scholarly journals Analysis of IgM, IgA, and IgG isotype antibodies Directed against SARS-CoV-2 spike glycoprotein and ORF8 in the course of COVID-19

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Denise Meinberger ◽  
Manuel Koch ◽  
Annika Roth ◽  
Gabriele Hermes ◽  
Jannik Stemler ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Clinical Parameters ◽  
Spike Glycoprotein ◽  
Reliability Of Results ◽  
Serum Samples ◽  
Reading Frame ◽  
Insight Into

AbstractImmunoassays are a standard diagnostic tool that assesses immunity in severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. However, immunoassays do not provide information about contaminating antigens or cross-reactions and might exhibit inaccurately high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS-CoV-2 patients by immunoblot analysis. We analyzed serum immunoglobulins IgM, -A, and -G directed against SARS-CoV-2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA and assessed the clinical parameters of inflammation status and kidney and liver injury. Unexpectedly, we found no correlation between the presence of antibodies and the future course of the disease. However, attention should be paid to the parameters CRP, IL-6, and LDH. We found evidence of antibody cross-reactivity, which questions the reliability of results for serum samples that tested negative for anti-SARS-CoV-2 antibodies when assessed by immunoassays. Nevertheless, for the detection of IgG anti-SARS-CoV-2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best way of detecting anti-SARS-CoV-2 IgM antibodies.

scholarly journals Analysis of IgM, IgA, and IgG Isotype Antibodies Directed Against SARS‑CoV‑2 Spike Glycoprotein and ORF8 in the Course of COVID‑19.

2020 ◽  
Author(s):  
Denise Meinberger ◽  
Manuel Koch ◽  
Annika Roth ◽  
Gabriele Hermes ◽  
Jannik Stemler ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Spike Glycoprotein ◽  
Serum Samples ◽  
Reading Frame ◽  
Reliable Assessment ◽  
Insight Into ◽  
Inflammation Parameters

Abstract Immunoassays are a standard diagnostic tool assessing immunity in severe acute respiratory syndrome coronavirus type 2 (SARS‑CoV‑2) infection. However, immunoassays do not provide information about contaminating antigens or cross‑reactions and might exhibit inaccurately high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS‑CoV‑2 patients by immunoblot analysis.We analyzed serum immunoglobulins IgM, ‑A, and ‑G directed against SARS‑CoV‑2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA and assessed the clinical parameters of inflammation status and kidney and liver injury.We found evidence for antibody cross‑reactivity, which calls into question a reliable assessment of serum samples tested negative for anti‑SARS‑CoV‑2 antibodies by immunoassays. Nevertheless, for the detection of IgG anti‑SARS‑CoV‑2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best for the detection of patients positive for anti‑SARS‑CoV‑2 IgM antibodies. We found that the antibody response alone is not decisive for the course of the disease, but inflammation parameters are promising indicators.

scholarly journals Analysis of IgM, IgA, and IgG Isotype Antibodies Directed Against SARS‑CoV‑2 Spike Glycoprotein and ORF8 in the Course of COVID‑19.

2020 ◽  
Author(s):  
Denise Meinberger ◽  
Manuel Koch ◽  
Annika Roth ◽  
Gabriele Hermes ◽  
Jannik Stemler ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Spike Glycoprotein ◽  
Serum Samples ◽  
Reading Frame ◽  
Reliable Assessment ◽  
Insight Into ◽  
Inflammation Parameters

Abstract Immunoassays are a standard diagnostic tool assessing immunity in severe acute respiratory syndrome coronavirus type 2 (SARS‑CoV‑2) infection. However, immunoassays do not provide information about contaminating antigens or cross‑reaction and might exhibit inaccurate high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS‑CoV‑2 patients by immunoblot analysis. We analyzed serum immunoglobulins IgM, ‑A, and ‑G directed against SARS‑CoV‑2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA, and assessed clinical parameters of inflammation status, kidney, and liver injury. We found evidence for antibody cross‑reactivity, which calls into question a reliable assessment of serum samples tested negative for anti‑SARS‑CoV‑2 antibodies by immunoassays. Nevertheless, for the detection of IgG anti‑SARS‑CoV‑2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best for the detection of patients positive for anti‑SARS‑CoV‑2 IgM antibodies. We found that the antibody response alone is not decisive for the course of the disease but the inflammation parameters are promising indicators.

scholarly journals EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION

2020 ◽  
Author(s):  
Beatriz Araujo Oliveira ◽  
Lea Campos de Oliveira ◽  
Franciane Mendes de Oliveira ◽  
Geovana Maria Pereira ◽  
Regina Maia de Souza ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Diagnostic Techniques ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
List Type ◽  
Rt Pcr ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Good Agreement

AbstractBackgroundCOVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population.MethodsHere we evaluate the sensitivity and specificity of eleven different immunochromatographic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests.ResultsOur results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%.ConclusionsWe found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.HighlightsImmunochromatographic assays demonstrated high sensitivity and specificity and good agreement with the gold-standard RT-PCR;Increase in sensitivity and specificity of assays using samples collected after the 10th day of symptoms;Cross-reaction with Dengue serology in evaluation of IgM.

