scholarly journals Characterization and Evaluation of a Recombinant Multiepitope Peptide Antigen MAG in the Serological Diagnosis of Toxoplasma Gondii Infection in Pigs

2021 ◽  
Author(s):  
Yongle Song ◽  
Yongjuan Zhao ◽  
Ke Pan ◽  
Bang Shen ◽  
Rui Fang ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Large Scale ◽  
Serological Test ◽  
Animal Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Method ◽  
Peptide Antigen ◽  
Diagnostic Kits ◽  
Specificity And Sensitivity ◽  
Antibody Elisa

Abstract Background Toxoplasmosis caused by toxoplasma gondii (T. gondii) is a serious disease threatening human and animal health. People could be infected with T. gondii by ingesting raw pig meat contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Since commercial pig toxoplasma antibody ELISA diagnostic kits are too expensive, it is difficult to use them widely, moreover, the native antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is novel diagnostic marker, and it has the potential to be developed into more accurate and inexpensive diagnostic kits. Methods The synthetic multiepitope antigen (MAG) gene encoding a protein with epitopes from 5 T. gondii dominant antigen (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii positive and negative serum, then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG level after artificially infection with RH tachyzoites was evaluated through MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA). Results MAG antigen could be specifically recognized by pig anti-T. gondii positive but not negative serum. MAG-ELISA possessed a high diagnostic performance in terms of specificity and sensitivity. The overall coincidence rate between MAG-ELISA and a commercial PrioCHECK Toxoplasma antibody ELISA was 78.47%. MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection). Conclusions Our results suggest that MAG antigen could be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale diagnosis of T. gondii infection in pig farms and intensive industries.

scholarly journals Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yongle Song ◽  
Yongjuan Zhao ◽  
Ke Pan ◽  
Bang Shen ◽  
Rui Fang ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Large Scale ◽  
Serological Test ◽  
Animal Health ◽  
Screening Tests ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Method ◽  
Artificial Infection ◽  
Peptide Antigen ◽  
Diagnostic Kits

Abstract Background Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits. Methods The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK®Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA). Results MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection). Conclusions Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries. Graphical abstract

scholarly journals Validation of a Novel Commercial ELISA Test for the Detection of Antibodies against Coxiella burnetii

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1075
Author(s):  
Salvatore Ledda ◽  
Cinzia Santucciu ◽  
Valentina Chisu ◽  
Giovanna Masala
Keyword(s):  
Diagnostic Accuracy ◽  
Phase Ii ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Animal Health ◽  
Elisa Test ◽  
Clinical Diagnostics ◽  
Complex Life Cycle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity

Q fever is a zoonosis caused by Coxiella burnetii, a Gram-negative pathogen with a complex life cycle and a high impact on public and animal health all over the world. The symptoms are indistinguishable from those belonging to other diseases, and the disease could be symptomless. For these reasons, reliable laboratory tests are essential for an accurate diagnosis. The aim of this study was to validate a novel enzyme-linked immunosorbent assay (ELISA) test, named the Chorus Q Fever Phase II IgG and IgM Kit (DIESSE, Diagnostica Senese S.p.A), which is performed by an instrument named Chorus, a new device in medical diagnostics. This diagnostic test is employed for the detection of antibodies against C. burnetii Phase II antigens in acute disease. Our validation protocol was performed according to the Italian Accreditation Body (ACCREDIA) (Regulation UNI CEI EN ISO/IEC 17025:2018 and 17043:2010), OIE (World Organization for Animal Health), and Statement for Reporting Studies of Diagnostic Accuracy (STARD). Operator performance was evaluated along with the analytical specificity and sensitivity (ASp and ASe) and diagnostic accuracy of the kit, with parameters such as diagnostic specificity and sensitivity (DSp and DSe) and positive and negative predictive values (PPV and NPV), in addition to the repeatability. According to the evaluated parameters, the diagnostic ELISA test was shown to be suitable for validation and commercialization as a screening method in human sera and a valid support for clinical diagnostics.

scholarly journals An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses

2004 ◽  
Vol 34 (5) ◽  
pp. 1525-1529 ◽  
Author(s):  
Cristiane Divan Baldani ◽  
Rosangela Zacarias Machado ◽  
Paulo de Tarso Landgraf Botteon ◽  
Felipe Santoro Takakura ◽  
Carlos Luiz Massard
Keyword(s):  
Immunosorbent Assay ◽  
Serological Test ◽  
Epidemiological Studies ◽  
Sao Paulo ◽  
Enzyme Linked Immunosorbent Assay ◽  
São Paulo ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Babesia Equi ◽  
Specificity And Sensitivity

A crude antigenic preparation of Babesia equi was used to develop and establish the suitability of an enzyme-linked immunosorbent assay (ELISA) for the detection of parasite carriers. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 90 serum samples were taken from horses from the Northeast region of São Paulo State and examined for diagnosis of equine B. equi infection by ELISA. Approximately 75% (n=67) of all the horses tested were found serologically positive for B. equi. These results suggest that the ELISA described may prove to be an appropriate serological test for epidemiological studies on B. equi infections in the field and that equine piroplasmosis is a cause for serious concern in the State of São Paulo, Brazil.

scholarly journals SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sharif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

Abstract As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

scholarly journals SARS-CoV-2 S1 and N-Based Serological Assays Reveal Rapid Seroconversion and Induction of Specific Antibody Response in COVID-19 Patients

2020 ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sherif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

As the coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses. The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

scholarly journals Seroepidemiological study of the exposure to Toxoplasma gondii among horses in Algeria and analysis of risk factors

