Characterization of the Diagnostic Performance of a Novel COVID-19 PETIA in Comparison to Four Routine N-, S- and RBD-Antigen Based Immunoassays

In 2019, a novel coronavirus emerged in Wuhan in the province of Hubei, China. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly spread across the globe, causing the neoteric COVID-19 pandemic. SARS-CoV-2 is commonly transmitted by droplet infection and aerosols when coughing or sneezing, as well as high-risk exposures to infected individuals by face-to-face contact without protective gear. To date, a broad variety of techniques have emerged to assess and quantify the specific antibody response of a patient towards a SARS-CoV-2 infection. Here, we report the first comprehensive comparison of five different assay systems: Enzyme-Linked Immunosorbent Assay (ELISA), Chemiluminescence Immunoassay (CLIA), Electro-Chemiluminescence Immunoassay (ECLIA), and a new Particle-Enhanced Turbidimetric Immunoassay (PETIA) for SARS-CoV-2. Furthermore, we also evaluated the suitability of N-, S1- and RBD-antigens for quantifying the SARS-CoV-2 specific immune response. Linearity and precision, overall sensitivity and specificity of the assays, stability of samples, and cross-reactivity of general viral responses, as well as common coronaviruses, were assessed. Moreover, the reactivity of all tests to seroconversion and different sample matrices was quantified. All five assays showed good overall agreement, with 76% and 87% similarity for negative and positive samples, respectively. In conclusion, all evaluated methods showed a high consistency of results and suitability for the robust quantification of the SARS-CoV-2-derived immune response.

Download Full-text

Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692831 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bochao Liu ◽

Ze Wu ◽

Chaolan Liang ◽

Jinhui Lu ◽

Jinfeng Li ◽

...

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Primary Screening ◽

Global Pandemic ◽

Novel Coronavirus ◽

Acid Test ◽

Control Samples

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors’ control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

Download Full-text

Multiple Indirect ELISAs for Serological Detection of SARS-CoV-2 Antibodies

10.20944/preprints202005.0188.v1 ◽

2020 ◽

Author(s):

Abdullah Algaissi ◽

Mohamed A. Alfaleh ◽

Sherif Hala ◽

Turki S. Abujamel ◽

Sawsan S. Alamari ◽

...

Keyword(s):

Antibody Response ◽

Large Scale ◽

Disease Onset ◽

High Specificity ◽

Epidemiological Studies ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serological Tests ◽

Specificity And Sensitivity ◽

Novel Coronavirus

As the coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus SARS-CoV-2, continues to spread rapidly around the world, there is an urgent need for validated serological assays to evaluate viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological tests to study the antibody response in COVID-19 patients. In order to validate the assays, we determined the cut-off values, sensitivity and specificity of the developed assays using sera collected from COVID-19 patients in Saudi Arabia at different time points after disease onset, as well as sera that are seropositive to other human CoVs; namely MERS-CoV, hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1. The SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N) ELISAs that we developed here not only showed high specificity and sensitivity, but also did not show any cross-reactivity with other CoVs. We also showed that all RT-PCR confirmed COVID-19 patients included in our study developed both virus specific IgM and IgG as early as one week after the onset of disease. The availability of these validated assays will enable us to determine the nature and duration of the antibody response mounted in response to SARS-CoV-2 infection. It will also allow conducting large-scale epidemiological studies to determine evidence of previous exposure to the virus and assess the true extent of virus spread within communities.

Download Full-text

Highly Specific Chemiluminescence Immunoassay for the Determination of Chloramphenicol in Cosmetics

International Journal of Analytical Chemistry ◽

10.1155/2019/7131907 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6

Author(s):

Qiyan Li ◽

Riran Zhu ◽

Jun Li ◽

Xiaobing Wang ◽

Lihua Xu ◽

...

