Method For Detection Of Antibodies Research Articles

Equine tapeworm (Anoplocephala spp.) infection: evaluation of saliva- and serum-based antibody detection methods and risk factor analysis in Slovak horse populations

A lack of accurate information on the prevalence and distribution of Anoplocephala spp. infections on horse farms has led to insufficient attention to tapeworm control and increasing horse anoplocephaloses in Europe. Our study aimed to examine the occurrence of Anoplocephala spp. infection using coprological, serum- and saliva-based antibody detection methods and to analyze the risk factors associated with tapeworm infection in domestic horses in Slovakia. Fecal, serum, and saliva samples were collected from 427 horses from 31 farms in Slovakia. Additionally, a questionnaire study was conducted to collect information on tapeworm distribution on horse farms and analyze risk factors associated with infection. Fecal samples were examined by the mini-FLOTAC and the double centrifugation/combined sedimentation-flotation techniques. Serum and saliva samples were analyzed by ELISA to determine antibody levels against Anoplocephala spp. The effects of variables associated with an individual horse were tested for the positive result of the saliva ELISA test on Anoplocephala spp. Cestode eggs were detected in 1.99% of fecal samples (farm prevalence 12.90%), with no differences between the two coprological methods. Serum-based tapeworm ELISA results revealed that 39.39% of horses tested positive (farm prevalence 83.87%); while saliva-based tapeworm ELISA results revealed 56.95% positive horses (farm prevalence 96.77%). Binary logistic regression analysis revealed four meaningful predictors that significantly impacted the likelihood of detecting tapeworm infection in horses: horse age, pasture size, anthelmintic treatment scheme, and access to pasture. The influences of other variables associated with an individual horse were not significantly associated with detecting tapeworm infection.

History and Utility of Specific IgE Cutoff levels: What is the Relevance for Allergy Diagnosis?

Allergy is defined clinically, by symptoms on allergen exposure. A patient is considered sensitized when allergen-specific IgE (sIgE) antibody can be detected in serum or plasma or a skin test result is positive, even if no clinical reaction has been experienced. Sensitization should be regarded as a requisite and risk factor for allergy but is not synonymous with an allergy diagnosis. To provide a correct allergy diagnosis, test results regarding allergen-sIgE must always be considered in view of the patient's case history and clinical observations. Correct assessment of a patient's sensitization to specific allergens relies on the use of accurate and quantitative methods for detection of sIgE antibodies. The evolution of sIgE immunoassays toward higher analytical performance and the use of different cutoff levels in the interpretation of test results sometimes cause confusion. Earlier versions of sIgE assays offered a limit of quantitation of 0.35 kilounits of sIgE per liter (kUA/L), which also became an established cutoff level for a positive test result in the clinical use of the assays. Current sIgE assays are capable of reliably measuring sIgE levels as low as 0.1 kUA/L and can thereby demonstrate sensitization in cases in which previous assays could not. When the outcome of sIgE test results is evaluated, it is critically important to distinguish between the analytical data as such and their clinical interpretation. Even though sIgE may be present in the absence of symptoms of allergy, available information suggests that sIgE concentrations between 0.1 kUA/L and 0.35 kUA/L may be clinically relevant in some individuals, not least among children, although this should be further evaluated for various allergies. Moreover, it is becoming widely adopted that nondichotomous interpretation of sIgE levels may offer a diagnostic benefit compared with using a predefined cutoff level.

