Detection Of Virus-specific Antibodies Research Articles

Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections.Key points• The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs.• Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs.• Inoculation of mutated SVPs induces antibodies with low cross-reactivity.

Prevalence of SARS-CoV-2 RNA and antibody-detection, and vaccination status in patients with ocular vascular occlusion.

To analyze the annual prevalence of ocular vascular occlusion in relation to COVID-19 infection and vaccination status in a prospective study. All patients were examined for an active SARS-CoV-2 infection by RNA detection, for a previous infection by virus-specific antibody detection (ECLIA), their vaccination status was documented. Data from pandemic year 2020 and previous years, before COVID-19 (2019, 2018, 2017), were retrospectively analyzed. In 2021, a total of 103 patients with the first diagnosis of ocular vascular occlusion were treated. Most frequent sub-diagnoses were central retinal vein occlusion (20.4%), non-arteriitic anterior ischemic optic neuropathy (18.4%), central retinal artery occlusion (13.6%) and branch retinal artery occlusion (12.6%). Thereof, only 3 patients (2.9%) presented with virus-specific SARS-CoV-2-antibodies, none was PCR-positive. Patients with preceded SARS-CoV-2 vaccination (59.2%) presented with comparable characteristics as unvaccinated patients with vascular occlusion regarding age, gender distribution, systemic risk factors, duration of symptoms, visual acuity and the present sub-diagnoses (p>0.05). The total number of cases in 2021 (103 cases) was comparable to the pandemic year 2020, at which no vaccination was available (114 cases), and to earlier years 2017, 2018, and 2019 without COVID pandemic (100, 120 and 119 cases). Furthermore, we did not reveal any differences between pandemic and reference years regarding patients' characteristics (p>0.05). Our study did not reveal an increased annual prevalence of ocular vascular occlusions during COVID-19 pandemic years 2020 and 2021. Patients with previous COVID vaccination did not present differences regarding the risk profile nor symptoms, compared to unvaccinated individuals.

Virus-like particles based on rotavarus A recombinant VP2/VP6 proteins for assessment the antibody immune response by ELISA

Rotavirus infection is one of the main concerns in infectious pathology in humans, mammals and birds. Newborn piglets or rodents are usually being used as a laboratory model for the evaluation of immunogenicity and efficacy for all types of vaccines against rotavirus A (RVA), and the use of ELISA for the detection of virus-specific antibodies of specific isotype is an essential step of this evaluation. Development of indirect solid-phase ELISA with VP2/VP6 rotavirus VLP as an antigen to detect and assess the distribution of RVA-specific IgG, IgM and IgA in the immune response to rotavirus A. VP2/VP6 rotavirus VLP production and purification, electron microscopy, PAGE, immunoblotting, ELISA, virus neutralization assay. The study presents the results of development of a recombinant baculovirus with RVA genes VP2-eGFP/VP6, assessment of its infectious activity and using it for VLP production. The morphology of the VP2/VP6 rotavirus VLPs was assessed, the structural composition was determined, and the high antigenic activity of the VLP was established. VLP-based ELISA assay was developed and here we report results for RVA-specific antibody detection in sera of different animals. The developed ELISA based on VP2/VP6 rotavirus VLP as a universal antigen makes it possible to detect separately IgG, IgM and IgA antibodies to rotavirus A, outlining its scientific and practical importance for the evaluation of immunogenicity and efficacy of traditional vaccines against rotavirus A and those under development.

Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence.

Tick-borne encephalitis virus (TBEV) is a flavivirus transmitted by ticks. Serological screenings in animals are performed to estimate the prevalence and distribution of TBEV. Most screenings consist of a primary screening by ELISA, followed by confirmation of positive samples by plaque reduction neutralization tests (PRNTs). In this study, 406 wild boar sera were tested with 2 regularly used commercial ELISAs for flavivirus screening in animals (Immunozym FSME (TBEV) IgG All Species (Progen) and ID Screen West Nile Competition (Innovative Diagnostics)) and PRNTs for TBEV and USUTU virus. The results showed that the Immunozym and IDScreen ELISAs had low relative sensitivities of 23% and 20%, respectively, compared to the PRNT results. The relative specificities were 88% and 84% due to cross reactions with USUTU virus-specific antibodies. The minimal TBEV prevalence in our sample set was 8.6% when determined by PRNT. When the screening approach of ELISA testing followed by PRNT confirmation was applied, a TBEV seroprevalence of only 2.0% and 1.7% was found. The suboptimal performance of the ELISAs was confirmed by testing sera collected from experimentally TBEV-infected sheep. While the PRNT detected TBEV specific antibodies in 94% of samples collected between 7 and 18 days post-infection, the ELISAs classified only 50% and 31% of the samples as positive. Both routinely used ELISAs for TBEV antibody screening in animal sera were shown to have a low sensitivity, potentially leading to an underestimation of the true prevalence, and furthermore cross-react with other flavivirus antibodies.

