Serological evidence for Japanese encephalitis virus and West Nile virus infections in water frequenting and terrestrial wild birds in Kolar District, Karnataka State, India. A retrospective study

In a serological survey of birds in a Japanese encephalitis (JE) endemic area of Kolar District, Karnataka State, India, 859 bird sera were tested by hemagglutination-inhibition test (HIT) for JE encephalitis and West Nile encephalitis (WNE) viruses. Only 2 (0.002%) and 178 (20.72%) sera were positive for JE virus (JEV) and WNE virus (WNV), respectively. Only 160 (18.63%) of 859 sera could be subjected to neutralizing test (NT). Of these, 20 (12.50%) and 62 (38.75%) were positive for JEV and WNV antibodies, respectively. These findings indicate that bird species such as Pond Herons and Little Egrets among ardeid birds and Grey Partridges and Quails among terrestrial birds are infected with JEV and WNV and play probably a role in the maintenance of these viruses in the abovementioned part of India.     

Screening of birds in the Volga delta (Astrakhan region, 2001) for the West Nile virus by reverse transcription-polymerase chain reaction

Infection of birds, residing in the Volga lower and middle delta, with West Nile Virus (WNV) genome was detected by the RT-PCR method. A total of 315 samples of bird organs, collected in the Astrakhan region in August 2001, were examined. Positive results, with various severity degrees, were found in coots (15.1%) and in cormorants (14.3%) in the lower delta. As for the middle delta, the infection rate among coots, herons, sea-gulls and terms was found to be identical and amounted to 8-13%, it was essentially higher in cormorants--42%. The infection rate of land-based birds did not exceed 5% in synanthropic biocenosis. According to a partial sequencing of the 5'-end region of WNV genome, all positive tests can be described as belonging to the 1st WNV genotype. The obtained results are indicative of a high activity of circulation of WNV among the birds of the water and near-water complexes in the mentioned region.     

Genetic analysis of West Nile virus isolates from US blood donors during 2002-2005

BACKGROUND: Since its introduction into North America in 1999, West Nile virus (WNV) has spread rapidly across United States (US). OBJECTIVE: To genetically analyze WNV isolates from US blood donors during 2002-2005. STUDY DESIGN: Full-length nucleotide (NT) sequences of WNV isolates from 23 US volunteer blood donors of different geographic areas from 2002 to 2005 were determined and analyzed. RESULTS: Results indicated an overall lack of geographic pattern to WNV in US. Analyses of the viral genetic diversity demonstrated that the WNV evolved at approximately five NT substitutions and 0.8 amino acid (AA) mutations per genome per year. Comparison of the functional sequences of WNV genome showed a higher evolution rate in the coding region than in the non-coding region. Furthermore, a greater diversity was observed in the nonstructural proteins as compared to the structural proteins. Sequence alignment analysis revealed that the rapid spread of WNV in US was accompanied by the establishment of a dominant genetic variant with 11 conserved NT mutations. CONCLUSIONS: The establishment of a dominant genotype across US and the displacement and possible extinction of earlier progenitor genotypes appears to have resulted from the accumulation and fixation of 11 nucleotide mutations throughout the coding region of WNV genome.     

Prevalence of Usutu and West Nile virus antibodies in human sera,Modena, Italy, 2012

A collection of 3069 human sera collected in the area of the municipality of Modena, Emilia Romagna, Italy, was retrospectively investigated for specific antibodies against Usutu (USUV) and West Nile viruses (WNV). All the samples resulting positive using a preliminary screening test were analyzed with the plaque reduction neutralization test. Overall, 24 sera were confirmed as positive for USUV (0.78%) and 13 for WNV (0.42%). The results suggest that in 2012, USUV was circulating more than WNV in North‐eastern Italy.     

Monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detecting and quantifying West Nile virus-neutralizing antibodies in horse sera

A rapid immunoassay for detecting and quantifying West Nile virus (WNV)-neutralizing antibodies in sera was developed as an alternative to the plaque reduction neutralization test (PRNT), the gold standard test for WNV. The assay is a competitive, enzyme-linked immunosorbent assay using neutralizing monoclonal antibody 5E8 (NT-ELISA). A cutoff percent inhibition (PI) value of 35% (mean PI plus 3 standard deviations), with a specificity of 99%, was established based on analysis of 246 serum samples from horses free of WNV. The NT-ELISA detected neutralizing antibodies in all sera collected 7 or 14 days postinoculation from mice (n = 11) infected with lineage I (strain NY385-99) or II (strain B956) WNV. When sera from WNV-vaccinated horses (n = 212) were tested by NT-ELISA and PRNT, the NT-ELISA gave a positive result for 96.1% (173/180) of the PRNT-positive sera and 3.1% (1/32) of the PRNT-negative sera. Discrepancies between the two tests were observed mainly with sera with low PRNT(90) titers (expressed as the reciprocal of the highest dilution yielding > or = 90% reduction in the number of plaques) for WNV or low PIs by NT-ELISA. The overall agreement (k value) between the two tests was 0.86. A good correlation (r(2) = 0.77) was also observed between the tests for endpoint titration of sera (n = 116). In conclusion, the newly developed NT-ELISA may be a good alternative serologic assay for detecting WNV that can be used for large-scale testing of WNV-neutralizing antibodies in multiple species.     

Evaluation of Cross-Protection of a Lineage 1 West Nile Virus Inactivated Vaccine against Natural Infections from a Virulent Lineage 2 Strain in Horses,under Field Conditions

Although experimental data regarding cross-protection of horse West Nile virus (WNV) vaccines against lineage 2 infections exist, the cross-protective efficacy of these vaccines under field conditions has not been demonstrated. This study was conducted to evaluate the capability of an inactivated lineage 1 vaccine (Equip WNV) to protect against natural infections from the Nea Santa-Greece-2010 lineage 2 strain. In total, 185 WNV-seronegative horses in Thessaloniki, Greece, were selected during 2 consecutive years (2011 and 2012); 140 were immunized, and 45 were used as controls. Horses were examined for signs compatible with WNV infection. Neutralizing antibody titers against the Greek strain and the PaAn001/France lineage 1 strain were determined in immunized horses. WNV circulation was detected during both years in the study area. It was estimated that 37% and 27% of the horses were infected during 2011 and 2012, respectively. Three control animals developed clinical signs, and the WNV diagnosis was confirmed. Signs related to WNV infection were not observed in the vaccinated animals. The nonvaccinated animals had a 7.58% ± 1.82% higher chance of exhibiting signs than immunized animals (P < 0.05). Neutralizing antibodies raised against both strains in all immunized horses were detectable 1 month after the initial vaccination course. The cross-protective capacity of the lowest titer (1:40) was evident in 19 animals which were subsequently infected and did not exhibit signs. Neutralizing antibodies were detectable until the annual booster, when strong anamnestic responses were observed (geometrical mean titer ratio [GMTR] for lineage 1 of 30.2; GMTR for lineage 2 of 27.5). The results indicate that Equip WNV is capable of inducing cross-protection against natural infections from a virulent lineage 2 WNV strain in horses.     

Baboon model for West Nile virus infection and vaccine evaluation

Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.     

Geographic factors contributing to a high seroprevalence of West Nile virus-specific antibodies in humans following an epidemic

Sera of 624 blood donors were evaluated to determine seroprevalence of West Nile virus (WNV) antibodies following the 2003 WNV epidemic in Nebraska. Geographic factors contributing to differences in WNV seropositivity were evaluated. The overall prevalence of WNV in Nebraska was higher than reported previously in other U.S. locations (9.5% WNV immunoglobulin G seroprevalence rate), with the highest prevalence identified in the western part of the state (19.7%), followed by the central (13.8%) and the eastern (4.2%) parts. Regions of the state with the highest WNV-positive mosquito rates correlated with the highest human WNV seroprevalence rates. The results showed that both the western and central parts of the state, where mosquito positivity rates were highest, had significantly higher seroprevalence rates than the eastern region. Additional studies are needed to determine whether the high prevalence rates in Nebraska will be reflected in other states and what impact environmental and geographical factors may have on future outbreaks of WNV infection.     

