Development and Evaluation of Recombinant GRA8 Protein for the Serodiagnosis of Toxoplasma Gondii Infection in Goats

Abstract Background The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).Results Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.Conclusion Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

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Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

10.21203/rs.3.rs-46170/v3 ◽

2020 ◽

Author(s):

Charoonluk Jirapattharasate ◽

Ruenruetai Udonsom ◽

Apichai Prachasuphap ◽

Kodcharad Jongpitisub ◽

Panadda Dhepakson

Keyword(s):

Toxoplasma Gondii ◽

Recombinant Protein ◽

Sensitivity And Specificity ◽

Western Blotting ◽

Sodium Dodecyl ◽

Laboratory Animals ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Field Samples ◽

Specific Igg

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Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

10.21203/rs.3.rs-46170/v2 ◽

2020 ◽

Author(s):

Charoonluk Jirapattharasate ◽

Ruenruetai Udonsom ◽

Apichai Prachasuphap ◽

Kodcharad Jongpitisub ◽

Panadda Dhepakson

Keyword(s):

Toxoplasma Gondii ◽

Recombinant Protein ◽

Sensitivity And Specificity ◽

Western Blotting ◽

Sodium Dodecyl ◽

Laboratory Animals ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Field Samples ◽

Specific Igg

Abstract BackgroundThe development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).ResultsWestern blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.ConclusionOur findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera

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Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

BMC Veterinary Research ◽

10.1186/s12917-020-02719-3 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Charoonluk Jirapattharasate ◽

Ruenruetai Udonsom ◽

Apichai Prachasuphap ◽

Kodcharad Jongpitisub ◽

Panadda Dhepakson

Keyword(s):

Toxoplasma Gondii ◽

Recombinant Protein ◽

Sensitivity And Specificity ◽

Western Blotting ◽

Sodium Dodecyl ◽

Laboratory Animals ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Field Samples ◽

Specific Igg

Abstract Background The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). Results Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. Conclusion Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

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Simple and Rapid Method for Production of Whole-Virus Antigen for Serodiagnosis of Caprine Arthritis-Encephalitis Virus by Enzyme-Linked Immunosorbent Assay

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.2.352-356.2001 ◽

2001 ◽

Vol 8 (2) ◽

pp. 352-356 ◽

Cited By ~ 4

Author(s):

Carole Simard ◽

Molly Twinomwe Kibenge ◽

Patricia Singh ◽

Phyllis Dixon

Keyword(s):

Tissue Culture ◽

Sodium Dodecyl Sulfate ◽

Western Blotting ◽

Sodium Dodecyl ◽

Virus Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Optimum Time ◽

Caprine Arthritis Encephalitis Virus ◽

Time Of Harvest

ABSTRACT Polyethylene glycol (PEG) was used to produce whole-virus antigen derived from tissue culture cells infected with a Canadian strain of caprine arthritis-encephalitis virus. PEG antigen batches were obtained after precipitation and concentration of infected tissue culture material with PEG 8000 and final treatment with sodium dodecyl sulfate. The optimum time of harvest of tissue culture extracted material to produce the maximum amount of viral proteins was determined in roller bottles, after cocultivation of infected and noninfected fetal lamb corneal cells. Samples from day 9 to day 25 postculture were collected and processed. By Western blotting, the optimum time of harvest was found to be day 25 following the coculture. Two large batches of PEG antigen were prepared at the optimum time of harvest. Both batches gave similar results when tested by Western blotting and enzyme-linked immunosorbent assay (ELISA), using reference control sera from infected and noninfected goats. For further testing in ELISA, cutoff values and ratios were determined for PEG batch 1, using 200 known serum samples from goats free of the disease. The PEG antigen batch was compared with an in-house ELISA antigen in a kinetic mode, using 498 serum samples from field goats. The in-house ELISA antigen was produced following two rounds of ultracentrifugation and treatment with sodium dodecyl sulfate (R. A. Heckert, W. B. McNab, S. M. Richardson, and M. R. Briscoe, Can. J. Vet. Res. 56:237–241, 1992). The PEG antigen batch was found suitable for ELISA, with a relative specificity of 100% and a relative sensitivity of 99.4% compared to the in-house ELISA antigen. This method of antigen production for ELISA was found to be rapid, inexpensive, and reliable for the diagnosis of caprine-arthritis encephalitis, without requiring the use of sophisticated laboratory equipment.