Crimean-Congo hemorrhagic fever virus-specific antibody detection in Equids

Kırım Kongo kanamalı ateşi (KKKA) virus enfeksiyonu, dünyanın geniş bir bölgesinde endemik olan ve özellikle insan sağlığını tehdit eden, potansiyel olarak ölümcül bir hastalık olan kene kaynaklı zoonotik bir hastalıktır. Hastalığın bir bölgede endemik olduğunun ana göstergelerinden biri, hayvan popülasyonlarında KKKAV'ye özgü antikorların varlığıdır. Birçok hayvan türü KKKAV'yi asemptomatik olarak taşıyabilir ve bu nedenle hastalığın bulaşma döngüsüne katılabilir. Serolojik çalışmalar, doğada KKKAV' nin hayatta kalması için tek tırnaklıların önemli olduğunu göstermiştir. Ancak ülkemizde bu konuda daha fazla çalışmaya ihtiyaç vardır. Araştırma için Afyonkarahisar ve Burdur illerinde çeşitli cinsiyet ve yaştaki 97 hayvandan kan örnekleri alındı. Yetiştiriciler tarafından çeşitli amaçlarla tutulan bu hayvanların kan serumlarında KKKV'ye özgü antikorların varlığı araştırıldı. Spesifik antikorların tespiti için hızlı ve güvenilir bir yöntem olan çift antijenli ELISA test yöntemi kullanıldı. Sonuç olarak toplamda %51.5 seropozitiflik tespit edildi. Bulgular, tek tırnaklı hayvanların KKKA'nın epidemiyolojisinde rezervuar olarak önemli bir rol oynayabileceğini göstermektedir.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus

BackgroundClassical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV.MethodsThe CSFV Erns and truncated E2 (tE2, residues 690–865) of BVDV were expressed in E. coli and purified by Ni–NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT).ResultsIndirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively.ConclusionThe newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

SARS-CoV-2 Infection Detection by PCR and Serologic Testing in Clinical Practice.

Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be diagnosed by PCR during acute infection or later in their clinical course by detection of virus-specific antibodies. While in theory complementary, both PCR and serologic tests have practical shortcomings. A retrospective study was performed in order to further define these limitations in a clinical context and to determine how to best utilize these tests in a coherent fashion. A total of 3,075 patients underwent both PCR and serology tests at University of California, Los Angeles (UCLA), in the study period. Among these, 2,731 (89%) had no positive tests at all, 73 (2%) had a positive PCR test and only negative serology tests, 144 (5%) had a positive serology test and only negative PCR tests, and 127 (4%) had positive PCR and serology tests. Approximately half of the patients with discordant results (i.e., PCR positive and serology negative or vice versa) had mistimed tests in reference to the course of their disease. PCR-positive patients who were asymptomatic or pregnant were less likely to generate a detectable humoral immune response to SARS-CoV-2. On a quantitative level, the log number of days between symptom onset and PCR test was positively correlated with cycle threshold (CT) values. However, there was no apparent relationship between PCR CT and serologic (arbitrary units per milliliter) results.

