Epitope identification of calcitonin gene-related peptide antibodies based on ELISA and NMR methods / 中国生物制品学杂志

@#Objective To develop an indirect ELISA-based peptide scanning method combined with nuclear magnetic resonance(NMR)technique for the epitope identification of calcitonin gene-related peptide(CGRP)antibodies.Methods The antigen binding activities of two antibodies(new CGRP antibody and control antibody)were determined by indirect ELISA using each truncated CGRP fragment as coating antigen,and the linear epitope was analyzed according to the EC50value of four-parameter curve. Two-dimensional hydrogen-nitrogen correlation(2D1H-15N HSQC)spectrum of CGRP were acquired by NMR technique,and the binding of antibodies to the arginine of CGRP were analyzed through the disturbance of the antibodies to CGRP signals. Specific arginine modifications were detected by liquid chromatography-mass spectrum(LCMS) and NMR technique,and two arginine resonances were assigned on CGRP by correlating the rank order of the modification rate.ResultsThe antigen binding activities of two antibodies with CGRP(1-37),CGRP(19-37)and CGRP(25-37)showed dose-response relationships,and were fitted with four-parameter equation. However,there were no significant antigen binding with CGRP(1-18),CGRP(19-24)and CGRP(25-37)without C-terminal amide. The linear epitopes of both antibodies were located at the C-terminal of CGRP. The resonances of arginine ε-NH in 2D1H-15N HSQC spectrum disappeared in the presence of the control antibody;and the resonances appeared in the presence of the new antibody. The arginine R11 and R18 of CGRP could bind to the control antibody,but not to the new antibody. The NMR assignment for the arginine resonances were made by correlating the relative ranking of the modification rate where signals A and B arose from R11 ε-NH and R18 ε-NH respectively.ConclusionIn this study,the linear and conformational epitopes of new CGRP antibody and control antibody were identified based on the methods of ELISA and NMR,which may provide a theoretical basis for the design of the candidate therapeutic CGRP antibodies.

Development and verification of an indirect ELISA method for determination of specific IgG antibody against SARS-CoV-2 variant strain in rhesus monkey serum / 中国生物制品学杂志

@#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.

Seroincidencia de anticuerpos IgG del SARS-CoV-2 y factores de riesgo en trabajadores sanitarios asintomáticos del hospital Departamental de Villavicencio

Objetivo: Estimar la seroincidencia acumulada de inmunoglobulinas (Ig) clase G (IgG) anti-SARS-CoV-2 en trabajadores de la salud asintomáticos y su asociación epidemiológica dentro de las áreas funcionales del Hospital Departamental de Villavicencio (HDV). Metodología: Se llevó a cabo un estudio observacional analítico longitudinal de una cohorte de trabajadores, donde cada 21 días, en tres oportunidades, se midieron IgG anti-SARS-CoV-2 en suero sanguíneo, a través de ELISA indirecto, en una muestra representativa aleatoria (n= 105) de trabajadores sanitarios del hospital (N= 756). Como instrumento de recolección de datos se utilizó una encuesta, donde cada trabajador sanitario declaró no haber sido diagnosticado con COVID-19, e igualmente registró la información sobre las variables independientes: sexo, edad, condición laboral, área funcional y comorbilidades. Resultados: La prevalencia inicial para SARS-CoV-2 entre los trabajadores sanitarios asintomáticos del HDV fue de 9,52 % (IC 95 % 5,25-16,65). La seroincidencia acumulada durante 42 días fue de 12,38 % (IC 95 % 7,38-20,04). El riesgo relativo (RR) se utilizó para establecer los factores de riesgo asociados a las variables independientes. El sexo masculino (RR ajustado = 3,34, IC 95 % 1,98-5,86), obesidad (RR ajustado = 10,98, IC 95 % 1,41-85,98) y sexo femenino (RR ajustado = 2,15, IC 95 % 1,12-4,31) en las áreas funcionales de Hospitalización, Medicina Crítica y Urgencias, respectivamente, son factores de riesgo en el HDV. Conclusión: Un total de 13 de 105 trabajadores sanitarios del hospital seroconvirtieron positivamente para SARS-CoV-2 y fueron asintomáticos durante 42 días de seguimiento epidemiológico. Además, existen factores de riesgo importantes en su exposición a este virus en el HDV.

