Recombinant Protein-based ELISA Research Articles

Antioxidant Defence in Labeo rohita to Biotic and Abiotic Stress: Insight from mRNA Expression, Molecular Characterization and Recombinant Protein-Based ELISA of Catalase, Glutathione Peroxidase, CuZn Superoxide Dismutase, and Glutathione S-Transferase.

Fish possess numerous enzymatic antioxidant systems as part of their innate immunity. These systems have been poorly studied in Labeo rohita (rohu). The present study characterized and investigated the role of antioxidant genes in the defence mechanisms against two types of stressors, including infection and ammonia stress. Four key genes associated with antioxidant activity-catalase, glutathione peroxidase, glutathione S-transferase, and CuZn superoxide dismutase were successfully cloned and sequenced. These genes were found to be expressed in different tissues and developmental stages of rohu. The expression levels of these antioxidant genes in the liver and anterior kidney tissues of rohu juveniles were modulated in response to bacterial infection (Aeromonas hydrophila), parasite infection (Argulus siamensis), poly I:C stimulation and ammonia stress. Additionally, the recombinant proteins derived from these genes exhibited significant antioxidant and antibacterial activities. These proteins also demonstrated a protective effect against A. hydrophila infection in rohu and had an immunomodulatory role. Furthermore, indirect ELISA assay systems were developed to measure these protein levels in healthy as well as A. hydrophila and ammonia-induced rohu serum. Overall, this study characterized and emphasised the importance of the antioxidant mechanism in rohu's defence against oxidative damage and microbial diseases.

Development of a recombinant protein-based ELISA for detection of antibodies against bovine herpesvirus 6 (BoHV6).

Bovine herpesvirus 6 (BoHV6) belongs to the Herpesviridae family, Gammaherpesvirinae subfamily and Macavirus genus. It is common in cattle, but was also detected in American bison (Bison bison) and water buffalo (Bubalus bubalis). The aim of the experiment was to develop an ELISA for serological examination of cattle sera for the presence of anti-BoHV6 specific antibodies. Viral DNA from a BoHV6-positive cow was amplified by qPCR and the resulting fragments of the gB and gH genes encoding glycoproteins B and H (gB and gH) were cloned into the pLATE52 vector to express recombinant gB (rgB) and gH (rgH) in Rosetta (DE3) E. coli. The expressed recombinant proteins were used as antigens in the developed ELISA. The proteins expressed had the expected molecular weight. A total of 143 sera were examined, and 141 of them were positive, according to the chosen cut-off values of 9% and 10% for the sample-to-positive ratios of the rgB and rgH antigens, respectively. The rgB and rgH recombinant antigens of BoHV6 were successfully expressed in E. coli and successfully used in a newly developed ELISA.

Early detection and persistent positivity of anti-Leishmania antibodies using a recombinant protein-based ELISA in naturally infected dogs in Brazil

BackgroundVisceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania infantum, for which dogs constitute the main urban parasite reservoir. Control measures and the treatment of canine visceral leishmaniasis (CVL) are essential to reduce VL cases. Early and accurate detection of L. infantum-infected dogs is crucial to the success of VL control. To improve the serological detection of L. infantum-exposed dogs, we evaluated the early diagnosis capacity of a recombinant protein (rLci5) in an immunosorbent assay (ELISA) to detect naturally infected dogs. Additionally, we evaluated the persistence of the positive results obtained by rLci5 ELISA in comparison to other conventional diagnostic test methods.MethodsSerum samples obtained from 48 L. infantum-infected dogs involved in a cohort study were evaluated using different diagnostic methods (qPCR, EIE-LVC, DPP-LVC and splenic culture). The results were compared to rLci5 ELISA to determine its capacity to diagnose L. infantum infection at earlier infection time points. The persistence of positive diagnostic test results was also compared for each dog evaluated.ResultsrLci5 ELISA presented higher rates of positive results at early time points compared to the other diagnostic tests employed in the cohort study, as early as 24 months prior to detection by other tests. rLci5 ELISA positivity was 52.1% (25/48) at baseline, while qPCR was 35.4% (17/48), DPP-LVC 27.1% (13/48), EIE-LVC 22.9% (11/48) and culture only 4.2% (2/48). In at least one of the time points of the 24-month cohort study, rLci5 ELISA was positive in 100% (48/48) of the dogs, versus 83% (40/48) for qPCR, 75% (36/48) for DPP-LVC, 65% (31/48) for EIE-LVC and 31% (15/48) for culture. Investigating clinical signs in association with diagnostic test positivity, rLci5 ELISA successfully detected CVL in 62.9% (95/151) of the clinical evaluations with a score of 0–3, 64.3% (45/70) with scores between 4 and 7, and 73.7% (14/19) with scores > 7, providing higher rates of positivity than all other methods evaluated. Moreover, rLci5 ELISA presented the greatest persistence with respect to test positivity: 45.8% of the dogs evaluated.ConclusionFour diagnostic tests were compared to rLci5 ELISA, which presented earlier infection diagnosis and a greater persistence of positive test results. Accordingly, the use of the rLci5 ELISA can improve CVL diagnostic performance by detecting infected dogs sooner than other testing methods, with enhanced persistence of positive results over the course of the infection.Graphic abstract