Keyword(s):

Quality Control ◽

Limit Of Detection ◽

Cross Reactivity ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa Method ◽

Chemiluminescence Immunoassay ◽

Detection Range ◽

Direct Immunoassay

A direct and highly specific chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) method for monitoring chloramphenicol (CAP) in cosmetics has been developed. The anti-chloramphenicol antibody (mAb) adopted in this work for direct immunoassay could bind to CAP specifically, with negligible cross-reactivity (CR) (less than 0.01%) with most CAP analogues, including structurally related thiamphenicol (TAP) and florfenicol (FF). The limit of detection (LOD), measured by IC10, was 0.0021 ng mL−1. The detection range (IC20-IC80) was ranged from 0.00979 to 0.12026 ng mL−1. In spiked cosmetics samples, mean recoveries ranged from 82.7% to 99.6%, with intraday and interday variation less than 9.8 and 8.2%, respectively. Moreover, with the help of HRP-labeled anti-CAP mAb, the method could be processed in fast direct immunoreaction mode. This CL-ELISA method could be applied for specific, rapid, semiquantitative, and quantitative detection of CAP in cosmetics, facilitating the precise quality control of CAP contamination.

Download Full-text

Egg-derived anti-SARS-CoV-2 immunoglobulin Y (IgY) with broad variant activity as intranasal prophylaxis against COVID-19: preclinical studies and randomized controlled phase 1 clinical trial

10.1101/2022.01.07.22268914 ◽

2022 ◽

Author(s):

Lyn Frumkin ◽

Michaela Lucas ◽

Curt Scribner ◽

Nastassja Ortega-Heinly ◽

Jayden Rogers ◽

...

Keyword(s):

Low Cost ◽

Phase 1 ◽

Cross Reactivity ◽

Vaccine Hesitancy ◽

Systemic Absorption ◽

Enzyme Linked Immunosorbent Assay ◽

Tolerability Profile ◽

Immunoglobulin Y ◽

Double Blind ◽

Novel Coronavirus

COVID-19 emergency use authorizations and approvals for vaccines were achieved in record time. However, there remains a need to develop additional safe, effective, easy-to-produce, and inexpensive prevention to reduce the risk of acquiring SARS-CoV-2 infection. This need is due to difficulties in vaccine manufacturing and distribution, vaccine hesitancy, and, critically, the increased prevalence of SARS-CoV-2 variants with greater contagiousness or reduced sensitivity to immunity. Antibodies from eggs of hens (immunoglobulin Y; IgY) that were administered receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were developed as nasal drops to capture the virus on the nasal mucosa. Although initially raised against the 2019 novel coronavirus index strain (2019-nCoV), these anti-SARS-CoV-2 RBD IgY surprisingly had indistinguishable enzyme-linked immunosorbent assay binding against variants of concern that have emerged, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529). This is distinct for sera from immunized or convalescent patients. Culture neutralization titers against available Alpha, Beta, and Delta were also indistinguishable from the index SARS-CoV-2 strain. Efforts to develop these IgY for clinical use demonstrated that the intranasal anti-SARS-CoV-2 RBD IgY preparation showed no binding (cross-reactivity) to a variety of human tissues and had an excellent safety profile in rats following 28-day intranasal delivery of the formulated IgY. A double-blind, randomized, placebo-controlled phase 1 study evaluating single-ascending and multiple doses of anti-SARS-CoV-2 RBD IgY administered intranasally for 14 days in 48 healthy participants also demonstrated an excellent safety and tolerability profile, and no evidence of systemic absorption. As these antiviral IgY have broad selectivity against many variants of concern, are fast to produce, and are a low-cost product, their use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation.

Download Full-text

753. Performance Characteristics of Diagnostic Assays in Blastomycosis

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.943 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S424-S424

Author(s):

Timothy O’Dowd ◽

Jack McHugh ◽

Nancy Wengenack ◽

Elitza Theel ◽

Paschalis Vergidis

Keyword(s):

