Capture ELISA Research Articles

Patient-derived monoclonal antibodies to SARS-CoV-2 nucleocapsid protein N-terminal and C-terminal domains cross-react with their counterparts of SARS-CoV, but not other human betacoronaviruses.

SARS-CoV-2 nucleocapsid (N) protein plays a key role in multiple stages of the viral life cycle such as viral replication and assembly. This protein is more conserved than the Spike protein of the virus and can induce both humoral and cell-mediated immune responses, thereby becoming a target for clinical diagnosis and vaccine development. However, the immunogenic characteristics of this protein during natural infection are still not completely understood. Patient-derived monoclonal antibodies (mAbs) against SARS-CoV-2 N protein were generated from memory B cells in the PBMCs using the antigen-specific B cell approach. For epitope mapping of the isolated hmAbs, a panel of series-truncated N proteins were used , which covered the N-terminal domain (NTD, aa 46-174 ) and C-terminal domain (CTD, aa 245-364 ), as well as the flanking regions of NTD and CTD. NTD- or CTD-specific Abs in the plasma from COVID-19 patients were also tested by ELISA method. Cross-binding of hmAbs or plasma Abs in COVID-19 patients to other human β-CoV N proteins was determined using the capture ELISA. We isolated five N-specific monoclonal antibodies (mAbs) from memory B cells in the peripheral blood of two convalescent COVID-19 patients. Epitope mapping revealed that three of the patient-derived mAbs (N3, N5 and N31) targeted the C-terminal domain (CTD), whereas two of the mAbs (N83 and 3B7) targeted the N-terminal domain (NTD) of SARS-CoV-2 N protein. All five patient-derived mAbs were cross-reactive to the N protein of SARS-CoV but showed little to no cross-reactivity to the N proteins of other human beta coronaviruses (β-CoVs). We also tested 52 plasma samples collected from convalescent COVID-19 patients for Abs against the N proteins of human β-CoVs and found that 78.8% of plasma samples showed detectable Abs against the N proteins of SARS-CoV-2 and SARS-CoV. No plasma sample had cross-reactive Abs to the N protein of MERS-CoV. Cross-reactive Abs to the N proteins of OC43 and HKU1 were detected in 36.5% (19/52) and 19.2% (10/52) of plasma samples, respectively. These results suggest that natural SARS-CoV-2 infection elicits cross-reactive Abs to the N protein of SARS-CoV and that the five patient-derived mAbs to SARS-CoV-2 N protein NTD and CTD cross-react with their counterparts of SARS-CoV, but not other human β-CoVs. Thus, these five patient-derived mAbs can potentially be used for developing the next generation of COVID-19 At-Home Test kits for rapid and specific screening of SARS-CoV-2 infection.

Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection.

Extracellular vesicles (EVs), produced during viral infections, are of emerging interest in understanding infectious processes and host-pathogen interactions. EVs and exosomes in particular have the natural ability to transport nucleic acids, proteins, and other components of cellular or viral origin. Thus, they participate in intercellular communication, immune responses, and infectious and pathophysiological processes. Some viruses are known to hijack the cell production and content of EVs for their benefit. Here, we investigate whether two pathogenic flaviviruses i.e., Zika Virus (ZIKV) and Dengue virus (DENV2) could have an impact on the features of EVs. The analysis of EVs produced by infected cells allowed us to identify that the non-structural protein 1 (NS1), described as a viral toxin, is associated with exosomes. This observation could be confirmed under conditions of overexpression of recombinant NS1 from each flavivirus. Using different isolation methods (i.e., exosome isolation kit, size exclusion chromatography, Polyethylene Glycol enrichment, and ELISA capture), we showed that NS1 was present as a dimer at the surface of excreted exosomes, and that this association could occur in the extracellular compartment. This finding could be of major importance in a physiological context. Indeed, this capacity of NS1 to address EVs and its implication in the pathophysiology during Dengue or Zika diseases should be explored. Furthermore, exosomes that have demonstrated a natural capacity to vectorize NS1 could serve as useful tools for vaccine development.