Objective: To estimate the cumulative seroincidence of antisars-CoV-2 immunoglobulin (Ig) class G (IgG) in asymptomatic health care workers and its epidemiological association within the functional areas of the Villavicencio Departmental Hospital (HDV). Methodology: A longitudinal analytical observational study of a cohort of workers was conducted in which anti- SARS-CoV-2 IgG levels in blood serum were measured every 21 days on three occasions using an indirect ELISA in a random representative sample (n = 105) of hospital health workers (N = 756). The data collection tool was a survey in which each healthcare worker indicated that they had not been diagnosed with COVID-19 and provided information on the independent variables: sex, age, job status, functional area, and comorbidities. Results: The baseline prevalence for SARS-CoV-2 among asymptomatic HDV healthcare workers was 9.52% (CI 95% 5.25-16.65). Cumulative seroincidence over 42 days was 12.38% (CI 95% 7.38-20.04). Relative risk (RR) was used to establish the risk factors associated with the independent variables. Male sex (adjusted RR 3.34, CI 95% 1.98-5.86), obesity (adjusted RR 10.98, CI 95% 1.41- 85.98) and female sex (adjusted RR 2.15, CI 95% 1.12-4.31) in the functional areas of Hospitalization, Critical Medicine and Emergency, respectively, are risk factors in the HDV. Conclusion: During 42 days of epidemiological follow-up, 13 out of 105 hospital healthcare workers seroconverted positively for SARS-CoV-2 and remained asymptomatic. Additionally, significant risk factors are associated with their exposure to this virus in the HDV.

Objetivo: Estimar a incidência zero acumulada de imunoglobulinas (Ig) classe G (IgG) anti-SARS-CoV-2 em profissionais de saúde assintomáticos e sua associação epidemiológica dentro das áreas funcionais do Hospital Estadual de Villavicencio (HDV). Metodologia: Foi realizado um estudo observacional analítico longitudinal de uma coorte de profissionais, no qual a cada 21 dias, em três ocasiões mediram-se IgG anti-SARS-CoV-2 em soro sanguíneo, através de ELISA indireto, em uma amostra representativa aleatória (n = 105) de profissionais de saúde do hospital (N =756). Como instrumento de recolecção de dados foi usada uma pesquisa, onde cada profissional de saúde declarou não ter sido diagnosticado com COVID-19, e igualmente registrou a informação sobre as variáveis independentes: sexo, idade, condições de trabalho, área de atuação e comorbidades. Resultados: A prevalência inicial para SARS-CoV-2 entre os profissionais de saúde assintomáticos do HDV foi de 9,52% (IC 95% 5,25-16,65). A incidência zero acumulada durante 42 dias foi de 12,38% (IC 95% 7,38-20,04). O risco relativo (RR) foi utilizado para estabelecer os fatores de risco associados às variáveis independentes. O sexo masculino (RR ajustado 3,34, IC 95% 1,98-5,86), obesidade (RR ajustado 10,98, IC 95% 1,41-85,98) e sexo feminino (RR ajustado 2,15, IC 95% 1,12-4,31) nas áreas funcionais de Internação, Unidade de Terapia Intensiva e Urgências, respectivamente, são fatores de risco no HDV. Conclusão: Um total de 13 de 105 profissionais de saúde do hospital foram detectados positivamente para SARS-CoV-2 e foram assintomáticos durante 42 dias de seguimento epidemiológico. Além disso, existem importantes fatores de risco na sua exposição a este vírus no HDV.

Epidemiology of canine visceral leishmaniasis in a vulnerable region in Brazil / Soroepidemiologia da leishmaniose visceral canina em uma região vulnerável do Brasil

Abstract Visceral leishmaniasis (VL) is a neglected and endemic zoonosis that occurs throughout Brazil; nevertheless, few studies have focused on the early detection of the disease. The municipality of Ourinhos is a non-receptive, silent and vulnerable area for VL, where the seroprevalence of this disease has so far not been investigated. The present study aimed to determine the seroprevalence of canine VL in Ourinhos-SP, and to identify the presence of risk factors. Blood samples were obtained from 604 dogs during a rabies vaccination campaign together with application of a socioeconomic questionnaire, environmental and animal characteristics and tutor's knowledge about the disease. The samples were subjected to indirect ELISA and new samples were collected from reactive and suspect animals, including whole blood and lymph node aspiration evaluated by parasitological method, complete blood count and PCR. No animal was diagnosed as positive based on the combination of direct and indirect tests and the tutors' answers indicated little knowledge about leishmaniasis, being often confused with other diseases transmitted by arthropods; hence, according to the proposed methods, the presence of canine leishmaniasis in the city of Ourinhos was not confirmed and health education campaigns about the disease should be carried out.