Realization of humoral immunity against SARS-CoV-2 infections

Abstract The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major ongoing challenge to global health. After being infected by SARS-CoV-2, a specific humoral immune response may be rapidly induced in the host to restrain the viral infection via the production of neutralizing antibodies (NAbs), which are also useful for preventing reinfection [ 1 , 2 ]. However, there is a lack of comprehensive understanding of the mechanisms underlying SARS-CoV-2 specific humoral immunity and alterations in immunoglobulin M (IgM) and G (IgG) levels in humans during viral infection. In this perspective, we have summarized several characteristics of the humoral immune response against SARS-CoV-2 infection and its potentially critical role in vaccine development. Additionally, we have discussed the antibody-dependent enhancement (ADE) phenomenon in antibody immunotherapy, the current status of vaccine development, and public health strategies aimed at ending the global COVID-19 pandemic. Since the novel coronavirus SARS-CoV-2 was reported as the causative agent of COVID-19 in December 2019, the subsequent spread of SARS-CoV-2 has led to the COVID-19 pandemic. SARS-CoV-2 continues to considerably impact human health and life expectancy and endangers the global economy and socio-economic stability. The gold standard technique used to confirm early SARS-CoV-2 infection is reverse transcription polymerase chain reaction (RT-PCR)-based viral nucleic acid testing. However, previous studies conducted by our colleagues and other researchers indicate that virus-specific antibody detection for COVID-19 may be used as a complement to viral RNA detection for the diagnosis of suspected cases with negative RT-PCR results and/or for surveying asymptomatic infection in close contacts [3] . For theemerging virus, the characteristics and specific detailed mechanisms of how protective humoral immunity is developed in COVID-19 patients are not clearly defined. Herein, we compared the characterization of SARS-CoV-2-specific antibody responses in the COVID-19 patients conducted in different studies worldwide and further discussed the critical roles of specific anti-viral humoral immunity in virus clearance and vaccine development.

Antikörpertests bei COVID-19 - Was uns die Ergebnisse sagen

IntroductionIn the context of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the detection of virus-specific antibodies (AB) will play an increasing role. The presence or absence of such antibodies can potentially lead to considerations regarding immunity and infection. IssueHow reliable are inferences from positive or negative test results regarding the actual presence of SARS-CoV-2 specific antibodies? MethodsCalculation of the probability that, depending on the pretest probability (prevalence of SARS-CoV-2 infection) and test properties, antibodies are present or absent in the case of positive or negative test results. ResultsSensitivity and specificity of different SARS-CoV-2 AB test systems vary between 53 % and 94 % and between 91 % and 99.5 %, respectively. When using a test with high test quality, the positive predictive value (PPV) is 42 % and 7 9%, respectively, with a pre-test probability of 1 % to 5 %, as can currently be assumed for the general population in Austria or Germany. For persons with an increased pre-test probability of 20 %, e. g. persons from high-risk professions, the PPW is 95 %, with a pre-test probability of 80 % the PPW is almost 100 %. The negative predictive value (NPV) is at least 99.7 % for persons with a low pre-test probability of up to 5 % and 79.1 % for persons with a pre-test probability of 80 %. When using test systems with lower sensitivity and specificity, the reliability of the results decreases considerably. The PPV is 5.9 % with a pre-test probability of 1 %. ConclusionsA sufficiently high sensitivity and specificity are prerequisites for the application of antibody test systems. Positive test results are often false if the pre-test probability is low. Depending on the assumed prevalence of a SARS-CoV-2 infection, there are substantial differences in the significance of a concrete test result for the respective affected persons.

Severe fever with thrombocytopenia syndrome in canines from the Republic of Korea

Severe fever with thrombocytopenia syndrome (SFTS) in a companion dog was confirmed based on clinical symptoms, virus isolation, and virus-specific antibody detection. Fever and anorexia began after tick bite. Viremia disappeared within two weeks and antibodies were detected one week after disease onset.

An interaction of Zika virus envelope fragments with serum antibodies derived from subjects after flavivirus infections