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Development of an Immunochromatographic Assay Based on Dense Granule Protein 7 for Serological Detection of Toxoplasma gondii Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.00747-12 ◽

2013 ◽

Vol 20 (4) ◽

pp. 596-601 ◽

Cited By ~ 32

Author(s):

Mohamad Alaa Terkawi ◽

Kyohko Kameyama ◽

Nazim Hamza Rasul ◽

Xuean Xuan ◽

Yoshifumi Nishikawa

Keyword(s):

Toxoplasma Gondii ◽

Neospora Caninum ◽

Latex Agglutination ◽

Dense Granule ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Content Type ◽

Specificity And Sensitivity ◽

Field Samples

ABSTRACTDense granule antigen proteins derived fromToxoplasma gondii(TgGRAs) are potential antigens for the development of diagnostic tools. TgGRA7 and TgGRA14 were detected in the peritoneal fluid ofT. gondii-infected mice, suggesting that TgGRAs may be highly antigenic proteins. Here, TgGRA7 and TgGRA14 were evaluated as candidates for the development of a marker for a rapid diagnostic test. The specificity and sensitivity of purified recombinant proteins of TgGRA7 and TgGRA14 were compared in an indirect enzyme-linked immunosorbent assay (iELISA) using a series of serum samples fromT. gondii-experimentally infected mice and using recombinantT. gondiimajor surface antigen 2 (TgSAG2) as a reference control. The iELISA with TgGRA7 showed the greatest diagnostic accuracy and could detect anti-TgGRA7 antibody in acute and chronic infections. A total of 59 field samples from pigs were also examined by the iELISAs, and the results compared with those of the latex agglutination test (LAT). Among the three recombinant antigens, TgGRA7 had the highest rates of positivity, with significant concordance (88.14) and kappa value (0.76) in comparison with the results using LAT. Furthermore, an immunochromatographic test (ICT) based on recombinant TgGRA7 was developed for rapid detection of antibodies to the infection. The ICT differentiated clearly between sera fromT. gondii-infected mice and uninfected orNeospora caninum-infected mice. Pig sera were examined with the ICT, and the results compared favorably with those of LAT and iELISA for TgGRA7, with kappa values of 0.66 and 0.70 to 0.79, respectively. These data suggest that the ICT based on TgGRA7 is a promising diagnostic tool for routine testing in the clinic and mass screening of samples in the field.

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Enzyme-Linked Immunosorbent Assay Using Recombinant SAG1 Antigen To Detect Toxoplasma gondii-Specific Immunoglobulin G Antibodies in Human Sera and Saliva

Clinical and Vaccine Immunology ◽

10.1128/cvi.00512-12 ◽

2013 ◽

Vol 20 (4) ◽

pp. 468-473 ◽

Cited By ~ 16

Author(s):

Nouha Chahed Bel-Ochi ◽

Aïda Bouratbine ◽

Mohamed Mousli

Keyword(s):

Toxoplasma Gondii ◽

Sensitivity And Specificity ◽

Specific Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Native Form ◽

Content Type ◽

Percent Agreement ◽

Human Sera ◽

Commercial Kits

ABSTRACTSerologic detection ofToxoplasma gondiiIgG antibodies is widely accepted as a means to determine immune status and susceptibility toToxoplasmainfection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinantT. gondiiSAG1 antigen (rSAG1) to assess its diagnostic performance inToxoplasmaIgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

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Development and evaluation of a colloidal gold immunochromatographic assay based on recombinant protein CatL1D for serodiagnosis of sheep fasciolosis

Journal of Helminthology ◽

10.1017/s0022149x19000919 ◽

2019 ◽

Vol 94 ◽

Author(s):

W. Xifeng ◽

Q. Mengfan ◽

Z. Kai ◽

Z. Guowu ◽

L. Jing ◽

...

Keyword(s):

Recombinant Protein ◽

Sensitivity And Specificity ◽

Colloidal Gold ◽

Fasciola Hepatica ◽

Fusion Gene ◽

Immunochromatographic Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Specificity And Sensitivity ◽

Overlap Extension

Abstract Fasciolosis is a zoonotic parasitic disease that seriously endangers the development of animal husbandry and human health. In order to develop a rapid serological diagnostic method for fasciolosis in ruminants, the CatL1D and CatB4 genes of Fasciola hepatica were amplified by reverse transcription polymerase chain reaction (PCR) and cloned, respectively, and then the CatL-B fusion gene (MeCatL-B) was constructed by gene splicing by overlap extension PCR technique. The recombinant rCatL1D, rCatB4 and rMeCatL-B proteins were then prepared by prokaryotic expression, respectively, and the recombinant protein with high specificity and sensitivity was screened via indirect enzyme-linked immunosorbent assay. Using the selected recombinant protein rCatL1D as a diagnostic antigen, we developed a colloidal gold immunochromatographic assay (CGIA) for detecting F. hepatica-specific antibodies, and 426 serum samples of slaughtered sheep were used to evaluate the sensitivity and specificity of F. hepatica CGIA assay. The results showed that the sensitivity and specificity of rCatL1D protein (100%, 96.67%) were higher than those of rCatB4 (94.29%, 80%) and rMeCatL-B (91.43%, 90%). Compared with the gold standard post-mortem inspection, the specificity and sensitivity of the CGIA method was 100% and 97%, respectively, and the consistency rate between these two methods was 99.3%. These results confirmed that the CGIA method based on rCatL1D protein could be a promising approach for rapid diagnosis of sheep fasciolosis because of its high sensitivity and specificity.