B-101 Aquaporin 4 and Neuromyelitis Optica Spectrum Disorders

Abstract Background Neuromyelitis óptica (NMO) is a rare demyelinating inflammatory disease of the central nervous system, characterized by acute optic neuritis (ON) and transverse myelitis (TM). Previously, it was considered as a variant of multiple sclerosis (MS), however, there are now several clinical, laboratory, neuroimaging and pathological anatomical features that distinguish it from MS as the presence of a specific autoantibody in blood called IgG-NMO or anti-AQP4 that binds to water channels named aquaporin-4 (AQP4) expressed on astrocytes and plays a key role in the pathogenesis of this disease. Some patients remain seronegative for AQP4-IgG despite a definite diagnosis of NMO and the use of the finest methods for antibody detection. In anti-AQP4 seronegative forms, antibodies against myelin oligodendrocyte glycoprotein (MOG) may be identified. We have carried out a review of those patients with anti-AQP4-IgG detected in the autoimmunity laboratory during the last 3 years. Methods Each year we receive an average of 260 samples from patients with suspected demyelinating pathology with or without visual impairment. For the detection of antibodies against AQP-4 specifically HEK293 cells transfected were used as the standard substrate. Anti-MOG antibody can be detected in the same test. Measurement range: The dilution starting point for this measurement system is 1:10 (serum/plasma) and 1:1 (CSF, undiluted). Results A total of 5 patients were positive for anti AQP-4 antibody in serum samples, all women aged between 23 and 78 years. All patients except the youngest, who presented to the neurology department for loss of visual acuity, had other autoimmune diseases: systemic lupus erythematosus (SLE) in conjunction with rheumatoid arthritis, SLE in conjunction with Graves’ disease and hypothyroidism, the two oldest (59 and 78 years), respectively. The oldest patient was admitted to the internal medicine department for persistent nausea, vomiting and hiccups and was diagnosed as NMO spectrum disorder. In all cases, the oligoclonal immunoglobulin G bands study in CSF was negative. None of the CSF samples tested were positive for anti AQP-4 antibody. All patients were treated with methylprednisolone, but treatment with azathioprine or rituximab was necessary in 3 patients. Conclusion Titers of antibodies against AQP-4 are significantly higher in serum than in CSF, with serum being the most sensitive simple. NMO is more prevalent in women than men, with a female predominance usually higher than observed in MS. The median age of onset is also higher than MS, with a median of 35–45 years. Since the discovery of AQ-4 antibodies, there has been an increase in the number of clinical and radiological manifestations of NMO beyond involvement of the optic nerve and the spinal cord, including manifestations in the brain. It is important to recognise them in order to make an early diagnosis, to avoid unnecessary complementary tests and to establish the most suitable treatment. Persistent and intractable hiccups and nausea may occur in 17%–43% as in our oldest patient’s case. Associations with organ-specific autoimmune diseases such as autoimmune thyroiditis, and non-organ-specific diseases such as SLE and Sjögren’s syndrome have been described.

Comparative Analysis of the Prevalence of Bovine Viral Diarrhea Virus in Cattle Populations Based on Detection Methods: A Systematic Review and Meta-Analysis.

Infectious diseases of cattle, including bovine viral diarrhea (BVD), pose a significant health threat to the global livestock industry. This study aimed to investigate the prevalence and risk factors associated with bovine viral diarrhea virus (BVDV) infections in cattle populations through a systematic review and meta-analysis. PubMed, Web of Science, and Scopus were systematically searched for relevant articles reporting the prevalence of and associated risk factors in studies published between 1 January 2000 and 3 February 2023. From a total of 5111 studies screened, 318 studies were included in the final analysis. BVDV prevalence in cattle populations was estimated using various detection methods. The analysis detected heterogeneity in prevalence, attributed to detection techniques and associated risk factors. Antibody detection methods exhibited a higher prevalence of 0.43, reflecting the cumulative effect of detecting both active and past infections. Antigen detection methods showed a prevalence of 0.05, which was lower than antibody methods. A prevalence of 0.08 was observed using nucleic acid detection methods. The health status of the examined cattle significantly influenced the prevalence of BVDV. Cattle with bovine respiratory disease complex (BRDC) exhibited higher antibody (prevalence of 0.67) and antigen (prevalence 0.23) levels compared to cattle with reproductive problems (prevalence 0.13) or diarrhea (prevalence 0.01). Nucleic acid detection methods demonstrated consistent rates across different health conditions. Age of cattle influenced prevalence, with higher rates in adults compared to calves. Risk factors related to breeding and reproduction, such as natural or extensive breeding and a history of abortion, were associated with increased prevalence. Coinfections with pathogens like bovine herpesvirus-1 or Neospora caninum were linked to higher BVDV prevalence. Management practices, such as commingling, introducing new cattle, and direct contact with neighboring farms, also influenced prevalence. Herd attributes, including larger herd size, and the presence of persistently infected cattle, were associated with higher prevalence. These findings indicated the importance of detection methods and risk factors in BVDV epidemiological studies.

