Antigen Enzyme-linked Immunosorbent Assay Research Articles

274 Evaluation of recombinant Capripoxvirus antigens in the development of indirect enzyme linked immunosorbent assays

Abstract Capripoxvirus is a genus of DNA viruses that includes sheep pox virus, goat pox virus and lumpy skin disease virus (LSDV). These viruses are considered high consequence viruses due to the severe clinical disease leading to economic losses to production and impact on trade. The recent spread of LSDV in Europe and Asia has demonstrated the transboundary nature of LSDV. The vaccines used to control Capripoxvirus infections currently do not have the ability to differentiate infected from vaccinated animals (DIVA) and the enzyme linked immunosorbent assays (ELISA) available for detection of antibodies to Capripoxvirus infections are not ideal. Previous research has been done to evaluate different Capripoxvirus antigens using E. coli expressed antigens; however, there has been no prior evaluation of baculovirus expressed Capripoxvirus antigens for use in ELISA. The objective of the current research was to produce 6 recombinant Capripoxvirus antigens using a baculovirus expression vector system (BEVS) and to evaluate these antigens in ELISAs to test bovine, sheep, and goat serum. The sequences used to produce the recombinant antigens were based on a consensus obtained using reference genomes consisting of all three viruses within the genus. All six antigens were produced and purified in-house using BEVS followed by benchtop or FPLC purification (Figure 1). To date, the antigens have been fully validated for performance in indirect ELISA using sheep sera (Figure 2) and partially using cattle sera, with goat sera validation to follow. The ELISAs were developed and optimized using known reference sera as controls. The performance of the ELISAs developed was assessed in comparison with the only commercially available Capripoxvirus serological test. The results demonstrate that some of the antigens used for the ELISAs completely outperformed the commercial test. Several antigens were found to be useful for ELISAs to detect responses in sheep and cattle sera. The sensitivities and specificities of the different ELISAs for sheep sera ranged from 99.54% to 53.57% sensitivity and 99.54 to 94.95% specificity with likelihood ratios above 10 using receiver operating characteristic curve analysis, while the commercial kit had a 42.85% sensitivity using the same positive sheep serum panel evaluated. The development of these serological assays allows for the simultaneous evaluation of immunogenicity of Capripoxvirus antigens through the testing of serum from experimentally infected animals. This study demonstrates the proof of principle, that sensitive and specific ELISAs can be developed for Capripoxviruses.

Revolutionizing ELISA: A one-step blocking approach using lipid bilayer coatings

Enzyme-linked immunosorbent assay (ELISA) is an essential technique for biomolecule detection in diagnostics and research, but traditional protocols are time-consuming and variable. Conventional blocking agents like bovine serum albumin (BSA) pose ethical issues and potential assay interference. This study presents a one-step lipid-blocking approach to simplify the ELISA procedure. Compared to conventional blockers, the Lipid Universal Coating Assembly (LUCA) antifouling blocker showed comparable sensitivity while significantly reducing processing time and steps. Furthermore, it showed improved co-blocking efficacy and signal intensity in conventional protocols when combined with minimal BSA concentrations. Stability tests under accelerated aging confirmed the robustness of the LUCA antifouling blocker. Additionally, it effectively facilitated antibody detection for COVID-19 antigens in direct ELISA and human IL-6 antigen in Sandwich ELISA, suggesting its versatile applicability. Taken together, our findings reveal that one-step lipid blocking not only streamlines the ELISA process but also maintains high sensitivity and specificity, providing an efficient, user-friendly, and environmentally friendly alternative to traditional ELISA blockers and a robust platform for rapid diagnostics.