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AN EVALUATION STUDY OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) USING RECOMBINANT PROTEIN GRA1 FOR DETECTION OF IgG ANTIBODIES AGAINTS TOXOPLASMA GONDII INFECTIONS

Indonesian Journal of Tropical and Infectious Disease ◽

10.20473/ijtid.v6i5.5903 ◽

2017 ◽

Vol 6 (5) ◽

pp. 105

Author(s):

Nina Difla Muflikhah ◽

Wayan Tunas Artama

Keyword(s):

Toxoplasma Gondii ◽

Recombinant Protein ◽

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Low Cost ◽

Evaluation Study ◽

Time Frame ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibody ◽

Elisa Kit

Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, an intracellular protozoan parasite that live inside the cells of the reticulo endothelial and parenchymal cells of human and animals (mammals and birds). Some cases of toxoplasmosis usually have no symptoms, but in any cases caused severe symptoms, such as hydrocephalus, microcephalus, intracranial calcification, retinal damage, brain abscess, mental retardation, lymphadenopathy, and others. Its severe symptoms usually showed a long time after first exposure, except symptoms showed by congenital transmission caused by infected mother. Early diagnosis is important to prevent the illness but methods for toxoplasmosis screening are still too expensive for developing country. Enzyme-linked immunosorbent assay (ELISA) allow the testing of a large number samples within short time frame and based on antibody or antigen detection. This study aimed to know the sensitivity and specificity of recombinat protein GRA1 as antigen using ELISA methods. We tested the sensitivity and spesificity of GRA1 protein as antigen in ELISA methods to diagnose toxoplasmosis and compared with ELISA Kit Commercial. Reliable laboratory testing is important to detect Toxoplasma gondii infection, and focused to improving the low cost and easy-to-use diagnostic instrument. Seventy sera collected and tested using both indirect ELISA, commercial ELISA kit and GRA1 protein coated as antigen. Fourty eight and fifty one samples showed positive IgG antibody result of ELISA-GRA1 and ELISA kit. Negative sample tested by ELISA-GRA1 was 22 samples and 19 sample tested by ELISA Kit. The sensitivity and specificity of GRA1-based on ELISA were 100% and 86.36%, positive prediction value (ppv) was 94.11%. These data indicate that the recombinant protein GRA1 is a highly immunogenic protein in human toxoplasmosis and become a promising marker for the screening of toxoplasmosis.

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Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba

Parasitology Research ◽

10.1007/s00436-021-07245-1 ◽

2021 ◽

Author(s):

Julio César Castillo-Cuenca ◽

Álvaro Martínez-Moreno ◽

José Manuel Diaz-Cao ◽

Angel Entrena-García ◽

Jorge Fraga ◽

...

Keyword(s):

Risk Factors ◽

Toxoplasma Gondii ◽

Meat Products ◽

Control Measures ◽

Health Concern ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Cross Sectional ◽

Weaning Pigs ◽

Associated Risk Factors

AbstractA cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1–16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm’s location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

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Potential linkage between Toxoplasma gondii Infection and physical education scores of College Students

10.1101/2020.10.20.346817 ◽

2020 ◽

Author(s):

Zhijin Sheng ◽

Yu Jin ◽

Yinan Du ◽

Xinlei Yan ◽

Yong Yao

Keyword(s):

College Students ◽

Physical Education ◽

Toxoplasma Gondii ◽

Protozoan Parasite ◽

Anhui Province ◽

Behavioral Changes ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

School Students ◽

Toxoplasma Gondii Infection

ABSTRACTObjectiveToxoplasma gondii is a worldwide protozoan parasite that could infect virtually all warm-blooded animals, including humans. Our study aimed to investigate the prevalence of T. gondii infection in college students at Anhui province, China. Moreover, growing studies demonstrated the association between T. gondii infection and host behavioral changes. We also studied the linkage between T. gondii and scores of college students.Methods2704 serum samples of medical school students attending physical education lessons were collected from September 2017 to September 2019 and evaluated for T. gondii IgG antibodies using an enzyme-linked immunosorbent assay (ELISA). We also analysed PE scores of T. gondii infected students and T. gondii uninfected students.ResultsThe overall seroprevalence of T. gondii was 11.5%. The main risk factors related to T. gondii infections were cat in the household and gardening or agriculture activity. Furthermore, in basketball group and football group, scores of T. gondii seropositive students were significantly higher than that of seronegative students, while in other sports there is no difference between scores of T. gondii infected students and T. gondii uninfected students.ConclusionThis is the first report of T. gondii seroprevalence in college students in Anhui province, China.

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