scholarly journals Obtaining Multiprotein Antigens Of Brucella And Study Of Their Immunoreactivity

2021 ◽  
Author(s):  
B.K. Ingirbai ◽  
A. Syzdykova ◽  
A.K. Kurmasheva ◽  
A.K. Bulashev
Keyword(s):  
Complement Fixation Test ◽  
Recombinant Dna ◽  
Outer Membrane Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Producer Strain ◽  
Recombinant Dna Technology ◽  
Serological Tests ◽  
Highly Sensitive ◽  
Antigenic Properties ◽  
Low Sensitivity

Rose-Bengal test, complement fixation test and the agglutination test are mainly used for the diagnosis of animal brucellosis. These tests are characterized by low sensitivity and specificity, which is one of the main reasons for the low effectiveness of measures aimed at eradicating brucellosis. The use of modern highly sensitive serological tests requires the availability of antigens specific to Brucella spp. The aim of the study was to obtain multiproteins of the pathogen by recombinant DNA technology and to study their antigenic properties. In the course of the study, three types of multiproteins were obtained, constructed from diagnostically important peptides that form B.abortus and B.melitensis outer membrane proteins. All target products were synthesized by the producer strain in a form that is authentic to natural proteins, and showed immunogenicity in mouse model. Antibodies produced against the multiproteins were specific for the single proteins of the pathogen's cell wall. The data obtained indicate the need to continue studies to determine the possibility of using multiproteins as an antigen in an enzyme-linked immunosorbent assay to detect anti-Brucella specific antibodies.

scholarly journals Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

2003 ◽  
Vol 10 (4) ◽  
pp. 658-663 ◽  
Author(s):  
A. Billecocq ◽  
D. Coudrier ◽  
F. Boué ◽  
B. Combes ◽  
H. Zeller ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Puumala Virus ◽  
Wild Rodents ◽  
Serological Tests ◽  
N Protein ◽  
Antigenic Properties ◽  
Native Antigen

ABSTRACT Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

Immunodiagnosis of human dirofilariasis by enzyme-linked immunosorbent assay using recombinant DNA-derived fusion protein

1992 ◽  
Vol 66 (3) ◽  
pp. 220-226 ◽  
Author(s):  
S. Sun ◽  
K. Sugane
Keyword(s):  
Fusion Protein ◽  
Immunosorbent Assay ◽  
Dirofilaria Immitis ◽  
Recombinant Dna ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Highly Sensitive ◽  
Human Sera ◽  
Human Dirofilariasis ◽  
Antibody Elisa

ABSTRACTAn enzyme-linked immunosorbent assay (ELISA) using antigenic β-galactosidase-Dirofilaria immitis recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human dirofilariasis. D. immitis-infected human sera reacted strongly with FP that was immobilized with anti-β-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other filariasis. In detection of anti-D. immitis IgG antibody. ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.

scholarly journals Recent Advances in the Evaluation of Serological Assays for the Diagnosis of SARS-CoV-2 Infection and COVID-19

2021 ◽  
Vol 8 ◽  
Author(s):  
Angela Chiereghin ◽  
Rocco Maurizio Zagari ◽  
Silvia Galli ◽  
Alessandra Moroni ◽  
Liliana Gabrielli ◽  
...  
Keyword(s):  
Point Of Care ◽  
Standard Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nasopharyngeal Swab ◽  
Pcr Assay ◽  
Serological Tests ◽  
Retrospective Diagnosis ◽  
Specific Igg ◽  
Low Sensitivity ◽  
Few Data

Introduction: Few data on the diagnostic performance of serological tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are currently available. We evaluated sensitivity and specificity of five different widely used commercial serological assays for the detection of SARS-CoV-2–specific IgG, IgM, and IgA antibodies using reverse transcriptase-PCR assay in nasopharyngeal swab as reference standard test.Methods: A total of 337 plasma samples collected in the period April–June 2020 from SARS-CoV-2 RT-PCR positive (n = 207) and negative (n = 130) subjects were investigated by one point-of-care lateral flow immunochromatographic assay (LFIA IgG and IgM, Technogenetics) and four fully automated assays: two chemiluminescence immunoassays (CLIA-iFlash IgG and IgM, Shenzhen YHLO Biotech and CLIA-LIAISON® XL IgG, DiaSorin), one electrochemiluminescence immunoassay (ECLIA-Elecsys® total predominant IgG, Roche), and one enzyme-linked immunosorbent assay (ELISA IgA, Euroimmune).Results: The overall sensitivity of all IgG serological assays was >80% and the specificity was >97%. The sensitivity of IgG assays was lower within 2 weeks from the onset of symptoms ranging from 70.8 to 80%. The LFIA and CLIA-iFlash IgM showed an overall low sensitivity of 47.6 and 54.6%, while the specificity was 98.5 and 96.2%, respectively. The ELISA IgA yielded a sensitivity of 84.3% and specificity of 81.7%. However, the ELISA IgA result was indeterminate in 11.7% of cases.Conclusions: IgG serological assays seem to be a reliable tool for the retrospective diagnosis of SARS-CoV-2 infection. IgM assays seem to have a low sensitivity and IgA assay is limited by a substantial rate of indeterminate results.

