 1/24.300 con un coeficiente de correlación R2≥0,98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (≤10 por ciento, ≤20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la respuesta a células enteras de Bordetella pertussis en ensayos clínicos(AU)
Bordetella pertussis is a pathogen exclusive to humans that causes pertussis, an acute respiratory disease that mainly affects the pediatric population. There are two types of vaccines commercially available against this pathogen: cellular and acellular. Cellular vaccines have been widely used and continue to be of great relevance. The aim of the present work was to standardize an ELISA for the quantification of IgG antibodies against whole cells of Bordetella pertussis. For this purpose, the coating concentration, the linear range of the curve, the intra- and inter-assay precision parameters, the specificity, the cut-off value and the detection limit were determined. The coating concentration was determined as 0.5 UO/mL of whole cells. The standard curve using an international reference serum presented a good fit to a polynomial function in a range between dilutions 1/100 and 1/24,300 with a correlation coefficient R2≥0.98. The coefficients of variation in the intra- and inter-assay precision tests were in the intervals established for each (≤10percent, ≤20percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of whole-cell response to Bordetella pertussis in clinical trials(AU)