Advances in Simple, Rapid, and Contamination-Free Instantaneous Nucleic Acid Devices for Pathogen Detection.

Pathogenic pathogens invade the human body through various pathways, causing damage to host cells, tissues, and their functions, ultimately leading to the development of diseases and posing a threat to human health. The rapid and accurate detection of pathogenic pathogens in humans is crucial and pressing. Nucleic acid detection offers advantages such as higher sensitivity, accuracy, and specificity compared to antibody and antigen detection methods. However, conventional nucleic acid testing is time-consuming, labor-intensive, and requires sophisticated equipment and specialized medical personnel. Therefore, this review focuses on advanced nucleic acid testing systems that aim to address the issues of testing time, portability, degree of automation, and cross-contamination. These systems include extraction-free rapid nucleic acid testing, fully automated extraction, amplification, and detection, as well as fully enclosed testing and commercial nucleic acid testing equipment. Additionally, the biochemical methods used for extraction, amplification, and detection in nucleic acid testing are briefly described. We hope that this review will inspire further research and the development of more suitable extraction-free reagents and fully automated testing devices for rapid, point-of-care diagnostics.

SARS-CoV-2 Genome Structure, Pathogenesis, Issues, and Challenges in Laboratory Diagnosis

Severe acute respiratory syndrome coronavirus 2 causing coronavirus disease 2019 pandemic disease is an enveloped virus, showing genome similarity with bat coronavirus. This virus initially infects the upper respiratory tract, with subsequent spread to the lower respiratory tract. Despite the availability of antigen and antibody detection methods, reverse transcription-polymerase chain reaction (RT-PCR) is the diagnostic test of choice for this novel coronaviral infection. Care must be taken while interpreting the RT-PCR results, as single RT-PCR, especially in early days of infection, maybe false negative. The availability of cartridge-based nucleic acid amplification test has improved the diagnostic facilities in a peripheral setting of developing countries.

Autoimmune Autonomic Ganglionopathy: Solving the 10 Assignments

Autoimmune autonomic ganglionopathy (AAG) is a rare acquired immune-mediated neurological disease that causes various autonomic symptoms. AAG is induced by autoantibodies for the α3 and β4 subunits of the ganglionic acetylcholine receptor (gAChR). gAChR antibodies mediate synaptic transmission in all autonomic ganglia, resulting in dysautonomia. Recent clinical and basic research topics in AAG include the following: 1)analysis of clinical features; 2)novel methods for gAChR antibody detection; 3)efficacy of combined immunotherapy; 4)novel experimental AAG; 5)COVID-19 and mRNA COVID-19 vaccination and its association with autonomic dysfunction; and 6)dysautonomia as an immune-related adverse event of immune checkpoint inhibitors in cancer therapy. The author and his collaborators have previously established "10 assignments" to understand the basic research and clinical issues of AAG. In this review, the author describes the current status of research on each of the "10 assignments," incorporating research trends over the last five years.

Development and Application of a Nanobody-Based Competitive ELISA for Detecting Antibodies against Hepatitis E Virus from Humans and Domestic Animals.

