Double-antigen Enzyme-linked Immunosorbent Assay Research Articles

Prevalence of SARS-CoV-2 in domestic cats (Felis catus) during COVID-19 pandemic in Latvia.

The causative agent of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is of zoonotic origin and has shown reverse zoonotic transmissibility. The aim of this cross-sectional study was to investigate the serological and molecular prevalence of SARS-CoV-2 infection in the domestic cat (Felis catus) population from Latvia in natural conditions and subsequently perform viral genome analysis. Oropharyngeal and rectal swabs and blood samples were collected from 273 domestic cats during the second wave of COVID-19 infection in Latvia. Molecular prevalence was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR). Serum samples were analysed via double antigen enzyme-linked immunosorbent assay targeting the antibody against the nucleocapsid protein of SARS-CoV-2. Positive swab samples were analysed using whole viral genome sequencing and subsequent phylogenetic analysis of the whole genome sequencing data of the samples was performed. The overall SARS-CoV-2 RT-PCR positivity and seroprevalence was 1.1% (3/273) and 2.6% (7/273), respectively. The SARS-CoV-2 genome sequences from three RT-PCR positive cats were assigned to the three common lineages (PANGOLIN lineage S.1.; B.1.177.60. and B.1.1.7.) circulating in Latvia during the particular period of time. These findings indicate that feline infection with SARS-CoV-2 occurred during the second wave of the COVID-19 pandemic in Latvia, yet the overall prevalence was low. In addition, it seems like no special 'cat' pre-adaptations were necessary for successful infection of cats by the common lineages of SARS-CoV-2.

Crimean-Congo hemorrhagic fever virus circulating among sheep of Portugal: a nationwide serosurvey assessment.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread zoonotic pathogen that can cause mild to severe hemorrhagic disease in humans. CCHFV may be transmitted through direct contact with tissue or blood of viremic animals; however, the primary transmission route is through infected tick bites. CCHFV RNA has been detected in ticks feeding on domestic and wild animals in western Spain, suggesting an established circulation of CCHFV in Western Europe. Ruminants have been recognized as important CCHFV reservoirs and have been linked to human cases in endemic regions. Given the emergence of CCHF in neighboring Spain, and a report of two CCHFV seropositive humans in southern Portugal in 1985, we investigated the potential circulation of this virus in the country by performing a nationwide anti-CCHFV IgG serosurvey in sentinel sheep of Portugal. Sera (n = 459) randomly selected from widely distributed farms (n = 20) of Portugal were tested using a commercial double-antigen enzyme-linked immunosorbent assay, yielding an overall seroprevalence of 0.4% (95% confidence interval [CI] 0.04-1.56%). Positive sheep were from the southern region of Portugal (Alentejo region), which raise the seroprevalence of this region to 0.74% (95% CI 0.09-2.66%). This is the first study reporting the presence of CCHFV antibodies in sheep of Portugal, thus suggesting a geographical expansion of CCHFV to this country. It seems likely that CCHFV may exist focally in southern Portugal.

First evidence of Crimean-Congo haemorrhagic fever virus circulation in Bosnia and Herzegovina.

BackgroundCrimean–Congo haemorrhagic fever (CCHF) is a widespread tick‐borne zoonosis with reported detection of virus and/or virus‐specific antibodies from over 57 countries across Africa, Asia, Europe and the Middle East and is endemic in the Balkans. Detection of Crimean–Congo Haemorrhagic Fever Virus (CCHFV) antibodies in domestic ruminants has been important in providing initial evidence of virus circulation and in localising CCHFV high‐risk spots for human infection.ObjectivesThe present study investigated the possible exposure of sheep to CCHFV in Bosnia and Herzegovina (B&H).MethodsTo investigate the presence of anti‐CCHFV antibodies in sheep, all sera (n = 176) were tested using multi‐species double antigen enzyme‐linked immunosorbent assay (ELISA). Reactive sera were further complementary tested by adapted commercial indirect immunofluorescence assay (IFA) using FITC‐conjugated protein G instead of anti‐human immunoglobulins.ResultsCCHFV specific antibodies were detected in 17 (9.66%) animals using ELISA test. All negative sera were determined as negative by both tests, while 13 out of 17 ELISA‐positive reactors were also determined as unambiguously positive by IFA test. The age group with the highest proportion of seropositive rectors were the oldest animals.ConclusionsThis is the first report of anti‐CCHFV antibodies in sheep from B&H providing the evidence of CCHFV circulation in the country's sheep population. So far, these findings indicate the circulation of the virus in the westernmost region of the Balkans and point to the potential CCHFV spread further out of this endemic area.