Resumo A leishmaniose visceral (LV) é uma zoonose negligenciada e endêmica presente em todas as regiões do Brasil, mas mesmo assim poucos estudos têm objetivado a detecção inicial da doença. O município de Ourinhos - SP é uma área não receptiva, silenciosa e vulnerável à LV, não havendo até o momento estudos que tenham investigado a soroprevalência no município. Nesse sentido, o presente estudo objetivou determinar a soroprevalência da LV canina em Ourinhos-SP, bem como associar a presença de fatores de risco. Amostras sanguíneas de 604 cães foram obtidas juntamente com a aplicação de questionário socioeconômico, características ambientais e dos animais e conhecimento sobre a doença. As amostras foram submetidas à sorologia por ELISA e novas amostras coletadas de cães reagentes ou suspeitos foram analisadas por método parasitológico direto, hemograma e PCR. Nenhum animal foi considerado positivo na combinação de testes direto e indireto, e as respostas dos tutores indicaram pouco conhecimento sobre a leishmaniose, sendo muitas vezes confundida com outras doenças transmitidas por artrópodes. Dessa forma, de acordo com os métodos propostos, a presença de leishmaniose canina, na cidade de Ourinhos, não foi confirmada. Por isso campanhas de educação em saúde sobre a doença deveriam ser realizadas.

Seroprevalence and associated factors of infectious bovine rhinotracheitis and bovine viral diarrhea in dairy cows in the Caparaó region, Espírito Santo, Brazil / Soroprevalência e fatores associados à rinotraqueíte infecciosa bovina e diarreia viral bovina em vacas leiteiras na região do Caparaó, Espírito Santo, Brasil

ABSTRACT: This study evaluated the seroprevalence and associated factors of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD), and to analyze the possible relationship between IBR, BVD, and the occurrence of mastitis. For this purpose, 854 crossbred dairy cows were evaluated from 69 properties allocated in the 12 municipalities that make up the Caparaó region, Espírito Santo (ES), Brazil. The seroprevalence of IBR and BVD was determined using the indirect ELISA test. Associations between variables were estimated using the prevalence ratio (PR) with a 95% confidence interval. The chi-square test was used to verify the significance of the associations (P < 0.05). The average prevalence of IBR and BVD was 48.59% and 26.46%, respectively. Animals seroreactive for IBR were more likely to develop subclinical mastitis (P < 0.01; PR: 1.27), and cows that were seroreactive for BVD were more likely to develop clinical mastitis (P < 0.01; PR: 2.24). Mechanical milking was considered a factor associated with IBR (P < 0.01; PR: 1.36) and BVD (P < 0.01; PR: 1.25). Reproductive management by natural breeding was considered a factor associated with IBR (P < 0.01; PR: 1.22). Animals seroreactive for BVD were more likely to develop reproductive problems (P < 0.01; PR: 1.34). It was concluded that the agents that cause IBR and BVD are widely disseminated in dairy cattle herds in the municipalities of the Caparaó region, ES, Brazil. The presence of IBR and BVD increased the chances of cows developing subclinical mastitis and clinical mastitis, respectively, and the cows that were mechanically milked were more likely to be seroreactive for IBR and BVD.