Hepatitis E virus (HEV) is a zoonotic pathogen that is widespread worldwide. At present, most enzyme-linked immunosorbent assay (ELISA) kits only detect antibodies against human HEV. In this study, a nanobody-horseradish peroxidase (HRP) fusion protein-based competitive ELISA (cELISA) with more convenience and spectral characteristics for HEV antibody detection was developed and used to detect HEV IgG in various species. First, 6 anti-swine HEV capsid protein nanobodies were screened using phage display technology from an immunized Bactrian camel. Then, HEV-Nb67-HRP fusions were expressed and used as a probe for developing a cELISA. The cutoff value of the cELISA was 17.8%, and there was no cross-reaction with other anti-swine virus antibodies, suggesting that the cELISA had good specificity. The intra-assay and interassay coefficients of variation (CVs) were 1.33 to 5.06% and 1.52 to 6.84%, respectively. The cELISA and Western blot showed a higher coincidence rate (97.14%, kappa value = 0.927) than cELISA and indirect ELISA (95.00%, kappa value = 0.876) in clinical swine serum samples. Finally, the seroprevalence of HEV IgG in humans, pigs, rabbits, cows, and goats was 30.67%, 19.26%, 8.75%, 27.59%, and 18.08%, respectively, suggesting that cELISA may have a broader scale for mammalian HEV antibody detection. These results suggest that the newly developed cELISA was rapid, low-cost, reliable, and useful for the serological evaluation of current HEV. IMPORTANCE HEV is thought to be a zoonotic infection and is widespread worldwide; it is beneficial to establish a more convenient and spectral method for HEV antibody detection. In this study, a convenient, time-saving, reproducible, highly sensitive, specific, and novel nanobody-based cELISA was developed and can be used to detect IgG antibodies against mammalian HEV. It provides a new technique for serological evaluation and ELISA-based diagnosis of HEV infection.

Detection of viral antibodies in camel sera using magnetic particle spectroscopy

Pandemics like SARS-Cov-2 very frequently have their origin in different animals and in particular herds of camels could be a source of zoonotic diseases. This study took advantage on a highly sensitive and adaptable method for the fast and reliable detection of viral antibodies in camels using low-cost equipment. Magnetic nanoparticles (MNP) have high variability in their functionalization with different peptides and proteins. We confirm that 3-aminopropyl triethoxysilane (APTES)-coated MNP could be functionalized with viral proteins. The protein loading could be confirmed by simple loading controls using FACS-analysis (p < 0.05). Complementary combination of antigen and antibody yields in a significant signal increase could be proven by both FACS and COMPASS. However, COMPASS needs only a few seconds for the measurement. In COMPASS, the phase φn on selected critical point of the fifth higher harmonic (n = 5th). Here, positive sera display highly significant signal increase over the control or negative sera. Furthermore, a clear distinction could be made in antibody detection as an immune response to closely related viruses (SARS-CoV2 and MERS). Using modified MNPs along with COMPASS offers a fast and reliable method that is less cost intensive than current technologies and offers the possibility to be quickly adapted in case of new occurring viral infections.Key points• COMPASS (critical offset magnetic particle spectroscopy) allows the fast detection of antibodies.• Magnetic nanoparticles can be adapted by exchange of the linked bait molecule.• Antibodies could be detected in camel sera without washing steps within seconds.

Serological diagnosis of myasthenia gravis and its clinical significance.

Myasthenia gravis (MG) is the most common immune-mediated disorder of the neuromuscular junction. Anti-acetylcholine receptor (anti-AChR), anti-muscle-specific kinase (anti-MuSK), and anti-lipoprotein receptor-related protein 4 (anti-LRP4) antibodies are the three well-defined pathogenic antibodies. Patients with MG can also have other antibodies, such as anti-titin, anti-ryanodine receptor (anti-RyR), anti-Agrin and anti-KV1.4 antibodies. Since MG is heterogeneous in terms of pathophysiology, antibody status, and other factors, serological tests are crucial for clinical diagnosis confirmation and treatment choice. Herein, we review the different methods for detection of various antibodies involved in MG and their sensitivity and specificity. The understanding of these elements should be useful for improving the diagnosis and determining how to adapt the existing therapies to the requirements of each patient.

