SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region

A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

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SARS-CoV-2 epitope mapping on microarrays highlights strong immune-response to N protein region

10.1101/2020.11.09.374082 ◽

2020 ◽

Author(s):

Angelo Musicò ◽

Roberto Frigerio ◽

Alessandro Mussida ◽

Luisa Barzon ◽

Alessandro Sinigaglia ◽

...

Keyword(s):

Immune Response ◽

Epitope Mapping ◽

Serum Samples ◽

Promising Candidate ◽

Strong Immune Response ◽

Serological Tests ◽

N Protein ◽

Peptide Microarrays ◽

Validation Phase ◽

Epitope Discovery

AbstractA workflow for SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 Spike (S), Nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-171) providing 92% sensitivity and 100% specificity of IgG detection in Covid-19 samples thus being a promising candidate for rapid implementation in serological tests.

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Evaluation of the Serologic Cross-Reactivity between Transmissible Gastroenteritis Coronavirus and Porcine Respiratory Coronavirus Using Commercial Blocking Enzyme-Linked Immunosorbent Assay Kits

mSphere ◽

10.1128/msphere.00017-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 4

Author(s):

Ronaldo Magtoto ◽

Korakrit Poonsuk ◽

David Baum ◽

Jianqiang Zhang ◽

Qi Chen ◽

...

Keyword(s):

Culture Medium ◽

Large Deletion ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Transmissible Gastroenteritis Virus ◽

Serum Samples ◽

Serological Tests ◽

Transmissible Gastroenteritis ◽

Respiratory Coronavirus ◽

Porcine Respiratory

ABSTRACTThis study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (TGEV/PRCV) blocking enzyme-linked immunosorbent assays (ELISAs) using serum samples (n = 528) collected over a 49-day observation period from pigs inoculated with TGEV strain Purdue (n = 12), TGEV strain Miller (n = 12), PRCV (n= 12), or with virus-free culture medium (n = 12). ELISA results were evaluated both with “suspect” results interpreted as positive and then as negative. All commercial kits showed excellent diagnostic specificity (99 to 100%) when testing samples from pigs inoculated with virus-free culture medium. However, analyses revealed differences between the kits in diagnostic sensitivity (percent TGEV- or PRCV-seropositive pigs), and all kits showed significant (P < 0.05) cross-reactivity between TGEV and PRCV serum antibodies, particularly during early stages of the infections. Serologic cross-reactivity between TGEV and PRCV seemed to be TGEV strain dependent, with a higher percentage of PRCV-false-positive results for pigs inoculated with TGEV Purdue than for TGEV Miller. Moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the ELISA kit evaluated.IMPORTANCECurrent measures to prevent TGEV from entering a naive herd include quarantine and testing for TGEV-seronegative animals. However, TGEV serology is complicated due to the cross-reactivity with PRCV, which circulates subclinically in most swine herds worldwide. Conventional serological tests cannot distinguish between TGEV and PRCV antibodies; however, blocking ELISAs using antigen containing a large deletion in the amino terminus of the PRCV S protein permit differentiation of PRCV and TGEV antibodies. Several commercial TGEV/PRCV blocking ELISAs are available, but performance comparisons have not been reported in recent research. This study demonstrates that the serologic cross-reactivity between TGEV and PRCV affects the accuracy of commercial blocking ELISAs. Individual test results must be interpreted with caution, particularly in the event of suspect results. Therefore, commercial TGEV/PRCV blocking ELISAs should only be applied on a herd basis.

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Retrospective study of COVID-19 seroprevalence among tissue donors at the onset of the outbreak before implementation of strict lockdown measures in France

10.1101/2020.09.11.20192518 ◽

2020 ◽

Author(s):

Nicolas Germain ◽

Stephanie Herwegh ◽

Anne Sophie Hatzfeld ◽

Laurence Bocket ◽

Brigitte Prevost ◽

...

