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

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 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

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/v1 ◽

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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Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfate-disrupted antigen.

Journal of Clinical Microbiology ◽

10.1128/jcm.25.4.633-636.1987 ◽

1987 ◽

Vol 25 (4) ◽

pp. 633-636 ◽

Cited By ~ 14

Author(s):

G C Winkler ◽

G M Brown ◽

H Lutz

Keyword(s):

Sodium Dodecyl Sulfate ◽

Immunosorbent Assay ◽

Sodium Dodecyl ◽

Anaplasma Marginale ◽

Enzyme Linked Immunosorbent Assay ◽

Dodecyl Sulfate

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“Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A

Analytical Biochemistry ◽

10.1016/0003-2697(81)90281-5 ◽

1981 ◽

Vol 112 (2) ◽

pp. 195-203 ◽

Cited By ~ 5126

Author(s):

W.Neal Burnette

Keyword(s):

Sodium Dodecyl Sulfate ◽

Western Blotting ◽

Protein A ◽

Sodium Dodecyl ◽

Polyacrylamide Gels ◽

Dodecyl Sulfate ◽

Electrophoretic Transfer

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Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blotting Analyses of Ureaplasmas Isolated from Dogs.

Journal of Veterinary Medical Science ◽

10.1292/jvms.54.325 ◽

1992 ◽

Vol 54 (2) ◽

pp. 325-328

Author(s):

Yumiko IMADA ◽

Ryo HARASAWA ◽

Hitoshi KOTANI ◽

Kaoru KOSHIMIZU

Keyword(s):

Sodium Dodecyl Sulfate ◽

Western Blotting ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Dodecyl Sulfate

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Serodiagnosis of Anthroponotic Cutaneous Leishmaniasis (ACL) Caused by Leishmania tropica in Sanliurfa Province, Turkey, Where ACL Is Highly Endemic

Clinical and Vaccine Immunology ◽

10.1128/cvi.00133-07 ◽

2007 ◽

Vol 14 (11) ◽

pp. 1409-1415 ◽

Cited By ~ 14

Author(s):

Fadile Yildiz Zeyrek ◽

Metin Korkmaz ◽

Yusuf Özbel

Keyword(s):

Cutaneous Leishmaniasis ◽

Western Blotting ◽

Blood Donors ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Western Blot Test ◽

Conventional Elisa ◽

Negative Controls ◽

Positive Controls

ABSTRACT In this study, we aimed to evaluate the validity of the conventional enzyme-linked immunosorbent assay (ELISA) and the Western blotting test for the diagnosis of anthroponotic cutaneous leishmaniasis (ACL) using serum samples obtained from 51 patients with parasitologically proven nontreated CL (NonT-CL patients) and 62 patients under treatment for CL (UT-CL patients). Additionally, 29 serum samples obtained from patients with parasitologically and serologically proven visceral leishmaniasis (VL) were also used as positive controls, and serum samples from 43 blood donors were used as negative controls. All sera were diluted to the same dilution (1/100). Leishmania infantum MON-1 was used as the antigen in the conventional ELISA. The sera of 27 (93.1%) of 29 VL patients were seropositive by ELISA, while the sera of 40 (78.4%) of 51 NonT-CL patients and 43 (69.3%) of 62 UT-CL patients were seropositive by the conventional ELISA. The absorbance values of the CL patients' sera were significantly lower than the absorbance values of the VL patients' sera. Bands between 15 and 118 kDa were detected in two groups of CL patients. Among all bands, the 63-kDa band was found to be more sensitive (88.5%). When we evaluated the Western blotting results for the presence of at least one of the diagnostic antigenic bands, the sensitivity was calculated to be 99.1%. By using serological tests, a measurable antibody response was detected in most of the CL patients in Sanliurfa, Turkey. It is also noted that this response can be changed according to the sizes, types, and numbers of lesions that the patient has. The Western blot test was found to be more sensitive and valid than the conventional ELISA for the serodiagnosis of ACL. In some instances, when it is very difficult to demonstrate the presence of parasites in the smears, immunodiagnosis can be a valuable alternative for the diagnosis of ACL.

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