IgM Antibody Capture ELISA Research Articles

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.

Intrathecal antibody production in two cases of yellow fever vaccine associated neurotropic disease in Argentina

During the period 2007-2008 several epizootics of Yellow fever with dead of monkeys occurred in southeastern Brasil, Paraguay, and northeastern Argentina. In 2008 after a Yellow fever outbreak an exhaustive prevention campaign took place in Argentina using 17D live attenuated Yellow fever vaccine. This vaccine is considered one of the safest live virus vaccines, although serious adverse reactions may occur after vaccination, and vaccine-associated neurotropic disease are reported rarely. The aim of this study was to confirm two serious adverse events associated to Yellow fever vaccine in Argentina, and to describe the analysis performed to assess the origin of specific IgM against Yellow fever virus (YFV) in cerebrospinal fluid (CSF). Both cases coincided with the Yellow fever vaccine-associated neurotropic disease case definition, being clinical diagnosis longitudinal myelitis (case 1) and meningoencephalitis (case 2). Specific YFV antibodies were detected in CSF and serum samples in both cases by IgM antibody-capture ELISA. No other cause of neurological disease was identified. In order to obtain a conclusive diagnosis of central nervous system (CNS) infection the IgM antibody index (AI(IgM) ) was calculated. High AI(IgM) values were found in both cases indicating intrathecal production of antibodies and, therefore, CNS post-vaccinal YFV infection could be definitively associated to YFV vaccination.

Validation of an IgM antibody capture ELISA based on a recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus

The presence of competent vectors in some countries currently free of Rift Valley fever (RVF) and global changes in climate, travel and trade have increased the risk of RVF spreading to new regions and have emphasised the need for accurate and reliable diagnostic tools for early diagnosis during RVF outbreaks. Highly sensitive viral detection systems like PCR have a limited use during outbreaks because of the short duration of viraemia, whereas antibodies like specific IgM which are serological indicators of acute infection, can be detected for up to 50 days after infection. Using the highly conserved and immunogenic recombinant nucleoprotein of RVF virus in an IgM capture ELISA, the risk of laboratory infection associated with traditional serological methods is avoided. The use of pre-coated/pre-blocked ELISA plates and the conjugation of the recombinant nucleoprotein with horseradish peroxidase simplified and shortened the assay procedure. Results showed the assay to be highly reproducible with a lower detection limit equal to that of a commercial competition ELISA. By receiver operating characteristic (ROC) curve analysis the area under curve (AUC) index was determined as 1.0 and the diagnostic sensitivity and specificity at a PP cut-off value of 4.1 as 100% and 99.78% respectively. The results of this study demonstrated that the IgM capture ELISA is a safe, reliable and highly accurate diagnostic tool which can be used on its own or in parallel with other methods for the early diagnosis of RVF virus infection and also for monitoring of immune responses in vaccinated domestic ruminants.

Clinical and Virological Factors Influencing the Performance of a NS1 Antigen-Capture Assay and Potential Use as a Marker of Dengue Disease Severity

BackgroundDetection of dengue NS1 antigen in acute infection has been proposed for early diagnosis of dengue disease. The aim of this study was to evaluate the clinical and virological factors influencing the performance of the Platelia NS1 Ag kit (BioRad) and to assess the potential use of NS1 antigen and dengue viral loads as markers of dengue disease severity.Methodology/Principal FindingsBlood specimens were collected from patients hospitalized at the Kampong Cham hospital during the 2006 and 2007 dengue epidemics in Cambodia. Dengue infection was confirmed in 243/339 symptomatic patients and in 17 asymptomatic individuals out of 214 household members tested. Overall sensitivity and specificity of Platelia NS1 Ag kit were 57.5% and 100% respectively. NS1 Ag assay combined with IgM antibody capture ELISA significantly increased the sensitivity for dengue diagnosis. NS1 Ag positivity rate was found significantly higher in DF than in DHF/DSS, in primary than in secondary infections, in patients with a high viremia (>5 log/mL) and in patients infected with DENV-1. In asymptomatic individuals, the NS1 Ag capture sensitivity tends to be lower than that in symptomatic patients. Milder disease severity was observed independently in patients with RNA copy number >5 log10 cDNA equivalents/mL or in high level of NS1 antigen ratio or in DENV-1 infection.ConclusionsOverall sensitivity of NS1 Ag detection kit varied widely across the various forms of dengue infection or disease. Sensitivity was highest in patients sampled during the first 3 days after onset of fever, in patients with primary infection, DENV-1 infection, with high level of viremia and in DF rather than DHF/DSS. In asymptomatic patients, RT-PCR assay has proved to be more sensitive than NS1 antigen detection. The NS1 antigen level correlated significantly with viremia and a low NS1 antigen ratio was associated with more severe disease.