Keyword(s):

Tissue Bank ◽

Transmission Probability ◽

Risk Level ◽

Intermediate Risk ◽

Serum Samples ◽

Tissue Donors ◽

Serological Tests ◽

Systematic Screening ◽

N Protein ◽

Selection Of

Background: The COVID-19 pandemic has altered organ and tissue donations as well as transplantation practices. SARS-CoV-2 serological tests could help in the selection of donors. We assessed COVID-19 seroprevalence in a population of tissue donors, at the onset of the outbreak in France, before systematic screening of donors for SARS-CoV-2 RNA. Methods: 235 tissue donors at the Lille Tissue bank between November 1, 2019 and March 16, 2020 were included. Archived serum samples were tested for SARS-CoV-2 antibodies using two FDA-approved kits. Results: Most donors were at higher risks for severe COVID-19 illness including age over 65 years (142/235) and/or presence of co-morbidities (141/235). According to the COVID-19 risk assessment of transmission, 183 out of 235 tissue donors presented with a low risk level and 52 donors with an intermediate risk level of donor derived infection. Four out of the 235 (1.7%) tested specimens were positive for anti-SARS-CoV-2 antibodies: 2 donors with anti-N protein IgG and 2 other donors with anti-S protein total Ig. None of them had both type of antibodies. Conclusion: Regarding the seroprevalence among tissue donors, we concluded that the transmission probability to recipient via tissue products was very low at the beginning of the outbreak.

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A filtration-based rapid test using a partially purified third-stage larval antigen to detect specific antibodies for the diagnosis of gnathostomiasis

Journal of Helminthology ◽

10.1017/s0022149x17001080 ◽

2017 ◽

Vol 93 (1) ◽

pp. 26-32 ◽

Cited By ~ 1

Author(s):

A. Ma ◽

Y. Wang ◽

X.L. Liu ◽

H.M. Zhang ◽

P. Eamsobhana ◽

...

Keyword(s):

Rapid Test ◽

Cross Reactivity ◽

Serum Samples ◽

Serological Tests ◽

Promising Tool ◽

Diagnostic Potential ◽

Food Borne ◽

Third Stage ◽

Diagnostic Sensitivity And Specificity ◽

Purified Antigen

AbstractHuman gnathostomiasis is an emerging food-borne parasitic disease caused by nematodes of the genusGnathostoma. Currently, serological tests are commonly applied to support clinical diagnosis. In the present study, a simple and rapid filtration-based test, dot immune–gold filtration assay (DIGFA) was developed using a partially purified antigen ofGnathostomathird-stage larvae (L3). A total of 180 serum samples were tested to evaluate the diagnostic potential of DIGFA for gnathostomiasis. The diagnostic sensitivity and specificity were 96.7% (29/30) and 100% (25/25), respectively. The cross-reactivity with sera from other helminthiasis patients ranged from 0 to 4%, with an average of 1.6% (2/125). DIGFA using a partially purified L3 antigen was not only simple and rapid, but also more accurate than standard assays for the diagnosis of human gnathostomiasis. DIGFA may represent a promising tool for application in laboratories or in the field, without requiring any instrumentation.

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DETECTION OF BAT CORONAVIRUS AND SPECIFIC ANTIBODIES IN CHESTNUT BAT (SCOTOPHILUS KUHLII) POPULATION IN CENTRAL TAIWAN

Taiwan Veterinary Journal ◽

10.1142/s1682648515500171 ◽

2016 ◽

Vol 42 (01) ◽

pp. 19-26 ◽

Cited By ~ 2

Author(s):

Bo-Gang Su ◽

Hong Chang Chen ◽

Hsi-Chi Cheng ◽

Yi-Ning Chen

Keyword(s):

