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1.
Anal Chem ; 94(10): 4446-4454, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1713092

ABSTRACT

The enrichment of co-reactants is one of the keys to improving the sensitivity of electrochemiluminescence (ECL) detection. This work developed a novel hydrophobic localized enrichment strategy of co-reactants utilizing the inner hydrophobic cavity of ß-cyclodextrin (ß-CD). Pt nanoparticles (Pt NPs) were grown in situ on the coordination sites for metal ions of ß-CD to prepare the ß-CD-Pt nanocomposite, which could not only enrich co-reactant 3-(dibutylamino) propylamine (TDBA) highly efficiently through its hydrophobic cavity but also immobilize TDBA via the Pt-N bond. Meanwhile, the carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] (PDP) polymer nanoparticles (PNPs) were developed as excellent ECL luminophores. With SARS-CoV-2 nucleocapsid protein (ncovNP) as a model protein, the TDBA-ß-CD-Pt nanocomposite combined PDP PNPs to construct a biosensor for ncovNP determination. The PDP PNPs were modified onto the surface of a glassy carbon electrode (GCE) to capture the first antibody (Ab1) and further capture antigen and secondary antibody complexes (TDBA-ß-CD-Pt@Ab2). The resultant biosensor with a sandwich structure achieved a highly sensitive detection of ncovNP with a detection limit of 22 fg/mL. TDBA-ß-CD-Pt shared with an inspiration in hydrophobic localized enrichment of co-reactants for improving the sensitivity of ECL detection. The luminophore PDP PNPs integrated TDBA-ß-CD-Pt to provide a promising and sensitive ECL platform, offering a new method for ncovNP detection.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Biosensing Techniques/methods , Electrochemical Techniques/methods , Humans , Limit of Detection , Luminescent Measurements/methods , Metal Nanoparticles/chemistry , Nucleocapsid Proteins , Polymers/chemistry , SARS-CoV-2
2.
PLoS Pathog ; 18(2): e1010265, 2022 02.
Article in English | MEDLINE | ID: covidwho-1686115

ABSTRACT

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.


Subject(s)
Biosensing Techniques/methods , COVID-19 Testing/methods , COVID-19/virology , Luminescent Measurements/methods , Peptide Hydrolases/analysis , SARS-CoV-2/enzymology , Viral Proteins/analysis , COVID-19/diagnosis , Cell Line , Humans , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication
4.
Molecules ; 26(15)2021 Jul 31.
Article in English | MEDLINE | ID: covidwho-1346517

ABSTRACT

Thin-layer chromatography (TLC) bioautography is an evolving technology that integrates the separation and analysis technology of TLC with biological activity detection technology, which has shown a steep rise in popularity over the past few decades. It connects TLC with convenient, economic and intuitive features and bioautography with high levels of sensitivity and specificity. In this study, we discuss the research progress of TLC bioautography and then establish a definite timeline to introduce it. This review summarizes known TLC bioautography types and practical applications for determining antibacterial, antifungal, antitumor and antioxidant compounds and for inhibiting glucosidase, pancreatic lipase, tyrosinase and cholinesterase activity constitutes. Nowadays, especially during the COVID-19 pandemic, it is important to identify original, natural products with anti-COVID potential compounds from Chinese traditional medicine and natural medicinal plants. We also give an account of detection techniques, including in situ and ex situ techniques; even in situ ion sources represent a major reform. Considering the current technical innovations, we propose that the technology will make more progress in TLC plates with higher separation and detection technology with a more portable and extensive scope of application. We believe this technology will be diffusely applied in medicine, biology, agriculture, animal husbandry, garden forestry, environmental management and other fields in the future.