A novel recombinant Theileria annulata surface protein as an antigen in indirect enzyme-linked immunosorbent assay for the serological diagnosis of tropical theileriosis

Background and Aim: Theileria annulata infection in cattle causes major economic losses in livestock production in many Central Asian countries, including the southern region of Kazakhstan. This study aimed to obtain a recombinant T. annulata surface protein (TaSP) and to investigate its possible use as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of bovine theileriosis. Materials and Methods: Recombinant TaSP was obtained by cloning a polymorphic region of the TaSP gene, expressing it in Escherichia coli strain BL21, and purifying it by metal chelating chromatography. An indirect ELISA using recombinant TaSP as an antigen was developed and evaluated for the detection of T. annulata-specific antibodies in plasma samples from 69 cows polymerase chain reaction (PCR)-positive or PCR-negative for T. annulata and/or Theileria orientalis from southern Kazakhstan. Results: The obtained recombinant protein had a molecular weight of 32 kDa, and mass spectrometry analysis of the purified protein identified it as a fragment of the surface protein of T. annulata. Initial testing of 69 field plasma samples from cattle showed that the results of indirect ELISA using TaSP as an antigen agreed substantially with those of T. annulata PCR (κ: 0.78). The relative sensitivity and specificity of indirect ELISA were 88.7% and 100%, respectively, using PCR as a reference. There was no evidence of cross-reaction with T. orientalis. Conclusion: Initial results using recombinant TaSP as an antigen in indirect ELISA are promising and support the widespread use of this assay for routine diagnosis and T. annulata seroprevalence studies in cattle in Kazakhstan and possibly neighboring countries. Keywords: cattle, indirect enzyme-linked immunosorbent assay, Kazakhstan, recombinant T. annulata surface protein, Theileria annulata.

Production and evaluation of a new set of recombinant antigens for the serological diagnosis of human cysticercosis

Human cysticercosis caused by Taenia soliun (T. soliun) is endemic in certain areas of Latin America, Asia and Sub-Saharan Africa. Neurocysticercosis (NCC) is mainly diagnosed by neuroimaging, which, in most cases, is unavailable in endemic areas. Due to their high sensitivity and specificity, serological tests such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) based on the glycosylated fraction of the cyst CS50 are widely used for the detection of the anti-cysticercus IgG antibodies despite their significant cost and the need of cysticercus material. Given their cost-effectivess and simplicity, immunoassays based on recombinant proteins could provide new alternatives for human cysticercosis diagnosis: such tests would be aimed at screening those people living in remote areas who need further examination. To date, however, no test using recombinant antigens is commercially available.Herein, five recombinant proteins (R14, R18, R93.1, R914.1, and R915.2) were produced, three of which (R93.1, R914.1, and R915.2) were newly identified from the cyst fluid. Evaluation of the diagnostic performance of these recombinant antigens by ELISA was done using sera from 200 epileptic and non-epileptic individuals in comparison with the WB-CS50 as the reference serological method.Recombinant proteins-based ELISA showed a level of diagnostic performance that is inferior than the reference serological method, but similar to that of the native antigen ELISA for human cysticercosis (commonly used for screening). Further optimization of expression conditions is still needed in order to improve proteins solubility and enhance diagnostic performance for human cysticercosis detection. However, this preliminary evaluation of the recombinant antigens has shown their potential valuable use for screening cysticercosis in patients with epilepsy attending dispensaries in remote areas. Future studies should be conducted to evaluate our recombinant antigens in a large group of patients with different stages of NCC, and in correlation with imaging findings.

Clinicomycological Study of the Spectrum of Pulmonary Aspergillosis at a Tertiary Care Hospital in Central India.

Knowing the spectrum, prevalence, and modes of diagnosis of pulmonary aspergillosis (PA) will be beneficial to clinicians for its early diagnosis and management. This study aims to estimate the prevalence, spectrum, and role of serological tests and radiological findings in the diagnosis of PA. A total of 150 patients were suspected of having PA after obtaining relevant clinical history and radiological imaging. The patients were grouped into each spectrum of PA as invasive PA (IPA), chronic necrotizing PA (CNPA), aspergilloma, allergic bronchopulmonary aspergillosis (ABPA) based on predisposing factors, clinical and radiological findings, and the guidelines of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG). Samples (bronchoalveolar lavage (BAL), sputum, blood) were collected from these patients and processed in a microbiology lab. BAL and sputum were subjected to microscopy by potassium hydroxide mount, calcofluor white mount, and culture. The serum was separated from blood by centrifugation and subjected to specific serological tests based on the spectrum of PA that the patient was suspected to have. For IPA, serum and BAL galactomannan antigen enzyme-linked immunosorbent assay (ELISA) was performed. For CNPA and aspergilloma, the anti-Aspergillus IgG antibody ELISA was performed. For ABPA, the tests performed were total immunoglobulin E (IgE) ELISA, Aspergillus fumigatus-specific IgE ELISA, and anti-Aspergillus immunoglobulin G (IgG) antibody ELISA. After compiling the clinical, radiological, culture, and serological findings, patients were diagnosed to have a particular spectrum of PA. The prevalence of IPA was 1.4%, CNPA was 4%, ABPA was 3.2%, and aspergilloma was 2.9%. CNPA was the predominant spectrum of PA in our study. Culture positivity for Aspergillus species was seen the highest in aspergilloma patients, followed by IPA, ABPA, and CNPA patients. A. fumigatus was the most common causative agent of PA, except for IPA for which Aspergillus flavus was the most common causative. Aspergillus niger and Aspergillus terreus were less the frequent causes of PA. A combination of radiological, microbiological, and serological tests along with clinical correlation is needed to confirm the diagnosis of PA.