Cross Reactivity ◽

Rdrp Gene ◽

Rt Pcr ◽

Protein Fragment ◽

Serum Samples ◽

Protein Fragments ◽

N Protein ◽

Polymerase Chain ◽

Central Taiwan ◽

Bat Coronavirus

Bats can serve as natural reservoirs for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome coronavirus (MERS-CoV). Investigating the prevalence of bat CoV is critical for assessing the risks of the outbreaks of emerging CoV. Chestnut bats (Scotophilus kuhlii) were captured in this study for detecting the partial RNA-dependent RNA polymerase (RdRp) gene in their feces through reverse transcription polymerase chain reaction (RT-PCR) and antibodies to the nucleocapsid (N) protein of bat CoV through western blotting (WB) analysis. Three recombinant N protein fragments (N1, N2, N3) of the isolated Scotophilus bat CoV/CYCU-S1/TW/2013 were expressed by Escherichia coli. WB analyses were performed with bat serum samples and the sera of a patient who recovered from a SARS-CoV infection. Fragment N2 contained a highly conserved motif among CoVs whereas N1 and N3 protein fragments were specific to the S. kuhlii bat CoV. A total of 32 fecal and 19 serum samples were collected in Changhua County and Yunlin County during 2013 and 2014. About 17 fecal samples tested positive for the RdRp gene with an overall prevalence of 53%. Sequences comparison showed that the Scotophilus bat CoV isolates in Taiwan belonged to the genus Alphacoronavirus and were closest to Scotophilus bat CoV/Hainan/China/2005 and Diliman1552G1/Philippines/2008, followed by porcine epidemic diarrhea coronavirus. Only one bat serum sample reacted positively to all 3[Formula: see text]N protein fragments. Cross-reactivity was observed between N2 protein fragment and the sera of a patient recovered from a SARS-CoV infection. The results indicated that Scotophilus bat CoV was circulating endemically in chestnut bat population in Taiwan.

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Test performance evaluation of SARS-CoV-2 serological assays

10.1101/2020.04.25.20074856 ◽

2020 ◽

Cited By ~ 52

Author(s):

Jeffrey D. Whitman ◽

Joseph Hiatt ◽

Cody T. Mowery ◽

Brian R. Shy ◽

Ruby Yu ◽

...

Keyword(s):

Test Performance ◽

Severe Disease ◽

Cross Reactivity ◽

Time Interval ◽

Rt Pcr ◽

Serum Samples ◽

Serological Tests ◽

Full Spectrum ◽

Assay Performance ◽

Lateral Flow Assays

ABSTRACTBackgroundSerological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data.MethodWe conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 130 plasma or serum samples from 80 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system.ResultsAmong specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.8-94.8%. No consistent cross-reactivity was observed.ConclusionOur evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

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Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/003418-0 ◽

2008 ◽

Vol 57 (12) ◽

pp. 1547-1552 ◽

Cited By ~ 12

Author(s):

Zhijun Bai ◽

Licheng Liu ◽

Zeng Tu ◽

Lisi Yao ◽

Jianwei Liu ◽

...

Keyword(s):

Dengue Virus ◽

Real Time ◽

Real Time Pcr ◽

Cross Reactivity ◽

Clinical Samples ◽

Sequencing Analysis ◽

Serum Samples ◽

Serological Tests ◽

Wide Range ◽

Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

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Evaluation of Recombinant Antigens for Serodiagnosis of Chagas’ Disease in South and Central America

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1554-1560.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1554-1560 ◽

Cited By ~ 80

Author(s):

Eufrosina S. Umezawa ◽

Sueli F. Bastos ◽

Mario E. Camargo ◽

Luci M. Yamauchi ◽

Márcia R. Santos ◽

...

Keyword(s):

Chagas Disease ◽

Recombinant Proteins ◽

High Sensitivity ◽

Cross Reactivity ◽

Recombinant Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Recombinant Antigens ◽

Fusion Products

The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions ofTrypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzirecombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.