RESUMO: O objetivo do presente estudo foi avaliar a soroprevalência e os fatores de risco associados à Rinotraqueíte Infecciosa Bovina (IBR) e Diarreia Viral Bovina (BVD), e analisar a possível relação entre IBR, BVD e à ocorrência de mastite. Para tanto, foram avaliadas 854 vacas leiteiras mestiças de 69 propriedades localizadas nos 12 municípios que compõem a região do Caparaó, Espírito Santo (ES), Brasil. A soroprevalência de IBR e BVD foram determinadas pelo teste ELISA indireto. As associações entre variáveis ​​foram estimadas pela razão de prevalência (PR) com intervalo de confiança de 95%. O teste do qui-quadrado foi utilizado para verificar a significância das associações (P < 0,05). A prevalência média de IBR e BVD foi de 48% e 26%, respectivamente. Os animais sororreagentes para IBR foram mais propensos a desenvolver mastite subclínica (P < 0,01; PR: 1,27), e as vacas sororreagentes para BVD foram mais propensas a desenvolver mastite clínica (P < 0,01; PR: 2,24). À ordenha mecânica foi considerada um fator associado a IBR (P < 0,01; PR: 1,36) e BVD (P < 0,01; PR: 1,25). O manejo reprodutivo por monta natural foi considerado um fator associado IBR (P < 0,01; PR: 1,22). Os animais sororreagentes para BVD foram mais propensos a desenvolverem problemas reprodutivos (P < 0,01; PR: 1,34). Concluiu-se que os agentes causadores de IBR e BVD estão amplamente disseminados em rebanhos leiteiros nos municípios da região de Caparaó, ES, Brasil. A presença de IBR e BVD aumentaram as chances das vacas desenvolverem mastite subclínica e mastite clínica, respectivamente, e as vacas que foram ordenhadas mecanicamente apresentaram maior probabilidade de serem sororreagente para IBR e BVD.

Comparison of the antigenicity of African swine fever virus p35 protein as diagnostic antigen / 生物工程学报

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.

Diagnóstico serológico de Neospora caninum en vacas del municipio de Tuta, Boyacá / Serological diagnosis of Neospora caninum in cows of Tuta, Boyacá

RESUMEN Neospora caninum es un parásito protozoario del filo Apicomplexa que ha sido reconocido como causante de abortos y fallas reproductiva en el ganado de todo el mundo. Aunque en Colombia existen algunos estudios sobre la seroprevalencia de esta enfermedad, la información sigue siendo limitada. Objetivo: establecer la seroprevalencia de N. caninum en vacas lecheras del municipio de Tuta (Boyacá, Colombia). Materiales y Métodos: se muestrearon 375 animales. Las muestras se procesaron bajo la técnica de ELISA indirecta y se realizó un análisis estadístico con la prueba de chi-cuadrado de asociación-independencia para determinar si existía relación entre la presencia de anticuerpos contra N. caninum y las diferentes variables reproductivas. Resultados: el 52% de los individuos fueron positivos a anticuerpos contra N. caninum y la única variable reproductiva que presentó relación estadística con la presencia del protozoo fue la repetición de celo; por otra parte, no existió relación entre edad y raza de los bovinos y la presencia de N. caninum. Conclusión: la seroprevalencia es elevada si se compara con los datos reportados con anterioridad en otras regiones del país; no obstante, estos resultados no demostraron la presencia de enfermedad en los animales analizados, pero sí la evidencia antigénica, lo que sugiere que en algún momento de la vida se infectaron con el agente y promovieron la formación de anticuerpos específicos.

ABSTRACT Neospora caninum is a protozoan parasite of the phylum Apicomplexa, and has been recognized as a major cause of abortion and reproductive failure in cattle in the world, Although in Colombia there are some studies on the seroprevalence of this disease, even so the information remains limited. Objective: the aim of this study was to establish the seroprevalence of N. caninum in dairy cows of Tuta, (Boyacá, Colombia). Materials and methods: 375 animals were sampled. The samples were processed under indirect ELISA technique, a statistical analysis was performed with the chi-square association independence test to determine if there was a relationship between the presence of antibodies against N. caninum and the different reproductive variables. Results: it was observed that 52% of the individuals were positive for antibodies against N. caninum. the only reproductive variable that presented a statistical relationship with the presence of the protozoan was repetition of estrus, there was no relationship between age and breed of the cattle and the presentation of N.caninum. Conclusions: the seroprevalence is high if they if you keep in mind the seroprevalence data previously reported in other regions of the country, however, these results do not demonstrate the presence of disease in the animals analyzed, but the antigenic evidence, which implies that at some point life they became infected with the agent and promoted the formation of specific antibodies.