The causative agent of Zika fever (ZIKV) belongs to the genus Flavivirus of the family Flaviviridae. The flavivirus genus consists of more than 70 members. Based on virion structural organization and amino acid composition of proteins, this virus resembles other flaviviruses such as dengue (DENV), yellow fever and West Nile (WNV) posing a threat to human health. ZIKV is an arbovirus and may be transmitted by diverse mosquito species of the genus Aedes. It is believed that the main carriers are also able to transmit dengue virus, yellow fever virus as well as other flavivirus infections. In 1947, ZIKV was isolated for the first time from blood samples obtained from rhesus macaques inhabiting the Zika Forest (Uganda). Long time this virus was not considered as a dangerous to human pathogen, as Zika fever mostly occurs asymptomatically. However, analysis of Zika fever course in pregnant women unveiled a link between this disease and severe congenital disorders of the nervous system, including microcephaly, that allowed to deal with it as a dangerous infection thereafter. Rapid ZIKV spread outlined a number of problems faced by medical doctors, among which the main issue was the lack of assays for its virus-specific diagnostics. ZIKV displays a marked antigenic similarity with other flaviviruses. The majority of dengue-specific monoclonal antibodies binds to Zika virus. It is expected given the high degree of amino acid sequence similarity found for flavivirus polyprotein. Several antigens bearing ZIKV E surface protein fragments were constructed to assess an opportunity for conducting differential diagnostics for distinct flaviviruses based on detection of virus-specific antibodies. Vector plasmid pET32 was selected for producing recombinant antigens in E. coli cells. After creating constructs encoding the ZEa187 and ZEa40 proteins, the chimeric proteins were produced in amount necessary for performing ELISA with blood serum samples. Protein samples were prepared by isolating them from bacterial biomass via lysis followed by chromatographic purification. Blood sera obtained from human subjects recovered after Zika, Dengue and West Nile fevers were used to examined immunochemical properties of chimeric proteins. Human sera containing no antibodies against flavivirus types were used as a negative control. It was found that serum IgM class antibodies derived from patients with flavivirus infections demonstrated a high level of cross-reactivity by interacting with ZEa187 and ZEa40. Upon that, despite increment of mean specific interaction signal observed for such proteins and IgG of ZIKV sera, a marked cross-reactivity with the IgG of WNV and DENV sera was found. Thus, with some certainty it may be concluded that in immunochemical assays use of natural amino acid sequence specific to Zika virus surface protein as antigenic material does not allow to achieve high specificity for antibody detection.

Crimean-Congo haemorrhagic fever (CCHF) virus-specific antibody detection in blood donors, Castile-León, Spain, summer 2017 and 2018.

BackgroundCrimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging or even a probable re-emerging pathogen in southern Europe. Presence of this virus had been reported previously in Spain in 2010.AimWe aimed to evaluate the potential circulation of CCHFV in western Spain with a serosurvey in asymptomatic adults (blood donors).MethodsDuring 2017 and 2018, we conducted a CCHFV serosurvey in randomly selected asymptomatic blood donors from western Spain. Three assays using specific IgG antibodies against CCHFV were performed: the VectoCrimea ELISA test, an in-house ELISA and indirect immunofluorescence (EuroImmun) test with glycoprotein and nucleoprotein.ResultsA total of 516 blood donors participated in this cross-sectional study. The majority of the study participants were male (68.4%), and the mean age was 46.3 years. Most of the participants came from rural areas (86.8%) and 68.6% had contact with animals and 20.9% had animal husbandry practices. One in five participants (109/516, 21.1%) were engaged in at-risk professional activities such as agriculture and shepherding, slaughtering, hunting, veterinary and healthcare work (mainly nursing staff and laboratory technicians). A total of 15.3% of the participants were bitten by ticks in the days or months before the date of sampling. We detected anti-CCHFV IgG antibodies with two diagnostic assays in three of the 516 individuals and with one diagnostic assay in six of the 516 individuals.ConclusionSeroprevalence of CCHFV was between 0.58% and 1.16% in Castile-León, Spain. This is the first study in western Spain that showed circulation of CCHFV in healthy people.

Crimean-Congo Hemorrhagic Fever Virus-Specific Antibody Detection in Cattle in Mauritania.