Safety and immunogenicity of Innovax bivalent human papillomavirus vaccine in girls 9–14 years of age: Interim analysis from a phase 3 clinical trial

BackgroundWorld Health Organization human papillomavirus (HPV) vaccination recommendations include a single- or two-dose schedule in individuals 9–20 years old and advice for generating data on single-dose efficacy or immunobridging. The ongoing Phase 3 trial of Innovax’s bivalent (types 16 and 18) HPV vaccine (Cecolin®) assesses in low- and middle-income countries alternative dosing schedules and generates data following one dose in girls 9–14 years old. Interim data for the 6-month dosing groups are presented. MethodsIn Bangladesh and Ghana, 1,025 girls were randomized to receive either two doses of Cecolin at 6-, 12-, or 24-month intervals; one dose of Gardasil® followed by one dose of Cecolin at month 24; or two doses of Gardasil 6 months apart (referent). Serology was measured by enzyme-linked immunosorbent assay (ELISA) and, in a subset, by neutralization assays. Primary objectives include immunological non-inferiority of the Cecolin schedules to referent one month after the second dose. Safety endpoints include reactogenicity and unsolicited adverse events for 7 and 30 days post-vaccination, respectively, as well as serious adverse events throughout the study. ResultsInterim analyses included data from the two groups on a 0, 6-month schedule with 205 participants per group. One month after Dose 2, 100% of participants were seropositive by ELISA and had seroconverted for both antigens. Non-inferiority of Cecolin to Gardasil was demonstrated. Six months following one dose, over 96% of participants were seropositive by ELISA for both HPV antigens, with a trend for higher geometric mean concentration following Cecolin administration. Reactogenicity and safety were comparable between both vaccines. ConclusionsCecolin in a 0, 6-month schedule elicits robust immunogenicity. Non-inferiority to Gardasil was demonstrated one month after a 0, 6-month schedule. Immunogenicity following one dose was comparable to Gardasil up to six months. Both vaccines were safe and well tolerated (ClinicalTrials.gov No. 04508309).

Development of a quantitative NS1 antigen enzyme-linked immunosorbent assay (ELISA) for Zika virus detection using a novel virus-specific mAb

Viruses from the Flaviviridae family, such as Dengue virus (DENV), Yellow fever virus (YFV), and Zika virus (ZIKV) are notorious global public health problems. ZIKV emergence in Polynesia and the Americas from 2013 to 2016 raised concerns as new distinguishing features set it apart from previous outbreaks, including its association with neurological complications and heightened disease severity. Virus detection is impaired as cross-reactivity to other closely related orthoflaviviruses is common among commercially available diagnostic kits. While non-structural protein 1 (NS1) has been used as an early marker of DENV and West Nile virus (WNV) infection, little is known about NS1 expression during ZIKV infection. In the present work, we developed a NS1 capture ELISA using a novel ZIKV-specific monoclonal antibody to study NS1 expression dynamics in vitro in mosquito and human cell lines. While detectable in culture supernatants, higher concentrations of NS1 were predominantly cell-associated. To our knowledge, this is the first report of NS1 detection in human cells despite viral clearance over time. Tests with human samples need to be conducted to validate the applicability of NS1 detection for diagnosis, but overall, the tools developed in this work are promising for specific detection of acute ZIKV infection.

