scholarly journals Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xiao Teng Ching ◽  
Yee Ling Lau ◽  
Mun Yik Fong ◽  
Veeranoot Nissapatorn ◽  
Hemah Andiappan
Keyword(s):  
Western Blot ◽  
Affinity Purification ◽  
Expression System ◽  
Economic Loss ◽  
Serum Samples ◽  
Serological Tests ◽  
Human Patient ◽  
Prokaryotic Expression System ◽  
Human Sera ◽  
Lysate Antigens

Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).

Expression of Recombinant Human Cytomegalovirus Fusion Proteins pp150-pp65 Fragments and its Application

2013 ◽  
Vol 634-638 ◽  
pp. 1313-1318
Author(s):  
Yu Fen Jin ◽  
Yan Lei Li ◽  
Yan Hua ◽  
Xiao Gang Zhang ◽  
Ting Yu
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Human Cytomegalovirus ◽  
Colloidal Gold ◽  
Fusion Proteins ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Serum Samples ◽  
Igg Antibody ◽  
Prokaryotic Expression System

Objective To evaluate the effectiveness of prokaryotic expression of fusion proteins pp150-pp65 of human cytomegalovirus (hCMV) for its application as antigen, the fusion protein of pp150-pp65 were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column for preparing the colloidal gold kit. Methods Using DNA from HCMV strain as template, the genes encoding pp150 and pp65 protein fragment were amplified by PCR technique, respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pET28a-pp150-pp65 was transformed into E.Coil.BL21(DE3) and induced to express with IPTG. The expressed fusion protein was characterized by SDS-PAGE and western blot after purified, then we used the purified fusion protein to develop combined detection kit of IgM/IgG antibody against HCMV (colloidal gold method) with Beijing Innovita Bio-tech Co., Ltd for detecting the samples, compared with the imported kits (ELISA). Results The gene of fusion fragment pp150-pp65 was correctly amplified and the recombinant vector was successfully constructed. The purified protein with the molecular weight of 45KD had good antigenicity by western blot. The protein was subjected to assay with an ELI SA capture kit in its specific and sensitive assay based on colloidal gold nanoparticles, testing of 600 serum samples indicated that this kit had a sensitivity of 92.7%;, specificity of 83.1%, crude consistency o f 90.2%, compared to the imported HCMV-IgG kit; the sensitivity of the kit was 88.1%, specificity was 89.2%, coarse consistency was 88.5%, compared to the imported HCMV-IgM kit; Conclusion In this experiment, the HCMV antigen with high purity and specificity (pp150-pp65 recombinant protein) was prepared effectively through genetic engineering technology. Compared to imported reagents, the colloidal gold kit consisting of fusion protein in a capture assay had high sensitivity and specificity. Preliminary clinical use warrants further development and use of this kit. Furthermore, it provides a technological basis for detection of HCMV in different stages of clinical infection.

scholarly journals Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria

2020 ◽  
Vol 8 (5) ◽  
pp. 780
Author(s):  
Armando Navarro ◽  
Delia Licona-Moreno ◽  
Alejandro Monsalvo-Reyes ◽  
Ulises Hernández-Chiñas ◽  
Carlos A. Eslava-Campos
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Amino Acid Sequences ◽  
Serum Samples ◽  
E Coli ◽  
Peptide Mimotopes ◽  
Human Sera ◽  
Shared Epitopes

Background: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. Aim: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. Methods: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. Results: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. Conclusion: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

scholarly journals Optimization and Validation of Indirect ELISA Using Truncated TssB Protein for the Serodiagnosis of Glanders amongst Equines

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Harisankar Singha ◽  
Praveen Malik ◽  
Sachin K. Goyal ◽  
Sandip K. Khurana ◽  
Chiranjay Mukhopadhyay ◽  
...  
Keyword(s):  
Indirect Elisa ◽  
Expression System ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diagnostic Efficacy ◽  
Diagnostic Potential ◽  
Prokaryotic Expression System ◽  
Control Serum

Objective. To express truncated TssB protein ofBurkholderia malleiand to evaluate its diagnostic efficacy for serological detection of glanders among equines.Materials and Methods. In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences ofB. malleiTssB protein—a type 6 secretory effector protein—were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n=49), negative (n=30), and field serum samples (n=1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum.Results. In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively.Conclusions. The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

scholarly journals Diagnostic-test evaluation of immunoassays for anti-Toxoplasma gondii IgG antibodies in a random sample of Mexican population