Crimean-Congo hemorrhagic fever virus (CCHFV) was detected for the first time in Mauritania in 1983 and several CCHFV outbreaks were reported in the following years. The last human case was diagnosed in 2015. However, no recent data exist about the prevalence of CCHFV in animals, although it is already described that prevalence studies in animals serve as good risk indicators. CCHFV can cause a severe hemorrhagic fever with a high case fatality rate in humans. Therefore, a precise risk assessment on the basis of updated data is very important. This article gives an overview about the current CCHFV prevalence in cattle in Mauritania. A seroprevalence study was carried out using 495 cattle sera from Mauritania, which were collected in the year 2013. The sera were analyzed by an inhouse CCHFV-IgG-ELISA. As second screening test, an adapted commercial CCHFV-IgG-ELISA was performed. Inconclusive sera were additionally tested by a modified commercial CCHFV-IgG-IFA. All assays showed high diagnostic sensitivity (>95%) and specificity (>98%). The overall prevalence of CCHFV-specific antibodies found in Mauritanian cattle was 67%, ranging from 56% to 90% in different provinces. This study shows a very high CCHFV-specific antibody prevalence in cattle in Mauritania. It is the highest seroprevalence detected in Mauritania so far. This strengthens the hypothesis that CCHFV is a serious and ongoing threat for public health in Mauritania.

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

Use of serologic tests to predict resistance to Canine distemper virus–induced disease in vaccinated dogs

The objective of the current study was to determine whether detection of Canine distemper virus (CDV)-specific serum antibodies correlates with resistance to challenge with virulent virus. Virus neutralization (VN) assay results were compared with resistance to viral challenge in 2 unvaccinated Beagle puppies, 9 unvaccinated Beagle dogs (4.4-7.2 years of age), and 9 vaccinated Beagle dogs (3.7-4.7 years of age). Eight of 9 (89%) unvaccinated adult dogs exhibited clinical signs after virus challenge, and 1 (13%) dog died. As compared to adult dogs, the 2 unvaccinated puppies developed more severe clinical signs and either died or were euthanized after challenge. In contrast, no clinical signs were detected after challenge of the 9 adult vaccinated dogs with post-vaccination intervals of up to 4.4 years. In vaccinated dogs, the positive and negative predictive values of VN assay results for resistance to challenge were 100% and 0%, respectively. Results indicate that dogs vaccinated with modified live CDV can be protected from challenge for ≤4.4 years postvaccination and that detection of virus-specific antibodies is predictive of whether dogs are resistant to challenge with virulent virus. Results also indicate that CDV infection in unvaccinated dogs results in age-dependent morbidity and mortality. Knowledge of age-dependent morbidity and mortality, duration of vaccine-induced immunity, and the positive and negative predictive values of detection of virus-specific serum antibodies are useful in development of rational booster vaccination intervals for the prevention of CDV-mediated disease in adult dogs.

Rapid and sensitive detection of norovirus antibodies in human serum with a biolayer interferometry biosensor

Here, we describe the use of a biolayer interferometry biosensor for the fast and sensitive detection of virus-specific antibodies from human serum samples. Norovirus-like particles and norovirus P-particles were used to functionalise the biosensor tip. The detection of antibodies directly from serum samples was challenging, but the addition of a metal chelator (DAB) combined with an anti-human horseradish peroxidase-tagged antibody enabled enhanced detection of virus-specific antibodies in serum dilutions up to 1:100,000. Biolayer interferometry provides results faster than an ELISA, with results in as little as 10–20min when using pre-functionalised sensors. Therefore, biolayer interferometry combined with DAB enhancement offers an attractive method for quick and sensitive quantification of biomolecules from complicated sample matrices.

Development and application of an indirect ELISA for the detection of antibodies to novel duck reovirus

A novel duck reovirus (N-DRV) disease emerged in China in 2000 and it has become an epidemic genotype. A test for detection of virus-specific antibodies in serum samples would be useful for epidemiological investigations. Currently, Currently, serological assays for N-DRV diagnosis are not available. A test for detection of virus-specific antibodies in serum samples would be useful for epidemiological investigations. In this study, a highly sensitive and specific indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to N-DRV was developed. The outer capsid (σC) of N-DRV was cloned and expressed in Escherichia coli as a coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. Furthermore, the specificity of σC-ELISA assay was confirmed by cross checking with other duck viral pathogens. In comparison with the western blot, the sensitivity and specificity of the σC-ELISA was 92.6% and 88.9%, respectively, and agreement of two tests was excellent with κ value of 0.786 (p<0.05). A serological survey was performed using the assay on serum samples from different age and species of duck flocks in the Zhejiang and Jiangsu Province, China. The seropositive rate of the 1209 serum samples was 57.7%. In conclusion, the developed σC-ELISA assay is a very specific and sensitive test that will be useful for large-scale serological survey in N-DRV infection and monitoring antibodies titers against N-DRV.