Development of an indirect ELISA system for diagnosis of porcine edema disease using recombinant modified Stx2e antigen.

This study aimed to develop a sensitive and specific recombinant antigen protein indirect enzyme-linked immunosorbent assay (ELISA) kit to detect the Shiga toxin (Stx)-producing Escherichia coli (STEC) antibodies against porcine edema disease (ED). The recombinant antigen was co-expressed with the STEC-derived Stx2e A2-fragment and Stx2e B protein in E. coli BL21(DE3) pLysS cells and purified using maltose-binding protein open columns. We used a Shiga-like toxin 2 antibody to test the specificity of the recombinant antigen in an indirect ELISA, which was detected in antigen-coated wells but not in uncoated wells. We tested the indirect ELISA system using samples from the STEC-immunized pig group, the commercial swine farm group, and healthy aborted fetal pleural effusion group; five and twenty samples, respectively, were positive for STEC in the former, whereas all three samples were negative for STEC in the latter. This newly developed indirect ELISA may be a specific method for diagnosing STEC infections in pigs.

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.

A single synthetic lipid antigen for improved serological diagnosis of Buruli ulcer

SETTING: The diagnosis of Buruli ulcer (BU) is frequently made by experienced health workers in rural regions. This leads to long turnaround times to confirm the diagnosis as it requires specialised laboratory infrastructure to perform confirmatory testing.BACKGROUND: Given the lack of success with protein antigens to detect BU in human sera, the aim of this study was to evaluate a range of single synthetic lipid antigens using an enzyme-linked immunosorbent assay (ELISA). The ELISA system used was initially developed to detect TB using single synthetic lipid antigens.METHODS: Thirty polymerase chain reaction (PCR) positive BU samples and 30 PCR-negative healthy contact samples collected from Asante Akim North and Ahafo Ano North Districts, Ghana, that are endemic for BU between 2013 and 2016 were used to evaluate the synthetic lipid antigen ELISA. A Quantikine ELISA was also conducted on a randomly blinded sub-set of 30 samples.RESULTS: The synthetic lipid ELISA evaluated here outperforms all other ELISA tests using protein antigens to detect BU to date and has shown potential as a fast (2 h) test for BU which may be adapted for use at the point of care. A sensitivity of 63% and specificity of 80% was observed for 30 BU-positive and 30 BU-negative samples, with significantly reduced interleukin-8 (IL-8) levels in a subset of patients with BU.CONCLUSION: A single lipid was shown for the first time to have the ability to distinguish between PCR-positive BU and negative sera using ELISA. The low lipid antibody load detected may be a result of immune suppression caused by the presence of mycolactone in patients with BU, given that levels of IL-8 were significantly reduced in patients with BU compared to the control serum samples.

Accuracy of a new rapid diagnostic test for urinary antigen detection and assessment of drug treatment in opisthorchiasis

BackgroundScreening for opisthorchiasis, a parasitic worm infection affecting many millions of people in Southeast Asia, has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique (FECT) and Kato-Katz method. Although the urinary enzyme-linked immunosorbent assay (ELISA) has been used more recently, we developed a urinary antigen-based rapid diagnostic test (RDT) to simplify diagnosis and as a point-of-care testing (POCT) and field applications for surveillance and control of opisthorchiasis.MethodsA urinary Opisthorchis viverrini (OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV. The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA (n = 493). Cross-reactivities of urinary OV-RDT with other helminthiases coexisted with O. viverrini were determined (n = 96). A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis (n = 1629). The McNemar chi-square, Kruskal–Wallis and Cohen’s kappa coefficient (κ-value) tests were used for statistical analyses.ResultsUrinary OV-RDT had sensitivity of 94.2% and specificity of 93.2%, compared to faecal FECT. Urinary OV-RDT had high diagnostic agreement (Kappa = 0.842–0.874, P < 0.001) and quantitative correlation with urinary antigen ELISA (Kruskal–Wallis tests = 316.2, P < 0.0001) and faecal FECT (Kruskal–Wallis tests = 362.3, P < 0.0001). The positive rates by OV-RDT, ELISA and FECT were 48.9%, 52.5% and 49.3%, respectively. Cross-reactions of urinary OV-RDT with other helminthiases were few (2%). Field trials of urinary OV-RDT yielded comparable prevalence of O. viverrini between urinary OV-RDT (53.2%) and urinary antigen ELISA (54.0%). OV screening showed high diagnostic agreement (kappa > 0.8, P < 0.0001) between urinary OV-RDT and urinary antigen ELISA. The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT (86.6%) and urinary antigen ELISA (80.5%) were similar (P > 0.05).ConclusionsThe urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis. The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening, control and elimination of opisthorchiasis, thereby contributing to a reduction in the disease burden in Southeast Asia.Graphical

