scholarly journals Development of a serological assay to identify SARS-CoV-2 antibodies in COVID-19 patients

2020 ◽  
Author(s):  
Angela Huynh ◽  
Donald M Arnold ◽  
John G Kelton ◽  
James W Smith ◽  
Jane C Moore ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Acute Infection ◽  
Genetic Material ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Global Pandemic ◽  
Triton X ◽  
High Throughput Assay ◽  
Ig G

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While molecular assays are used to detect viral genetic material for the diagnosis of acute infection, reliable serological assays are needed to measure immunity against SARS-CoV-2. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) that detects antibodies against the following SARS-CoV-2 recombinant proteins: the full-length spike (S) protein and the receptor-binding domain (RBD). Our assay is sensitive and specific for immunoglobulin (Ig) G, IgA and IgM anti-S protein and anti-RBD antibodies. Samples were pre-treated with Triton X-100 to inactivate potential virus without affecting antibody detection. Our in-house ELISA performed as well as the commercial EUROIMMUN and Ortho assays for anti-SARS-CoV-2 antibodies. This method provides a high-throughput assay that does not require specialized instrumentation and can be widely used to determine immunity and the dynamic range of antibodies found within SARS-CoV-2.

scholarly journals Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 697
Author(s):  
Angela Huynh ◽  
Donald M. Arnold ◽  
James W. Smith ◽  
Jane C. Moore ◽  
Ali Zhang ◽  
...  
Keyword(s):  
Population Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Rt Pcr ◽  
S Protein ◽  
Viral Neutralization ◽  
Humoral Immune ◽  
Global Pandemic ◽  
Polymerase Chain ◽  
Ig G

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

scholarly journals Rapid, Simple, Quantitative, and Highly Sensitive Antibody Detection for Lyme Disease

2010 ◽  
Vol 17 (6) ◽  
pp. 904-909 ◽  
Author(s):  
Peter D. Burbelo ◽  
Alexandra T. Issa ◽  
Kathryn H. Ching ◽  
Jeffrey I. Cohen ◽  
Michael J. Iadarola ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Dynamic Range ◽  
Independent Set ◽  
Antibody Responses ◽  
Peptide Sequence ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Characteristic Analysis ◽  
Serum Samples ◽  
Serological Tests

ABSTRACT There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n = 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n = 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

scholarly journals Computationally Designed Peptides for Zika Virus Detection: An Incremental Construction Approach

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 498 ◽  
Author(s):  
Marcello Mascini ◽  
Emre Dikici ◽  
Marta Robles Mañueco ◽  
Julio A. Perez-Erviti ◽  
Sapna K. Deo ◽  
...  
Keyword(s):  
Zika Virus ◽  
Dynamic Range ◽  
Acute Infection ◽  
Detection Limits ◽  
Analytical Signal ◽  
Cross Reactivity ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intact Virus

Herein, and in contrast to current production of anti-Zika virus antibodies, we propose a semi-combinatorial virtual strategy to select short peptides as biomimetic antibodies/binding agents for the detection of intact Zika virus (ZIKV) particles. The virtual approach was based on generating different docking cycles of tetra, penta, hexa, and heptapeptide libraries by maximizing the discrimination between the amino acid motif in the ZIKV and dengue virus (DENV) envelope protein glycosylation site. Eight peptides, two for each length (tetra, penta, hexa, and heptapeptide) were then synthesized and tested vs. intact ZIKV particles by using a direct enzyme linked immunosorbent assay (ELISA). As a reference, we employed a well-established anti-ZIKV antibody, the antibody 4G2. Three peptide-based assays had good detection limits with dynamic range starting from 105 copies/mL of intact ZIKV particles; this was one order magnitude lower than the other peptides or antibodies. These three peptides showed slight cross-reactivity against the three serotypes of DENV (DENV-1, -2, and -3) at a concentration of 106 copies/mL of intact virus particles, but the discrimination between the DENV and ZIKV was lost when the coating concentration was increased to 107 copies/mL of the virus. The sensitivity of the peptides was tested in the presence of two biological matrices, serum and urine diluted 1:10 and 1:1, respectively. The detection limits decreased about one order of magnitude for ZIKV detection in serum or urine, albeit still having for two of the three peptides tested a distinct analytical signal starting from 106 copies/mL, the concentration of ZIKV in acute infection.