Subject(s)
Chromatography, Thin Layer/methods , Drug Discovery/methods , Luminescent Measurements/methods , Animals , Anti-Infective Agents/isolation & purification , Antineoplastic Agents/isolation & purification , Antioxidants/isolation & purification , Enzyme Inhibitors/isolation & purification , Humans , Microbial Sensitivity Tests/methods , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plants, Medicinal/chemistry , Sensitivity and Specificity
5.
Viruses ; 13(8)2021 07 30.
Article in English | MEDLINE | ID: covidwho-1335234

ABSTRACT

The development of rapid serological detection methods re urgently needed for determination of neutralizing antibodies in sera. In this study, four rapid methods (ACE2-RBD inhibition assay, S1-IgG detection, RBD-IgG detection, and N-IgG detection) were established and evaluated based on chemiluminescence technology. For the first time, a broadly neutralizing antibody with high affinity was used as a standard for the quantitative detection of SARS-CoV-2 specific neutralizing antibodies in human sera. Sera from COVID-19 convalescent patients (N = 119), vaccinated donors (N = 86), and healthy donors (N = 299) confirmed by microneutralization test (MNT) were used to evaluate the above methods. The result showed that the ACE2-RBD inhibition assay calculated with either ACE2-RBD binding inhibition percentage rate or ACE2-RBD inhibiting antibody concentration were strongly correlated with MNT (r ≥ 0.78, p < 0.0001) and also highly consistent with MNT (Kappa Value ≥ 0.94, p < 0.01). There was also a strong correlation between the two evaluation indices (r ≥ 0.99, p < 0.0001). Meanwhile, S1-IgG and RBD-IgG quantitative detection were also significantly correlated with MNT (r ≥ 0.73, p < 0.0001), and both methods were highly correlated with each other (r ≥ 0.95, p < 0.0001). However, the concentration of N-IgG antibodies showed a lower correlation with the MNT results (r < 0.49, p < 0.0001). The diagnostic assays presented here could be used for the evaluation of SARS-CoV-2 vaccine immunization effect and serological diagnosis of COVID-19 patients, and could also have guiding significance for establishing other rapid serological methods to surrogate neutralization tests for SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19 Vaccines/immunology , COVID-19/virology , Immunoassay/methods , Luminescent Measurements/methods , SARS-CoV-2/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Serological Testing/instrumentation , COVID-19 Vaccines/administration & dosage , Humans , SARS-CoV-2/genetics , Vaccination
6.
Ann Clin Biochem ; 58(5): 496-504, 2021 09.
Article in English | MEDLINE | ID: covidwho-1255782

ABSTRACT

STUDY OBJECTIVE: SARS-CoV-2, which causes coronavirus disease (COVID-19), continues to cause significant morbidity and mortality. The diagnosis of acute infection relies on reverse transcription-polymerase chain reaction (RT-PCR)-based viral detection. The objective of this study was to evaluate the optimal serological testing strategy for anti-SARS-CoV-2 antibodies which provides an important indicator of prior infection and potential short-term immunity. METHODS: The sensitivity and specificity of four different ELISA assays (Euroimmun IgG, Euroimmun NCP-IgG, Fortress and DIAsource) and one CLIA assay (Roche ELECSYS) were evaluated in 423 samples; 137 patients with confirmed RT-PCR COVID-19 infection (true positives), and 100 pre-pandemic samples collected prior to October 2019 (true negatives). A further 186 samples were collected from health-care staff and analysed by all five assays. RESULTS: The Fortress ELISA assay demonstrated the highest sensitivity and specificity followed by the Roche ECLIA assay. The highest overall sensitivity came from the assays that measured total antibody (IgM-IgG combined) and the three assays that performed the best (Fortress, Roche, Euroimmun IgG) all have different antigens as their target proteins which suggests that antigen target does not affect assay performance. In mildly symptomatic participants with either a negative RT-PCR or no RT-PCR performed, 16.76% had detectable antibodies suggesting previous infection. CONCLUSIONS: We recommend a combined testing strategy utilizing assays with different antigenic targets using the fully automated Roche ECLIA assay and confirming discordant samples with the Fortress Total Antibody ELISA assay. This study provides an important indicator of prior infection in symptomatic and asymptomatic individuals.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Pandemics , SARS-CoV-2 , Antibodies, Viral/blood , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing/statistics & numerical data , Electrochemical Techniques/methods , Electrochemical Techniques/statistics & numerical data , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Female , Health Personnel , Humans , Immunoglobulin G/blood , Ireland/epidemiology , Luminescent Measurements/methods , Luminescent Measurements/statistics & numerical data , Male , Pregnancy , Sensitivity and Specificity
7.
Virol Sin ; 35(6): 752-757, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1217477