WITHDRAWN: Development of a double-antigen sandwich ELISA for African swine fever virus antibody detection based on K205R protein

African swine fever (ASF) is a highly fatal infectious disease that affects swine caused by the African swine fever virus (ASFV) and poses a significant threat to the swine industry, and its effective diagnosis is crucial due to the current lack of commercially available vaccine for disease prevention and control. In this regard, the K205R protein is a suitable diagnostic antigen for enzyme-linked immunosorbent assay (ELISA) owing to its continuous expression throughout all ASFV replication stages, high immunogenicity, and sequence conservation. The double-antigen sandwich ELISA (DAgS-ELISA) is capable of detecting all immunoglobulins present in the serum, and outperforms alternative methods by enabling earlier antibody detection. Therefore, in this study, the K205R protein was prokaryotically expressed, and the K205R protein digested His-tag was labeled with horseradish peroxidase (HRP) and named K205R-HRP. The DAgS-ELISA was conducted using a combination of digested His-tag proteins and K205R-HRP. This method exhibits exceptional properties, including high sensitivity and no cross-reactivity with other porcine antibodies, with both the intra- and inter-assay coefficients of variation (CV) below 10 %. Antibody kinetics experiments have demonstrated that this method is capable of detecting antibodies as early as 7 to 9 days post-infection, which is a significant improvement compared to the commercial kit that can only detect antibodies from 11 to 13 days post-infection. In 120 clinical serum tests, the Kappa value between the DAgS-ELISA and the commercial kit was 0.96 (almost perfect). Owing to its high sensitivity, impressive repeatability, and strong specificity, this method enables earlier antibody detection and is suitable for ASFV antibody testing.

Seroprevalence of Human Immunodeficiency Virus, Hepatitis C Virus, and Hepatitis B Virus Among Blood Donors in Sidi Bel Abbes, West Algeria.

Introduction Blood transfusions can transmit various viruses. Among them, the most common are hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). These viruses can cause fatal and life-threatening diseases. Worldwide, many people are infected with these viruses. Blood safety has made majorprogress in recent years. Yet, blood-borne viruses continue to be a major concern for patients, physicians, and policymakers. The aim of this study was to assessthe prevalence of HIV, HBV, and HCV in blood donors. Methods A cross-sectional study was performed to assess HIV, HBV, and HCV seropositivity in blood donors. This research was carried out at the blood bank of Sidi Bel Abbes University Hospital. This was a retrospective study based on records of blood donors. All data of 10,386 donors were analyzed from January 2015 to December 2015. Biological screening was performed by enzyme-linked immunosorbent assay (ELISA) using antibodies and/or antigens. The combined HCV and HIV antigen and antibody ELISA test was utilized. To confirm the results, the blood bank and the virology laboratory used the same technique in duplicate. Results The overall seroprevalence of blood-borne viral infections (HIV, HBV, and HCV) in blood donors was 0.8%. The prevalence of HIV was found to be 0.1%, while the prevalence of HBV and HCV was 0.4%. Coinfection was rare with only one case of HBV with HIV.There was a significant difference in seroprevalence rates among blood donors compared to the general population. Significant variations were observed between the prevalence of this study and those conducted in West, East, Central, and South African countries but not with those of neighboringNorth African countries. The study found no association between seropositivity in blood donors and factors like age, gender, donor status, type of donation, or site. Besides, HIV, HBV, or HCV prevalence was not influenced by ABO and Rhesus blood group. Conclusion The study showed that blood donors in Algeria have a lower prevalence of blood-borne viral infections than the general population. The seropositivity rate of viral markers was similar throughout North African countries. This rate remained low compared to other African countries. Residual risk of infection persists. There is a need to increase blood safety for recipients. This report is the first comprehensive overview of blood-borne viruses among Algerian blood donors. There is a need for further nationwide studies to get a whole picture of the situation.

Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR)

Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.

Prevalence of Different Courses of Feline Leukaemia Virus Infection in Four European Countries.

Prevalence of progressive feline leukaemia virus (FeLV) infection is known to still be high in cats in Europe, especially in Southern Europe, but the prevalence of other outcomes of FeLV infection has not been determined in most countries. The present study aimed to investigate the prevalence of progressive, regressive, abortive, and focal infection in four European countries, two with a high (Italy, Portugal) and two with a low expected prevalence (Germany, France). Blood samples of 934 cats (Italy: 269; Portugal: 240; France: 107; Germany: 318) were evaluated for the p27 antigen, as well as anti-whole virus, anti-SU, and anti-p15E antibodies by enzyme-linked immunosorbent assay (ELISA) in serum and for proviral DNA by quantitative polymerase chain reaction (qPCR) in whole blood. Positive p27 antigen ELISA results were confirmed by reverse transcriptase-qPCR (RT-qPCR) detecting viral RNA in saliva swabs and/or blood. The outcome of FeLV infection was categorised as progressive (antigen-positive, provirus-positive), regressive (antigen-negative, provirus-positive), abortive (antigen- and provirus-negative, antibody-positive), and focal (antigen-positive, provirus-negative) infection. Overall FeLV prevalence was 21.2% in Italy, 20.4% in Portugal, 9.5% in Germany, and 9.3% in France. Prevalence of progressive, regressive, abortive, and focal infection in Italy was 7.8%, 4.5%, 6.3%, and 2.6%; in Portugal 3.8%, 8.3%, 6.7%, and 1.7%; in Germany 1.9%, 1.3%, 3.5%, and 2.8%; in France 1.9%, 3.7%, 2.8%, and 0.9%, respectively. In conclusion, overall FeLV prevalence is still very high, especially in Southern European countries. Therefore, testing, separation of infected cats, and vaccination are still important measures to reduce the risk of FeLV infection.

Development of an instrument-free and low-cost ELISA dot-blot test to detect antibodies against SARS-CoV-2.

Most laboratory tests to detect the presence of anti-SARS-CoV-2 antibodies use enzyme-linked immunosorbent assays (ELISA) or chemiluminescence immunoassays (CLIA); however, equipment for these immunoassays is unavailable in many areas of low- and middle-income countries. Rapid lateral flow immunoassay (LFIA) tests are an equipment-free option, but their high price may make them less suitable for conducting seroprevalence surveys. Here, we describe a simple dual antigen ELISA dot-blot test to detect anti-SARS-CoV-2 IgG antibodies with high sensitivity (94-98%) and specificity (92-100%), compared to commercially available ELISA and CLIA options. Additionally, this ELISA dot-blot test can be completed in one hour using minimal laboratory equipment. Importantly, this immunoassay is significantly more affordable than most LFIA tests available on the global market. The dot-blot strips may be stored for up to 7 days under freezing conditions. This ELISA dot-blot test is a cost-effective option for conducting seroprevalence screenings in areas lacking ELISA or CLIA facilities, compared to LFIA tests.

Development and application of HPV31-specific monoclonal antibodies

Human papillomavirus (HPV) is the infectious etiological agent for diseases such as cervical cancer. Developing HPV vaccines is of great significance for cancer prevention. During the development of polyvalent HPV vaccines, such as 9-valent and 15-valent HPV vaccines, specifical identification of different HPV antigen is very important. The homemade HPV31 L1 VLPs was used as an immunogen to generate HPV 31 monoclonal antibodies (mAbs) through hybridoma cell culture. We generated several mAbs through hybridoma cell culture and selected two specific neutralizing antibodies, 19B2 and 19C4, which were further analyzed and characterized for binding activity, neutralizing activity, and specificity by enzyme-linked immunosorbent assays (ELISA). It is shown that both 19B2 and 19C4 could specifically recognize HPV31 L1 virus-like particles (VLPs). These two specific mAbs were further used to assemble a HPV31 antigen ELISA test kit. The assembled kit with high detection performance could be widely applied for the quantitative detection of HPV31 antigen during vaccine production as well as in the preliminary analysis of HPV31 infection.