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Development of a ery-C recombinant protein-based ELISA approach for differentiating brucellosis infected cattle from vaccinated ones

Development of a ery-C recombinant protein-based ELISA approach for differentiating brucellosis infected cattle from vaccinated ones

Development of a Recombinant Protein-based ELISA for Detection of Antibodies Against Bovine Foamy Virus.

IntroductionInfections with bovine foamy virus (BFV) were found in many countries but there is a lack of large-scale surveys on the prevalence of BFV among dairy cattle. The aim of this study was to develop and validate the recombinant Gag protein-based ELISA and to estimate the prevalence of antibodies against BFV.Material and MethodsGag coding region from BFV was cloned into expression vector pT7Arg-STOP, which expressed a high level of recombinant Gag protein from E.coli. The ELISA was standardised, and the cut-off value and sensitivity and specificity of the test were calculated using a receiver operating characteristic and Bayesian estimation.ResultsA total of 3,051 serum samples were tested by ELISA and 939 (30.8%) sera were recognised as positive. When Bayesian approach was used, the overall true BFV prevalence was 29.7% (95% CI: 25.9–33.4%).ConclusionExpressed Gag protein of BFV has been used successfully as an antigen for ELISA. Eventually, this study provides basic information about the epidemiological status of infection with BFV in dairy cattle in Poland, which can be used for further studies on dissemination and transmission of BFV infection.

Evaluation of six novel antigens as potential biomarkers for the early immunodiagnosis of schistosomiasis.

BackgroundEarly diagnosis of schistosomiasis, prior to egg laying, would enable earlier treatment and help interrupt the transmission cycle of the parasite and the progress of the disease. Previously we identified six novel antigens with potential as diagnostic markers for human Schistosoma japonicum infections. In this study, we evaluated these antigens as candidate biomarkers for the early diagnosis of schistosomiasis in mice and rabbits.MethodsThe transcriptional profiles of the six antigens (SjSP-13, SjSP-23, SjSP-160, SjSP-164, SjSP-189 and SjSP-216) at different developmental stages were analyzed by quantitative PCR. The recombinant proteins were expressed in E. coli and purified with nickel chelate affinity chromatography. We then developed recombinant protein-based ELISA kits to analyze the kinetics of antigen-specific antibodies during the course of infection in mice and rabbits. The early diagnostic validity of the candidate SjSP-216 was further evaluated in mice and rabbits infected with S. japonicum.ResultsOf the six antigens, SjSP-13, SjSP-160 and SjSP-216 were highly expressed in 21-day old young worms, while SjSP-23, SjSP-164 and SjSP-189 were highly expressed in eggs. In the mouse model, we detected a significant increase in antibodies against SjSP-13 and SjSP-216 at 3 weeks post-infection. However, in the rabbit model, only anti-SjSP-216 antibody showed a significant increase at this time point. We recorded 100 % diagnostic sensitivity and specificity of SjSP-216-based ELISA in both infected mice and rabbits, 3 weeks after infection.ConclusionsThis study strongly suggests that SjSP-216, a highly expressed gene in the young worms, could serve as a potential biomarker for the early immunodiagnosis of S. japonicum infections in vertebrate hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1048-2) contains supplementary material, which is available to authorized users.