ABSTRACT

The immense patient number caused by coronavirus disease 2019 (COVID-19) global pandemic brings the urge for more knowledge about its immunological features, including the profile of basic immune parameters. In this study, eighty-eight reported COVID-19 patients in Wuhan were recruited from January to February, 2020, including 32 severe/critical cases and 56 mild/moderate cases. Their mean age was 56.43 years (range 17-83) and gender ratio (male/female) was 43:45. We tested SARS-CoV-2 RNA with commercial kits, investigated the level of serologic IgM and IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using magnetic particle chemiluminescence immunoassays, and compared the results of serologic tests and nucleic acid test (NAT). Among 88 patients, 95.45% were confirmed as positive by the combination of NAT and antibody test, which was significantly higher (P < 0.001) than by single nucleic acid test (73.86%) or serologic test (65.91%). Then the correlation between temporal profile and the level of antibody response was analyzed. It showed that seroconversion started on day 5 after disease onset and IgG level was rose earlier than IgM. Comparison between patients with different disease severity suggested early seroconversion and high antibody titer were linked with less severe clinical symptoms. These results supported the combination of serologic testing and NAT in routine COVID-19 diagnosis and provided evidence on the temporal profile of antibody response in patients with different disease severity.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , Antibody Formation , COVID-19/blood , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/methods , China/epidemiology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Luminescent Measurements/methods , Male , Middle Aged , Pandemics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Young Adult
8.
J Med Virol ; 93(3): 1805-1809, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196506

ABSTRACT

Plasma specimens from coronavirus disease 2019 patients were double-tested for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies by two different batches of MAGLUMI 2019-nCov immunoglobulin M/immunoglobulin G (IgM/IgG) assays to evaluate IgM/IgG levels, qualitative interpretation, antibody kinetics, and linearity of diluted specimen. Here we show that (i) high-level IgM specimens need to be diluted with negative human plasma but not kit diluents and (ii) measured anti-SARS-CoV-2 IgM/IgG concentrations are substantially higher with later marketed immunoassay batch leading to (iii) the change of qualitative interpretation (positive vs. negative) in 12.3% of specimens measured for IgM, (iv) the informative time-course pattern of antibody production only when data from different immunoassay batches are not combined.


Subject(s)
COVID-19/blood , COVID-19/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , SARS-CoV-2/immunology , Antibodies, Viral/immunology , COVID-19 Testing/methods , Humans , Immunoassay/methods , Luminescence , Luminescent Measurements/methods , Sensitivity and Specificity
9.
J Med Virol ; 93(3): 1465-1477, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196453

ABSTRACT

Since December 2019, we have been in the battlefield with a new threat to the humanity, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), characterized by viral pneumonia. It may be asymptomatic or cause various symptoms, ranging from flu-like symptoms to acute respiratory distress syndrome and eventually death. At present, the only reliable test for COVID-19 diagnosis is quantitative reverse transcriptase-polymerase chain reaction. Assessing the immune response against SARS-CoV-2 could increase the detection sensitivity of infected population. Hereby, we report the performances of a fully automated chemiluminescent immunoassay (CLIA) on 276 serum samples. One hundred samples obtained from COVID-19 negative subjects (COVID-19 free) were analyzed to evaluate the diagnostic specificity of antibody (Ab) detection. Thereafter, 176 samples obtained from 125 patients with confirmed COVID-19 (COVID-19 patients) were selected to assess the diagnostic sensitivity of the CLIA. All samples were analyzed on MAGLUMI 800 platform. All COVID-19 free samples had Ab levels below the cutoff values. Hence, the diagnostic specificity was estimated at 100% (95% confidence interval [CI] = 96.3-100.0; positive predictive value = 100%). By the 18th day from the onset of symptoms, we reached an optimal diagnostic sensitivity (more than 95.0%) In fact, the diagnostic sensitivity increased over time and between 15 and 25 days after symptoms onset, reached 95.5% (95% CI = 84.9-99.2). The new automated CLIA analyzer appeared to be a robust and reliable method to measure specific Ab against COVID-19 at high throughput. Our data suggest that combining Ab and nucleic acid detection could increase diagnostic sensitivity.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , Luminescent Measurements/methods , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Young Adult
10.
J Med Virol ; 93(2): 916-923, 2021 02.
Article in English | MEDLINE | ID: covidwho-1196420