Serologic evidence of West Nile Virus in wild ducks captured in major inland resting sites for migratory waterfowl in South Korea

The rapid global expansion of West Nile virus (WNV) has recently raised concerns regarding its possible spread into South Korea. To date, WNV infection in wild birds in South Korea has not been identified. Bird migration is thought to be involved in spreading WNV, and wild birds are the possible routes of introduction of WNV infection. To assess the risk of WNV infection in South Korea, we conducted a nationwide WNV surveillance of wild birds, with an emphasis on migratory ducks from WNV-affected areas. Our chief aim was to determine whether birds with the potential to introduce WNV are present in South Korea by testing migrating and resident wild birds for WNV antibodies. We collected blood samples from 1531 wild birds representing 57 bird species at several major inland resting sites for migratory waterfowl in South Korea. A seroepidemiological analysis of WNV and Japanese encephalitis virus (JEV) infections was conducted using plaque-reduction neutralization tests (PRNTs) for each virus. To search for recent WNV infections, sera were also evaluated by IgM antibody capture ELISA. Of the 1531 serum samples, 5 (0.3%) tested positive for WNV-specific antibodies, and 70 (4.6%) tested positive for JEV-specific antibodies. A total of 9 (0.6%) samples were positive for both WNV and JEV antibodies; these samples were interpreted as having a flavivirus exposure. All birds that had neutralizing antibodies specific to WNV were negative for IgM, which indicates the likelihood of a relatively old infection. Along with the recognized distribution of flaviviruses along several duck species’ migratory routes, our findings strongly suggest that some of the birds captured in this study had been exposed to WNV or JEV.

Co-infection with Dengue Virus and Pandemic (H1N1) 2009 Virus

Co-infection with Dengue Virus and Pandemic (H1N1) 2009 Virus

Hantavirus pulmonary syndrome in Brasilia periphery: a diagnostic challenge

Hantavirus pulmonary syndrome (HPS) is an emerging rodent-borne disease in the American continent, characterized by acute respiratory distress and a high case-fatality ratio. The present work describes a case of HPS, with favorable outcome, whose initial features were mistaken for leptospirosis or other less severe acute infections. The case of a 32-year-old male with an uneventful course of HPS is reported. He was inadvertently infected at work by exposure to a rodent-contaminated environment in Brasília, Federal District vicinity, during May 2008. Fever, headache and myalgia after exposure to a rodent-contaminated environment raised clinical suspicion. Non-cardiac pulmonary edema, hydrothorax, neutrophilia with band forms 26%, high hematocrit, thrombocytopenia, and elevated liver enzymes were observed. Leptospirosis and dengue were the main differential diagnoses because both pathogens are endemic in the area. Hantavirus IgM antibody-capture ELISA was positive, while tests for dengue, leptospirosis and yellow fever were negative. The prognosis for HPS is ominous and misdiagnoses may increase mortality. Better chances of survival depend on prompt intensive care support. Reports of moderate or less typical cases can raise the suspicion index among primary care and hospital-based physicians about this uncommon but severe condition that often affects previously healthy young individuals from developing countries, and subjects who interact with rodent-infested environments in North America. High awareness of HPS allowed successful management of the patient, even before establishing the diagnosis, by serological tests at the reference laboratory of the Ministry of Health. Clinical suspicion favored warning local health authorities about a new case of HPS.

Dengue encephalopathy in children in Northern India: Clinical features and comparison with non dengue

Acute febrile encephalopathy (AFE) is a common cause of childhood hospital admissions in Lucknow. In recent years, many patients have been hospitalized with AFE and hemorrhagic manifestations, some of whom were proven to have dengue viral infection. To (i) define the role of dengue encephalopathy (DE) as a cause of AFE in children in Lucknow, (ii) document features of dengue hemorrhagic fever (DHF) in them and (iii)compare clinical features of definite dengue and non dengue AFE. Prospective study at a teaching hospital in northern India. Children between 1-12 years of age hospitalized with fever and altered consciousness of 2 weeks or less duration were enrolled after excluding bacterial and tuberculous meningitis and frank hepatic encephalopathy. Clinical and laboratory details were charted. Haemagglutination inhibition (HI) test for dengue and Japanese encephalitis viruses in paired sera and IgM antibody capture ELISA for dengue were done. Real time PCR was done in those samples testing positive for dengue IgM. Those with either positive HI test or positive dengue PCR in CSF or serum were considered definite dengue infection and features of DHF were charted in them. Those negative for IgM antibodies after 5 days of illness or whenever done, HI test, were considered definite non dengue. Clinical and laboratory features were compared between definite dengue and non dengue groups. A total of 265 patients of AFE were enrolled over a 2 year period. HI test was positive in 15/49 (30.6%) and IgM in 52/238 (21.8%) patients thus tested. A total of 62 patients were positive for dengue antibodies by either test. Real time PCR assay for dengue virus genome was positive in 28/42 (69%) tested -- 21/29 (72.4%) in CSF and 9/15 (60%) in serum. A total of 39 patients met the criteria for definite dengue infection of which only 2 fulfilled the WHO criteria for DHF. Comparing DE and non DE, rash, bleeding, swelling over body, and hepatomegaly were significantly more common and meningeal signs less frequent in DE. Mean platelet counts and serum albumen were lower and liver enzymes and INR were significantly higher in DE. Dengue viral infection is a cause of AFE in children in this region. Majority of DE here appears to be due to viral invasion of brain as suggested by high PCR positivity in CSF and lack of WHO criteria for DHF. Differentiating features of DE include swelling and hepatic dysfunction.

MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination

The IgM antibody capture ELISA (MAC-ELISA) and ELISA inhibition methods for the detection of antibodies against dengue virus were modified to detect antibodies against yellow fever virus. Tests were carried out in 21 persons vaccinated with 17D and compared with the Plaque reduction neutralizing test. Of 17 naive subjects vaccinated, 16 (94%) seroconverted using the MAC-ELISA test and 14 (82%) seroconverted (or ≥fourfold titer increase) in the ELISA inhibition method. Cross-reactivity was evaluated by both tests and resulted in a high specificity to IgM antibodies against yellow fever, when all the samples from vaccinated individuals were negative by MAC-ELISA using dengue antigen. However, 10.7% of the positive dengue sera from the Santiago de Cuba epidemic cross-reacted by MAC-ELISA using yellow fever antigen. ELISA inhibition method showed high cross-reactivity when the 21 sera pairs were worked with yellow fever and dengue antigens. The MAC-ELISA and ELISA inhibition methods have become indispensable tools in our laboratory in order to maintain a surveillance system for dengue and dengue hemorrhagic fever. They are relatively rapid, simple, and they do not require sophisticated equipment. Both MAC-ELISA and ELISA inhibition methods for yellow fever could be useful for diagnosis, surveillance and yellow fever vaccine evaluation.

Viral encephalitis of public health significance in India: current status.

Japanese encephalitis (JE) and rabies are 2 viral encephalitis that are of public health importance in India. JE is a zoonosis with the primary cycle occurring in arthropods (mosquito vectors) and vertebrate animals (primarily the pig), man being only an incidental 'dead end' host. Out-breaks have been seen in most parts of India except the north west. The disease presents with a prodromal stage, an acute encephalitic stage with coma, convulsions and variable deficits and a convalescent stage. Diagnosis can be made by viral isolation from CSF or brain, or serologic tests such as haemagglutination inhibition test and IgM antibody capture ELISA in CSF and blood. There is no specific treatment. Mortality ranges from 20-50% and almost half the survivors have sequelae. The most effective control measure besides control of mosquitos is vaccination. A killed mouse brain vaccine is being prepared in India and is safe and effective but expensive. Rabies is a highly fatal encephalomyelitis primarily occurring in urban dogs and wild animals especially canines. It is endemic in India and affects an estimated 3 per 100,000 persons annually. The patient initially may display bizarre combative behaviour. The disease can be effectively prevented by post exposure vaccination. The nervous tissue vaccine is no longer recommended because of unacceptable neurotoxicity. Three cell culture vaccines are presently available with about equal efficacy.

A poliovirus type-specific IgM antibody-capture enzyme-linked immunosorbent assay for the rapid diagnosis of poliomyelitis

Objectives: To develop and evaluate an IgM antibody-capture ELISA for the rapid detection of poliovirus type-specific IgM antibodies in serum and CSF of patients suspected of poliomyelitis. Study design: The assay uses monoclonal antibody to human IgM as catching antibody, monovalent IPV as antigen and type-specific monoclonal antibodies labeled with horseradish peroxidase as detecting antibody. Results: Sera from 21 of 24 patients, contacts of these patients or recently vaccinated children were positive in the IgM assay. Sera from 100 healthy individuals were negative in all three poliovirus type-specific ELISAs. Sera from 5 of 81 patients with non-polio viral infections showed (weak) reactivity in one or more of the IgM assays. In a prospective study, sera and/or CSFs from 72 patients with a recent clinical diagnosis of poliomyelitis were tested. A homotypic IgM antibody response was found in 51 of the 55 patients which shedded wild poliovirus in stool or throat. Poliovirus type-specific IgM antibodies were also detected in serum from 13 of the 17 patients, from whom no poliovirus was isolated. Poliovirus type-specific IgM-antibodies could be detected from the second day up till at least 28 days after onset of paralysis. Background: Poliovirus infections still create measurable morbidity and mortality in the world. Rapid diagnosis methods are needed to detect infection.

Dot-ELISA for serodiagnosis of human infections due to Western equine encephalitis virus

A standard dot-ELISA (enzyme-linked immunosorbent assay) was modified for use in detecting IgM and IgG class antibodies to Western equine encephalitis (WEE) virus in serum samples from humans infected with this virus. Nitrocellulose membranes were soaked in supernatant fluid from WEE virus-infected cell cultures, air dried, and blocked with bovine protein. Serum samples were pipetted onto sections of the nitrocellulose, incubated, and washed. Addition of antibody to human immunoglobulin conjugated to alkaline phosphatase and enzyme substrate were used to detect the antibodies. Of 13 samples positive for IgM antibody to WEE virus by IgM antibody capture ELISA, 12 were positive by IgM dot-ELISA. IgG antibody to WEE virus was detected by dot-ELISA in 7/8, 10/14 and 7/10 samples with neutralizing, hemagglutination-inhibiting, or complement-fixing antibodies, respectively.

Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies

This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells are used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice as sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent Mayaro infection. IgG EIA-ICC showed high sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by EIA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA), for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection.