Recombinant ELISA Research Articles

Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

Efficiency of CMV serodiagnosis during pregnancy in daily laboratory routine

BackgroundMaternal acute primary cytomegalovirus (CMV) infection during the first trimester may cause severe long-term sequelae in newborns. For risk assessment, serological screening is routinely performed in pregnant women based on IgM, IgG and avidity tests using whole-virus antigen. A recent study evaluated the diagnostic value of recombinant protein-based ELISAs as second-line tests in pregnancy CMV screening, including anti-p52 IgM and anti-gB IgG as markers defining the early and late phase of infection, respectively. In the present study, these recombinant ELISAs were used as first-line screening tests in daily laboratory routine and compared to lysate-based assays with respect to [i] the number of conclusive results obtained with the initial sample and [ii] the underlying workload. Methods553 unselected routine serum samples from pregnant women were tested for anti-CMV IgM and IgG antibodies using lysate-based ELISAs and avidity testing. Anti-CMV IgM antibodies against recombinant p52 and anti-CMV IgG antibodies against recombinant glycoprotein B (gB) were also determined by ELISA. All assays were performed and interpreted according to the manufacturer’s instructions. ResultsFor lysate-based IgM, IgG and avidity testing, 84.6 % of samples yielded conclusive results in a total of 1156 tests, while 15.4 % needed follow-up testing of a consecutive sample. Anti-p52 CMV IgM and anti-gB CMV IgG testing produced conclusive results for 92.8 % of samples in a total of 1026 tests, while 7.2 % samples required follow-up testing. ConclusionsThe first-line use of ELISAs measuring anti-p52 CMV IgM and anti-gB CMV IgG antibodies to test for maternal CMV infection increases the number of conclusive results derived from an initial serum sample while requiring a considerably lower number of tests compared to the lysate-based approach. For day-to-day routines in a diagnostic laboratory, this high efficiency of the recombinant testing approach has significant practical relevance.

Chagas Disease: Seroprevalence and Associated Factors in Indigenous Communities of the Southern Limit of Argentine Chaco.

Chagas disease is more prevalent in socially vulnerable communities in the Gran Chaco Eco-region. The study evaluated the seroprevalence of Chagas disease and associated factors between May 2014 and September 2015, in indigenous communities of Santa Fe, Argentina, in the southern Chaco. Lysate ELISA and indirect hemagglutination tests were used to detect antibodies against Trypanosoma cruzi, and recombinant ELISA was used in the case of disagreement. Household surveys were conducted with the head of household about risk factors for the disease. Serological tests were conducted on 298 people from three indigenous communities, 127 male and 171 female. Seroprevalence was 18.5%. A total of 64 surveys were conducted; 82.8% of the heads of household were male, with a median age of 39 years, and 61.0% had not completed primary school. In 35.9% of the households, there was at least one member of the cohabiting group infected with T. cruzi. The level of education of the head of household showed a statistically significant association with Chagas disease (OR = 3.43), among all the risk factors studied. The prevalence of infection is lower than that of other indigenous communities of the Gran Chaco, probably because environmental conditions are moderating and disfavoring the establishment of the insect vector in homes, but also because of socioeconomic differences with the rest of the eco-region. Beyond this, serological controls are needed to prevent vertical transmission.

Human Infections with Borna Disease Virus 1 (BoDV-1) Primarily Lead to Severe Encephalitis: Further Evidence from the Seroepidemiological BoSOT Study in an Endemic Region in Southern Germany

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

Comparative diagnostic efficacy of Avidin-Biotin recombinant nucleoprotein competitive ELISA for serosurveillance and monitoring of peste des petits ruminants in sheep and goats