Seropositivity of dengue cases at a tertiary care centre in Chhindwara, Madhya Pradesh: A three year trend

BackgroundDuring the recent decades, dengue virus infection has been emerged as a major public health problem. Dengue is one of the important mosquito borne infections causing high mortality and morbidity of humans. Methods: This study was carried out from October 2018 to December 2020. A total of 354 serum samples of clinically suspected dengue patients were tested for immunoglobulin M (IgM) anti-dengue antibodies by dengue monoclonal antibody capture enzyme-linked immunosorbent assay (NIV DEN IgM Capture MAC ELISA). Seasonal variations, age and sex wise incidences were also determined. ResultsTotal of 354 serum samples were processed from October 2018 to December 2020. Each year males were mostly infected with Dengue 08, 10, and 03 in 2018, 2019, and 2020, respectively. Age group 11–20 was found to be mostly infected by Dengue in case of both male and female. The prevalence of Dengue in each year was from July to November might be due to the rainy season. Clinical characteristics of patients such as fever, headache, abdominal pain and nausea/vomiting, retro-orbital pain, epistaxis, petechiae, altered sensorium, positive tourniquet test were reported. ConclusionsContinuous dengue virus surveillance is required for monitoring of dengue virus so that early detection can be carried out. Effective vector control measures should be implemented for early detection of impending outbreak and to initiate timely control measures.

CO-INFECTION OF DENGUE AND CHIKUNGUNYA VIRUSES: A SEROLOGICAL STUDY IN A TERTIARY CARE HOSPITAL IN WARANGAL, TELANGANA, INDIA

Background: All around India, Dengue and Сhikungunya (CHIK) diseases seem to be rising. Aedes aegypti mosquitoes frequently transmit the CHIK virus and Dengue virus (DENV). Both viruses can spread together in regions where they cocirculate. Objectives: The objective of the current investigation is to determine the seroprevalence of coinfection with Dengue and Сhikungunya. Methods: The present study was conducted at the Virus Research and Diagnostic Laboratory, Kakatiya Medical College, Warangal, Telangana, India, from January to December 2022. The district health authority and the admitted cases provided the samples. The patients had symptoms typical of CHIK and DEN infections, including high fever (>39°C), chills, rashes, joint pain, joint swelling, nausea, and headache, myalgia, and pain behind the eyes. Results: Between January 2022 and December 2022, a total of 4892, 3344, and 2103 hospitalised patients underwent serological testing for the Dengue virus (DENV), the Сhikungunya virus (CHIKV), and both Dengue and Сhikungunya virus for co-infection, respectively. IgM Capture ELISA was used for all tests. 153 (7.8%) of the cases had both Dengue virus (DENV) and Сhikungunya virus IgM positivity. (CHIKV). Dengue virus (DENV) monoinfection, or 703 (14.3%), denotes the presence of Dengue virus. 313 (9.3%) individuals tested positive for the Сhikungunya virus, or mono-infected with the virus (CHIKV). Chikungunya IgM, Dengue IgM, and Co-infection Conclusion: Dengue and Chikungunya virus co-infections are clinically more serious than a single infection. However, in endemic regions, particularly during the monsoon season, clinically suspected cases should be tested for both viruses. To determine the severity and clinical result of co-infection, more thorough investigations are needed to analyse the pathophysiology and complications of co-infection.

Hepatitis E virus seroprevalence among employees in catering and public place industries in Nanjing, China.