Circulation of West Nile virus (Flaviviridae, Flavivirus) and some other arboviruses in the ecosystems of Volga delta, Volga-Akhtuba flood-lands and adjoining arid regions (2000-2002)

Comprehensive virological, serological as well as genetic studies of the ecology of West Nile Virus (WNV) as well as of some other arboviruses were undertaken in different ecosystems in the territories of the Astrakhan Region and of the Kalmyk Republic. The main carriers (mosquitoes, ticks, birds and mammals) were defined as involved in the circulation of viruses within the natural and anthropogenic biocenosis. Phylogenetic examinations of isolated strains and samples, which were positive in RT-PCR, showed an absolute predominance of genotype I virus that was most closely related to American and Israeli strains. At the same time, epidemic strains had up to 6% of nucleotide differences versus the historic strains isolated in the same region 20-30 years ago. Besides, the circulation of genotype IV was discovered; it was characterized by a lower pathogenicity, which, possibly, ensures the shaping of a pronounced immune interlayer bearing no epidemic consequences. An analysis of the study results on the WNV ecology denotes the epicenter of the endemic territory located in the middle part of the Volga delta.     

Use of immunoglobulin G avidity assays for differentiation of primary from previous infections with West Nile virus

Since its introduction in 1999, West Nile virus (WNV) infections have spread rapidly across the North American continent. Diagnosis of acute WNV infection by detection of WNV-specific immunoglobulin M (IgM) is complicated by the persistence of detectable IgM for more than 1 year in some patients. IgG antibody avidity testing was assessed as a supplemental assay in the diagnosis of current infections. Three groups of serum samples were assayed in parallel by two different IgG avidity test systems (indirect immunofluorescence test [IIFT] and prototype enzyme-linked immunosorbent assay [ELISA]; EUROIMMUN, Luebeck, Germany). Group I (40 sera taken between 2 and 9 days after the onset of influenza-like symptoms) and group II (40 sera taken between 10 and 43 days after onset) were acute and convalescent specimens from patients with a positive anti-WNV IgM test (ELISA; Focus Diagnostics, Cypress, CA). Group III consisted of 43 patient sera collected between 6 and 12 months after infection. IgG antibodies specific for WNV were detected in 38% (ELISA) and 50% (IIFT) of group I sera, in 90% (ELISA and IIFT) of group II sera, and in 100% (ELISA and IIFT) of group III sera. Low-avidity IgG antibodies were demonstrated in 86% (ELISA) and 95% (IIFT) of IgG-positive patient samples taken between 2 and 43 days after the onset of symptoms (groups I and II). High-avidity IgG antibodies were detected in 100% of group III sera obtained 6 months or more after the onset of symptoms (ELISA and IIFT). IgG avidity tests for WNV infections are rapid and simple to perform. The determination of IgG avidity provides additional diagnostic certainty in differentiating between recently acquired and previous infections with WNV.     

Morphological changes in the murine medulla during experimental West Nile virus (strain 986) infection

The trends in antigen expression and structural changes in the medulla of albino mice infected by West Nile virus (WNV) were studied in different periods of an infectious process, by using monoclonal antibodies to WNV. The maximum amount of immunoreactive material was found in the damaged neurons of dead animals, although WNV antigen expression in the perikaryonic cytoplasm of individual neurons was observed in all the experimental groups. Small amount of neuronal immunoreactive material and its significant levels in the glial cells of symptomatic animals are regarded as a manifestation of individual differences in the neuronal microelement.     

Seroprevalence of West Nile and dengue virus in the human population of the Bolivian Chaco

To determine the seroprevalence of antibodies against dengue virus (DENV) and West Nile virus (WNV) in the human population of the Bolivian Chaco, we tested 256 inhabitants of two rural communities. The seroprevalence, confirmed by plaque reduction neutralization test, was 7.8% and 2.7% for DENV and WNV, respectively.     