Granulomatous Inflammation ◽

Cross Reactivity ◽

Performance Characteristics ◽

Enzyme Linked Immunosorbent Assay ◽

Fungal Culture ◽

Noninvasive Test ◽

Serum Antigen ◽

Urine Antigen ◽

Antigen Tests ◽

Urine And Serum

Abstract Background Blastomycosis has historically been a difficult diagnosis to establish, often initially misdiagnosed as bacterial pneumonia. Serologic assays and polymerase chain reaction (PCR) tests are available, but their performance is not well defined. The objective of this study was to characterize their performance. Methods Subjects were identified via chart review of patients diagnosed with blastomycosis from 2005 to 2020. A definitive diagnosis was based on fungal culture, histopathology, or cytology. Performance characteristics of the Blastomyces antibody enzyme linked immunosorbent assay (ELISA), immunodiffusion (ID), complement fixation (CF), urine and serum antigen ELISAs, and PCR were evaluated in patients with confirmed blastomycosis. Data on patient demographics, location of disease, and mortality was also collected. Results We identified 193 patients with blastomycosis. The mean age was 51.8 years (range, 11-84) and 73.6% of patients were male. 42.5% resided in Minnesota, 18.1% in Wisconsin, and 12.9% in Iowa. Diagnosis was based on culture in 142 (73.2%) or histopathology/cytology in 67 (34.7%) patients. Granulomatous inflammation was present in 73.1% (38/52) while 21.2% (41/193) had evidence of extrapulmonary dissemination. The antibody, ID, and CF assays were positive in 43.5% (37/85), 35.1% (33/94) and 20.5% (8/39) of patients, respectively. Sensitivity of Blastomyces PCR was 40% (4/10) in sputum and 75% (21/28) in bronchoalveolar lavage (BAL) fluid. Blastomyces urine and serum antigen tests were positive in 68% (34/50) and 50% (9/18) of cases, respectively, while the urine antigen was positive in 63.6% (7/11) of disseminated cases. Patients had a positive Histoplasma urine antigen test in 54.1% (20/37) and Aspergillus galactomannan in BAL in 34.8% (8/23) of cases. Serum beta-D-glucan test was positive in 16.7% (2/12). 90-day mortality was 21/193 (10.9%) and median time from diagnosis to death was 18 days. Conclusion In this cohort, Blastomyces urine antigen was the most sensitive noninvasive test, with similar performance in pulmonary and disseminated disease. However, its utility is limited by poor specificity due to cross-reactivity. Blastomyces PCR from BAL fluid demonstrated the highest sensitivity. Blastomyces antibody, ID, and CF had poor sensitivity. Disclosures All Authors: No reported disclosures

Download Full-text

Aptamer BC 007’s Affinity to Specific and Less-Specific Anti-SARS-CoV-2 Neutralizing Antibodies

Viruses ◽

10.3390/v13050932 ◽

2021 ◽

Vol 13 (5) ◽

pp. 932

Author(s):

Annekathrin Haberland ◽

Oxana Krylova ◽

Heike Nikolenko ◽

Peter Göttel ◽

Andre Dallmann ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Resonance Spectroscopy ◽

Plastic Plate ◽

Calorimetric Titration ◽

Background Signal ◽

High Background ◽

Specific Virus ◽

Binding Sequence

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0–21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low.

Download Full-text

Dogs as Sentinels for Flavivirus Exposure in Urban, Peri-Urban and Rural Hanoi, Vietnam

Viruses ◽

10.3390/v13030507 ◽

2021 ◽

Vol 13 (3) ◽

pp. 507

Author(s):

Long Pham-Thanh ◽

Thang Nguyen-Tien ◽

Ulf Magnusson ◽

Vuong Bui-Nghia ◽

Anh Bui-Ngoc ◽

...

Keyword(s):

Mosquito Control ◽

Risk Mitigation ◽

Significant Risk ◽

Cross Sectional Study ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Cross Sectional ◽

Major Health ◽

Household Level

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.