scholarly journals A serological assay to detect SARS-CoV-2 seroconversion in humans

2020 ◽  
Author(s):  
Fatima Amanat ◽  
Daniel Stadlbauer ◽  
Shirin Strohmeier ◽  
Thi H.O. Nguyen ◽  
Veronika Chromikova ◽  
...  
Keyword(s):  
Adult Population ◽  
Acute Infection ◽  
Genetic Material ◽  
Negative Control ◽  
Viral Spread ◽  
Global Pandemic ◽  
Care Workers ◽  
General Adult Population ◽  
Screening And Identification ◽  
Antibody Titers

AbstractSARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.

scholarly journals Exploratory field study on the effects of porcine circovirus 2 (PCV-2) sow vaccination at different physiological stages mimicking blanket vaccination

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Patricia Pleguezuelos ◽  
Marina Sibila ◽  
Raúl Cuadrado ◽  
Rosa López-Jiménez ◽  
Diego Pérez ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Negative Control Group ◽  
Negative Control ◽  
Late Gestation ◽  
Antibody Detection ◽  
Porcine Circovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Physiological Status ◽  
Porcine Circovirus 2

Abstract Background The objective of the present study was to explore the benefits of Porcine circovirus 2 (PCV-2) blanket vaccination in a sow herd on productive parameters, PCV-2 infection and immune status in sows and their progeny. For this purpose, 288 sows were distributed among four balanced experimental groups. One group remained as negative control group and the other three received 1 mL of PCV-2 Ingelvac Circoflex® intramuscularly at different productive cycle moments: before mating, mid gestation (42–49 days post-insemination) or late gestation (86–93 days post-insemination); phosphate buffered saline (PBS) was used as negative control item. Reproductive parameters from sows during gestation and body weight of their progeny from birth to weaning were recorded. Additionally, blood was collected from sows at each vaccination time and piglets at 3 weeks of age. Moreover, up to 4 placental umbilical cords (PUC) per sow were taken at peri-partum. Sera from sows and piglets were analysed for PCV-2 antibody detection using an enzyme-linked immunosorbent assay (ELISA). Sera from sows and PUC were tested to quantify viraemia using a real time quantitative polymerase chain reaction (qPCR) assay. Results Globally, results indicated that vaccinated sows showed heavier piglets at birth and at weaning, less cross-fostered piglets, lower viral load at farrowing as well as in PUC, and higher antibody levels at farrowing, compared to non-vaccinated ones. When all groups were compared among them, sows vaccinated at mid or late gestation had heavier piglets at birth than non-vaccinated sows, and lower proportion of PCV-2 positive PUC. Also, cross-fostering was less frequently practiced in sows vaccinated at pre-mating or mid gestation compared to non-vaccinated ones. Conclusions In conclusion, the present study points out that PCV-2 sow vaccination at different time points of their physiological status (mimicking blanket vaccination) offers benefits at production and serological and virological levels.

scholarly journals Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuan Li ◽  
Hongliu Ye ◽  
Meng Liu ◽  
Suquan Song ◽  
Jin Chen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Large Scale ◽  
Operation Time ◽  
Reference Method ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Concentration

Abstract Background H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. Results The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. Conclusions In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

scholarly journals Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Kevin Kuhlmann ◽  
Melanie Cieselski ◽  
Julia Schumann
Keyword(s):  
Dynamic Range ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Test Procedures ◽  
Experimental Conditions ◽  
Rna Quantification ◽  
The Stability ◽  
Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

scholarly journals Quantitative Assessment of Periodontal Bacteria Using a Cell-Based Immunoassay with Functionalized Quartz Crystal Microbalance

Chemosensors ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 159
Author(s):  
Satit Rodphukdeekul ◽  
Miyuki Tabata ◽  
Chindanai Ratanaporncharoen ◽  
Yasuo Takeuchi ◽  
Pakpum Somboon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Quartz Crystal Microbalance ◽  
Quantitative Assessment ◽  
Quartz Crystal ◽  
Dynamic Range ◽  
Gold Surface ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammatory Disorder ◽  
Label Free ◽  
Periodontal Bacteria