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Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle

Veterinary World ◽

10.14202/vetworld.2021.2187-2196 ◽

2021 ◽

pp. 2187-2196

Author(s):

Aitbay K. Bulashev ◽

Bakytkali K. Ingirbay ◽

Kanatbek N. Mukantayev ◽

Alfiya S. Syzdykova

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Cross Reactivity ◽

Accurate Diagnosis ◽

Expression Vectors ◽

Enzyme Linked Immunosorbent Assay ◽

Chimeric Proteins ◽

Serum Samples ◽

Serological Tests

Background and Aim: An accurate diagnosis of Brucella-infected animals is one of the critical measures in eradication programs. Conventional serological tests based on whole-cell (WC) antigens and detecting antibodies against pathogen-associated lipopolysaccharide might give false-positive results due to the cross-reactivity with other closely related bacteria. This study evaluated the serological potential of Brucella spp. chimeric outer membrane proteins (Omps) as antigens in an indirect enzyme-linked immunosorbent assay (i-ELISA). Materials and Methods: The chimeric gene constructs of the most immunodominant regions of Brucella Omps 25+31, 25+19, and 19+31 were cloned into the pET28a expression vectors and transformed into Escherichia coli BL21 (DE3). The serological potential of chimeric proteins compared with single recombinant Omps (rOmps)19, 25, and/or 31 were studied on blood serum samples of (i) a rabbit immunized with killed Brucella abortus 19WC, (ii) mice immunized with single rOmps, (iii) cows seropositive for brucellosis by rose Bengal test, and (iv) cattle naturally and/or experimentally infected with brucellosis. Results: E. coli BL21 actively produced Brucella chimeric rOmps, the concentration of which reached a maximum level at 6 h after isopropyl-β-D-1-thiogalactopyranoside stimulation. Target proteins were antigenic and expressed in an active state, as recognized by rabbit anti-B. abortus antibodies in an i-ELISA and western blotting. Murine antibodies against the single rOmps reacted with chimeric antigens, and conversely, antichimeric antibodies found their epitopes in single proteins. Brucella chimeric rOmps showed higher antigenicity in blood sera of seropositive cattle kept in the hotbed of the infection and/or experimentally challenged with brucellosis than single proteins. Conclusion: Brucella chimeric recombinant outer membrane proteins could be a potential antigen candidate for developing an ELISA test for accurate diagnosis of bovine brucellosis.

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Evaluation of 18 commercial serological assays for the detection of antibodies against SARS-CoV-2 in paired serum samples

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04220-7 ◽

2021 ◽

Author(s):

Daniëlle A. T. Hanssen ◽

Michiel Slaats ◽

Marlies Mulder ◽

Paul H. M. Savelkoul ◽

Inge H. M. van Loo

Keyword(s):

Healthcare Workers ◽

Cross Reactivity ◽

Serum Samples ◽

Serological Tests ◽

Past Infection ◽

Icu Patients ◽

Test Characteristics ◽

Antibody Assays ◽

Reactivity Test ◽

Paired Serum

AbstractA variety of serological tests have been developed to detect the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the performance of 18 commercially available SARS-CoV-2 antibody assays. Early (6–8 days after the start of symptoms) and late sera (>14 days) from ICU patients (n=10 andn=16, respectively) and healthcare workers (n=5 andn=9, respectively) were included. Additionally, 22 sera were included to detect potential cross-reactivity. Test characteristics were determined for the 18 assays. In >14 days samples, the Vircell IgG and Wantai Ig ELISAs had superior sensitivity compared to the other ELISAs (96%). Furthermore, the Roche Ig, the Epitope Diagnostics IgM, Wantai IgM, Euroimmun IgG, and IgA all showed a specificity of 100%. The POCTs of Boson Biotech and ACRO Biotech showed the highest sensitivities: 100% and 96% (83.5–99.8), respectively. The POCT of Orient Gene Biotech, VOMED Diagnostics, and Coris-Bioconcept showed highest specificities (100%). For the IgM and IgA assays, the Euroimmun IgA test showed the highest sensitivity in early samples: 46.7% (23.5–70.9) to 53.3% (29.1–76.5). In general, all tests performed better in patients with severe symptoms (ICU patients). We conclude that the Wantai Ig and Vircell IgG ELISAs may be suitable for diagnostic purposes. The IgM/IgA tests performed poorer than their IgG/Ig counterparts but may have a role in diagnoses of SARS-CoV-2 in a population in which the background seroprevalence of IgG high, and IgM and/or IgA may distinguish between acute or past infection.

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