Download Full-text

A Summary of the Third Global Interferon-γ Release Assay Symposium

Infection Control and Hospital Epidemiology ◽

10.1086/670634 ◽

2013 ◽

Vol 34 (6) ◽

pp. 619-624 ◽

Cited By ~ 2

Author(s):

Antonino Catanzaro ◽

Charles Daley

Keyword(s):

Immune Response ◽

Tuberculin Skin Test ◽

Skin Test ◽

Interferon Γ ◽

Enzyme Linked Immunosorbent Assay ◽

In Vitro Tests ◽

The Third ◽

Specific Antigens ◽

Ifn Γ

Studies over the past several decades have dramatically increased our understanding of the immune response to Mycobacterium tuberculosis infection, and advances in proteomics and genomics have led to a new class of immune-diagnostic tests, termed interferon-γ (IFN-γ) release assays (IGRAs), which appear to obviate many of the problems encountered with the tuberculin skin test (TST). Worldwide, 2 IGRAs are currently commercially available. QuantiFERON-TB Gold In-Tube (Cellestis) is a third-generation product that uses an enzyme-linked immunosorbent assay to measure IFN-γ generated in whole blood stimulated with M. tuberculosis–specific antigens. T-Spot-TB (Oxford Immunotec) employs enzyme-linked immunosorbent spot technology to enumerate the number of purified lymphocytes that respond to M. tuberculosis–specific antigens by producing IFN-γ. These in vitro tests measure the host immune response to M. tuberculosis–specific antigens, which virtually eliminates false-positive cross reactions caused by bacillus Calmette-Guérin vaccination and/or exposure to environmental nontuberculous mycobacteria that plague the interpretation and accuracy of the tuberculin skin test (TST). The high specificity of IGRAs, together with sensitivity commensurate with or better than that of the TST, promises an accurate diagnosis and the ability to focus tuberculosis-control activities on those who are actually infected with M. tuberculosis. The Third Global Symposium was held over a 3-day period and was presented by the University of California, San Diego, Continuing Medical Education department; slides and sound recordings of each presentation are available at http://cme.ucsd.edu/igras/syllabus.html. A moderated discussion is also available at http://cme.ucsd.edu/igrasvideo. This document provides a summary of the key findings of the meeting, specifically focusing on the use of IGRAs in screening healthcare worker populations.

Download Full-text

Dairy Heifers Naturally Exposed toFasciola hepaticaDevelop a Type 2 Immune Response and Concomitant Suppression of Leukocyte Proliferation

Infection and Immunity ◽

10.1128/iai.00607-17 ◽

2017 ◽

Vol 86 (1) ◽

Cited By ~ 3

Author(s):

John Graham-Brown ◽

Catherine Hartley ◽

Helen Clough ◽

Aras Kadioglu ◽

Matthew Baylis ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Peripheral Blood ◽

Serum Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Mixed Effect ◽

Content Type ◽

Grazing Season ◽

Type 2 Immune Response

ABSTRACTFasciola hepaticais a parasitic trematode of global importance in livestock. Control strategies reliant on anthelmintics are unsustainable due to the emergence of drug resistance. Vaccines are under development, but efficacies are variable. Evidence from experimental infection suggests that vaccine efficacy may be affected by parasite-induced immunomodulation. Little is known about the immune response toF. hepaticafollowing natural exposure. Hence, we analyzed the immune responses over time in calves naturally exposed toF. hepaticainfection. Cohorts of replacement dairy heifer calves (n= 42) with no prior exposure toF. hepatica, on three commercial dairy farms, were sampled over the course of a grazing season. Exposure was determined through anF. hepatica-specific serum antibody enzyme-linked immunosorbent assay (ELISA) and fluke egg counts. Concurrent changes in peripheral blood leukocyte subpopulations, lymphocyte proliferation, and cytokine responses were measured. Relationships between fluke infection and immune responses were analyzed by using multivariable linear mixed-effect models. All calves from one farm showed evidence of exposure, while cohorts from the remaining two farms remained negative over the grazing season. A type 2 immune response was associated with exposure, with increased interleukin-4 (IL-4) production, IL-5 transcription, and eosinophilia. Suppression of parasite-specific peripheral blood mononuclear cell (PBMC) proliferation was evident, while decreased mitogen-stimulated gamma interferon (IFN-γ) production suggested immunomodulation, which was not restricted to parasite-specific responses. Our findings show that the global immune response is modulated toward a nonproliferative type 2 state following natural challenge withF. hepatica. This has implications in terms of the timing of the administration of vaccination programs and for host susceptibility to coinfecting pathogens.

Download Full-text