Periodontal disease is an inflammatory disorder that is triggered by bacterial plaque and causes the destruction of the tooth-supporting tissues leading to tooth loss. Several bacteria species, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, are considered to be associated with severe periodontal conditions. In this study, we demonstrated a quartz crystal microbalance (QCM) immunoassay for quantitative assessment of the periodontal bacteria, A. actinomycetemcomitans. An immunosensor was constructed using a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA) on the gold surface of a QCM chip. The 11-MUA layer was evaluated using a cyclic voltammetry technique to determine its mass and packing density. Next, a monoclonal antibody was covalently linked to 11-MUA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to act as the biorecognition element. The specificity of the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. A calibration curve, for the relationship between the frequency shifts and number of bacteria, was used to calculate the number of A. actinomycetemcomitans bacteria in a test sample. Based on a regression equation, the lower detection limit was 800 cells, with a dynamic range up to 2.32 × 106 cells. Thus, the QCM biosensor in this study provides a sensitive and label-free method for quantitative analysis of periodontal bacteria. The method can be used in various biosensing assays for practical application and routine detection of periodontitis pathogens.

scholarly journals Prevalence and clinical correlation of hepatitis E virus antibody in the patients’ serum samples from a tertiary care hospital in Thailand during 2015–2018

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Atiporn Boonyai ◽  
Anchalee Thongput ◽  
Thidarat Sisaeng ◽  
Parisut Phumchan ◽  
Navin Horthongkham ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Tertiary Care ◽  
Laboratory Data ◽  
Organ Transplant ◽  
Hospital Setting ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Care Hospital ◽  
Serum Samples ◽  
Igm Antibodies

Abstract Background Prevalence and incidence of hepatitis caused by HEV infection are usually higher in developing countries. This study demonstrated the HEV seroprevalence and incidence of HEV infection in patients with clinical hepatitis in a tertiary hospital in Thailand. Methods A laboratory-based cross-sectional study was conducted using 1106 serum samples from patients suspected of HEV infection sent to the Serology laboratory, Siriraj Hospital, for detecting HEV antibodies during 2015–2018. Prevalence of anti-HEV IgG and IgM antibodies in general patients, including organ transplant recipients and pregnant women in a hospital setting, were determined using indirect enzyme-linked immunosorbent assay (ELISA) kits. Comparison of laboratory data between groups with different HEV serological statuses was performed. Results HEV IgG antibodies were detected in 40.82% of 904 serum samples, while HEV IgM antibodies were detected in 11.75% of 1081 serum samples. Similar IgG and IgM antibody detection rates were found in pregnant women. Interestingly, anti-HEV IgM antibodies were detected in 38.5% of patients who underwent organ transplantation. Patients who tested positive for anti-HEV IgM antibodies had higher alanine aminotransferase levels than those who had not. In contrast, patients who tested positive for anti-HEV IgG had more elevated levels of total bilirubin than those who tested negative. Conclusions HEV seroprevalence and incidence in patients with clinical hepatitis were relatively high in the Thai population, including the pregnancy and organ transplant subgroups. The results potentially benefit the clinicians in decision-making to investigate HEV antibodies and facilitating proper management for patients.

scholarly journals Safety and immunogenicity of an mRNA-lipid nanoparticle vaccine candidate against SARS-CoV-2

2021 ◽  
Author(s):  
Peter G. Kremsner ◽  
Philipp Mann ◽  
Arne Kroidl ◽  
Isabel Leroux-Roels ◽  
Christoph Schindler ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vaccine Candidate ◽  
Phase 1 ◽  
Lipid Nanoparticles ◽  
Enzyme Linked Immunosorbent Assay ◽  
Receptor Binding Domain ◽  
Igg Antibodies ◽  
Phase 1 Study ◽  
S Protein ◽  
Dosage Escalation

Summary Background We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV‑2 spike (S) protein encapsulated in lipid nanoparticles (LNP). Methods This is an interim analysis of a dosage escalation phase 1 study in healthy 18–60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV‑2 S‑protein and receptor binding domain (RBD), and SARS-CoV‑2 neutralizing titers (MN50). Results In 245 volunteers who received 2 CVnCoV vaccinations (2 μg, n = 47, 4 μg, n = 48, 6 μg, n = 46, 8 μg, n = 44, 12 μg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S‑protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S‑protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 μg group. Responses to 12 μg were comparable to those observed in convalescent sera from known COVID-19 patients. Conclusion In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 μg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.

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