scholarly journals Dengue pre-vaccination screening test evaluation for the use of dengue vaccine in an endemic area

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257182
Author(s):  
Umaporn Limothai ◽  
Sasipha Tachaboon ◽  
Janejira Dinhuzen ◽  
Taweewun Hunsawong ◽  
Prapapun Ong-ajchaowlerd ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Geometric Mean ◽  
Dengue Infection ◽  
Reference Method ◽  
Elisa Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dengue Vaccine ◽  
Serum Samples ◽  
Serological Tests ◽  
Low Sensitivity

Background The dengue vaccine (Dengvaxia) is only recommended for individuals with prior dengue infection (PDI). This study aimed to perform a serosurvey to inform decision-making for vaccine introduction and identify appropriate target populations. We also evaluated the performance of the serological tests using plaque reduction neutralization test (PRNT) as a reference test in identifying PDI to determine suitability for pre-vaccination screening. Methods We enrolled 115 healthy individuals between 10 and 22 years of age living in the Ratchaburi province of Thailand. The serum samples were tested by PRNT to measure the prevalence and concentration of serotype-specific neutralizing antibodies. The performance of the IgG rapid diagnostic test (RDT, SD Bioline, Korea) and IgG enzyme-linked immunosorbent assay (ELISA, EUROIMMUN, Germany) in identifying PDI were evaluated by using PRNT as a reference method. Results Ninety-four (81.7%) individuals neutralized one or more dengue serotypes at a titer threshold greater than or equal to 10. Multitypic profiles were observed in 70.4% of the samples which increased to 91.9% in subjects aged 19–22. Among monotypic samples, the highest proportion was reactive against DENV-1 followed by DENV-2, DENV-3, and DENV-4. The highest anti-dengue antibody titers were recorded against DENV-1 and increased with age to a geometric mean NT50 titer (GMT) of 188.6 in the 19–22 age group. While both RDT and ELISA exhibited 100% specificity, RDT demonstrated low sensitivity (35%) with ELISA displaying much greater sensitivity (87%). Conclusions Almost 80% of adolescents and youth in Ratchaburi province had already been exposed to one or more of the dengue virus serotypes. The dengue IgG RDT displayed low sensitivity and is likely not be suitable for dengue pre-vaccination screening. These results support the use of IgG ELISA test for dengue vaccination in endemic areas.

scholarly journals Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle

2021 ◽  
pp. 2187-2196
Author(s):  
Aitbay K. Bulashev ◽  
Bakytkali K. Ingirbay ◽  
Kanatbek N. Mukantayev ◽  
Alfiya S. Syzdykova
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Cross Reactivity ◽  
Accurate Diagnosis ◽  
Expression Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chimeric Proteins ◽  
Serum Samples ◽  
Serological Tests

Background and Aim: An accurate diagnosis of Brucella-infected animals is one of the critical measures in eradication programs. Conventional serological tests based on whole-cell (WC) antigens and detecting antibodies against pathogen-associated lipopolysaccharide might give false-positive results due to the cross-reactivity with other closely related bacteria. This study evaluated the serological potential of Brucella spp. chimeric outer membrane proteins (Omps) as antigens in an indirect enzyme-linked immunosorbent assay (i-ELISA). Materials and Methods: The chimeric gene constructs of the most immunodominant regions of Brucella Omps 25+31, 25+19, and 19+31 were cloned into the pET28a expression vectors and transformed into Escherichia coli BL21 (DE3). The serological potential of chimeric proteins compared with single recombinant Omps (rOmps)19, 25, and/or 31 were studied on blood serum samples of (i) a rabbit immunized with killed Brucella abortus 19WC, (ii) mice immunized with single rOmps, (iii) cows seropositive for brucellosis by rose Bengal test, and (iv) cattle naturally and/or experimentally infected with brucellosis. Results: E. coli BL21 actively produced Brucella chimeric rOmps, the concentration of which reached a maximum level at 6 h after isopropyl-β-D-1-thiogalactopyranoside stimulation. Target proteins were antigenic and expressed in an active state, as recognized by rabbit anti-B. abortus antibodies in an i-ELISA and western blotting. Murine antibodies against the single rOmps reacted with chimeric antigens, and conversely, antichimeric antibodies found their epitopes in single proteins. Brucella chimeric rOmps showed higher antigenicity in blood sera of seropositive cattle kept in the hotbed of the infection and/or experimentally challenged with brucellosis than single proteins. Conclusion: Brucella chimeric recombinant outer membrane proteins could be a potential antigen candidate for developing an ELISA test for accurate diagnosis of bovine brucellosis.