Serological diagnosis of myasthenia gravis and its clinical significance

: Myasthenia gravis (MG) is the most common immune-mediated disorder of the neuromuscular junction. Anti-acetylcholine receptor (anti-AChR), anti-muscle-specific kinase (anti-MuSK), and anti-lipoprotein receptor-related protein 4 (anti-LRP4) antibodies are the three well-defined pathogenic antibodies. Patients with MG can also have other antibodies, such as anti-titin, anti-ryanodine receptor (anti-RyR), anti-Agrin and anti-KV1.4 antibodies. Since MG is heterogeneous in terms of pathophysiology, antibody status, and other factors, serological tests are crucial for clinical diagnosis confirmation and treatment choice. Herein, we review the different methods for detection of various antibodies involved in MG and their sensitivity and specificity. The understanding of these elements should be useful for improving the diagnosis and determining how to adapt the existing therapies to the requirements of each patient.

Application of a Novel Aptamer Beacon for Rapid Detection of IgG1 Antibody Drugs.

Antibody drugs have been widely used to treat many diseases and are the fastest-growing drug class. IgG1 is the most common type of antibody because of its good serum stability; however, effective methods for the rapid detection of IgG1-type antibodies are lacking. In this study, we designed two aptamer molecules derived from the reported aptamer probe that has been proven to bind to the Fc fragment of the IgG1 antibody. The results showed that Fc-1S could specifically bind to the human IgG1 Fc proteins. In addition, we modified the structure of Fc-1S and constructed three aptamer molecular beacons that could quantitatively detect IgG1-type antibodies within a short time. Furthermore, we unveiled that the Fc-1S37R beacon has the highest sensitivity for IgG1-type antibodies with a detection limit of 48.82813ng/mL and can accurately detect serum antibody concentrations in vivo with consistent results to ELISA. Therefore, Fc-1S37R is an efficient method for the production monitoring and quality control of IgG1-type antibodies to enable the large-scale production and application of antibody drugs.

Evaluation of glutamic acid decarboxylase (GAD) 65 antibody detection methods for neurological and diabetic investigation in an Australian diagnostic laboratory

The role of anti-glutamic acid decarboxylase (GAD) 65 autoantibodies in autoimmune neurological conditions is evolving, but testing recommendations remain unchanged in Australia with GAD enzyme-linked immunosorbent assay (ELISA) and immunoblot as the only two Therapeutic Goods Administration approved testing methods available in Australia. Common practice is for use of ELISA in diagnosis of type 1 diabetes mellitus (T1DM) and use of immunoblot for diagnosis of GAD65-associated neurological disease. We observed a cohort of patients with negative immunoblot results and positive ELISA in the context of GAD-associated neurological disease without T1DM. In the absence of robust consensus guidelines on preferred testing modalities, we sought to determine if ELISA could have a superior role in the diagnosis of GAD-associated neurological disease when compared to immunoblot in this paper. We tested for anti-GAD65 autoantibodies on 55 patient samples, 40 samples requested for neurological disease and 15 type 1 diabetes samples with detectable anti-GAD65, using two testing platforms: Euroimmun anti-GAD enzyme-linked immunosorbent assay (ELISA) and. Euroimmun EuroLine immunoblot for paraneoplastic neurologic syndromes. These results were correlated against the clinical scenario. Positive ELISA results had a sensitivity of 100% and specificity of 91% for GAD65-related neurological disease. Immunoblot showed sensitivity of 43% and specificity of 76% for GAD65-related neurological disease. ELISA proved more sensitive and specific for GAD65-related neurological disease compared to immunoblot, raising questions about the role of this testing modality in neurological disease. We propose that ELISA should be used as a sole diagnostic method for all GAD65 antibody-related neurological disease over immunoblot. The presence of anti-GAD65 antibody on immunoblot is of doubtful clinical significance.

Modeling the accuracy of a novel PCR and antibody ELISA for African swine fever virus detection using Bayesian latent class analysis.