In this study extensive evaluation of Avidin-Biotin recombinant nucleoprotein competitive ELISA (ABrC-ELISA) was carried out by mass screening of a large number of sera to make use of this assay for serosurveillance and seromonitoring of peste des petits ruminants (PPR) in sheep and goats to evaluate its diagnostic efficacy value and strengthen findings associated with the assay. The recombinant PPR virus (PPRV) nucleoprotein was over-expressed in E. coli, Ni-NTA affinity-purified, and characterized and used as coating diagnostic antigen in ABrC-ELISA, and evaluated using the field sera from animals. On evaluation of the diagnostic performance or efficacy of this assay using the pre-vaccinated and post-vaccinated sera of sheep and goats (n = 1437), the ABrC-ELISA showed a relative diagnostic sensitivity of 87.2% (95% CI: 84.1–90%) and diagnostic specificity of 92.0% (95% CI: 90—93.7%), against well-established existing indigenous H protein-specific PPR competitive ELISA kit with an accuracy of 90.1% (95% CI: 88.5–91.7%) and good or substantial agreement of Cohen's Kappa value of 0.79 ± 0.017 SE (95% CI: 0.76 to 0.82). These findings suggest that the ABrC-ELISA is a potential additional diagnostic tool of a rapid, sensitive, and specific assay for the detection of the PPRV nucleoprotein antibodies in sera of sheep and goats. This PPR Ab Chek kit can be used extensively under field conditions for serosurveillance, and seromonitoring of PPR in sheep and goats at the eradication /post-eradication phase in disease-controlled countries or PPR non-enzootic countries.

Establishment of an indirect ELISA method for antibody detection of porcine pseudorabies by recombinant gB, gC, and gD proteins.

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide.This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET-28a+ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichiacoli.The results of thepolymerase chain reactionand enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme-linked immunosorbent assay(ELISA) analysis of 184 PRV-positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D-ELISAhas the highest specificity and sensitivity among the ELISA methods developed in this study.We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein-based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.

Seroprevalence of Getah virus in Pigs in Eastern China Determined with a Recombinant E2 Protein-Based Indirect ELISA.

Getah virus (GETV), in the genus Alphavirus and the family Togaviridae, has been detected throughout the world. GETV causes high morbidity and mortality in newborn piglets, entailing serious economic losses. Therefore, the experimental work on GETV detection is necessary. However, due to the influence of a variety of unavoidable factors, the ELISA test for the primary screening of animal diseases has low accuracy in detection results. Therefore, we optimized a recombinant E2 (rE2) protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of GETV antibodies in pig serum. The E2 protein was successfully expressed and purified with SDS-PAGE. A Western blotting analysis of sera from infected pigs showed strong reaction with a viral antigen of ~46 KDa corresponding to the E2 glycoproteins. By using chessboard titration and comparing the P/N values, we found that the optimal concentration of coated antigen was found to be 24.5 μg/mL, and the optimal dilution of serum specimens was 1:100. The best working dilution of the horseradish peroxidase (HRP)-conjugated goat anti-pig immunoglobulin (IgG) was 1:5000. The optimal coating conditions were 12 h at 4 °C. The optimal incubation conditions for serum specimens, blocking, and reaction with the secondary antibody were all 1 h at 37 °C. We also investigated the seroprevalence of GETV in 133 serum specimens collected in Eastern China, and 37.59% of the samples tested positive for anti-GETV IgG antibodies, indicating that the seroprevalence of GETV is high in pig populations in China. The seroprevalence was significantly lower in spring (April; 24.24%, 16/66) than in autumn (October; 50.75%, 34/67), which suggested that the presence of anti-GETV antibodies in pigs was seasonal. In conclusion, we improved an rE2 ELISA that detected pig antibodies against GETV after experimental and natural infections. This should be useful in the diagnosis and surveillance of GETV infections.

Development and Optimization of an Immunoassay for the Detection of Antibodies Against SARS-CoV-2 with In-house Recombinant RBD Protein

COVID-19 caused by SARS-CoV-2 poses a major threat to the global community, particularly in Indonesia. Countermeasures to prevent the spread of this disease have also been implemented, including the implementation of a vaccination program. An immunoassay technique that can be used to analyze antibodies that might develop following vaccination is the indirect enzyme-linked immunosorbent assay (ELISA). We produced the recombinant spike protein used in this study. The optimization comprised adjusted concentrations of spike recombinant protein (5 and 10 ng/mL), blocking agent (2.5% and 5%), and conjugate (1:1000 and 1:5000). The optimal conditions in this study included a spiked concentration of 10 ng/mL, a blocking agent concentration of 5%, sample dilution of 1:33, and a conjugate concentration of 1:1000. The intra-assay value of this optimized indirect ELISA was 7.3, and the inter-assay value was 5.3. The commercial MyBioSource kit and immunodiagnostic were utilized as a reference in the T-test, with P-values of 0 and 0.313, indicating that the recombinant protein in-house ELISA kit in this study demonstrated the same ability as the commercial immunodiagnostic kit in detecting SARS-CoV-2 antibodies, allowing it to be used for post-vaccination efficacy evaluation.