scholarly journals Detection by Two Enzyme-Linked Immunosorbent Assays of Antibodies to Ehrlichia ruminantium in Field Sera Collected from Sheep and Cattle in Ghana

2003 ◽  
Vol 10 (5) ◽  
pp. 917-925 ◽  
Author(s):  
Lesley Bell-Sakyi ◽  
Enoch B. M. Koney ◽  
Otilia Dogbey ◽  
Keith J. Sumption ◽  
Alan R. Walker ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Amblyomma Variegatum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Ehrlichia Ruminantium ◽  
Ruminant Species ◽  
Cutoff Values ◽  
Highly Sensitive ◽  
Percent Positivity ◽  
Cutoff Levels

ABSTRACT Two serological tests for detection of antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, were compared by using field sera collected from sheep and cattle as part of serosurveys in Ghana. Sera selected as either negative or positive by a new polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) were tested by the indirect MAP1-B ELISA. Cutoff values of 14 percent positivity (14 PP) for both ruminant species were obtained for the MAP1-B ELISA by using preseroconversion Ghanaian sera and were compared with previously recommended cutoff values of 29 PP for sheep and 38 PP for cattle. With the 14-PP cutoff, of 151 sheep sera which tested negative by PC-ELISA, 89% were also negative by MAP1-B ELISA, while of 419 sheep sera positive by PC-ELISA, 98% were also positive by MAP1-B ELISA. Of 261 bovine sera negative by PC-ELISA, 82% were also negative by MAP1-B ELISA. Of 511 bovine sera positive by PC-ELISA, only 47% were positive by MAP1-B ELISA; these included 168 sera collected from cattle following first seroconversion as detected by both tests, with 125 of these sera positive by PC-ELISA but only 59 and 5 positive by MAP1-B ELISA with the 14- and 38-PP cutoff levels, respectively. These results indicate that both assays are highly sensitive and specific for detection of E. ruminantium exposure in sheep but that the MAP1-B ELISA lacks sensitivity for postseroconversion bovine sera in comparison to the PC-ELISA. Both tests confirm E. ruminantium seroprevalence of at least 70% in Ghanaian sheep; levels of exposure among Amblyomma variegatum-infested Ghanaian cattle are likely to be higher than the seroprevalence value of 66% obtained with the PC-ELISA.

scholarly journals Use of recombinant Brucella outer membrane proteins 19, 25, and 31 for serodiagnosis of bovine brucellosis

2020 ◽  
Vol 13 (7) ◽  
pp. 1439-1447
Author(s):  
Aitbay Bulashev ◽  
Orken Akibekov ◽  
Alfiya Syzdykova ◽  
Zhanbolat Suranshiyev ◽  
Bakytkali Ingirbay
Keyword(s):  
Outer Membrane ◽  
Single Injection ◽  
Outer Membrane Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Inoculation ◽  
Serological Testing ◽  
Bovine Brucellosis ◽  
Serum Samples ◽  
Serological Tests ◽  
Time Point

Background and Aim: Brucellosis remains one of the most common zoonoses. The current anti-brucellosis measures are largely deemed ineffective due to a lack of specificity of conventional serological tests. This study evaluated the use of Brucella outer membrane protein (Omp)19 for serodiagnostic testing. Materials and Methods: The antigenicity of recombinant Brucella Omp19, Omp25, and Omp31 was examined in serum samples from mice and rabbits immunized with Omp19 or Brucella abortus 19 whole cell (WC) and 12 and 152 cows experimentally or naturally infected with brucellosis, respectively. Serum samples were collected from 151 cows that were vaccinated with B. abortus 19 and 12 unvaccinated heifers that were maintained on a brucellosis-free farm. Results: Immunization with Omp19 resulted in antibody production in mice after a single injection without the use of adjuvant. Serum antibodies obtained from rabbits immunized with inactivated B. abortus strain 19 WC targeted Omps by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibodies targeting Omp19 were identified in all B. abortus strain 544 experimentally infected cows at day 14 post-inoculation (p.i.); Omp25 was detected by ELISA at day 28 p.i., while an ELISA targeting Omp31 was negative for 25% of cows at this time point. Omp19 and Omp25 were readily detected by sera from cows from a new epizootic focus. Antibodies recognizing Omps were also detected in >50% of the animals maintained in a brucellosis-free herd at 10 months after vaccination. Conclusion: Brucella Omp19 in combination with Omp25 and Omp31 may be utilized as target antigens in an ELISA designed for serological testing of unvaccinated cattle.