ABSTRACT

Serology testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly being used during the current pandemic of coronavirus disease 2019 (COVID-19), although its clinical and epidemiologic utilities are still debatable. Characterizing these assays provides scientific basis to best use them. The current study assessed one chemiluminescent assay (Abbott COVID-2 IgG) and two lateral flow assays (STANDARD Q [SQ] IgM/IgG Duo and Wondfo total antibody test) using 113 blood samples from 71 PCR-confirmed COVID-19 hospitalized patients, 119 samples with potential cross-reactions, and 1068 negative controls including 942 pre-pandemic samples. SARS-CoV-2 IgM antibodies became detectable 3-4 days post-symptom onset using SQ IgM test and IgG antibodies were first detected 5-6 days post-onset using SQ IgG. Abbott IgG and Wondfo Total were able to detect antibodies 7 to 8 days post-onset. After 14 days post-symptom onset, the SQ IgG, Abbott IgG and Wondfo Total tests were able to detect antibodies from 100% of the PCR-confirmed patients in this series; 87.5% sensitivity for SQ IgM. Overall agreement was 88.5% between SQ IgM/IgG and Wondfo Total and 94.6% between SQ IgG and Abbott IgG. No cross-reaction due to recent sera with three of the endemic coronaviruses was observed. Viral hepatitis and autoimmune samples were the main source of limited cross-reactions. The specificities were 100% for SQ IgG and Wondfo Total, 99.62% for Abbott IgG, and 98.87% for SQ IgM. These findings demonstrated high sensitivity and specificity of appropriately validated SARS-CoV-2 serologic assays with implications for clinical use and epidemiological seroprevalence studies.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/immunology , Aged , COVID-19/diagnosis , Cross Reactions , Female , Humans , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , Luminescent Measurements/methods , Male , Middle Aged , Reagent Kits, Diagnostic , Sensitivity and Specificity
11.
Curr Med Sci ; 41(2): 318-322, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1193161

ABSTRACT

Serology tests for viral antibodies provide an important tool to support nucleic acid testing for diagnosis of the novel coronavirus disease 2019 (COVID-19) and is useful for documenting previous exposures to SARS-CoV-2, the etiological agent of COVID-19. The sensitivities of the chemiluminescent SARS-CoV-2 IgG/IgM immunoassay were assessed by using serum samples collected from 728 patients testing positive for SARS-CoV-2 RNA. The specificity was evaluated on a panel of 60 serum samples from non-COVID-19 patients with high levels of rheumatoid factor, antinuclear antibody, or antibodies against Epstein-Barr virus (EBV), cytomegalovirus (CMV), mycoplasma pneumonia, human respiratory syncytial virus (RSV), adenovirus, influenza A or influenza B. The imprecision and interference were assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A2 and EP7-A2, respectively. Sensitivities between 1 and 65 days after onset of symptoms were 94.4% and 78.7%, for IgG and IgM test, respectively. The sensitivity increased with the time after symptom onset, and rose to the top on the 22nd to 28th days. The total imprecision (CVs) was less than 6.0% for IgG and less than 6.5% for IgM. Limited cross-reactions with antibodies against EBV, CMV, mycoplasma pneumonia, human RSV, adenovirus, influenza A or influenza B were found. These data suggested the chemiluminescent SARS-CoV-2 IgG and IgM, assay with reliable utility and sensitivity, could be used for rapid screening and retrospective surveillance of COVID-19.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/blood , SARS-CoV-2/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/pathology , COVID-19/virology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Luminescent Measurements/methods , Male , Middle Aged , RNA, Viral/blood , Retrospective Studies , SARS-CoV-2/pathogenicity , Young Adult
12.
PLoS One ; 16(4): e0249796, 2021.
Article in English | MEDLINE | ID: covidwho-1183675