Persistence of antibodies to West Nile virus nonstructural protein 5

BACKGROUND: Because IgM antibody against West Nile virus (WNV) pre-membrane/envelope (preM/E) recombinant protein may persist for more than 1 year, an assay distinguishing recent from past WNV infection would be useful. Published findings for a single patient suggest that the presence of antibody against WNV nonstructural protein 5 (NS5) indicates recent infection. OBJECTIVES: To compare the persistence of WNV NS5 antibodies and preM/E IgM using plasma samples from blood donors who were viremic at the time of donation. STUDY DESIGN: Follow-up plasma samples from 35 viremic donors were tested for WNV NS5 antibodies using a microsphere immunoassay, and compared to WNV preM/E IgM antibodies determined on the same samples using an enzyme-linked immunosorbent assay (ELISA). RESULTS: At 90+/-14 days of follow-up, 20/26 donors (77%) were positive for NS5 antibodies; 6/25 (24%) were positive at 180+/-27 days, and 3/23 (13%) were positive at 365+/-55 days. The comparable values for preM/E IgM antibodies were 77%, 32% and 17%, respectively. CONCLUSION: Persistence of WNV NS5 antibody in plasma is similar to that of preM/E IgM antibody. WNV NS5 antibody cannot be used to distinguish recent from past WNV infection.     

Evaluation of tests for rabies antibody and analysis of serum responses after administration of three different types of rabies vaccines.

Humoral antibody response to three types of rabies vaccines were assayed by the neutralization (NT), the mixed hemadsorption (MH), and the indirect immunofluorescence (IF) tests. The NT and MH tests were used to detect antibodies combining with antigens at the surface of virions and infected cells, whereas the indirect IF test measured antibodies mainly to the rabies nucleocapsid antigen. After immunization with a human diploid cell vaccine, antibodies were detected by both the NT and the MH test in the 14th- and 30th-day serum samples from each of eight vaccinated persons. There was a good correlation between titers obtained with the two tests in this group of vaccinees. Antibodies elicited by duck embryo and nervous tissue vaccines occurred less frequently and in lower titers. In these groups of vaccinees, 5 of 14 and 5 of 10, respectively, had antibodies detectable by the NT test in the 14th- and 30th-day sera but were negative by the MH test. It is suggested that this was due to the high levels of immunoglobulin M antibodies, which are known to be elicited by daily injections of vaccine. Since antibodies of the immunoglobulin M class are considered to be less important for protection against rabies, the MH test is recommended for immunity determinations. Compared with the NT test, this test also offers the advantage of being technically more convenient because of its capacity for testing numerous sera in a single run. Antibody titers obtained by the indirect IF test in the human diploid cell vaccine group were relatively low. Titers in the duck embryo and nervous tissue vaccine groups were higher but did not correlate with the results of the NT test.     

Evaluation of a poliovirus-binding inhibition assay as an alternative to the virus neutralization test.

An enzyme-linked immunosorbent assay (ELISA)-based poliovirus-binding inhibition (PoBI) test to detect and quantify antibodies to polioviruses was optimized and evaluated for use in population studies as an alternative to the virus neutralization test (NT) in tissue culture. The sensitivities of the inhibition ELISA compared with the NT in an inactivated poliovirus vaccine (IPV)-vaccinated population were 98.6, 97.4, and 92.1% for serotypes 1, 2, and 3, respectively. The specificities of the PoBI test, as determined with sera from nonvaccinated persons, were also high for all three serotypes (99.0, 95.8, and 100%, respectively). Antibodies to other enteroviruses did not cross-react in the serotype 1 and 3 PoBI, and only levels of cross-reactivity were found for serotype 2. We found high correlations between the PoBI and NT titers for serotypes 1 and 2 in IPV-vaccinated blood donors (0.97 and 0.95), in oral poliovirus vaccine (OPV)-vaccinated blood donors (0.91 and 0.95), and in naturally immune persons (0.90 and 0.87). The correlation coefficient for serotype 3, however, was significantly lower in OPV-vaccinated blood donors (0.73) and in naturally immune persons (0.76) than in IPV-vaccinated persons (0.94; P < 0.01). These results indicate that the antibody response to serotype 3 poliovirus in IPV recipients is different from that in OPV recipients and naturally infected persons. We conclude that the PoBI test is a suitable alternative to the NT for estimating the seroprevalence of neutralizing antibodies to poliovirus, especially in large-scale population studies.     

Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: effects of WN virus NS1 antibodies induced by inactivated WN vaccine

Antibodies to non-structural protein 1 (NS1) of West Nile virus (WNV) have been used to differentiate WNV infection from infection by serologically cross-reactive flaviviruses, including Japanese encephalitis virus (JEV), in horses. However, since the inactivated West Nile (WN) vaccine has been reported to induce NS1 antibodies, there is concern about the reliability of using NS1-based assays for testing vaccinated horses. Therefore, the effect of inactivated WN vaccine-induced antibodies on an epitope-blocking ELISA and complement-dependent cytotoxicity (CDC) assay were investigated. Both assays are based on NS1 antibodies and were established previously to differentiate WNV from JEV infections in horses. Groups of three horses were vaccinated with two or three doses of a commercial inactivated WN vaccine and NS1 antibodies were detected by a conventional ELISA after the second vaccination. Vaccine-induced NS1 antibodies were also detected by blocking ELISA and a CDC assay and affected the ability of these assays to differentiate WNV from JEV infections. However, the effect was less significant in the CDC assay, where use of a low serum concentration ensured effective differentiation. The more efficient detection of infection-induced antibodies over vaccine-induced antibodies by the CDC assay was potentially attributable to the different IgG isotype profiles of these antibodies.     

Assays for detecting West Nile Virus antibodies in human serum, plasma, and cerebrospinal fluid

The rapid spread of West Nile Virus (WNV) across the North American continent has led to a need to understand what assays for WNV antibodies are available and how they are used as diagnostic and epidemiologic tools. In this article, we review six methods for measuring WNV antibodies in human serum, plasma, and cerebrospinal fluid. The complement fixation and hemagglutination inhibition assays were historically important; however, due to their low sensitivity, low specificity, and complex technical and reagent production issues, they are no longer in common use. The plaque reduction neutralization test is the gold standard for WNV antibody detection; due to its complexity and long turnaround time, however, it is increasingly reserved for establishing the presence of WNV infection in a geographic area and characterizing problematic samples. The immunofluorescence assay measures both IgG and IgM antibodies to WNV. Although historically considered insensitive, recent studies using commercially available slides have shown acceptable performance; the immunofluorescence assay is thus a cost-effective way to measure WNV antibodies in laboratories that routinely test small numbers of samples. The enzyme-linked immunosorbent assay (ELISA) format is the most popular method currently used to detect WNV IgG and IgM. Both indirect and monoclonal antibody-mediated antigen capture formats of IgG ELISAs have been described, whereas nearly all IgM ELISAs utilize the IgM capture format. Before 2000, WNV antibody ELISAs employed native WNV antigens; since then, there has been a dramatic shift toward using recombinant WNV antigens, particularly subviral particles containing the envelope protein. Like in the other assays mentioned, however, antibodies induced by other flavivirus infections may crossreact with both native and recombinant WNV antigens, necessitating concurrent measurement of antibodies to flaviviruses endemic in a given geographic area. The new microsphere immunoassay shows great promise as a sensitive, specific, and cost-effective method for simultaneously measuring antibodies to multiple flaviviruses. This method has also been used to characterize antibodies to nonstructural WNV proteins; these antibodies appear to be highly specific for WNV, and their measurement may soon be the test of choice for diagnosing WNV infection.     