Diagnostic evaluation of two enzyme-linked immunosorbent assay tests for Trypanosoma cruzi infection in a Colombian population

Objective: To evaluate the diagnostic accuracy of two enzyme-linked immunosorbent assay tests to determine Trypanosoma cruzi infection in a Colombian population. Material and methods: Analytical study with case-control design of a diagnostic evaluation. Sensitivity, specificity and diagnostic efficacy of total CHAGATEK ELISA and recombinant CHAGATEK ELISA were determined. Positive predictive value, negative predictive value, and positive and negative likelihood ratios were calculated. The kappa coefficient was found to estimate the concordance between the index and reference tests. The discriminatory capacity of the tests was expressed by the area under the ROC curve. Results: Of the 184 samples included in the study, 99 were negative for T. cruzi and 85 were positive according to the reference tests used. Sensitivity for total and recombinant ELISA was 97.6 % and 98.8 %, respectively; while specificity was 97.0% and 94.9%. The level of concordance of the total and recombinant ELISA was almost perfect and the discriminatory ability of the two tests were good. Conclusions: It was demonstrated that the two tests can confirm and exclude the diagnosis of Chagas disease and have a good diagnostic performance.

Serological survey for maternal antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum in dogs from endemic and non-endemic regions of the United States

In North America, the tick-borne pathogens Borreliella burgdorferi (Lyme disease; LD) and Anaplasma phagocytophilum (anaplasmosis) are a significant health threat to dogs. Little is known regarding the seroprevalence of maternal antibodies (Abs) to these pathogens in young dogs. The analysis of maternal antibody (Ab) profiles is important as it could bear on the interpretation of currently available diagnostic assays and the potential for vaccine interference in pups. In this pilot study, sera from 32 client-owned dogs (6–24 weeks of age; 3 serum samples per dog) from four veterinary hospitals in the United States were screened for IgG against B. burgdorferi and A. phagocytophilum using whole cell lysate immunoblots and recombinant protein-based ELISAs. As a control, the sera were also screened for Abs to canine parvovirus and canine distemper virus using a commercially available colorimetric assay. Maternally derived Abs against B. burgdorferi including the diagnostic antigen VlsE were detected in 2 of the 32 dogs, accounting for 12.5 % of dogs from LD endemic regions, and as expected, the Ab levels declined over time. Differentiating between maternal Ab and infection-induced Ab is of importance in interpreting serological tests for tick-borne diseases in young dogs and in making decisions regarding treatment and timing of vaccination.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Serological Evidence of Scrub Typhus in Mizoram, North Eastern Region of India.

Differentiating scrub typhus from other acute febrile illnesses is difficult due to non-specificity of clinical symptoms and relative absence of eschar in Indian population. Antibody based serological tests are mainstay of scrub typhus diagnosis. To order to determine the performance of immunochromatography and IgM ELISA, immunochromatography and scrub typhus IgM ELISA were performed to detect the presence of antibodies against Orientia tsutsugamushi in acute serum of patients with acute febrile illness. A cross-sectional study was carried out in the Department of Microbiology, Civil Hospital Aizawl and Department of Microbiology, Civil Hospital Lunglei, Mizoram over a period of one year from October 1, 2018 to September 31, 2019. Serum samples from patients with acute febrile illness were processed for the detection of scrub typhus. A total of 253 samples were subjected to immunochromatography and IgM ELISA. Using scrub typhus IgM ELISA as gold standard, the sensitivity, specificity and negative predictive value were calculated. Of the 253 serum samples tested, 36 were positive by both scrub typhus rapid test and IgM ELISA. 27 samples which were negative by scrub typhus rapid test reacted positive by IgM ELISA. 66 samples which were positive by scrub typhus rapid test were negative by IgM ELISA. Owing to the limitations of scrub typhus rapid test and immunfluorescence assay (IFA), commercially available recombinant IgM ELISA which has a good sensitivity and specificity may be an alternative in laboratories with moderate set up.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a devastating disease of domestic and wild pigs. There is no effective vaccine, and the control of the disease relies mainly on surveillance and early detection of infected pigs. Previously, serological assays, such as ELISA, have been developed mainly based on recombinant structural viral proteins of ASFV, including p72, p54, and p30. However, the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs. Therefore, new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required. In this study, we expressed and purified a recombinant pB602L protein. The purified pB602L protein was then used as an antigen to develop an indirect ELISA assay. This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China, such as classical swine fever virus, pseudorabies virus, and porcine parvovirus. This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples, including an unknown number of anti-ASFV sera. The coincidence of the two assays was 95%. Furthermore, the pB602L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7GD in pigs. The results showed that the antibody levels in all vaccinated pigs, starting from the 10th day post-inoculation, have increased continuously during the observation period of 45 days. Our results indicate that this pB602L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Development of a recombinant nucleocapsid protein-based ELISA for the detection of IgM and IgG antibodies to SARS-CoV-2.