This study aimed to assess the prevalence of anti-hepatitis E virus (HEV) immunoglobulin (Ig) M and elevated serum alanine aminotransferase (ALT) levels among employees in catering and public place industries. Blood samples were collected between January and December 2020 from 26,790 employees working in the Qinhuai district of Nanjing, China. Anti-HEV IgM in the serum samples was tested by the capture ELISA method and ALT was tested by the IFCC method. Samples positive for anti-HEV IgM or with ALT levels over 200U/L were subjected to PCR screening of HEV RNA. The overall seroprevalence of anti-HEV IgM was 0.41%, and the seroprevalence was slightly higher in males (0.47%) than in females (0.37%); however, the difference was not substantial (p=0.177). Seroprevalence of anti-HEV IgM increased with age, reaching its peak level after 48years of age. The prevalence of elevated ALT levels was 4.24%, and males exhibited a higher prevalence than females (6.78% vs 2.65%, p<0.001). Prevalence of elevated ALT levels differed in age groups and the 26-36-year-old group had the highest rate of elevated ALT levels. Employees with elevated ALT levels had a higher prevalence of positive anti-HEV IgM than those with normal ALT (0.57% vs 0.31%, p<0.001). Positive HEV RNA was detected in one anti-HEV IgM-negative employee with ALT higher than 200U/L. In our study, all the HEV RNA-positive and IgM-positive individuals are asymptomatic, and a combination of ALT tests, serological methods, and molecular methods is recommended to screen asymptomatic HEV carriers and reduce the risk of transmission.

Development and evaluation of recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (ELISA) and double antigen sandwich ELISA for detection of antibodies to Chikungunya virus

Chikungunya virus (CHIKV) reemerged and caused millions of human infections since 2004. The disease could be established, when the virus has been introduced to areas where the appropriate vectors are endemic. The differential diagnosis of CHIKV infection varies based on place of residence, travel history, and exposures. Serological tests are commonly used to diagnose CHIKV infection, but their availability and assessments of the performance of the diagnostics have been limited. To develop and evaluate antibodies detection methods for chikungunya diagnosis and serological investigation. Recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (Mac-ELISA) and double antigen sandwich ELISA (Das-ELISA) for detection of antibodies to Chikungunya virus were developed and evaluated. The repeatability was evaluated by testing of three reference sera at single dilutions in triplicated for 5 times. The sensitivity, specificity, accuracy, and agreement of the MAC-ELISA and Das-ELISA were obtained by comparing the detection results of 225 serum samples (45 positive; 180 negative) with a real-time RT-PCR assay and an IFA commercial tests manufactured by Euroimmun. The established ELISA assays were standardized by determining the optimal concentrations of the key reagents. The coefficient values of repeat testing were within 10% and 20% for intraassay and interassay precision, respectively. A sensitivity of 60.0% and 52.5%, a specificity of 96.2% and 96.8%, and an accuracy of 89.8% and 88.9% were obtained for the Mac-ELISA and Das-ELISA, respectively, when compared to a CHIKV qRT-PCR method. And a sensitivity of 100%, a specificity of 97.5% and 99.5%, and an accuracy of 97.8% and 99.6% were yielded respectively when using the IIFT as a reference method, which showed a highly consistence to the commercial IIFT assay with a Kappa value greater than 0.90. The Mac-ELISA and Das-ELISA based on recombinant E2 protein of CHIKV were developed and standardized, which could detect IgM or total antibodies against CHIKV in 2-3 hours with acceptable sensitivities and specificities. These assays can be used for laboratory diagnosis and serological investigation of CHIKV infections to evaluate the risk of CHIKV transmission.

Induction of protective interferon-β responses in murine osteoblasts following Staphylococcus aureus infection.

The refractory and recurrent nature of chronic staphylococcal osteomyelitis may be due, at least in part, to the ability of Staphylococcus aureus to invade and persist within bone-forming osteoblasts. However, osteoblasts are now recognized to respond to S. aureus infection and produce numerous immune mediators and bone regulatory factors that can shape the host response. Type I interferons (IFNs) are best known for their antiviral effects, but it is becoming apparent that they impact host susceptibility to a wide range of pathogens including S. aureus. Here, we have assessed the local expression of IFN-β by specific capture ELISA in an established in vivo mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis, specific capture ELISAs, and/or immunoblot analyses, were then used to assess the expression of type I IFNs and select IFN stimulated genes (ISGs) in S. aureus infected primary murine osteoblasts. The effect of IFN-β on intracellular S. aureus burden was assessed in vitro following recombinant cytokine treatment by serial colony counts of liberated bacteria. We report the presence of markedly elevated IFN-β levels in infected bone tissue in a mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed enrichment of genes associated with type I IFN signaling and ISGs, and elevated expression of mRNA encoding IFN-β and ISG products. IFN-β production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. Furthermore, we showed increased protein expression of the ISG products IFIT1 and IFIT3 by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-β by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-β markedly reduces the number of viable bacteria harbored by these cells. Together, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-β that can act in an autocrine and/or paracrine manner to elicit ISG expression and mitigate S. aureus infection.

Antigenicity and immunogenicity of chikungunya virus-like particles from mosquito cells.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level-2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. KEY POINTS: • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Optimization of Capture ELISAs for Chicken Cytokines Using Commercially Available Antibodies.

Cytokines like interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-10, and IL-12p40 are important biomarkers for characterizing the nature and strength of immune responses. It is important to be able to quantify the cytokines at the protein level in biological samples. Quantification of chicken cytokines is generally performed on the level of messenger RNA (mRNA) by quantitative polymerase chain reaction (qPCR) because very few capture ELISAs for the quantification of chicken cytokine proteins are commercially available. Here, we describe the optimization and validation of capture ELISAs for chicken IL-2, IL-6, IL-10, IL-12p40, and IFN-γ using commercially available antibodies and reagents. First, we determined the optimal concentrations of the antibodies. We then verified the ELISAs' performance and established that the lower limit of detection (LLOD) for all cytokines was below 32 pg/mL. The ELISAs show the same binding characteristics for recombinant and native cytokines (parallelism was &lt;15.2% CV). Values for inter-assay variation were consistently low and mostly &lt;20% CV. Overall, the optimized capture ELISAs are sensitive (&lt;32 pg/mL) and reliable tools to quantify chicken cytokines. These ELISAs can easily and inexpensively be utilized in any immunological lab and may therefore have wide applicability in immunological research for poultry.

Phenotype and Neuronal Cytotoxic Function of Glioblastoma Extracellular Vesicles.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.

Overcome low levels of detection limit and choice of antibody affects detection of lipoarabinomannan in pediatric tuberculosis.

The World Health Organization (WHO) emphasizes that tuberculosis (TB) in children and adolescents is often overlooked by healthcare providers and difficult to diagnose. As childhood TB cases rise, finding a diagnostic high in sensitivity and specificity is critical. In this study 91 urine samples from children aged 1-10 years were analyzed for tuberculostearic acid (TBSA) by gas chromatography/mass spectrometry (GC/MS) and capture ELISA (C-ELISA). In C-ELISA the CS35/A194-01 antibody performed very poorly with both curve-based and model-based cutoffs. The area under the ROC curve (AUC) of the CS35 OD450 values was only 0.60. Replacing the capture antibody with BJ76 gave a better performance in both sensitivity and specificity (AUC = 0.95). When these samples were analyzed by GC/MS, 41 classified as 'probable/possible' for TB were distinctly TBSA positive with ten samples having <3 ng/mL LAM. However, from the 50 samples with 'unlikely' TB classification, 36 were negative but 7 had >3 ng/mL and were designated as LAM positive. This experimental assay assessment study signifies that i) the antibody pair CS35/A194-01 that has been successful for adult active TB diagnosis is not adequate when LAM level is low as in pediatric TB; ii) no one mAb appears to recognize all TB-specific LAM epitopes.

Prevalence of microcephaly and Zika virus infection in a pregnancy cohort in Kenya, 2017–2019

BackgroundZika virus (ZIKV), first discovered in Uganda in 1947, re-emerged globally in 2013 and was later associated with microcephaly and other birth defects. We determined the incidence of ZIKV infection and its association with adverse pregnancy and fetal outcomes in a pregnancy cohort in Kenya.MethodsFrom October 2017 to July 2019, we recruited and followed up women aged ≥ 15 years and ≤ 28 weeks pregnant in three hospitals in coastal Mombasa. Monthly follow-up included risk factor questions and a blood sample collected for ZIKV serology. We collected anthropometric measures (including head circumference), cord blood, venous blood from newborns, and any evidence of birth defects. Microcephaly was defined as a head circumference (HC) < 2 standard deviations (SD) for sex and gestational age. Severe microcephaly was defined as HC < 3 SD for sex and age. We tested sera for anti-ZIKV IgM antibodies using capture enzyme-linked immunosorbent assay (ELISA) and confirmed positives using the plaque reduction neutralization test (PRNT90) for ZIKV and for dengue (DENV) on the samples that were ZIKV neutralizing antibody positive. We collected blood and urine from participants reporting fever or rash for ZIKV testing.ResultsOf 2889 pregnant women screened for eligibility, 2312 (80%) were enrolled. Of 1916 recorded deliveries, 1816 (94.6%) were live births and 100 (5.2%) were either stillbirths or spontaneous abortions (< 22 weeks of gestation). Among 1236 newborns with complete anthropometric measures, 11 (0.9%) had microcephaly and 3 (0.2%) had severe microcephaly. A total of 166 (7.2%) participants were positive for anti-ZIKV IgM, 136 of whom became seropositive during follow-up. Among the 166 anti-ZIKV IgM positive, 3 and 18 participants were further seropositive for ZIKV and DENV neutralizing antibodies, respectively. Of these 3 and 18 pregnant women, one and 13 (72.2%) seroconverted with antibodies to ZIKV and DENV, respectively. All 308 samples (serum and urine samples collected during sick visits and samples that were anti-ZIKV IgM positive) tested by RT-PCR were negative for ZIKV. No adverse pregnancy or neonatal outcomes were reported among the three participants with confirmed ZIKV exposure. Among newborns from pregnant women with DENV exposure, four (22.2%) were small for gestational age and one (5.6%) had microcephaly.ConclusionsThe prevalence of severe microcephaly among newborns in coastal Kenya was high relative to published estimates from facility-based studies in Europe and Latin America, but little evidence of ZIKV transmission. There is a need for improved surveillance for microcephaly and other congenital malformations in Kenya.

Prevalence of African Horse Sickness in Donkeys in Three Northern Regions of Cameroon

African Horse Sickness (AHS) is a non-contagious viral disease that is endemic in sub-Saharan Africa countries and poses an important production and health threats of equines. AHS can induce up to 95% mortality in horses and 10% in donkeys. However, there is dearth of information of its epidemiology in donkeys in Cameroon. This study was therefore carried out to determine the prevalence and associated risk factors of AHS in donkey in three Northern regions of Cameroon. Donkey sera were subjected to the ELISA blocking test for the determination of antibodies against African Horse Sickness Virus (AHSV) and positive samples were subjected to Capture-ELISA to determine the presence of antigens. Spleen samples were collected from fifteen donkey carcasses, of unknown causes of death, were screened using capture ELISA and positive reactors confirmed using conventional PCR. Potential risk factors associated with AHS were assessed based on the information collected in the field. The Chi-Square test and the Odd ratio were used to test the association between prevalence and the different variables. Of the 266 sera obtained, 219 were positive for anti-AHS virus antibodies corresponding to a sero-prevalence of 82.33% [95%CI: 77.30 – 86.44] and no antigen detected from the corresponding whole blood. Locality (p&lt;0.001, χ2= 22.96) and age (p&lt;0.001, χ2= 24.31) significantly influenced sero-positivity of the donkeys. The sero-prevalence was higher (p&lt;0.05) in the North (87.18% [95%CI: 81.03 – 91.55]) and Far North regions (84.81% [95%CI: 75.30 – 91.09]) than in the Adamawa region (51.61% [95%CI: 34.84 – 68.03]). Donkeys that were at least 3 years old (89.42% [95%CI: 84.22 – 93.05]) were at higher risk of AHS (p&lt;0.0001, χ2 = 22.54, OR: 4.56 [95%CI: 2.36 – 8.82]) compared to young animals of less than 3 years old (32.14% [95%CI: 27.15-37.13, OR: 0.15]). The study highlights that AHS is highly endemic in all age groups and both sexes of donkey populations in Northern Cameroon and donkeys maybe playing important roles in the maintenance of the disease in equine populations in the country.