Evaluation of crude hydatid cyst fluid antigens for the serological diagnosis of hydatidosis in cattle

2010 ◽  
Vol 85 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Lemu Golassa ◽  
Tamrat Abebe ◽  
Asrat Hailu
Keyword(s):  
Hydatid Cyst ◽  
Addis Ababa ◽  
Cyst Fluid ◽  
Epidemiological Surveillance ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Efficiency ◽  
Infected Cattle ◽  
Serological Tests ◽  
Antigenic Properties ◽  
Hydatid Cyst Fluid

AbstractEchinococcosis is a zoonotic infection caused by adult or larval (metacestode) stages of cestodes belonging to the genusEchinococcus. The purpose of this study was to evaluate the antigenic ability of hydatid cyst fluid antigen for the diagnosis of hydatidosis in cattle using enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination test (IHA). The source of the antigens for the serological tests was fertile crude cyst fluids collected from naturally infected sheep at the Addis Ababa abattoir. A total of 502 sera were collected from 329 uninfected cattle and 173 hydatid-infected cattle which were confirmed by post-mortem examination. Most cysts were sterile and multiple organ infection predominated. Of 173 infected cattle, 166 (96.0%; confidence interval (CI) 91.8–98.4) were positive using ELISA while 7 (4.0%) were negative. Of 329 sera from uninfected cattle, 274 (83.3%; CI 78.8–87.2) were found to be negative and the remaining 55 (16.7%) were positive by ELISA. Of 173 infected cattle, 151 (87.3%; CI 81.4–91.9) were positive and 22 (12.7%) were negative by IHA. Of 329 negative sera tested using IHA, 266 (80.9%; CI 76.2–85.0) were negative and the remaining 63 (19.1%) were positive. The false positive and negative values of ELISA were 4.0 and 16.7%, respectively, and the corresponding values of IHA were 12.7 and 19.1%. The sensitivity and diagnostic efficiency of IHA were 87.2 and 83.6%, respectively. Crude hydatid cyst fluid antigen seems to have reasonable antigenic properties and hence could be employed for epidemiological surveillance of cattle hydatidosis.

Validity of a Serological Diagnostic Kit for SARS-CoV-2 Available in Iran

2020 ◽  
Vol 23 (9) ◽  
pp. 629-632
Author(s):  
Hamid Reza Shamsollahi ◽  
Mostafa Amini ◽  
Shaban Alizadeh ◽  
Saharnaz Nedjat ◽  
Ali Akbari-Sari ◽  
...  
Keyword(s):  
Diagnostic Techniques ◽  
Effective Control ◽  
Molecular Technique ◽  
Size Estimation ◽  
Medical Sciences ◽  
Serological Tests ◽  
Standard Case ◽  
Epidemic Size ◽  
Low Sensitivity ◽  
Negative Controls

Background: The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic broke out in December 2019 and is now characterized as a pandemic. Effective control of this infectious disease requires access to diagnostic techniques, for both case finding and epidemic size estimation. The molecular technique is routinely used worldwide. Although it is the "standard" case detection and management method, it has its own shortcomings. Thus, some easy-to-use rapid serological tests have been developed. Methods: One hundred and fourteen positive RT-PCR-diagnosed patients were tested by VivaDiag Kit, a brand of rapid serological kits available in hospitals affiliated to Tehran University of Medical Sciences (TUMS), Tehran, Iran. Frozen serum specimens taken from healthy people in summer and fall 2019 were also tested as negative controls. Results: Test sensitivity was 47.9% (95% confidence interval [CI]: 38.8-56.9) for IgM and 47.0% (95% CI: 38.0–56.0) for IgG. There was no difference between IgG and IgM seropositivity except in one case. Specificity was calculated as 99.0% (95% CI: 96.4–99.9) for IgM and of 100.0% (95% CI: 0.98.2–100.0) for IgG. Sensitivity was higher in men and older participants. Conclusion: This test can be used for epidemiological investigations, especially for the estimation of the level of infection in the community, after it is properly corrected for sensitivity and specificity. The low sensitivity could be attributed to the technical limitations of the kit or low levels of antibodies after infection. The different sensitivity in age and sex groups supports the hypothesis that different people show different immune responses to this virus.

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