2019 ◽  
Vol 12 (12) ◽  
pp. 2007-2016 ◽  
Author(s):  
Sabrine Fazia Ouslimani ◽  
Safia Tennah ◽  
Naouelle Azzag ◽  
Salima Yamina Derdour ◽  
Bernard China ◽  
...  
Keyword(s):  
Risk Factors ◽  
Toxoplasma Gondii ◽  
Coat Color ◽  
Fluorescent Antibody ◽  
Animal Health ◽  
Significant Risk ◽  
Reference Method ◽  
Latex Agglutination ◽  
Enzyme Linked Immunosorbent Assay ◽  
Toxoplasma Infection

Aim: The aim of this study was to assess the seroprevalence of the Toxoplasma gondii in horses in different parts of Algeria and to determine risk factors for the infection. Materials and Methods: A total of 736 blood samples were collected from horses of various breeds, gender, coat colors, and ages. All horses came from various farms, racecourses, and equestrian centers. The seroprevalence was investigated by three different methods: Indirect fluorescent antibody test (IFAT) as reference method, enzyme-linked immunosorbent assay (ELISA), and latex agglutination test (LAT). Results: Out of the 736 sera, 178 (24.18%) were positive for IFAT, 133 (18.07%) for LAT, and 317 (43.07%) for ELISA. It was found that IFAT and LAT were in high agreement (Kappa 0.79), indicating that LAT and IFAT had similar capabilities in the detection of anti-T. gondii antibodies from horse sera. Risk factors analysis based on IFAT results indicated that the habit of the animals was significant risk factors (p≤0.05) for Toxoplasma infection. The seroprevalence was significantly higher in horses living on farms. Moreover, a higher seroprevalence was found in older animals compared to younger ones. Furthermore, the seroprevalence in females was significantly higher than that in males and gelding. Breed, coat color, and water sources are also important factors to influence the seroprevalence of T. gondii. Conclusion: The results indicated that T. gondii is present in horses throughout Algeria and thus represents a risk for both human and animal health. These results underline the need to increase the vigilance and the preventive measures against this disease not only to protect the horses but also to limit the spread of the parasite.

scholarly journals Detection of Bovine Antibodies against a Conserved Capsid Epitope as the Basis of a Novel Universal Serological Test for Foot-and-Mouth Disease

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
A. S. Asfor ◽  
N. Howe ◽  
S. Grazioli ◽  
S. Berryman ◽  
K. Parekh ◽  
...  
Keyword(s):  
Large Scale ◽  
Serological Test ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nonstructural Proteins ◽  
Fmd Virus ◽  
Foot And Mouth ◽  
Serological Surveillance ◽  
The Individual

ABSTRACT Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents. As a consequence, FMD reference laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and postvaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterized using a panel of intertype-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide enzyme-linked immunosorbent assay (VP2-ELISA) was optimized using experimental and reference antisera from immunized, convalescent, and naïve animals (n = 172). The VP2-ELISA is universal and simple and provided sensitive (99%) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for serosurveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in countries where the disease is endemic.

scholarly journals Comparative analysis of antigen-specific anti-SARS-CoV-2 antibody isotypes in COVID-19 patients

2020 ◽  
Author(s):  
Hidetsugu Fujigaki ◽  
Masato Inaba ◽  
Michiko Osawa ◽  
Saya Moriyama ◽  
Yoshimasa Takahashi ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Serological Test ◽  
Polymerase Chain Reaction Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
S Protein ◽  
Neutralizing Activity ◽  
N Protein ◽  
Specificity And Sensitivity ◽  
Antibody Isotypes

AbstractSerological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

scholarly journals Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and PCR in diagnostics of enzootic bovine leukosis

2005 ◽  
Vol 59 (3-4) ◽  
pp. 363-370
Author(s):  
Tadej Malovrh ◽  
M. Pate ◽  
M. Ocepek ◽  
B. Krt
Keyword(s):  
Antibody Response ◽  
Immunosorbent Assay ◽  
Large Scale ◽  
Serological Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Agar Gel ◽  
Bovine Leukaemia Virus ◽  
Leukaemia Virus ◽  
Elisa Kit ◽  
Pcr Method

Bovine leukaemia virus (BLV) is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID) test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA) has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR) method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gpSI) served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELiSA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV.

scholarly journals Multiple Indirect ELISAs for Serological Detection of SARS-CoV-2 Antibodies

2020 ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sherif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamari ◽  
...  
Keyword(s):  
Antibody Response ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Specificity And Sensitivity ◽  
Novel Coronavirus

As the coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus SARS-CoV-2, continues to spread rapidly around the world, there is an urgent need for validated serological assays to evaluate viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological tests to study the antibody response in COVID-19 patients. In order to validate the assays, we determined the cut-off values, sensitivity and specificity of the developed assays using sera collected from COVID-19 patients in Saudi Arabia at different time points after disease onset, as well as sera that are seropositive to other human CoVs; namely MERS-CoV, hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1. The SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N) ELISAs that we developed here not only showed high specificity and sensitivity, but also did not show any cross-reactivity with other CoVs. We also showed that all RT-PCR confirmed COVID-19 patients included in our study developed both virus specific IgM and IgG as early as one week after the onset of disease. The availability of these validated assays will enable us to determine the nature and duration of the antibody response mounted in response to SARS-CoV-2 infection. It will also allow conducting large-scale epidemiological studies to determine evidence of previous exposure to the virus and assess the true extent of virus spread within communities.

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