Recent Advances in Dengue Diagnosis

Dengue virus is a mosquito-borne flavivirus that causes a spectrum of illness ranging from asymptomatic infection to dengue fever to dengue haemorrhagic fever or dengue shock syndrome. Dengue virus has four distinct serotypes: DEN-1, DEN-2, DEN-3 and DEN-4. Dengue virus is endemic in many parts of the world, where it is responsible for significant morbidity and mortality. Laboratory confirmation of dengue infection relies on isolation of the virus in cell culture, identification of viral nucleic acid or antigens, detection of virus specific antibodies or a combination of these techniques. Timing and appropriate use of these diagnostic assays has to be understood.

Diagnostics of Human Middle-East Respiratory Syndrome

Middle-East respiratory syndrome is a human disease caused by a new coronavirus. In December, 2012 WHO published draft regulatory document on diagnostics of the virus. It was recommended to use two methods of disease diagnostics - two-phase reverse-transcription real-time PCR and enzyme immunoassay. The first phase of the PCR-diagnostics should include reverse-transcription real-time PCR targeted on the genome fragment upwards of upE. The second (control) PCR-test may be alternatively targeted within the bonds of the genome, its target being non-crisscross with upE gene. It should include sequencing of, at least, a segment of one of the viral genomes and comparative analysis of the obtained sequence along with the like ones deposited in the GenBank. Enzyme immunoassay is retrospectively used for virus-specific antibody detection in convalescents’ blood sera. Examined are the key specifications of the methods for the detection of ethiological agent or specific antibodies to it, and WHO methodological recommendations in case of Middle-East respiratory syndrome diagnostics.

Rapid detection of different human anti-HCV immunoglobulins on electrical biochips

Rapid detection of different human anti-HCV immunoglobulins on electrical biochips Lars Blohm,1,2,§ Christiane Püttmann,3,§ Simone Holz,1 Gundula Piechotta,1,2 Jörg Albers,1,2 Christina Dammers,4–6 Michael Kleines,7,8 Alexander Krüttgen,8,9 Georg Melmer,2,10 Jörg Nähring,2,5 Stefan Barth,3,5,§ Eric Nebling,1,2,§ 1Department of Biotechnical Microsystems, Fraunhofer Institute for Silicon Technology, Itzehoe, Germany; 2POCDIA GmbH, Itzehoe, Germany; 3Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, Uniklinik RWTH Aachen, Germany; 4Institute of Biology VII, Molecular Biotechnology, RWTH Aachen University, Aachen, Germany; 5Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany; 6Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, Jülich, Germany; 7Department of Hygiene, Microbiology, Social Medicine, Division of Virology, Medical University IBK, Innsbruck, Austria; 8Medizinisches Versorgungszentrum Dr Stein und Kollegen, Mönchengladbach, Germany; 9Department of Medical Microbiology, Division of Virology, Uniklinik RWTH Aachen, Germany; 10Pharmedartis GmbH, Aachen, Germany §These authors contributed equally Abstract: The detection of hepatitis C virus (HCV) in the blood of patients is currently based on immunological assays (enzyme-linked immunosorbent assay [ELISA] and recombinant immunoblot assay) that use different HCV epitopes to detect anti-HCV antibodies, and these tests usually require laboratories and trained personnel. The ELISA-based systems are also time consuming. Portable diagnostic devices offering rapid test results would therefore be advantageous in the field of medical care. To facilitate the fast and reliable diagnosis of HCV, we used a miniaturized automated system based on a cartridge with an integrated electrical biochip for the decentralized detection of anti-HCV antibodies against the Core, NS3, and NS4A proteins. This system allows the detection of virus-specific antibodies in 2 µL of serum or whole blood within 15 minutes using an ELISA directly on a gold electrode array containing HCV proteins as the capture antigen. The sensitivity of this system is comparable with standard microtiter plate ELISAs, but the duration of the novel assay is 5%–6% that of standard ELISAs. Keywords: ELISA, point-of-care, cartridge, lab-on-chip