Seroprevalence of Rift Valley fever virus in domestic ruminants of various origins in two markets of Yaoundé, Cameroon.

BackgroundRift Valley fever (RVF) is a mosquito-borne zoonosis endemic in Africa. With little known of the burden or epidemiology of RVF virus (RVFV) in Cameroon, this study aimed to determine the seroprevalence of RVFV in domestic ruminants of various origins in two markets of Yaoundé, Cameroon.Methodology/Principal findingsThe origin of animals randomly sampled at two livestock markets in Yaoundé were recorded and plasma samples collected for competitive and capture Enzyme-linked Immunosorbent Assay (ELISA) to determine the prevalence of Immunoglobulins G (IgG) and Immunoglobulins M (IgM) antibodies. Following ELISA IgM results, a real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed to detect RVFV RNA. In June-August 2019, February-March 2020, and March-April 2021, 756 plasma samples were collected from 441 cattle, 168 goats, and 147 sheep. RVFV IgG seroprevalence was 25.7% for all animals, 42.2% in cattle, 2.7% in sheep, and 2.4% in goats. However, IgM seroprevalence was low, at 0.9% in all animals, 1.1% in cattle, 1.4% in sheep, and 0% in goats. The seroprevalence rates varied according to the animal’s origin with the highest rate (52.6%) in cattle from Sudan. In Cameroon, IgG and IgM rates respectively were 45.1% and 2.8% in the North, 44.8% and 0% in the Adamawa, 38.6% and 1.7% in the Far-North. All IgM positive samples were from Cameroon. In cattle, 2/5 IgM positive samples were also IgG positive, but both IgM positive samples in sheep were IgG negative. Three (42.9%) IgM positive samples were positive for viral RVFV RNA using qRT-PCR but given the high ct values, no amplicon was obtained.Conclusion/SignificanceThese findings confirm the circulation of RVFV in livestock in Cameroon with prevalence rates varying by location. Despite low IgM seroprevalence rates, RVF outbreaks can occur without being noticed. Further epidemiological studies are needed to have a broad understanding of RVFV transmission in Cameroon.

Isolation and characterization of phage display-derived scFv antibodies against human parechovirus 1 VP0 protein

Human parechoviruses (PeVs) are common viruses that are associated with a variety of diseases from mild gastrointestinal and respiratory symptoms to severe central nervous system infections. Until now there has not been antibodies for visualizing parechovirus infection. We used E. coli recombinant PeV-A1-VP0 protein as a target in phage display single chain variable fragment (scFv) antibody library panning. Three rounds of panning allowed identification and isolation of several candidate scFv clones, which tested positive in enzyme-linked immunosorbent assay (ELISA) against VP0. Three scFv clones (scFv-55, -59 and -71) with different CDR-3 sequences were further purified and tested in ELISA, Western blot and immunofluorescence microscopy (IFA) against a set of PeV-A1 isolates and a few isolates representing PeV types 2–6. In IFA, all three scFv binders recognized twenty PeV-A1 isolates. ScFv-55 and -71 also recognized clinical representatives of PeV types 1–6 both in IFA and in capture ELISA, while scFv-59 only recognized PeV-A1, -A2 and -A6. PeV-A1-VP0 (Harris strain) sequence was used to generate a peptide library, which allowed identification of a putative unique conformational antibody epitope with fully conserved flanking regions and a more variable core VVTYDSKL, shared between the scFv antibodies. Sequencing of the VP0 region of virus samples and sequence comparisons against parechoviral sequences in GenBank revealed 107 PeV-A1, -A3, -A8, -A17, -A (untyped) sequences with this exact epitope core sequence, which was most dominant among PeV-A1 isolates. These data suggest the first-time isolation of broad range phage display antibodies against human parechoviruses that may be used in diagnostic antibody development.