Detection of anti-SFTSV nuclear protein antibody in the acute phase sera of patients using double-antigen ELISA and immunochromatography

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening febrile illness that is caused by the SFTS virus (SFTSV). The diagnosis of SFTS is usually performed by detecting viral RNA. However, it has been reported that viral RNA is no longer detectable at 6–12 days after the onset of disease. In the current study, we have constructed a plasmid to express the recombinant nuclear protein (NP) based on the Japanese strain of SFTSV (J1). We developed a double-antigen enzyme-linked immunosorbent assay (ELISA) and immunochromatography (IC) assay using recombinant NP to detect antibody against SFTSV-NP. When we tested time-sequential samples from four patients with SFTS, antibody to SFTSV-NP were detectable not only during the recovery phase (days 10–622) but also during the acute phase (days 4–7) of the disease using both of a double-antigen ELISA and IC assay. SFTSV-RNA was detected until 8–11 days after onset, thus suggesting the coexistence of the virus and antibody during the acute phase of SFTS. These data suggest that assays for detecting antibody against SFTS-NP described in the current study may be applicable not only for the epidemiological studies but also for the diagnosis of SFTS.

Severe Fever with Thrombocytopenia Syndrome Virus Infection in Minks in China.

We analyzed the seroprevalence of tick-borne severe fever with thrombocytopenia syndrome virus (SFTSV) in farm-raised minks using double antigen ELISA (enzyme-linked immunosorbent assay) kit and indicated that 8.4% (15/178) of the minks had antibodies to the nucleoprotein of SFTSV and 72.7% (8/11) of mink farms had minks positive to SFTSV. The ELISA results were further confirmed by presence of neutralization to SFTSV in the mink sera. Our results suggested that minks were widely infected with SFTSV in China.

Tetanus Immunity among Women Aged 15 to 39 Years in Cambodia: a National Population-Based Serosurvey, 2012

To monitor progress toward maternal and neonatal tetanus elimination (MNTE) in Cambodia, we conducted a nationwide serosurvey of tetanus immunity in 2012. Multistage cluster sampling was used to select 2,154 women aged 15 to 39 years. Tetanus toxoid antibodies in serum samples were measured by gold-standard double-antigen enzyme-linked immunosorbent assay (DAE) and a novel multiplex bead assay (MBA). Antibody concentrations of ≥0.01 IU/ml by DAE or the equivalent for MBA were considered seroprotective. Estimated tetanus seroprotection was 88% (95% confidence interval [CI], 86 to 89%); 64% (95% CI, 61 to 67%) of women had antibody levels of ≥1.0 IU/ml. Seroprotection was significantly lower (P < 0.001) among women aged 15 to 19 years (63%) and 20 to 24 years (87%) than among those aged ≥25 years (96%), among nulliparous women than among parous women (71 versus 97%), and among those living in the western region than among those living in other regions (82 versus 89%). The MBA showed high sensitivity (99% [95% CI, 98 to 99%]) and specificity (92% [95% CI, 88 to 95%]) compared with DAE. Findings were compatible with MNTE in Cambodia (≥80% protection). Tetanus immunity gaps should be addressed through strengthened routine immunization and targeted vaccination campaigns. Incorporating tetanus testing in national serosurveys using MBAs, which can measure immunity to multiple pathogens simultaneously, may be beneficial for monitoring MNTE.

Analytical and clinical evaluation of a new immunoassay for therapeutic drug monitoring of infliximab and adalimumab