Expression of the VP1 protein and establishment of indirect ELISA detection method of Coxsackievirus B4 / 中华实验和临床病毒学杂志

Objective@#To detect antibodies to Coxsackievirus B4 (CV-B4), the indirect enzyme-linked immunosorbent assay (ELISA) method was established and optimized using the recombinant VP1 protein expressed in the prokaryote system as the envelope antigen.@*Methods@#The VP1 gene of CV-B4 was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). It was ligated into the expression vector pET32a(+ ) to obtain the recombinant plasmid pET32(+ )-VP1 and was then transformed into E. coli expression strain Rosetta (DE3). The recombinant VP1 protein was induced by IPTG, which was verified using SDS-PAGE electrophoresis and mass spectrometry. The establishment and optimization of the indirect ELISA reaction system was based on the purified recombinant protein mentioned above as the coating antigen.@*Results@#The CV-B4 VP1 gene was stably expressed in E. coli in the form of inclusion body. The optimal coating concentration of antigen was 7.5 μg per well and the optimal serum dilution was 1∶100. The threshold for determining the negative and positive result of the serum samples was the optical density value of ≥ 0.376 at 450 nm. The purified recombinant protein could be specifically recognized by CV-B4 positive serum without cross-reaction with Coxsackievirus (CV)-A, CV-B1 and CV-B5, indicating that it has good immunogenicity. However, it can cross-react with CV-B3 serum samples.@*Conclusions@#The indirect ELISA detection method based on the CV-B4 VP1 protein could be used in the detection of serum antibody to CV-B4 infection with good sensitivity, specificity and repeatability.

Multi-Epitope Fusion Protein Eg mefAg-1 as a Serodiagnostic Candidate for Cystic Echinococcosis in Sheep

Cystic echinococcosis (CE) in sheep is a hazardous zoonotic parasitic disease that is caused by Echinococcus granulosus (Eg). At present, serological test is an important diagnostic method for Eg infection in domestic animals. Here, a fusion protein Eg mefAg-1 harboring 8 dominant B-cell epitopes of Eg such as antigen B, tetraspanin 1, tetraspanin 6, reticulon and Eg95 was produced in E. coli and evaluated for CE in sheep by indirect ELISA. Eg mefAg-1 showed in ELISA a high sensitivity (93.41%) and specificity (99.31%), with a coincidence rate of 97.02%. Overall, it is suggested that the Eg mefAg-1 could be a potential antigen candidate for CE serodiagnosis in sheep.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Specific sIgA antibodies against swine influenza virus H3N2 infection / 中国人兽共患病学报

To study different breed pigs reply the swine flu virus infections,specific antibody of sIgA secretion regularity of respiratory tract and the differences of sIgA antibody according to different antigen proteins were detected.A/swine/Nanjing/ 51/2010(H3N2) was intranasally infected pigs (1 × 107 TCID50/mL and 2 mL/pig),and then the nasal swab samples were collected at different time points within 21 days after infection.M1,NS1 and PB1 recombinant protein,respectively,were used to establish indirect ELISA method for detecting specific antibody of sIgA,and to analyze its secretion regularity.Results displayed that there was no significant difference among three kinds of recombinant protein in the whole test,characterizing by specificity sIgA antibody levels rising rapidly after 5 infection days and reaching peak at day 14,then began to decline.Among different varieties of pigs,sIgA antibody production of PB1 protein in Obama group was significantly higher than that in binary pigs at 14th and 21st day (P＜0.05).It had no significant difference between M1 group and the NS1 group (P＞0.05).This experiment preliminary explores the secretion regularity of specificity sIgA antibody after infected swine flu virus,which laid a foundation for further study of SIV mucosal antibody diagnostic reagents.

An international collaborative research to evaluate the reference substance of anti-human EPO monoclonal antibody / 中国药学杂志

OBJECTIVE: To assess a panel of 9 human monoclonal antibodies against human erythropoietin (EPO) with defined characteristics (non-neutralizing, neutralizing, affinities) for suitability for EPO antibody assays. METHODS: A multi-center collaborative study involving three different laboratories and different assay platforms was carried out. Direct ELISA was used to test the affinities of the samples by Shenyang Sunshine pharmaceutical Co., Ltd and National Institutes for Food and Drug control, while indirect ELISA was used by Xiamen amoytop biotech Co., Ltd. The neutralizing assays were performed using UT-7 cell line by all the three laboratories. RESULTS: The properties of the 9 human monoclonal antibodies against human erythropoietin were assessed efficiently by all the three laboratories using different METHODS. CONCLUSION: It is suggested that the EPO antibody panel is established to enable the evaluation of the performance of different EPO antibody assays and thus the selection of appropriate assay for clinical use.