Detection and Quantification of the Capsular Polysaccharide of Burkholderia pseudomallei in Serum and Urine Samples from Melioidosis Patients.

ABSTRACTBurkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease common in Southeast Asia and northern Australia. Melioidosis often presents with nonspecific symptoms and has a fatality rate of upwards of 70% when left untreated. The gold standard for diagnosis is culturing B. pseudomallei from patient samples. Bacterial culture, however, can take up to 7 days, and its sensitivity is poor, at roughly 60%. The successful administration of appropriate antibiotics is reliant on rapid and accurate diagnosis. Hence, there is a genuine need for new diagnostics for this deadly pathogen. The Active Melioidosis Detect (AMD) lateral flow immunoassay (LFI) detects the capsular polysaccharide (CPS) of B. pseudomallei. The assay is designed for use on various clinical samples, including serum and urine; however, there are limited data to support which clinical matrices are the best candidates for detecting CPS. In this study, concentrations of CPS in paired serum and urine samples from melioidosis patients were determined using a quantitative antigen capture enzyme-linked immunosorbent assay. In parallel, samples were tested with the AMD LFI, and the results of the two immunoassays were compared. Additionally, centrifugal concentration was performed on a subset of urine samples to determine if this method may improve detection when CPS levels are initially low or undetectable. The results indicate that while CPS levels varied within the two matrices, there tended to be higher concentrations in urine. The AMD LFI detected CPS in 40.5% of urine samples, compared to 6.5% of serum samples, suggesting that urine is a preferable matrix for point-of-care diagnostic assays.IMPORTANCE Melioidosis is very challenging to diagnose. There is a clear need for a point-of-care assay for the detection of B. pseudomallei antigen directly from patient samples. The Active Melioidosis Detect lateral flow immunoassay detects the capsular polysaccharide (CPS) of B. pseudomallei and is designed for use on various clinical samples, including serum and urine. However, there are limited data regarding which clinical matrix is preferable for the detection of CPS. This study addresses this question by examining quantitative CPS levels in paired serum and urine samples and relating them to clinical parameters. Additionally, centrifugal concentration was performed on a subset of urine samples to determine whether this might enable the detection of CPS in samples in which it was initially present at low or undetectable levels. These results provide valuable insights into the detection of CPS in patients with melioidosis and suggest potential ways forward in the diagnosis and treatment of this challenging disease.

Trypanosoma evansi RoTat 1.2 variant surface antigen mimotopes selected by panning of the random peptide phage-display library against monoclonal antibodies.

Mimotope peptides of native antigens are valuable for diverse applications such as diagnostics, therapeutics and modern vaccine design. Here, we report for the first time the selection and identification of peptide mimotopes of Trypanosoma evansi RoTat 1.2 variant surface glycoprotein (VSG) for their potential uses in surra diagnostics and multi-epitope vaccine research. First, we produced the mouse monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (IgG1), against the antigens in T. evansi RoTat 1.2 lysates. We then used 2E11 mAb to immunoprecipitate the target antigen. The immunoprecipitated antigen was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in immunoblots. The surface plasmon resonance immunosensors developed with both the mAbs detected VSG in the parasite lysates as well as in the rodent sera. Further, the mAbs were biotinylated and used in three rounds of panning to select peptide mimotopes from the random peptide phage display library (PhD-12; New England Biolabs, USA). The phage clones selected against each mAb were amplified and tested by phage capture ELISA for specificity. The peptide coding regions of the selected phages were sequenced and the protein blast search of the deduced amino acid sequences was performed by accessing the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/. The conformational B epitope prediction of the selected mimotope sequences was done by using 3D Pepitope algorithms accessed at: http://pepitope.tau.ac.il/. The potential applications of the selected mimotopes in surra diagnostics and research are being explored.

Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development.

Fasciola hepatica, the common liver fluke and causative agent of zoonotic fasciolosis, impacts on food security with global economic losses of over $3.2 BN per annum through deterioration of animal health, productivity losses, and livestock death and is also re-emerging as a foodborne human disease. Cathepsin proteases present a major vaccine and diagnostic target of the F. hepatica excretory/secretory (ES) proteome, but utilization in diagnostics of the highly antigenic zymogen stage of these proteins is surprisingly yet to be fully exploited. Following an immuno-proteomic investigation of recombinant and native procathepsins ((r)FhpCL1), including mass spectrometric analyses (DOI: 10.6019/PXD030293), and using counterpart polyclonal antibodies to a recombinant mutant procathepsin L (anti-rFhΔpCL1), we have confirmed recombinant and native cathepsin L zymogens contain conserved, highly antigenic epitopes that are conformationally dependent. Furthermore, using diagnostic platforms, including pilot serum and fecal antigen capture enzyme-linked immunosorbent assay (ELISA) tests, the diagnostic capacities of cathepsin L zymogens were assessed and validated, offering promising efficacy as markers of infection and for monitoring treatment efficacy.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

IntroductionSevere fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. MethodsTo develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. ResultsThe MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. ConclusionsThe MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Highly Prevalent SARS-CoV-2 Antigenemia in COVID-19 Patients.

Background:Many issues, such as severity assessment and antibody responses, remain to be answered eagerly for evaluation and understanding of COVID-19. Immune lesion is one of key pathogenesis of the disease. It would be helpful to understand the disease if an investigation on antigenemia and association was conducted in the patients with SARS-CoV-2 infection.Methods:A total of 156 patients admitted to the First People's Hospital of Hefei or Anhui Provincial Hospital on January to February 2020 were involved in this study. SARS-CoV-2 nucleocapsid (NP) antigen, specific IgM/IgG antibodies, and RNA were detected in sequential sera from three COVID-19 patients, and additional 153 COVID-19 patients by means of NP-antigen capture enzyme-linked immunosorbent assay, colloidal gold quick diagnosis, and real-time RT-PCR, respectively. The clinical types of COVID-19 patients were classified into asymptomatic, mild, moderate, severe, and critical, following on the Chinese guideline of COVID-19 diagnosis and treatment. The demographic and clinical data of patients were obtained for comparable analysis.Results:NP antigen was detected in 5 of 20 sequential sera collected from three COVID-19 patients with typically clinical symptoms, and 60.13% (92/153) expanded samples collected within 17 days after illness onset. No SARS-CoV-2 RNA segment was detected in these sera. The NP positive proportion reached a peak (84.85%, 28/33) on 6 to 8 days after illness onset. Both NP concentration and positive proportion were increased with the increase of clinical severity of COVID-19. Compared to NP negative patients, NP positive patients had older age [years, medians (interquartile ranges (IQR)), 49 (6) vs. 31 (11)], lower positive proportion of NP specific IgM [27.17% (25/92) vs. 59.02% (36/61)], and IgG [21.74% (20/92) vs. 59.02% (36/61)] antibodies, and longer duration [days, medians (IQR), 24 (10) vs. 21 (13)] from illness to recovery.Conclusions:SARS-CoV-2 NP antigenemia occurred in COVID-19, and presented highly prevalent at early stage of the disease. The antigenemia was related to clinical severity of the disease, and may be responsible for the delay of detectable SARS-Cov-2 IgM.