A rapid, sensitive enzyme-linked immunoassay for human thyrotropin.

1985 ◽  
Vol 31 (7) ◽  
pp. 1131-1134 ◽  
Author(s):  
Y C Tseng ◽  
K D Burman ◽  
J R Baker ◽  
L Wartofsky
Keyword(s):  
Color Change ◽  
Clinical Laboratory ◽  
High Sensitivity ◽  
Normal Subjects ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Assay Sensitivity ◽  
Nitrophenyl Phosphate ◽  
Set Up

Abstract In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.

scholarly journals Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

2002 ◽  
Vol 9 (2) ◽  
pp. 374-377 ◽  
Author(s):  
Daniela A. Cunha ◽  
Roseli M. Zancopé-Oliveira ◽  
M. Sueli ◽  
S. Felipe ◽  
Silvia M. Salem-Izacc ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Paracoccidioides Brasiliensis ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
Healthy Individuals ◽  
Immunological Reactivity ◽  
Serum Samples ◽  
Recombinant Antigens

ABSTRACT The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

scholarly journals Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

2009 ◽  
Vol 16 (12) ◽  
pp. 1774-1780 ◽  
Author(s):  
Eduardo A. F. Coelho ◽  
Laura Ramírez ◽  
Mariana A. F. Costa ◽  
Vinicio T. S. Coelho ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Ribosomal Proteins ◽  
High Sensitivity ◽  
Leishmania Species ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

scholarly journals Immunodiagnosis of Human Granulocytic Ehrlichiosis by Using Culture-Derived Human Isolates †

1998 ◽  
Vol 36 (6) ◽  
pp. 1480-1488 ◽  
Author(s):  
M. Dana Ravyn ◽  
Jesse L. Goodman ◽  
Carrie B. Kodner ◽  
Deborah K. Westad ◽  
Lisa A. Coleman ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Etiologic Agent ◽  
Immunoglobulin M ◽  
Test Method ◽  
Serum Samples ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by an Ehrlichia species closely related toEhrlichia equi and Ehrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused byEhrlichia chaffeensis, Rickettsia rickettsii, and Coxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples from R. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests for Borrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n = 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure to Ehrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.

scholarly journals Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257351
Author(s):  
Carolina de la Guardia ◽  
Giselle Rangel ◽  
Alcibiades Villarreal ◽  
Amador Goodridge ◽  
Patricia L. Fernández ◽  
...  
Keyword(s):  
Area Under The Curve ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Close Relative ◽  
Rt Pcr ◽  
Characteristic Analysis ◽  
Serum Samples ◽  
Health Authorities ◽  
Significant Difference

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase–quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98–0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24–0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92–0.99), specificity (range 0.93–0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87–0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.

scholarly journals A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0248444
Author(s):  
Dayu Zhang ◽  
Tianyang Xu ◽  
Eric Chu ◽  
Aiguo Zhang ◽  
Jinwei Du ◽  
...  
Keyword(s):  
High Throughput ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Serum Samples ◽  
Igg Antibody ◽  
Igm Antibody ◽  
Percent Agreement ◽  
Novel Coronavirus ◽  
The Individual

The pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0–7 days, 8–14 days, and 2–8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.

scholarly journals A High-throughput Microsphere-based Immunoassay of Anti-SARS-CoV-2 IgM Testing for COVID-19 Diagnostics

2021 ◽  
Author(s):  
Dayu Zhang ◽  
Tianyang Xu ◽  
Eric Chu ◽  
Aiguo Zhang ◽  
Jinwei Du ◽  
...  
Keyword(s):  
High Throughput ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Serum Samples ◽  
Igg Antibody ◽  
Igm Antibody ◽  
Percent Agreement ◽  
Novel Coronavirus ◽  
The Individual

ABSTRACTThe pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0-7 days, 8-14 days, and 2-8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.

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