Coronavirus 2019 (COVID‐19) is a global concern for public health. Thus, early and accurate diagnosis is a critical step in management of this infectious disease. Currently, RT‐PCR is routine diagnosis test for COVID‐19, but it has some limitations and false negative results. enzyme‐linked immunosorbent assay (ELISA) against SARS‐CoV‐2 antigens seems to be an appropriate approach for serodiagnosis of COVID‐19. In the current study, an ELISA system, using a recombinant nucleocapsid (N) protein, was developed for the detection of IgM and IgG antibodies to SARS‐CoV‐2. The related protein was expressed, purified, and used in an ELISA system. Sera samples (67) for COVID‐19 patients, as well as sera samples from healthy volunteers (112), along with sera samples from non‐COVID‐19 patients were examined by the ELISA system. The expression and purity of the recombinant N protein were approved by SDS‐PAGE and Western blotting. The sensitivity of ELISA system was 91.04 and 92.53% for the detection of IgG and IgM antibodies, respectively. Moreover, the specificity of the developed ELISA system for IgG and IgM were 98.21 and 97.32%, respectively. Our developed ELISA system showed satisfactory sensitivity and specificity for the detection of antiSARS‐CoV‐2 IgM and IgG antibodies and could be used as a complementary approach for proper diagnosis of COVID‐19.

Challenges Encountered When Evaluating an Antibody-Detecting Point-of-Care Test for Taeniosis in an Endemic Community in Zambia: A Prospective Diagnostic Accuracy Study.

Taenia solium taeniosis diagnosis is challenging because current tests perform sub-optimally and/or are expensive, require sophisticated equipment, infrastructure and trained manpower, and therefore are not community deployable. A recently-developed, multi-strip, T. solium point-of-care test (TS POC) for simultaneous detection of tapeworm (TS POC T) and cysticercus (TS POC CC) human antibodies was evaluated for diagnostic accuracy on consecutively recruited community participants in Sinda district, Zambia. All participants were tested using the TS POC test. All test-positives and 20% of the test-negative participants were invited to give a blood and stool sample for reference testing. Three different reference tests were used for taeniosis diagnosis: recombinant rES33 enzyme-linked immunoelectrotransfer blot (rES33 EITB), copro PCR and copro Ag ELISA. Bayesian analysis with probabilistic constraints was used to estimate sensitivity and specificity. In total, 1254 participants were tested with the TS POC test, of whom 13 tested positive using the TS POC T. Based on 161 participants with complete data, the estimated sensitivity and specificity for the TS POC T test were 38% (95% CI: 5–93%) and 99% (95% CI: 98–100%), respectively. The challenge of highly variable inter-assay performance is highlighted. We recommend either increasing the sensitivity or redesigning the test.

Validation of New ELISA Technique for Detection of Aflatoxin B1 Contamination in Food Products versus HPLC and VICAM.

Toxin-contaminated foods and beverages are a major source of illness, may cause death, and have a significant negative economic impact worldwide. Aflatoxin B1 (AFB1) is a potent toxin that may induce cancer after chronic low-level exposure. This study developed a quantitative recombinant AflR gene antiserum ELISA technique for aflatoxin B1 detection in contaminated food products. Aflatoxin B1 residuals from 36 food samples were analyzed with HPLC and VICAM. DNA was extracted from aflatoxin-contaminated samples and the AflR gene amplified using PCR. PCR products were purified and ligated into the pGEM-T vector. Recombinant plasmids were sequenced and transformed into competent E. coli (BL21). Molecular size and B-cell epitope prediction for the recombinant protein were assessed. The purified protein was used to induce the production of IgG antibodies in rabbits. Serum IgG was purified and labeled with alkaline phosphatase. Finally, indirect-ELISA was used to test the effectiveness of polyclonal antibodies for detection of aflatoxin B1 in food samples.