ABSTRACT

The Japanese Ministry of Health requires large-scale cooking facilities to use sodium hypochlorite aqueous solution (HYP) on food preparation tools, equipment, and facilities to prevent secondary contamination. This study aimed to compare the disinfecting effect of HYP and surfactant using adenosine triphosphate (ATP) swab testing on large-scale equipment and facilities that could not be disassembled and disinfected in hospital kitchen. From May 2018 to July 2018, ATP swab tests were performed on the following six locations in the Shizuoka Cancer Centre Dietary Department Kitchen: cooking counter, mobile cooking counter, refrigerator handle, conveyor belt, tap handle, and sink. Six relative light unit (RLU) measurements were taken from each location. The log10 values of the RLU measurements were evaluated by dividing the samples into two groups: the control group (surfactant followed by HYP swabbing) and the HYP group (HYP swabbing only). The results showed that the RLUs (log10 values) in both the groups improved after disinfection (p<0.05), except for the RLUs (log10 values) of the mobile cooking counter, tap handle, and sink in the control group after the HYP swab. The changes in the RLU (log10 value) did not differ between the two groups for all locations of the kitchen. Hence, HYP swabbing of large-scale equipment and facilities provides the same level of disinfection as surfactants and can be as beneficial.


Subject(s)
Adenosine Triphosphate/analysis , Disinfection/methods , Food Industry/standards , Luminescent Measurements/methods , Sodium Hypochlorite/pharmacology , Surface-Active Agents/pharmacology , Disinfectants/pharmacology , Food Industry/methods , Hospitals , Humans
13.
J Mol Biol ; 433(13): 166983, 2021 06 25.
Article in English | MEDLINE | ID: covidwho-1174385

ABSTRACT

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.


Subject(s)
Antibodies, Viral/chemistry , Antigens, Viral/immunology , COVID-19/diagnosis , COVID-19/virology , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antigens, Viral/chemistry , Antigens, Viral/isolation & purification , Epitopes/immunology , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Luciferases , Luminescent Measurements/methods , Protein Engineering , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
14.
J Clin Virol ; 139: 104820, 2021 06.
Article in English | MEDLINE | ID: covidwho-1174355

ABSTRACT

BACKGROUND: Neutralization tests (NT) are the gold standard for detecting and quantifying anti-SARS-CoV-2 neutralizing antibodies (NAb), but their complexity restricts them to research settings or reference laboratories. Antibodies against S protein receptor binding domain (RBD) have been shown to confer a neutralizing activity against SARS-CoV-2. Assays quantitatively measuring anti-S1-RBD-SARS-CoV-2 antibodies could be of great value for NAb screening of potential donors for convalescent-phase plasma therapy, assessing natural or vaccine-induced immunity, stratifying individuals for vaccine receipt, and documenting vaccine response. METHODS: Elecsys Anti-SARS-CoV-2 S (Elecsys-S), a high-throughput automated electrochemiluminescence double-antigen sandwich immunoassay for quantitative measurement of pan-anti-S1-RBD-SARS-CoV-2 antibodies, was evaluated against NT on 357 patients with PCR-confirmed SARS-CoV-2 infection. NT was performed in a BSL-3 laboratory using a Slovenian SARS-CoV-2 isolate; the NT titer ≥1:20 was considered positive. RESULTS: Elecsys-S detected pan-anti-S1-RBD-SARS-CoV-2 antibodies in 352/357 (98.6 %) samples. NAb were identified by NT in 257/357 (72 %) samples. The Elecsys-S/NT agreement was moderate (Cohen's kappa 0.56). High NT titer antibodies (≥1:160) were detected in 106/357 (30 %) samples. Elecsys-S's pan-anti-S1-RBD-SARS-CoV-2 antibody concentrations correlated with individual NT titer categories (the lowest concentrations were identified in NT-negative samples and the highest in samples with NT titer 1:1,280), and the Elecsys-S cutoff value for reasonable prediction of NAb generated after natural infection was established (133 BAU/mL). CONCLUSION: Although NT should remain the gold standard for assessing candidates for convalescent-phase plasma donors, selected commercial anti-SARS-CoV-2 assays with optimized cutoff, like Elecsys-S, could be used for rapid, automated, and large-scale screening of individuals with clinically relevant NAb levels as suitable donors.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Immunoassay/methods , Luminescent Measurements/methods , Neutralization Tests , Spike Glycoprotein, Coronavirus/immunology , COVID-19 Serological Testing , High-Throughput Screening Assays , Humans , Spike Glycoprotein, Coronavirus/chemistry
15.
J Virol Methods ; 292: 114121, 2021 06.
Article in English | MEDLINE | ID: covidwho-1120750