Diagnostic test evaluation for African swine fever (ASF) in field settings like Vietnam is critical to understanding test application in intended populations for surveillance and control strategies. Bayesian latent class analysis (BLCA) uses the results of multiple imperfect tests applied to an individual of unknown disease status to estimate the diagnostic sensitivity and specificity of each test, forgoing the need for a reference test. Here, we estimated and compared the diagnostic sensitivity and specificity of a novel indirect ELISA (iELISA) for ASF virus p30 antibody (Innoceleris LLC.) and the VetAlert™ ASF virus DNA Test Kit (qPCR, Tetracore Inc.) in field samples from Vietnam by assuming that disease status 1) is known and 2) is unknown using a BLCA model. In this cross-sectional study, 398 paired, individual swine serum/oral fluid (OF) samples were collected from 30 acutely ASF-affected farms, 37 chronically ASF-affected farms, and 20 ASF-unaffected farms in Vietnam. Samples were tested using both diagnostic assays. Diagnostic sensitivity was calculated assuming samples from ASF-affected farms were true positives and diagnostic sensitivity by assuming samples from unaffected farms were true negatives. ROC curves were plotted and AUC calculated for each test/sample combination. For comparison, a conditionally dependent, four test/sample combination, three population BLCA model was fit. When considering all assumed ASF-affected samples, qPCR sensitivity was higher for serum (65.2%, 95% Confidence Interval [CI] 58.1-71.8) and OF (52%, 95%CI 44.8-59.2) compared to the iELISA (serum: 42.9%, 95%CI 35.9-50.1; OF: 33.3%, 95%CI 26.8-40.4). qPCR-serum had the highest AUC (0.895, 95%CI 0.863-0.928). BLCA estimates were nearly identical to those obtained when assuming disease status and were robust to changes in priors. qPCR sensitivity was considerably higher than ELISA in the acutely-affected population, while ELISA sensitivity was higher in the chronically-affected population. Specificity was nearly perfect for all test/sample types. The effect of disease chronicity on sensitivity and specificity could not be well characterized here due to limited data, but future studies should aim to elucidate these trends to understand the best use of virus and antibody detection methods for ASF. Results presented here will help the design of surveillance and control strategies in Vietnam and other countries affected by ASF.

Current Methods for the Detection of Antibodies of Varicella-Zoster Virus: A Review

Infection with the varicella-zoster virus (VZV) causes chickenpox and shingles, which lead to significant morbidity and mortality globally. The detection of serum VZV-specific antibodies is important for the clinical diagnosis and sero-epidemiological research of VZV infection, and for assessing the effect of VZV vaccine immunization. Over recent decades, a variety of methods for VZV antibody detection have been developed. This review summarizes and compares the current methods for detecting VZV antibodies, and discussed future directions for this field.

Analysis of Antibodies Induced after SARS-CoV-2 Vaccination Using Antigen Coded Bead Array Luminex Technology.

Objectives. Since the outbreak of SARS-CoV-2 in late 2019, nearly 12.2 billion doses of the COVID-19 vaccine have been administered worldwide; however, the humoral immune responses induced by different types of vaccines are yet to be fully validated. Methods. We analyzed antibody levels in 100 serum samples after vaccination with different types of COVID-19 vaccines and their reactivity against the RBD antigen of Delta and Omicron variants using a bead-based microarray. Results. Elevated levels of anti-wild-type (WT)-RBD IgG and anti-WT-NP IgG were detected in participants who received two doses of the inactivated vaccines (CoronaVac or BBIBP-CorV) and three doses of the recombinant spike protein vaccine (ZF2001), indicating that antibody responses to SARS-CoV-2 were generated regardless of the vaccine administered. We found highly correlated levels of serum anti-RBD IgG and anti-NP IgG (r = 0.432, p < 0.001). We observed that the antibodies produced in vivo after COVID-19 vaccination still reacted with variants of SARS-CoV-2 (p < 0.0001). Conclusions. Our results show that high levels of specific antibodies can be produced after completion of COVID-19 vaccination (two doses of the inactivated vaccines or three doses of ZF2001), with some degree of cross-reactivity to the RBD antigen of Delta and Omicron variants, and provide an accessible and practical experimental method for post-vaccination antibody detection.

Preparation and Identification of a Monoclonal Antibody against the Pseudorabies Virus gE Glycoprotein through a Novel Strategy.