Development of a recombinant ELISA for ovine herpesvirus 2, suitable for use in sheep

The minor capsid protein of ovine herpesvirus 2, identified as a potential antigen for serological testing, was over-expressed and purified to allow its assessment in ELISA. The corresponding gene sequence (OvHV-2 orf65, Ov65) was modified to incorporate epitope tags and internal restriction enzyme sites in an E. coli codon-optimised version of the gene. This codon-optimised gene was then subject to internal deletions to identify regions of the protein that could be removed while maintaining protein solubility and antigenicity. It was found that a derivative with deletion of the conserved 5’-end of the gene (Ov65delB) expressed a polypeptide that was soluble when over-expressed in bacteria and was detected by OvHV-2 specific sera. Proteomic analysis of the affinity purified Ov65delB showed that it contained multiple predicted Ov65 tryptic peptides but also showed contamination by co-purifying E. coli proteins.An indirect ELISA, based on this affinity-purified OV65delB, was optimised for use with sheep and cattle samples and cut-off values were established based on known negative serum samples. Analysis of groups of samples that were either presumed infected (UK sheep) or tested OvHV-2 positive or negative by PCR (cattle MCF diagnostic samples) showed that the assay had 95 % sensitivity and 96 % specificity for sheep serum; and 80 % sensitivity and 95 % specificity for cattle serum. The lower sensitivity with cattle samples appeared to be due to a lack of serological response in some MCF-affected cattle.This recombinant antigen therefore shows promise as the basis of an inexpensive, simple and reliable test that can be used to detect OvHV-2-specific antibody responses in both MCF-affected animals and in OvHV-2 reservoir hosts.

Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA.

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV (p = 0.03) and MLV-PRV (p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA (n = 428), serum iELISA (n = 426), and oral fluid iELISA (n = 247) showed no significant differences in performance (p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE 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

Serum vascular endothelial growth factor as a tumor marker for hepatocellular carcinoma in hepatitis C virus-related cirrhotic patients.

BACKGROUNDHepatocellular carcinoma (HCC) accounts for 8.2% of all cancer-related deaths worldwide. Being a vascular tumor, vascular endothelial growth factor (VEGF) plays a vital role in HCC pathogenesis, growth, and spread.AIMTo determine the accuracy of serum VEGF and VEGF/platelet (PLT) as tumor markers in the early detection of HCC cases in patients with hepatitis C virus (HCV)-related liver cirrhosis.METHODSWe conducted a case-control study with HCV patients from the outpatient and inpatient hepatology clinics. Patients were classified into three groups: (1) HCC group; (2) Cirrhosis group; and (3) HCV without cirrhosis (control group). Patients were clinically evaluated, and blood samples were drawn for the analysis; serum VEGF levels were measured by a specific VEGF human recombinant enzyme-linked immunosorbent assay kit. Data from the three study groups were compared by the one-way analysis of variance or Kruskal-Wallis test. Receivers operating characteristic curves were constructed to determine the optimal cut-off values of alpha fetoprotein (AFP), VEGF, and VEGF/PLT that provided the best diagnostic accuracy. The sensitivity and specificity at the optimal cut-off value of each biomarker were then calculated. RESULTSThis study included one hundred patients (HCC, cirrhosis, and control groups: n = 40, 30, 30, respectively). HCC patients had significantly higher serum VEGF and VEGF/PLT levels than the non-HCC groups (P = 0.001). Serum VEGF and VEGF/PLT showed significant positive correlations with and HCC tumor size, stage, vascular invasion, and Child-Pugh classification. Moreover, a VEGF cut-off the value of 250 pg/mL provided 80% sensitivity and 81.7% specificity for discriminating HCC patient from non-HCC patients. Similarly, the ratio of VEGF/PLT provided sensitivity and specificity of 77.5% and 80%, respectively which is higher than the accuracy provided by AFP. The combination of AFP, VEGF, and VEGF/PLT increases the accuracy of diagnosing HCC to > 95%.CONCLUSIONIn HCV patients, serum VEGF and VEGF/PLT separately or in combination with AFP are reliable biomarkers for early and accurate HCC diagnosis.