Establishment of a high sensitive indirect ELISA for detecting specific antibodies against H9 subtype avian influenza virus / 生物工程学报

H9 subtype avian influenza virus causes worldwide epidemic, resulting in enormous economic losses of poultry production. In the present study, an indirect ELISA method was established for more accurate and specific detection. The recombinant protein of the globular head domain of HA of H9 subtype avian influenza virus was used as antigen. Specific blocking buffers and dilution buffers were determined to increase the sensitivity and specificity. The sensitivity of ELISA was higher than that of hemagglutination inhibition (HI) test. The coating antigen is very specific and no cross-reactivity with positive serum against H3N2, H5N2 and H7N9 subtype influenza viruses, Newcastle disease virus, avian infectious bronchitis virus, avian infectious disease virus, and egg drop syndrome virus. Two hundred of clinical sera samples were examined. The results indicate the coincidence rate between ELISA and HI test reached 97%. In addition, there was a positive correlation between OD450 values and the logarithm of HI titer to the base 2 of an individual serum sample (R2=0.981 1).

Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus / 生物工程学报

Porcine deltacoronavirus (PDCoV) has been recently recognized as an emerging viral pathogen that causes diarrhea in newborn piglets. A total of 254 small intestinal or fecal samples collected from 10 provinces including Henan, Hunan, Zhejiang, Jiangxi, Anhui, Hebei, Heilongjiang, Jiangsu, Shandong and Shanghai between 2014 and 2015, were screened by quantitative RT-PCR targeting the viral M gene. Eleven PDCoV positive samples were identified with a total positive rate of 4.33%. An indirect enzyme-linked immunosorbent assay (ELISA) was developed based on the recombinant S1 protein of PDCoV. This assay was used to test 609 serum samples of pigs with diarrhea symptoms collected from 10 provinces between 2015 and 2016. The positive rate of PDCoV antibody was 44.17% (269/609). The two methods can be used to monitor the PDCoV epidemiology in the levels of PDCoV specific RNA or antibody, helping better prevent and control PDCoV.

Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.

Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.

Estudio epidemiológico de paratuberculosis bovina en hatos lecheros del sur de Nariño, Colombia / Epidemiological Study of Bovine Paratuberculosis in Dairy Herds in Southern Narino, Colombia / Estudo epidemiológico de paratuberculose bovina em gado leiteiro do sul de Nariño, na Colômbia

Introducción: la paratuberculosis (PTBC), también llamada enfermedad de Johne, es un trastorno gastrointestinal crónico de rumiantes domésticos y silvestres causado por Micobacterium avium subespecie paratuberculosis (MAP). Está distribuida mundialmente y genera un alto impacto en la ganadería, debido a que disminuye la producción, se pierde potencial genético por reemplazos tempranos de animales infectados y se incrementa la mortalidad. Objetivos: evaluar la presencia de anticuerpos anti-MAP en fincas lecheras del sur de Nariño y describir su distribución de acuerdo con características poblacionales. Métodos: se realizó un estudio transversal descriptivo en 958 vacas mayores de 2 años en 16 fincas lecheras. Para determinar la presencia de anticuerpos, se usó la prueba diagnóstica de Elisa indirecto (kit comercial Svanova®). La seropositividad fue asociada con las variables de raza, edad, ubicación, condición corporal, número de lactancias y estadio clínico mediante la prueba estadística de chi cuadrado. Resultados: se encontraron 15 fincas (94 %) con al menos un animal positivo y 77 vacas (8 %) con anticuerpos anti-MAP. Se estableció una asociación significativa (p < 0,05) con la condición corporal del animal. Conclusiones: en los principales municipios lecheros se encontraron vacas con anticuerpos anti-MAP y no se encontró relación entre edad, raza, ubicación y estatus clínico con la seroprevalencia-MAP, pero sí con la condición corporal.

Introduction: Paratuberculosis (PTB), or Johne's disease, is a chronic gastrointestinal disorder in domestic and wild ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). It is distributed worldwide and generates a high impact on livestock, due to a decrease in production, loss of genetic potential by early replacement of infected animals, and increase in mortality. Objectives: To evaluate the presence of anti-MAP antibodies in dairy farms in southern Narino and to describe their distribution according to population characteristics. Methods: A descriptive cross-sectional study was performed on 958 cows over 2 years of age in 16 dairy farms. To determine the presence of antibodies, the indirect Elisa diagnostic test was used (Svanova® commercial kit). Seropositivity was associated with variables of race, age, location, body condition, lactation number, and clinical state by using the chi-square test statistic. Results: There were found 15 farms (94%) with at least one positive animal and 77 cows (8%) with anti-MAP antibodies. A significant association (p < 0.05) with the animal's body condition was established. Conclusions: Cows with anti-MAP antibodies were found in major dairy municipalities; it was found that MAP-seroprevalence is not connected to age, race, location and clinical status, but it is linked to body condition.

