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

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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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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Development of Immunochromatographic Assay for the Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae Antibodies

Frontiers in Microbiology ◽

10.3389/fmicb.2021.743980 ◽

2022 ◽

Vol 12 ◽

Author(s):

Zhen Zhu ◽

Guanggang Qu ◽

Changjiang Wang ◽

Lei Wang ◽

Jige Du ◽

...

Keyword(s):

Immunoglobulin G ◽

Colloidal Gold ◽

Complement Fixation Test ◽

Complement Fixation ◽

Fixation Test ◽

Immunochromatographic Assay ◽

Economic Losses ◽

Serum Samples ◽

Mycoplasma Capricolum ◽

Prokaryotic Expression System

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

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

Ciência Rural ◽

10.1590/s0103-84782004000500031 ◽

2004 ◽

Vol 34 (5) ◽

pp. 1525-1529 ◽

Cited By ~ 12

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.

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Taenia saginata Metacestode Antigenic Fractions without Affinity to Concanavalin A Are an Important Source of Specific Antigens for the Diagnosis of Human Neurocysticercosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00516-09 ◽

2010 ◽

Vol 17 (4) ◽

pp. 638-644 ◽

Cited By ~ 9

Author(s):

Heliana B. Oliveira ◽

Gleyce A. Machado ◽

José R. Mineo ◽

Julia M. Costa-Cruz

Keyword(s):

Sensitivity And Specificity ◽

Concanavalin A ◽

Taenia Solium ◽

Specific Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Taenia Saginata ◽

Serum Samples ◽

Unbound Fraction ◽

Immunoblot Assay ◽

Homologous Antigen

ABSTRACT Taenia saginata metacestode antigens have been constituted a useful alternative antigen for neurocysticercosis (NC) serodiagnosis, particularly due to an increasing difficulty to obtain Taenia solium homologous antigen. Cross-reactivity with Echinococcus granulosus infection occurs in homologous and heterologous antigens and could be avoided by using different purified methods. The present study evaluated antigen fractions obtained from saline extracts of T. saginata metacestodes purified by affinity chromatography with jacalin or concanavalin A (ConA) lectins to detect IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis to diagnose human NC. Serum samples were collected from 142 individuals: 40 of them were diagnosed with NC, 62 presented Taenia sp. and other parasites, and 40 were apparently healthy individuals. The jacalin- and ConA-unbound fractions demonstrated sensitivity and specificity higher than those of bound fractions. Among unbound fractions, ConA demonstrated statistically higher sensitivity and specificity by ELISA (90% and 93.1%, respectively). By immunoblot assay, the 64- to 68-kDa component from the ConA-unbound fraction showed 100% sensitivity and specificity, making this component suitable for use as a specific antigen for diagnosis of NC. To our knowledge, this is the first report showing the relevance of using the unbound ConA fraction of T. saginata metacestodes to diagnose NC. In conclusion, the results obtained herein clearly demonstrate that antigenic fractions without affinity to ConA, obtained from T. saginata metacestodes, are an important source of specific peptides and are efficient in the diagnosis of NC when tested by immunoblot assay.

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Diagnosis of Visceral Leishmaniasis by Enzyme-Linked Immunosorbent Assay Using Urine Samples

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.4.789-794.2002 ◽

2002 ◽

Vol 9 (4) ◽

pp. 789-794 ◽

Cited By ~ 3

Author(s):

Mohammad Zahidul Islam ◽

Makoto Itoh ◽

S. M. Shamsuzzaman ◽

Rusella Mirza ◽

Farzana Matin ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Leishmania Donovani ◽

Laboratory Diagnosis ◽

Urine Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Healthy Controls ◽

Serum Samples ◽

Crude Antigen

ABSTRACT A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.

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Evaluation of a Blocking ELISA Using a Urease Conjugate for the Detection of Antibodies to Pseudorabies Virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200313 ◽

2000 ◽

Vol 12 (3) ◽

pp. 266-268 ◽

Cited By ~ 3

Author(s):

Maria Gabriela Echeverría ◽

Edgardo Omar Nosetto ◽

Maria Elisa Etcheverrigaray

Keyword(s):

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Pseudorabies Virus ◽

Visual Assessment ◽

Field Conditions ◽

Virus Neutralization ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Blocking Elisa ◽

Elisa Format

A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) was evaluated for screening sera for antibodies to pseudorabies virus under field conditions. A total of 764 serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing visual assessment of results.

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