scholarly journals Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis

2020 ◽  
Vol 40 (12) ◽  
pp. 977-984
Author(s):  
Cláudia F. Resende ◽  
Grazielle C. F. Galinari ◽  
Raphael M. Victor ◽  
Telissa C. Kassar ◽  
Thiago L.M. Arcebispo ◽  
...  
Keyword(s):  
Predictive Value ◽  
Indirect Elisa ◽  
Leukemia Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Kappa Index ◽  
Tadarida Brasiliensis ◽  
Diarrhea Virus ◽  
Bovine Leukosis ◽  
Specific Reactions ◽  
Serum Elisa

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.

scholarly journals Sensitivity and specificity of indirect ELISA for the detection of antibody titers against BVDV from beef cattle raised in Pará State

2017 ◽  
Vol 38 (5) ◽  
pp. 3049
Author(s):  
Rinaldo Batista Viana ◽  
Aline do Socorro Lima Kzam ◽  
Bruno Moura Monteiro ◽  
Cláudio Cabral Campello ◽  
Eliomar De Moura Sousa ◽  
...  
Keyword(s):  
Beef Cattle ◽  
Predictive Value ◽  
False Negative ◽  
Foot And Mouth Disease ◽  
Metropolitan Region ◽  
Enzyme Linked Immunosorbent Assay ◽  
Significance Level ◽  
Bovine Kidney ◽  
Diarrhea Virus ◽  
Positive Diagnosis

The aims of this study were to establish the prevalence of anti-bovine viral diarrhea virus (BVDV) antibodies (Ab) in beef cattle raised in Pará state, to compare the prevalence of seropositive animals to BVDV using a commercial indirect enzyme-linked immunosorbent assay kit (iELISA) and the virus neutralization (VN) test, and finally, to determine the sensitivity (Se) and specificity (Sp) of the iELISA for the detection of anti-BVDV Ab using VN as a gold standard. A total of 400 serum blood samples from Nelore cows aged at least 24 months from five farms in the Pará state from two mesoregions (Metropolitan Region of Belem and Northeast of Pará) were analyzed. All animals were vaccinated against brucellosis and foot-and-mouth disease. The examination of anti-BVDV Ab with VN was performed in the Laboratory of Bovine Viruses of the Biological Institute of Sao Paulo as described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. For VN, bovine kidney epithelial cells from the Madin Darby Bovine Kidney (MDBK) strain were used. The determinations of anti-BVDV Ab were performed with the iELISA test at the Laboratory of Immunology and Microbiology of the Federal Rural University of Amazonia according to the manufacturer's recommendations. The results were classified as follows: (a) correct positive diagnosis, (b) incorrect positive diagnosis, (c) correct negative diagnosis, and (d) incorrect negative diagnosis, according to the results obtained from VN. From the values obtained from VN and iELISA, Se [(a ÷ a + d) × 100], Sp [(c ÷ c + b) × 100], positive predictive value [(a ÷ a + B) × 100], and negative predictive value [(c ÷ c + d) × 100] were calculated for iELISA. The frequencies (%) of seropositive animals were determined and compared both between the different tests (iELISA and VN) and between the different farms (1, 2, 3, 4, and 5). The statistical analysis was performed with a significance level of 5%. The prevalence of seropositive animals was found to be different (P < 0.0001) using VN (39.25% [157/400]) and iELISA (54.50% [218/400]). It was observed that the Se and Sp of the iELISA assay were 98.72% and 74.07%, respectively. Of the total, 25.93% (63/243) of the samples were considered false-positive and 1.27% false-negative (2/157). It was concluded that the BVDV infection is present in beef cattle herds of the state of Para. Based on the speed of execution, ease of handling, and high Se of the iELISA, it is suggested that this assay can be used as a screening test for the detection of anti-BVDV Ab with the aim of eliminating infected animals from large herds of beef cattle.

scholarly journals Enzootic bovine Leukosis: development of an indirect enzyme linked immunosorbent assay (I-Elisa) in seroepidemiological studies

1999 ◽  
Vol 30 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Ester Teresa González ◽  
Estela Beatriz Bonzo ◽  
María Gabriela Echeverría ◽  
María Licursi ◽  
María Elisa Etcheverrigaray
Keyword(s):  
Core Protein ◽  
Leukemia Virus ◽  
Test Procedure ◽  
Immunological Response ◽  
Negative Control ◽  
Etiologic Agent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Enzootic Bovine Leukosis ◽  
Bovine Leukosis