Specific antibody responses against Neospora caninum recombinant rNcGRA7, rNcSAG4, rNcBSR4 and rNcSRS9 proteins are correlated with virulence in mice

The intraspecific diversity of Neospora caninum is a determinant for in vivo parasite virulence and in vitro parasite behaviour. The relationship between isolate virulence and specific antibody responses against key parasite proteins has not been well characterized. The response kinetics and the differences in specific anti-rNcGRA7, -rNcSAG4, -rNcBSR4 and -rNcSRS9 antibody levels were analysed by recombinant protein-based ELISA in groups of mice inoculated with 10 different N. caninum isolates that differ in their virulence. The majority of the virulence parameters analysed correlated with the specific antibody levels against the 4 recombinant proteins. The antibodies developed against the highly immunogenic protein NcGRA7 were significantly higher in mice inoculated with high virulence isolates than in those inoculated with low-to-moderate virulence isolates in both non-pregnant and pregnant mouse models. Moreover, these levels were correlated with the anti-N. caninum IgG1 and IgG2a responses and the in vitro tachyzoite yield at 56 h. The antibodies directed against the bradyzoite-specific proteins were not detected in a non-pregnant mouse model. However, some seropositive mice were found in groups inoculated with high virulence isolates in a pregnant mouse model. NcGRA7 and NcSAG4 are proteins clearly correlated with virulence, and to a lesser extent NcBSR4 and NcSRS9 proteins. Moreover, antibodies to bradyzoite-specific proteins appear to also be related to virulence in mice. Further analyses should be performed in order to verify the usefulness of these proteins as predictive markers for virulence in an experimental bovine model of neosporosis.

Field application of a recombinant protein-based ELISA during the 2010 outbreak of foot-and-mouth disease type A in South Korea

A recombinant protein-based enzyme-linked immunosorbent assay (RP ELISA) exists for the detection of antibodies to foot-and-mouth disease virus (FMDV) type A. In this study, the efficacy of the RP ELISA was compared to that of other current tests by examining sera collected in the field during an FMD type A outbreak in South Korea in 2010. The RP ELISA detected early antibodies to FMDV with the same sensitivity as the liquid-phase blocking ELISA (LPB ELISA), identifying FMD farm outbreaks correctly on a herd basis. In addition, the two assays exhibited a high correlation coefficient (γ(2)=0.83) when testing thirty seven sera from one outbreak farm exhibiting various antibody titers. The sensitivity and specificity of the RP ELISA relative to the LPB ELISA were 84% and 97%, respectively, and excellent agreement (kappa=0.82) was observed between the two tests. Taken together, the RP ELISA should be a useful alternative to the LPB ELISA for the detection of early antibodies to FMDV type A during an outbreak.

A recombinant protein-based ELISA for detecting antibodies to foot-and-mouth disease virus serotype Asia 1

A recombinant protein-based ELISA was evaluated for detecting antibodies to foot-and-mouth disease virus (FMDV) serotype Asia 1. The recombinant protein (rP13C) was derived from the P1 precursor and 3C protease genes that were cloned into a single expression vector and expressed in insect cells. This protein elicited a low titer of FMDV neutralizing antibodies in pigs. Its utility as a diagnostic antigen was explored in a blocking ELISA using monoclonal antibodies. The rP13C ELISA yielded higher endpoint titers than the liquid phase blocking (LPB) ELISA and virus neutralization test performed on sera from goats challenged with FMDV post-vaccination. The rP13C ELISA correctly scored the FMD international reference weak positive serum. The relative sensitivity between the rP13C ELISA and LPB ELISA was equivalent for vaccinated sera. With this comparable sensitivity, the rP13C ELISA exhibited a specificity of 99.7% for domestic naive swine, bovine and caprine sera. This report demonstrates that an ELISA using recombinant proteins has the potential to replace the LPB ELISA using an inactivated FMDV antigen as a simple and robust serological tool for screening antibodies to FMDV serotype Asia 1.

An immunoproteomic approach for identification of clinical biomarkers of Whipple's disease

Whipple's disease (WD) is a chronic multisystemic infection, caused by the bacterium Tropheryma whipplei. The main clinical presentations are classic WD (CWD) with histologic lesions in the gastrointestinal tract, endocarditis, and isolated neurologic infection. The current strategy for diagnosis remains invasive.The present study aimed to select the protein candidates for serological diagnosis of WD. The first step was to identify candidate proteins by an immunoproteomic approach combining 2-DE using a total extract of a T. whipplei, immunoblotting, and MS. The second step was to validate the discovered biomarkers using a recombinant protein-based ELISA. Serum samples from 18 patients with WD and from 54 control individuals were tested. Asugar ABC transporter, TWT328 (sensitivity (Se) 61%, specificity (Sp) 87%, positive predictive value (PPV) 61%, negative predictive value (NPV) 87%, and positive likelihood ratio (PLR) 4.69) was the best marker for development of serodiagnosis for CWD. We also obtained a reproducible immunoreactive protein pattern for patients with isolated neurological infection due to T. whipplei (Se 100%, Sp 93%, PPV 55.5%, NPV 100%, and PLR 13.51) as an encouraging step towards noninvasive diagnosis of this particular manifestation. Nine recombinant candidates have been successfully screened with serum samples. Results from these ELISA assays skewed with those obtained with immunoblots.