Importance of recrudescent avian infection in West Nile virus overwintering: incomplete antibody neutralization of virus allows infrequent vector infection

After the acute infection period, birds persistently infected with West Nile virus (family Flaviviridae, genus Flavivirus, WNV) occasionally shed virus into the bloodstream, but these virions normally are inactivated by neutralizing antibody. The current work tested the hypothesis that these host neutralizing antibodies protect mosquito vectors from WNV infection and reevaluated the minimum WNV infectious dose necessary to infect Culex tarsalis Coquillett. To determine whether host antibodies protect mosquitoes from infection, Cx. tarsalis and Culex stigmatosoma Dyar were fed bloodmeals containing avian blood, WNV, and sera with or without WNV-specific neutralizing antibodies. When viral particles were completely bound by antibody, mosquitoes were protected from infection; however, when incompletely bound, WNV titers as low as 10(2.3) plaque-forming units (pfu)/ml resulted in 5% infection. These data indicated that avian antibodies were protective to mosquito vectors and were not dissociated during digestion. Because recrudescent viremias may not attain the same magnitude as initial acute viremias, Cx. tarsalis vector competence was reevaluated focusing on the fate of low-titered bloodmeals. Females were evaluated for vector competence after ingesting bloodmeals containing 10(2.2), 10(3.4), 10(4.5), 10(5.5), or 10(6.5) WNV pfu/ml. Infection increased with bloodmeal titer, with 1% of the mosquitoes ingesting 10(3.4) pfu/ml and 45% of the mosquitoes ingesting 10(6.5) pfu/ml developing disseminated infections. The incomplete neutralization of recrudescent virus may be sufficient to infect a low proportion of competent blood-feeding Culex mosquitoes and perhaps allow persistently infected birds to provide a mechanism for arbovirus overwintering.     

Étude séroépidémiologique de la circulation du virus West Nile chez l’Homme en Tunisie

West Nile virus (WNV) is an arbovirus classified into the family of Flaviviridae, genus Flavivirus. It is responsible for neurological diseases that occurred frequently as outbreaks and considered as an emerging infection in different regions of the world. In Tunisia, two outbreaks of meningoencephalitis due to this virus occurred, in 1997 and 2003. The virus circulation is studied only in Sahel, region affected by the two epidemics. The aim of this study is to determine if WNV is present in other regions of the country where, up to now, no data are available. A total of 1,854 sera collected from healthy patients were investigated by ELISA to detect specific IgG, during January to December 2007. Patients included are from three governorates: Kairouan, Bizerte, and Sfax. The governorate of Sfax (center of Tunisia) was affected by the two outbreaks, whereas only two cases were observed previously at Kairouan and no cases at Bizerte. Specific IgG were detected in 12.5% of studied population. This seroprevalence varied largely between the three governorates studied. Globally, three regions with different endemicity were described: high endemicity at Kairouan (27.7%), moderate at Sfax (7.5%), and low at Bizerte (0.7%). At Kairouan, the seroprevalence is significantly higher in individuals aged over 40. Our results suggest that WNV circulates in Tunisia; it has a high risk not only in regions affected by previous outbreaks but throughout the country. An active surveillance should be instituted in the country. It must target individuals, and animals, which can be vectors or reservoirs for the virus.     

Comparison of two polyclonal antibodies for the detection of 19‐Nortestosterone in Bovine Bile by ELISA

19‐Nortestosterone (ß‐NT) is banned for use as a growth promoter in food animals within the European Union. For regulatory control purposes, urine and bile samples are routinely screened by immunoassay. The aim of the present study was to compare the ability of two immunoassays, using two rabbit polyclonal antibodies raised against two different NT derivatives, to detect NT residues in bovine bile. One antiserum cross‐reacted with both α‐NT and ß‐NT (α/ß‐NT), whereas the other was specific for α‐NT. Bile samples from 266 slaughtered cattle were deconjugated and analyzed using both antibodies, with all screening positives (>2 ng ml^?1) confirmed by high resolution gas chromatography mass spectrometry. The α /ß‐NT and α‐NT antibody‐based ELlSAs screened 39 and 44 samples positive, respectively, with NT confirmed in 22 and 39, respectively. The α/ß‐NT antibody‐based ELISA produced a false‐negative rate of 44% compared to 0% for the α‐NT antibody‐based ELISA. Supplementary investigations concluded that a matrix effect was a major cause of the marked differences in false‐negative rates. This result underlines the necessity to validate immunoassays in the sample matrix.