Bovine Leukemia Virus (BLV) is the etiologic agent of Enzootic Bovine Leukosis, a retrovirus exogenous to the bovine species. Once infected, there is no detectable viraemia but instead there is a strong and persistent immunological response to BLV structural proteins, essentially the gp51 envelope glycoprotein and the mayor core protein p24. We describe the test procedure of an indirect ELISA (I-ELISA) using polyclonal reagents for the detection of antibodies to BLV. For comparison, the sera were simultaneously tested by agar gel immunodiffussion (AGID) test, which is currently used as diagnostic standard for BLV infection. The antigen applied does not require a high degree of purification and the data from the analysis of the negative sera showed that the establishment of a cut-off level corresponding to 3 times the standard deviation (SD) above the mean for the negative control set of sera provided acceptable specificity, reducing the risk of false positives results. A comparison of the results obtained by AGID test and I-ELISA showed that considering a total of 465 serum samples, all of the 234 samples (50%) that were positive by AGID were positive to the I-ELISA. Of 225 serum samples which yielded negative results in the AGID test, 69 (15%) were found to be positive by the I-ELISA and 156 (33%) were negative by both techniques. Few sera (2%) that were non-specific by AGID were defined as negative or positive by I-ELISA.

scholarly journals Prevalence of Bovine viral diarrhea virus in cattle herds from Basrah and Nassirya Provinces by direct and indirect Elisa and Real time qPCR

2012 ◽  
Vol 11 (2) ◽  
pp. 1
Author(s):  
B. A. Jarullah, J. Aed Gati, and A. Saleh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Indirect Elisa ◽  
Positive Sample ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Virus Rna ◽  
Two Samples ◽  
Cattle Herds

The current study was conducted to investigate the prevalence of BVD virus in Basrah and Nassirya city by using ELISA and RT-PCR. Two hundreds and eighty two samples of non vaccinated cattle sera samples collected from two regions of Iraq (188 samples from Nassirya city and 92 samples from Basrah city). Samples tested by Enzyme Linked Immunosorbent Assay (ELISA) antigen capture. Positive results were 20 samples ( 8 sample in Thi-Qar and 12 positive samples from Basrah). All samples submitted to indirect ELISA(IDEXX HerdCheck ELISA )for detect BVDV antibodies .Genotyping of all 20 positive samples to antigen detection were tested by Real time PCR, using Cador BVDV ½ kit, after extraction of virus RNA by QIAamp mini kit. The results revealed that there were 20 positive sample according to direct ELISA(Ag detection), while 66 sample were positive to indirect ELISA, as well as, the result of RT-PCR showed that there were two sample positive to BVDV type-1 (one sample form each city).Key words: BVDV, Genotype, ELISA, Iraq, Real time PCR.

scholarly journals Sero-prevalence of bovine Leukemia virus in cattle from Caquetá state, Colombia

2019 ◽  
Vol 11 (2) ◽  
pp. 722
Author(s):  
Pablo Andrés Motta-Delgado ◽  
Luis Gabriel Rivera-Calderón ◽  
Wilmer Herrera-Valencia ◽  
Ricardo Alberto Martínez-Tovar ◽  
Marliyanini Londoño-Sánchez ◽  
...  
Keyword(s):  
Bovine Leukemia Virus ◽  
Indirect Elisa ◽  
Leukemia Virus ◽  
Elisa Test ◽  
Dual Purpose ◽  
Positive Ratio ◽  
Dual Purpose Cattle ◽  
Bovine Leukosis ◽  
Bovine Species ◽  
Municipal Level

Bovine Leukemia Virus (BLV) is the agent of enzootic bovine leukosis (EBL), this disease is a neoplasm of lymphatic tissue in bovine species.  The aim of this article was determinate the prevalence of bovine leukemia virus in dual-purpose cattle from nine municipalities that produce the 98% of milk in the Caquetá state, Colombia. Blood samples were collected in 100 herds dedicates to dual purpose cattle, obtained blood serum from 1000 animals, of which 893 corresponding to cows. Indirect Elisa test for detection of antibodies anti-GP51 of BLV was performance and the positive cases were considered if the serum-to-positive ratio with percentages of M/N lower than 40%. Categorized data were analyzed by contingency tables and ANOVA at the significant level of p<0.05 by DGC test was performed. The overall sero-prevalence of BLV in Caquetá state was of 25.18% (95%, CI: 21.9-28.46%), in males 26.25% and females 25.37% respectively. At municipal level the sero-prevalence varied of 7.12 to 41.81%. The prevalence of BLV at herd level was of 67% (95%, CI: 57.24-76.76%). In conclusion, the sero-prevalence of BLV in the dual-purpose livestock system over 36 months of age in Caquetá state is moderate, do not exist statistical difference between sero-prevalence of cows and bulls. At level of herds the prevalence of BLV is high. Improving strategies of control and managements in the herds, as well as implement policies of sanitary management are necessary.

scholarly journals Performance assessment of imported ELISA in the serodiagnosis of the enzootic bovine leukosis in herds of Pernambuco state, Brazil

2019 ◽  
Vol 86 ◽  
Author(s):  
Luiz Carlos Fontes Baptista Filho ◽  
Artur Cesar de Carvalho Fernandes ◽  
Tamyres Izarelly Barbosa da Silva ◽  
Taciana Rabelo Ramalho Ramos ◽  
Lúcio Esmeraldo Honório de Melo
Keyword(s):  
Leukemia Virus ◽  
High Sensitivity ◽  
Serum Levels ◽  
Diagnostic Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Enzootic Bovine Leukosis ◽  
Efficient Test ◽  
Bovine Leukosis ◽  
Low Prevalence

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease of cosmopolitan distribution and chronic character caused by a virus of the Retroviridae family, bovine leukemia virus (BLV). The epidemiological situation of EBL in Brazil has motivated studies to improve its diagnosis, based on the recommended serological techniques: agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). This study was designed to evaluate the use of imported ELISA for the detection of BLV in dairy herds raised in Pernambuco, Brazil, comparing it to AGID. Blood serum samples from 327 dairy cattle from the state of Pernambuco were tested to AGID and the imported commercial ELISA CHEKIT-Leucose-serum, produced by the IDEXX® laboratory for the diagnosis of EBL. Discarding 25 inconclusive samples from one or both tests, 302 samples were analyzed, being 24.1% positive (73/302) in the AGID and 45% (136/302) in the ELISA, which compared to the AGID, a technique considered standard, presented sensitivity of 98.6%, specificity of 72% and Kappa coefficient of 0.55. The lack of agreement in the diagnostic methods was probably due to the high sensitivity of the ELISA, which makes it possible to detect antibodies even in situations with low serum levels. Although AGID has been shown to be an efficient test so far, in more advanced stages of an EBL control and eradication program, with low prevalence rates, ELISA will present better performance, due to its higher sensitivity, avoiding the permanence of animals that spread the disease in the herds.

Development of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virus

2015 ◽  
Vol 61 (11) ◽  
pp. 811-817 ◽  
Author(s):  
Yufeng Li ◽  
Fangyuan Zheng ◽  
Baochao Fan ◽  
Hassan Mushtaq Muhammad ◽  
Yao Zou ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Virus Neutralization ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Diarrhea Virus ◽  
Coefficients Of Variation ◽  
Porcine Epidemic Diarrhea ◽  
The Relationship

Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by the porcine epidemic diarrhea virus (PEDV). To find a suitable ELISA method to assess the infection of PEDV and the effectiveness of vaccines, we developed and evaluated an indirect enzyme-linked immunosorbent assay (iELISA) based on a truncated recombinant spike (S) protein expressed in Escherichia coli. The parameters of the iELISA were optimized, and the cutoff value determined as 0.259 by analyzing optical density (OD) values of 80 PEDV negative sera confirmed by western blot. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were both less than 10%. Cross-reactivity assays demonstrated that iELISA was PEDV-specific. A virus neutralization test with sera of 7 different OD values showed a positive correlation between the OD values and virus neutralization. The results suggest this iELISA is specific, sensitive, and repeatable. Further studies should focus on the relationship between OD values of sera and its virus neutralization.

Tissue plasminogen activator as a novel diagnostic aid in acute pulmonary embolism

VASA ◽  
2014 ◽  
Vol 43 (6) ◽  
pp. 450-458 ◽  
Author(s):  
Julio Flores ◽  
Ángel García-Avello ◽  
Esther Alonso ◽  
Antonio Ruíz ◽  
Olga Navarrete ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Negative Predictive Value ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Predictive Value ◽  
Acute Pulmonary Embolism ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Utility ◽  
Tissue Plasminogen ◽  
D Dimer

Background: We evaluated the diagnostic efficacy of tissue plasminogen activator (tPA), using an enzyme-linked immunosorbent assay (ELISA) and compared it with an ELISA D-dimer (VIDAS D-dimer) in acute pulmonary embolism (PE). Patients and methods: We studied 127 consecutive outpatients with clinically suspected PE. The diagnosis of PE was based on a clinical probability pretest for PE and a strict protocol of imaging studies. A plasma sample to measure the levels of tPA and D-dimer was obtained at enrollment. Diagnostic accuracy for tPA and D-dimer was determined by the area under the receiver operating characteristic (ROC) curve. Sensitivity, specificity, predictive values, and the diagnostic utility of tPA with a cutoff of 8.5 ng/mL and D-dimer with a cutoff of 500 ng/mL, were calculated for PE diagnosis. Results: PE was confirmed in 41 patients (32 %). Areas under ROC curves were 0.86 for D-dimer and 0.71 for tPA. The sensitivity/negative predictive value for D-dimer using a cutoff of 500 ng/mL, and tPA using a cutoff of 8.5 ng/mL, were 95 % (95 % CI, 88–100 %)/95 % (95 % CI, 88–100 %) and 95 % (95 % CI, 88–100 %)/94 %), respectively. The diagnostic utility to exclude PE was 28.3 % (95 % CI, 21–37 %) for D-dimer and 24.4 % (95 % CI, 17–33 %) for tPA. Conclusions: The tPA with a cutoff of 8.5 ng/mL has a high sensitivity and negative predictive value for exclusion of PE, similar to those observed for the VIDAS D-dimer with a cutoff of 500 ng/mL, although the diagnostic utility was slightly higher for the D-dimer.

scholarly journals Pestivirus infections in cervids from the Czech Republic

2009 ◽  
Vol 54 (No. 4) ◽  
pp. 191-193
Author(s):  
K. Sedlak ◽  
T. Girma ◽  
J. Holejsovsky
Keyword(s):  
Czech Republic ◽  
Cervus Elaphus ◽  
Red Deer ◽  
Competitive Inhibition ◽  
Enzyme Linked Immunosorbent Assay ◽  
Border Disease Virus ◽  
Serum Samples ◽  
The Czech Republic ◽  
Diarrhea Virus ◽  
Border Disease

372 sera of cervids from the Czech Republic were examined for antibodies to the bovine viral diarrhea virus (BVDV) and border disease virus (BDV) by competitive-inhibition enzyme-linked immunosorbent assay (ELISA), and for the presence of the BVDV by AgELISA. Antibodies to BVDV/BDV were found in 0.6% (two positive/305 tested) red deer (<I>Cervus elaphus</I>). BVDV/BDV antibodies were not found in four sika deer (<I>Cervus Nippon</I>) and 63 fallow deer (<I>Dama dama</I>). All serum samples were BVDV antigen negative. Our results confirmed that red deer in the Czech Republic are only rarely infected with Pestiviruses. This was the first survey of pestiviruses in farmed and wild cervids in the Czech Republic.

scholarly journals PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 303
Author(s):  
Wei-Ting Hsu ◽  
Chia-Yu Chang ◽  
Chih-Hsuan Tsai ◽  
Sung-Chan Wei ◽  
Huei-Ru Lo ◽  
...  
Keyword(s):  
Recombinant Baculovirus ◽  
Immunocytochemical Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Perfect Agreement ◽  
Specific Antibodies ◽  
Diarrhea Virus ◽  
Serological Studies ◽  
A Cell ◽  
Infected Insect

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen’s kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.

Diagnosis of alveolar echinococcosis using immunoblotting with plural low molecular weight antigens

2009 ◽  
Vol 83 (1) ◽  
pp. 57-61 ◽  
Author(s):  
K. Yamano ◽  
A. Goto ◽  
M. Miyoshi ◽  
K. Furuya ◽  
Y. Sawada ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Alveolar Echinococcosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serological Screening ◽  
Screening Process ◽  
Medical Practitioners ◽  
Specific Reactions ◽  
Hospital Outpatients ◽  
Higher Sensitivity

AbstractAlveolar echinococcosis (AE) is endemic to Hokkaido, Japan. For the past 20 years, detection of AE among inhabitants has involved serological screening using an enzyme-linked immunosorbent assay (ELISA) followed by Western blotting (WB). Between the years 1987 and 2000, antigens targeted on 66, 55 and 30–35 kDa bands were routinely used in the WB step of AE diagnosis. However, since 2001 diagnosis has been dependent on three smaller molecular weight antigens (26–28, 18 and 7–8 kDa). Due to its higher sensitivity, this improved WB approach has been used as a confirmation step in the screening process and also for the testing of suspected AE cases in hospital outpatients. Using the improved WB technique, a total of 1745 serum samples were examined in 2001–2006 with 81 patients detected and registered with AE. Interestingly, sera from 76 of the 81 diagnosed AE patients (93.8%) demonstrated reactivity with all three antigens. However, sera from the remaining five patients (6.2%) demonstrated no reactivity with the 18 kDa antigen, even though they exhibited clearly detectable levels of reactivity with the 26–28 and 7–8 kDa bands. These results suggest that medical practitioners need to pay particular attention to the specific reactions to some different diagnostic antigens to minimize the risk of misdiagnosing AE patients. In turn, these results may also provide important diagnostic information for cystic echinococcosis (CE).

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