Introdução: a paratuberculose (PTBC), também conhecida como doença de Johne, é um transtorno gastrointestinal crônico de ruminantes domésticos e silvestres causado por Mi-cobacterium avium subespécieparatuberculose (MAP). Está distribuída mundialmente e gera um alto impacto na pecuária, devido a que diminui a produção, se perde potencial genético por substituições precoces de animais infectados e aumenta a mortalidade. Objetivos: avaliar a presença de anticorpos anti-MAP em fazendas de gado do sul de Nariño e descrever sua distribuição de acordo com características populacionais. Métodos: realizou-se um estudo transversal descritivo em 958 vacas maiores de 2 anos em 16 fazendas produtoras de gado. Para determinar a presença de anticorpos, se usou a prova diagnóstica de Elisa indireto (kit comercial Svanova®). A soro positividade foi associada com as variáveis de raça, idade, localização, condição corporal, número de lactancia e estádio clínico através da prova estatística de chi quadrado. Resultados: se encontraram 15 fazendas (94 %) com pelo menos um animal positivo e 77 vacas (8 %) com anticorpos anti-MAP. Estabeleceu-se uma associação significativa (p < 0,05) com a condição corporal do animal. Conclusões: nos principais municípios produtores de leite se encontraram vacas com anticorpos anti-MAP e não se encontrou relação entre idade, raça, localização e status clínico com a soro prevalência-MAP, mas sim com a condição corporal.

Screening and identification of immunodominant epitope of human serum albumin / 国际药学研究杂志

Objective To select the immunodominant epitope of human serum albumin (HSA) and provide the basis for setting up a special and rapid detecting method of HSA. Methods Bioinformatic method was used to compare protein sequences of human, pig, horse, ox, and ovine, and the immunodominant epitopes of HSA were predicted. The E.coli preferred codons were used todesign the DNA sequences of the selected epitopes. The genes of the epitopes were expressed after they were inserted into the PGEX-4T-2 vector. The recombinant antigens were identified and valued by HSA antibody with indirect ELISA. Results The length of the selected epitopes was H1 [126-162 amino acid (aa)], H2 (314-355aa), and H3 (373-424aa) and the relative molecular weight was 3.01×104, 3.06×104 and 3.17×104, respectively. The epitope of 373-424 aa were more active and its cross-reactivity IC50 of enzymelabeled antibody was 1.635 mg/L, which was higher than HSA (P0.05). Conlusion The immunodominant epitope of HSA is obtained, which is significant for developing a rapid and special reagent of HSA.

Development of a Novel PmpD-N ELISA for Chlamydia psittaci Infection / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Chlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen.</p><p><b>METHODS</b>The antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens.</p><p><b>RESULTS</b>The sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively.</p><p><b>CONCLUSION</b>These data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.</p>

Screening and identification of immunodominant epitope of human serum albumin / 国际药学研究杂志

Objective To select the immunodominant epitope of human serum albumin(HSA)and provide the basis for set?ting up a special and rapid detecting method of HSA. Methods Bioinformatic method was used to compare protein sequences of hu?man,pig,horse,ox,and ovine,and the immunodominant epitopes of HSA were predicted. The E.coli preferred codons were used to design the DNA sequences of the selected epitopes. The genes of the epitopes were expressed after they were inserted into the PGEX-4T-2 vector. The recombinant antigens were identified and valued by HSA antibody with indirect ELISA. Results The length of the selected epitopes was H1〔126-162 amino acid(aa)〕,H2(314-355aa),and H3(373-424aa)and the relative molecular weight was 3.01×104,3.06×104 and 3.17×104,respectively. The epitope of 373-424 aa were more active and its cross-reactivity IC50 of enzyme-labeled antibody was 1.635 mg/L,which was higher than HSA(P<0.05). Conlusion The immunodominant epitope of HSA is obtained, which is significant for developing a rapid and special reagent of HSA.