2014 ◽  
Vol 8 (05) ◽  
pp. 642-647 ◽  
Author(s):  
Heriberto Caballero-Ortega ◽  
Rocío Castillo-Cruz ◽  
Sandra Murieta ◽  
Luz Belinda Ortíz-Alegría ◽  
Esther Calderón-Segura ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Western Blot ◽  
Latin American ◽  
High Sensitivity ◽  
Reference Standards ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Serological Tests ◽  
Western Blots ◽  
Open Population

Introduction: There are few articles on evaluation of Toxoplasma gondii serological tests. Besides, commercially available tests are not always useful and are expensive for studies in open population. The aim of this study was to evaluate in-house ELISA and western blot for IgG antibodies in a representative sample of people living in Mexico. Methodology: Three hundred and five serum samples were randomly selected from two national seroepidemiological survey banks; they were taken from men and women of all ages and from all areas of the country. ELISA cut-off was established using the mean plus three standard deviations of negative samples. Western blots were analysed by two experienced technicians and positivity was established according to the presence of at least three diagnostic bands. A commercial ELISA kit was used as a third test. Two reference standards were built up: one using concordant results of two assays leaving the evaluated test out and the other in which the evaluated test was included (IN) with at least two concordant results to define diagnosis. Results: the lowest values of diagnostic parameters were obtained with the OUT reference standards: in-house ELISA had 96.9% sensitivity, 62.1% specificity, 49.6% PPV, 98.1% NPV and 71.8% accuracy, while western blot presented 81.8%, 89.7%, 84.0%, 88.2% and 86.6% values and the best kappa coefficient (0.72-0.82). Conclusions: The in-house ELISA is useful for screening people of Mexico, due to its high sensitivity, while western blot may be used to confirm diagnosis. These techniques might prove useful in other Latin American countries.

scholarly journals Serodiagnosis of Visceral Leishmaniasis in Northeastern Italy: Evaluation of Seven Serological Tests

2020 ◽  
Vol 8 (12) ◽  
pp. 1847
Author(s):  
Margherita Ortalli ◽  
Daniele Lorrai ◽  
Paolo Gaibani ◽  
Giada Rossini ◽  
Caterina Vocale ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Western Blot ◽  
Rapid Test ◽  
Antibody Test ◽  
Screening Tests ◽  
Serum Samples ◽  
Serological Tests ◽  
Northeastern Italy ◽  
Indirect Immunofluorescent ◽  
Immunofluorescent Antibody Test

This study compares the performance of seven assays, including two ELISA (Leishmania ELISA IgG + IgM, Vircell Microbiologists; Leishmania infantum IgG ELISA, NovaTec), three rK39-based immunochromatographic tests (rK39-ICTs) (Leishmania Dipstick Rapydtest, Apacor; On Site Leishmania IgG/IgM Combo Rapid Test, CTK Biotech; LEISHMANIA Strip quick Test, Cypress Diagnostic), one indirect immunofluorescent antibody test (IFAT) (Leishmania-Spot IF, BioMérieux), and one western blot (WB) (Leishmania WESTERN BLOT IgG, LDBio Diagnostics) for serodiagnosis of visceral leishmaniasis (VL). Serum samples from 27 VL patients living in northeastern Italy were analyzed, as well as the serum samples from 50 individuals in whom VL diagnosis was excluded. The WB and the IFAT had 96% sensitivity, followed by the ELISA (63% and 74%, respectively). The rK39-ICT exhibited the worst performance among the serological tests, with sensitivities ranging from 52% to 70%. By combining selected ELISA/ICT, the sensitivity of VL detection reached 89%. IFAT and WB outperformed ELISA and rK39-ICT by possessing optimal sensitivity, but their high cost and complexity of execution would not allow their employment as screening tests. In conclusion, the combination of easy-to-perform tests, such as ICT and ELISA, could improve sensitivity in the serodiagnosis of Mediterranean VL.

scholarly journals Antigenicity of Leishmania braziliensis Histone H1 during Cutaneous Leishmaniasis: Localization of Antigenic Determinants

2002 ◽  
Vol 9 (4) ◽  
pp. 808-811 ◽  
Author(s):  
Emma Carmelo ◽  
Enrique Martínez ◽  
Ana Cristina González ◽  
José Enrique Piñero ◽  
Manuel E. Patarroyo ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Chagas Disease ◽  
Expression System ◽  
Protein A ◽  
Histone H1 ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leishmania Braziliensis ◽  
Serum Samples ◽  
Prokaryotic Expression System

ABSTRACT The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that L. braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L. braziliensis histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant L. braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.