In the last decade, biological agents have revolutionised rheumatology. Tumour necrosis factorα (TNF α ) neutralising antibody constructs as infl iximab and adalimumab are widely used as treatment for rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and psoriasis. Infl iximab is a chimeric (mouse-human) monoclonal antibody against TNF α . It has been shown to induce the formation of human antichimeric antibodies. Adalimumab is a fully human antibody against TNF α and therefore thought to be less immunogenic than chimeric antibodies. However, even fully human antibodies may lead to the production of human antihuman antibodies. Unfortunately, despite the overall effectiveness of these biological agents, a signifi cant proportion of patients do not respond or lose response over time. An explanation may be that antibodies are formed against these therapeutic agents. Another reason could be the necessity of achieving trough anti-TNF α drug concentrations in a target range (1, 2) . In addition, the incidence of anti-drug antibodies could be associated with adverse events. Higher concentrations of anti-infl iximab antibodies were associated with a higher risk of infusion reactions (3) , and severe arterial and/or venous thromboembolic events have been related with the presence of anti-adalimumab antibodies (4) . Until now, in the absence of direct measurement of drug levels and anti-drug antibodies, decision-making in the case of failure to anti-TNF α treatment was based on clinical outcome alone. Recently, a new enzyme linked immunosorbent assay (ELISA) has been developed to detect soluble drug levels and antibody formation among patients receiving infl iximab or adalimumab (Promonitor ® , Proteomika, Derio, Vizcaya, Spain, distributed by Menarini Diagn o stics S.A. ® , Badalona, Barcelona, Spain). The aim of this work was to evaluate the analytical performance and the clinical validation of these four assays with the purpose of testing the hypothesis that they are suitable for therapeutic drug monitoring of infl iximab and adalimumab in treated patients. Following the recommendations for the optimisation of immunoassays (5) , we performed our evaluation of these assays during a period of 6 months and two different reagent lots were used. Intraand inter-assay variability were evaluated on three different days with two replicates of the same sample in each assay. The linearity was performed by diluting different proportions of a standard from the upper and lower end of the dynamic range for each analyte to be evaluated. For the clinical validation study, serum was collected from 69 patients with rheumatic diseases treated with infl iximab (Remicade ® , Schering-Plough) or adalimumab (Humira ® , Abbott Laboratories). Informed consent of the patients was required. Before each infusion of infl iximab or subcutaneous injection of adalimumab, 10 mL of serum was collected and stored at – 20 ° C. All sera were tested under standardised conditions specifi ed by the fabricant. Six dilutions of serum samples and 10 for each standard curve were made. All the analytical development was carried out without knowledge of clinical data. Briefl y, in the anti-TNF α drug assay, recombinant human TNF α was coated overnight at room temperature on the solid phase and recognised by infl iximab or adalimumab in each case. The therapeutic monoclonal antibody was detected by an anti-human inmunoglobulin specifi c antibody conjugated to biotine. Serum concentrations of antibodies against these drugs were measured using a double-antigen ELISA based on their capture by drug-coated microplates and their detection by biotine-coupled anti-TNF α drug. This ELISA has a detection limit of 2 ng/mL for infl i ximab and 0.4 ng/mL for adalimumab. And the cut-off value for the drug is 0.053 mg/L for infl iximab and 0.002 mg/L for adalimumab. And for antibodies, the cut-off value is 37 UA/mL for infl iximab and 8 UA/mL for adalimumab. Descriptive statistics were provided using median (range) or percentages. Statistical analysis was performed using the Sigma Plot ® program. Differences between independent groups were traced with the use of Student t-test for normally distributed values. p-Values < 0.05 were considered statistically signifi cant. *Corresponding author: Francisca Llinares-Tello c/o Col o n 120 2 ° A, 03570, Villajoyosa, Alicante, Spain E-mail: paquillinares@eresmas.com Received January 25, 2012; accepted March 9, 2012 ; previously published online March 26, 2012

Clinical utility of antihuman lambda chain-based enzyme-linked immunosorbent assay (ELISA) versus double antigen ELISA for the detection of anti-infliximab antibodies

Anti-infliximab antibodies (ATIs) are associated with lower serum infliximab (IFX) trough levels and diminished clinical response. The current most prevalent method for detection of ATI is a double-antigen (DA) enzyme-linked immunosorbent assay (ELISA) utilizing IFX for ligand and detection antibody. Serum IFX interferes with ATI measurement in this method. An alternative ELISA using antihuman lambda chain (AHLC) antibody for ATI detection may be less amenable to this interference. The aim of our study was to compare the performance of AHLC-ATI versus DA-ATI for prediction of clinical response and evaluate the clinical significance of positive ATI in the presence of detectable IFX levels in IFX-treated inflammatory bowel disease (IBD) patients. In all, 63 patients' sera were analyzed for IFX levels and antibody levels by AHLC and DA. The results were compared with the clinical response to IFX. Percentage of patients with IFX+ATI+ status among IFX-treated patients and the clinical outcome of IFX+ATI+ patients were assessed. ATIs were demonstrated in 22/63 (34.9%) and 18/63 (28.5%) sera of patients by AHLC and DA assay, respectively (P = 0.6). Detectable ATI and in IFX was detected in four patients (6.3%) by AHLC but not by DA assay. IFX+ATI+ status was documented in 8.7% of available sera and was associated with a trend for loss of response. AHLC and DA ELISA are equally effective for ATI detection in patients with undetectable serum IFX. AHLC ELISA detects ATI in some patients with detectable serum IFX. This IFX+ATI+ status may be a harbinger of evolving loss of response to the drug.

External Quality Assessment for the Determination of Diphtheria Antitoxin in Human Serum

Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro "gold standard." The performance of laboratories using NT was generally very good, while the laboratories' performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

Determination of tetanus antibodies by a double-antigen enzyme-linked immunosorbent assay in individuals of various age groups

In this study, tetanus immunity was determined in 549 randomly chosen individuals of various age groups in Ankara, Turkey. Antibody levels in sera of the individuals were measured using a double-antigen enzyme-linked immunosorbent assay. Overall, 66.5% (95%CI, 62.4-70.4) of the population studied was found to have basic protection (>or=0.01 IU/ml) against tetanus. Protective levels of tetanus antibodies declined progressively with age. The rate of protection in children and adolescents (aged<20 years) exceeded 90%, while only 16.3% (95%CI, 8.9-26.2) of those over 60 years of age were protected. Females over 60 years of age were less immune than males of the same age group (p=0.034). Although the rates of protection in children and adolescents are regarded as satisfactory, the rates among adults are low. Preventive measures against tetanus should therefore focus on scheduled booster immunization for adults as well as children.

European Sero-Epidemiology Network: standardisation of the results of diphtheria antitoxin assays

A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.