Comparison of sensitivity and specificity in three commercial foot-and-mouth disease virus non-structural protein ELISA kits with swine sera in Taiwan

Three commercialized ELISA kits for the detection of antibodies to the non-structural proteins (NSPs) of FMD virus were compared, using sera from uninfected, vaccinated, challenged and naturally infected pigs. The kinetics of the antibody response to NSPs was compared on sequential serum samples in swine from challenge studies and outbreaks. The results showed that ELISA A (UBI) and ELISA B (CEDI) had better sensitivity than that of the 3ABC recombinant protein-based ELISA C (Chekit). The peak for detection of antibodies to NSPs in ELISA C was significantly delayed in sera from natural infection and challenged swine as compared to the ELISA A and B. The sensitivity of the three ELISAs gradually declined during the 6-month post-infection as antibodies to NSP decline. ELISA kits A and B detected NSP antibody in 50% of challenged pigs by the 9–10th-day and 7–8th-day post-challenge, respectively. ELISA B and C had better specificity than ELISA A on sequential serum samples obtained from swine immunized with a type O FMD vaccine commercially available in Taiwan. Antibody to NSPs before vaccination was not detected in swine not exposed to FMD virus, however, antibody to NSPs was found in sera of some pigs after vaccination. All assays had significantly lower specificity when testing sera from repeatedly vaccinated sows and finishers in 1997 that were tested after the 1997 FMD outbreak. However, when testing sera from repeatedly vaccinated sows or finishers in 2003–2004, the specificity for ELISAs A, B and C were significantly better than those in 1997. This effect was less marked for ELISA A. The ELISA B was the best test in terms of the highest sensitivity and specificity and the lowest reactivity with residual NSP in vaccinates.

Recombinant protein‐based ELISA and immuno‐cytochemical assay for the diagnosis of SARS

A new Coronavirus (SARS‐CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in‐house ELISA based on SARS‐CoV recombinant antigens was developed. The SARS‐CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine‐tagged proteins. Six reference sera from SARS patients were used to detect virus‐specific IgG in an ELISA using each recombinant protein as coating antigen. High‐titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non‐SARS, low‐tract respiratory infections. Specific hyper‐immune sera to SARS‐CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS‐CoV infected and uninfected cells. J. Med. Virol. 76:137–142, 2005. © 2005 Wiley‐Liss, Inc.

Des corps élémentaires aux protéines recombinantes : comparaison des techniques sérologiques utilisées pour la détection des anticorps spécifiques de Chlamydia pneumoniae

Different serological techniques were evaluated using serological samples and throat swabs from patients with acute bronchitis episodes, in order to assess the diagnostic value of anti- Chlamydia pneumoniae antibodies. Throat swabs were used for PCR amplification of C. pneumoniae DNA with the PCR ServiAmpli C. pneumoniae technique. Serums were used for detection of specific anti- C. pneumoniae antibodies with four different ELISA techniques, and with the microimmunofluorescence (MIF) technique. Results show comparable sensitivities between MIF and Elementary Body-based ELISA techniques, and superior specificity for recombinant protein-based ELISA.

Serologic Diagnosis of Caprine Arthritis-Encephalitis by Elisa with Two Recombinant Proteins in a Parallel Testing Format

A scheme for screening sera for antibodies to caprine arthritis-encephalitis virus (CAEV) was evaluated for its ability to identify positive and negative samples in a population with heterogeneous risk factors, using the criteria of sensitivity, specificity and positive predictive value. Five hundred caprine serum samples were tested using a transmembrane recombinant-based ELISA. Those that gave positive results were considered positive, while those with equivocal or negative results were retested with a core recombinant protein-based ELISA. Equivocal results after the second test were considered indeterminate and retesting is advised. Using this approach, a sensitivity, specificity and positive predictive value of 98.8%, 97.2% and 98.6% were obtained. These values are superior to those obtained by these tests used individually. The high sensitivity, specificity and predictive value of this new scheme of CAEV screening make it an attractive addition to any control or eradication program.