scholarly journals Western Immunoblotting for Bartonella Endocarditis

2003 ◽  
Vol 10 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Pierre Houpikian ◽  
Didier Raoult
Keyword(s):  
Western Blot ◽  
Cross Reactivity ◽  
Molecular Techniques ◽  
Cat Scratch Disease ◽  
Serum Samples ◽  
Western Immunoblotting ◽  
Serological Tests ◽  
Heterologous Antigen ◽  
Bartonella Quintana ◽  
Causative Species

ABSTRACT To differentiate infectious endocarditis (IE) from other Bartonella infections and to identify infecting Bartonella bacteria at the species level on a serological basis, we used Western immunoblotting to test sera from 51 patients with Bartonella IE (of which 27 had previously benefited from species identification by molecular techniques), 11 patients with chronic Bartonella quintana bacteremia, and 10 patients with cat scratch disease. Patients with IE were Western blot positive in 49 of 51 cases, and significant cross-reactivity with three heterologous Bartonella antigens was found in 45 of 49 cases. Sera from bacteremic patients did not react with more than one heterologous antigen, and sera from patients with cat scratch disease gave negative results. Sera reacted only with B. henselae in four cases of IE, including one with a positive PCR result for valve tissue. Western blot and cross-adsorption performed on serum samples from patients with IE (the identity of the causative species having been determined by PCR) were demonstrated to identify efficiently the causative species in all cases. When applied to patients diagnosed on the basis of serological tests only, this technique allowed identification of the causative species in 20 of 22 cases. The results were in accordance with epidemiological features. Six reactive bands of B. quintana (of molecular sizes from 10 to 83 kDa) demonstrated significant association with sera from patients with B. quintana endocarditis. Overall, Western blotting and cross-adsorption made it possible to identify the causative species in 49 of 51 (96%) IE cases.

scholarly journals A novel multi-epitope recombinant protein for diagnosis of African swine fever

2020 ◽  
Author(s):  
ZHAN GAO ◽  
JUNJUN SHAO ◽  
GUANGLEI ZHANG ◽  
SUDAN GE ◽  
YANYAN CHANG ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
African Swine Fever Virus ◽  
Expression System ◽  
African Swine Fever ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Swine Diseases

Abstract Background: African swine fever(ASF) is an acute, severe and highly fatal infectious disease of pigs. The disease spreads rapidly, causing huge economic losses to the pig industry in infected areas. The structural proteins p30 and p54 in African swine fever virus(ASFV) have been verified as diagnostic antigens.Methods: In this study, we constructed a novel multi-epitope fusion antigen gene based on P30 and P54 proteins, induced expression in a prokaryotic expression system and analyzed the reactivity of the recombinant fusion protein. The purified recombinant protein m35 was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (ELISA) detection method for ASFV. 116 serum samples and positive sera of other swine diseases were detected by indirect ELISA.Results: Our results indicate that the m35 gene fragment with a length of 558bp was successfully constructed. SDS-PAGE and Western Blotting analysis showed that the protein had a band at 22kDa, proving its good reactogenicity. ROC analysis was performed to validate the assay, the area under the ROC curve is 0.9738 (95% confidence interval, 0.9336 to 1.014), and does not cross-react with other swine diseases.Conclusion: Our results show that its sensitivity and specificity were highly accurate. It is feasible to use this recombinant protein as a diagnostic antigen to distinguish ASFV infection.

scholarly journals New Coupled-Particle Light-Scattering Assay for Detection of Ro/SSA (52 and 60 Kilodaltons) and La/SSB Autoantibodies in Connective Tissue Diseases

2001 ◽  
Vol 8 (5) ◽  
pp. 922-925 ◽  
Author(s):  
Nicola Bizzaro ◽  
Fabrizio Bonelli ◽  
Elio Tonutti ◽  
Renato Tozzoli ◽  
Danilo Villalta
Keyword(s):  
Light Scattering ◽  
Connective Tissue ◽  
Lupus Erythematosus ◽  
Prokaryotic Expression ◽  
Viral Infections ◽  
Recombinant Dna ◽  
Expression System ◽  
Connective Tissue Diseases ◽  
Serum Samples ◽  
Prokaryotic Expression System

ABSTRACT The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjögren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.

scholarly journals The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica

2021 ◽  
Vol 120 (3) ◽  
pp. 979-991
Author(s):  
Rebekah B. Stuart ◽  
Suzanne Zwaanswijk ◽  
Neil D. MacKintosh ◽  
Boontarikaan Witikornkul ◽  
Peter M. Brophy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Liver Fluke ◽  
Glutathione Transferase ◽  
Fasciola Hepatica ◽  
Affinity Purification ◽  
Economic Loss ◽  
Differential Abundance ◽  
Parasitic Helminths

AbstractFasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

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