Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.     

Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.     

Comparison of diagnostic performances of ten different immunoassays detecting anti-CCHFV IgM and IgG antibodies from acute to subsided phases of Crimean-Congo hemorrhagic fever

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary.A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: μ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection.In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection.Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection.     

Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination

A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.     

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.     

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Development of multiple elisas for the detection of antibodies against classical swine fever virus in pig sera

The major immunogenic proteins (Ems,E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E.coli and purified by affinity chromatography.The recombinant antigens were appl...     

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.     

Serological reactivity of baculovirus-expressed Ebola virus VP35 and nucleoproteins

Ebola virus (EBOV) is a member of the family Filoviridae and is classified as a biosafety level 4 virus. This classification makes the preparation of antigen and performance of diagnostic assays time-consuming and complicated. The objective of this study was to evaluate the value of EBOV immunoassays based on recombinant nucleoprotein (r-NP) and recombinant VP35 (r-VP35) using large serum panels of African origin and from primates. Furthermore, we investigated whether the results obtained with EBOV r-VP35 enzyme-linked immunosorbent assay (ELISA) could improve on the findings obtained with the EBOV r-NP ELISA. The full-length EBOV NP and VP35 of the EBOV subtype Zaire were expressed as histidine-tagged recombinant proteins in the baculovirus expression system. The antigenic reactivity and specificity of these recombinant proteins were determined by Western blotting and ELISA using EBOV specific monoclonal antibodies. The results obtained with the r-NP and r-VP35 ELISAs were compared with the results obtained in an indirect immunofluorescence assay based on native EBOV subtype Zaire. EBOV specific monoclonal antibodies reacted specifically with the respective proteins in both Western blot and ELISA. Five hundred and twenty six samples from humans and primates were tested with r-NP and r-VP35 ELISAs. Monkey serum samples positive for EBOV subtype Reston and Zaire were both positive in the EBOV r-NP ELISA, whereas only the EBOV Zaire infected monkeys were positive in the r-VP35 ELISA. The sensitivity and specificity values of the EBOV recombinants' ELISAs compared to those of the immunofluorescence assay were 92% and 99% for r-NP and 44% and 100% for r-VP35. r-NP ELISA proved to be a sensitive and specific assay for EBOV diagnosis and for epidemiological studies for both EBOV subtypes Reston and Zaire. The use of r-VP35 in an ELISA format has no additional value for EBOV serodiagnosis.     

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep

Caseous lymphadenitis (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false-negative results. This study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.     

Seroprevalence study of Tick-borne encephalitis, Borrelia burgdorferi, Dengue and Toscana virus in Turin Province

Tick borne encephalitis virus (TBEV) is present in some European countries and it is transmitted by a tick bite. Ixodes ricinus is the main vector of the infection in Italy, where fortunately clinical neurological manifestations, typical of the more serious phase of the disease, are very rarely observed. This behaviour is different from other endemic Euroasiatic areas where numerous cases of encephalitis are described. However TBE transmission has not been widely investigated in Italy and available epidemiological data have been obtained only by studies performed in Central and Northern Regions of the country. In addition seroepidemiological researches were made prevalently on subjects at high risk of tick bite, such as hunters or forest guards from Trentin and Central Italy. No precise information about TBE virus diffusion was available in the Piedmont before our investigations. We found that hunters and wild boar breeders seem to be particularly exposed to the risk of TBE virus infection in Turin Province and in particular in the Susa valley, although no neurological involvement was observed in our population. In particular a seroprevalence of about 5% was detected by the use of purified antigens ELISA test, amongst the subjects at high risk of tick bite. Moreover low risk individuals showed a seroprevalence of below 2%. In addition a parallel seroepidemiological study was performed in Turin Province for Borrelia burgdorferi, the aetiological agent of Lyme disease, also transmitted by tick bite (e.g. Ixodes ricinus), for Dengue and Toscana (TOS) arboviruses, respectively transmitted by Aedes mosquitoes and phlebotomes. Data reported here demonstrate only a sporadic presence in our population of antibodies against Borrelia and Dengue infection. Moreover using an ELISA test performed with viral purified nucleoprotein, we reported a total percentage of about 3% of subjects positive for TOSV.     

乙型脑炎病毒（JEV）ELISA检测方法的建立

目的建立猪乙型脑炎病毒（JEV）抗体ELISA检测方法。方法培养BHK21细胞,接种JEV病毒,制备BHK21正常抗原和JEV特异抗原,滴定酶结合物和抗原最佳工作浓度,并进行精密性、敏感性、稳定性、特异性实验。结果正常、特异抗原和酶结合物最佳工作浓度分别为0.2μg/mL、10μg/mL和1∶20000;正常、特异抗原批内变异系数分别为8.3%和6.4%,批间平均变异系数分别为9.7%和11.5%;检测灵敏度为1∶1280;与猪瘟病毒（CSFV）、猪细小病毒（PPV）均无交叉反应。稳定性试验相对偏差小于25%。结论建立的ELISA方法重复性、稳定性好,特异性、敏感性强。可用于猪JEV抗体的检测。     

Strongy Detect: Preliminary Validation of a Prototype Recombinant Ss-NIE/Ss-IR Based ELISA to Detect Strongyloides stercoralis Infection

BackgroundStrongyloides stercoralis (Ss) is the etiological agent of strongyloidiasis, a neglected tropical disease of global concern. Laboratory diagnosis of strongyloidiasis is most often based on detection of antibodies against antigens in an enzyme linked immunosorbent assay (ELISA). Herein, we report a preliminary validation study of newly developed IgG4- and/or IgG- based ELISAs to detect strongyloidiasis (Strongy Detect, InBios) incorporating a cocktail of 2 previously described recombinant antigens, Ss-NIE and Ss-IR.MethodsThe sensitivity and specificity were determined by using the assay in 150 cryopreserved serum samples from humans known to be Ss infected (n = 74), helminth uninfected (n = 47), or infected with a helminth other than Ss [n = 29). The treatment associated dynamics of antibody detection were then assessed using 35 paired samples obtained before and after definitive therapy.ResultsThe IgG and IgG4 assays were 99% and 96% sensitive, respectively, and 99% and 100% specific, respectively. Neither the IgG or IgG4 assay showed cross reactions with sera from those infected with other helminths. Although ELISA values did decline post-treatment few returned to levels below the cutoff for infection.ConclusionStrongy Detect is the most sensitive and specific commercialized immunoassay for detection of strongyloidiasis. The assay remains positive for greater than a year post-treatment.     

Development and application of a fecal antigen diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in central Java, Indonesia

The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti-F. gigantica antibodies in serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood, feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than the anti-F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The coproantigen ELISA was able to detect 100% of the cattle with >15 flukes. A survey of 305 cattle in central Java over a 10-mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful diagnostic method to determine patent F. gigantica infections in cattle.     

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.     

Are Patients with Erythema Migrans Who Have Leukopenia and/or Thrombocytopenia Coinfected with Anaplasma phagocytophilum or Tick-Borne Encephalitis Virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.     

Serodiagnosis of infectious diseases with antigen microarrays

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.     

Results of using the ELISA method in studying tick-borne encephalitis and rabies

The results of using the indirect variant of ELISA for the study of serum samples from humans and white mice for the presence of antibodies to tick-borne encephalitis virus and rabies virus are presented. The high sensitivity and specificity of this method have been confirmed.     

Improved methods for the diagnosis of African trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.