Since 2011, pseudorabies virus (PRV) has recurred in several vaccinated pig farms in China. PRV variants with high virulence were found to be the main cause of the outbreaks. In the face of the PRV epidemic, detection of the wild strain is as important as vaccine immunization, so we hoped to achieve differential diagnosis of PRV by obtaining a monoclonal antibody (mAB) that could be used to identify the wild strain. In this study, we used a novel immunization and screening strategy to prepare an mAB and obtained mAB 1H5 against the gE glycoprotein. An immunofluorescence assay (IFA) revealed that this mAB was specific to both classic and variant strains of PRV. Subsequently, we further identified the linear epitopes of B cells recognized using the mAB. The mAB 1H5 bound at 67RRAG70, which is a novel epitope and is conserved in almost all PRV strains. These findings provide novel insight into the structure and function of PRV proteins, the analysis of antigenic epitope characteristics, and the establishment of antigen or antibody detection methods.

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.

Development and evaluation of an indirect enzyme-linked immunosorbent assay based on a recombinant SifA protein to detect Salmonella infection in poultry

Salmonella is an important zoonotic pathogen that not only endangers food safety and human health, but also causes considerable economic losses to the poultry industry. Therefore, it is essential to establish a rapid, sensitive, and specific diagnostic method for the early detection of Salmonella infection in poultry. In this study, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Salmonella antibodies using a recombinant SifA protein. Amino acid sequence comparison revealed that SifA is a relatively conserved secretory protein across Salmonella serotypes. Therefore, we hypothesized that SifA can serve as a detection antigen for diagnostic testing. The SifA protein was expressed in Escherichia coli and used as a coating antigen to establish an SifA-ELISA. Control sera from specific-pathogen-free (SPF) chickens infected with Salmonella or several other non-Salmonella pathogens were then tested using the SifA-ELISA. Specificity testing demonstrated that the SifA-ELISA could detect antibodies against 3 different serotypes of Salmonella, whereas antibodies against other non-Salmonella pathogens could not be detected. Compared to the SifA-ELISA, the Salmonella plate agglutination test (PAT) failed to detect antibodies in serum samples from chickens infected with Salmonella Typhimurium. This result suggests that our SifA-ELISA may be better than PAT at detecting Salmonella infection. Comparing clinical sera, we observed a similar rate of Salmonella positivity between SifA-ELISA and PAT (92.6%). In addition, anti-SifA antibodies were continuously detected during Salmonella infection of SPF chickens, demonstrating that SifA-ELISA could consistently detect high levels of antibodies for at least 8 wk. Furthermore, the intra-assay and interassay coefficients of variation (CV) of the SifA-ELISA were below 10%, which is considered acceptable. In summary, the SifA-ELISA established here is a promising and reliable method for detection of anti-Salmonella antibodies in poultry and may contribute to the early diagnosis of Salmonella infection.

Impedimetric Biosensor Coated with Zinc Oxide Nanorods Synthesized by a Modification of the Hydrothermal Method for Antibody Detection

Impedimetric biosensors are used for detecting a wide range of analytes. The detection principle is a perspective for the development of new types of analytical devices for biomolecular diagnosis of diseases. Of particular interest are biosensors with very high sensitivities, capable of detecting trace amounts of biomarkers or drugs in biological fluids. Impedimetric biosensors possess a potential for increased sensitivity, since their electrodes can be modified with nanostructured materials, in particular zinc oxide. In this work, a miniature biosensor with an array of zinc oxide nanorods synthesized by the hydrothermal method has been created. Protein A was immobilized on the resulting structure, which was previously tested for binding to omalizumab by capillary electrophoresis. Using impedance spectroscopy, it was possible to detect the binding of omalizumab at concentrations down to 5 pg/mL. The resulting structures are suitable for creating reusable biosensor systems, since ZnO-coated electrodes are easily cleaned by photocatalytic decomposition of the bound molecules. The biosensor is promising for use in Point-of-Care systems designed for fast, multimodal detection of molecular markers of a wide range of diseases.