An epidemiological survey of porcine cysticercosis in Rungwe District, Tanzania

Taeniasis/cysticercosis caused by tapeworms belonging to the genus Taenia, poses serious veterinary and public health problems, resulting in economic burden in endemic low-income countries worldwide. However, little epidemiological data exist on infection status among pigs in many areas in Tanzania. We conducted a cross-sectional survey in Rungwe District, Mbeya Region, Tanzania, to define the prevalence and risk factors associated with porcine cysticercosis transmission. One-hundred sixty-nine pigs from 152 households were examined for circulating taeniid antigens by cysticercosis antigen (Ag) enzyme-linked immunosorbent assay (ELISA). Agarose gel electrophoresis was used to differentially diagnose Taenia species-specific cysticerci DNA bands. Structured questionnaires were administered in the surveyed households to collect information on risk factors for porcine cysticercosis transmission. Sera from eleven household pigs tested positive for porcine cysticercosis in the Ag-ELISA with an apparent prevalence of 6.5 % (95 % C.I. 3.8–11.3 %) and estimated true prevalence of 6.1 % (95 % C.I. 3.3–10.9 %). DNA Gel electrophoresis confirmed that 100 % of cysticerci isolated amongst pigs slaughtered in the study area belonged to T. solium. Of the five surveyed wards, positive household pigs were from Bulyaga, Kiwira, and Mpuguso. Lack of knowledge on porcine cysticercosis among household members was found to be significantly associated with positivity of Taenia species antigen in pigs sera (OR = 7.742, p = 0.017). Our results show that porcine cysticercosis is prevalent in Rungwe. There is a definite need to establish control measures against this potential zoonosis to safeguard veterinary and public health in the Rungwe District.

Evaluation of a mimotope of the Rickettsia outer membrane protein A (OmpA) as an antigen in enzyme-linked immunosorbent assay to detect rickettsiosis in capybaras (Hydrochoerus hydrochaeris), horses (Equus caballus), and opossums (Didelphis sp.).

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever, which is an important tick-borne zoonosis and, in Brazil, it causes Brazilian spotted fever, which has high lethality rate. This study aimed to evaluate a synthetic peptide corresponding to a segment of the outer membrane protein A (OmpA) as an antigen in a serological test for the diagnosis of rickettsial infections. The amino acid sequence of the peptide was selected by predicting B cell epitopes using B Cell Epitope Prediction (Immune Epitope Database and Analysis Resource) and Epitopia and OmpA sequences of Rickettsia rickettsii strain 'Brazil' and Rickettsia parkeri strains 'Maculatum 20' and 'Portsmouth'. A peptide with amino acid sequence common to both Rickettsia species was synthesized and arbitrarily named OmpA-pLMC. To evaluate this peptide in enzyme-linked immunosorbent assay (ELISA), serum samples of capybara (Hydrochoerus hydrochaeris), horse (Equus caballus), and opossum (Didelphis albiventris) that had been previously tested by indirect immunofluorescence assay (IFA) for rickettsial infection were separated into IFA-positive and IFA-negative groups and used in the assay. There were no significant differences in ELISA optical density (OD) values between IFA-positive and IFA-negative groups with horse samples. The mean OD values were significantly higher in the IFA-positive capybara serum samples (IFA-pos vs. IFA-neg = 2.389 ± 0.761 vs. 1.760 ± 0.840). However, receiver operating characteristic (ROC) curve analysis did not show significant diagnostic parameters. On the other hand, 12 out of 14 (85.7%) opossum samples of the IFA-positive group showed reactivity in ELISA, and this was significantly higher than of the IFA-negative group (0.7196 ± 0.440 vs. 0.2318 ± 0.098, respectively; 85.7% sensitivity, 100% specificity). Therefore, our results show that OmpA-pLMC has a potential to be used in immunodiagnostic assays to detect spotted fever group rickettsial infections.

Von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS

BackgroundType 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. ObjectiveThe objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. MethodsAn indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. ResultsIn total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). ConclusionsAnti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.