ABSTRACT

BACKGROUND: Serological test is an essential surveillance tool to track down the extensiveness of SARS-CoV-2 transmission and subsequently to move out from the enforced lockdown stage. OBJECTIVE: The study measures the diagnostic accuracy of three popular chemiluminescent immunoassay (CLIA) based automated platforms for the detection of anti-SARS-CoV-2 antibodies and compares their agreements. STUDY DESIGN: Serum samples of 594 COVID-19 positive patients and 100 samples from pre-COVID cases were tested by three CLIA based automated platforms: Abbott architect i2000SR, Roche cobas e411 and Yhlo iFlash 1800 and their diagnostic accuracy were compared by the area under the curves (AUC) value obtained from receiver operator characteristic (ROC) curves. Cohen's kappa statistic and McNemar's test were used to interpret the agreement between the platforms. RESULTS: All three platforms showed high specificity as claimed by the manufacturer. Sensitivity was calculated as 64.48 % (58.67-70.3) for Abbott, 80.48 % (76.62-84.34) for Roche and 76.94 % (72.65-81.23) for Yhlo. AUC was maximum for Roche (0.929). The Cohen's kappa value was determined in between 0.69-0.89 as the inter-rater agreements. CONCLUSION: The overall statistical analysis demonstrated cobas e411 as the diagnostically most accurate platform among the three.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoassay/methods , Luminescent Measurements/methods , SARS-CoV-2/immunology , Humans
16.
PLoS One ; 16(3): e0247711, 2021.
Article in English | MEDLINE | ID: covidwho-1117485

ABSTRACT

PCR methods are presently the standard for the diagnosis of Coronavirus disease 2019 (COVID-19), but additional methodologies are needed to complement PCR methods, which have some limitations. Here, we validated and investigated the usefulness of measuring serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the iFlash3000 CLIA analyzer. We measured IgM and IgG titers against SARS-CoV-2 in sera collected from 26 PCR-positive COVID-19 patients, 53 COVID-19-suspected but PCR-negative patients, and 20 and 100 randomly selected non-COVID-19 patients who visited our hospital in 2020 and 2017, respectively. The repeatability and within-laboratory precision were obviously good in validations, following to the CLSI document EP15-A3. Linearity was also considered good between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG, while the linearity curves plateaued above the upper measurement range. We also confirmed that the seroconversion and no-antibody titers were over the cutoff values in all 100 serum samples collected in 2017. These results indicate that this measurement system successfully detects SARS-CoV-2 IgM/IgG. We observed four false-positive cases in the IgM assay and no false-positive cases in the IgG assay when 111 serum samples known to contain autoantibodies were evaluated. The concordance rates of the antibody test with the PCR test were 98.1% for SARS-CoV-2 IgM and 100% for IgG among PCR-negative cases and 30.8% for SARS-CoV-2 IgM and 73.1% for SARS-CoV-2 IgG among PCR-positive cases. In conclusion, the performance of this new automated method for detecting antibody against both N and S proteins of SARS-CoV-2 is sufficient for use in laboratory testing.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/blood , Immunoglobulin M/blood , SARS-CoV-2/isolation & purification , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/epidemiology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Japan/epidemiology , Luminescent Measurements/methods , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification
17.
Biosens Bioelectron ; 180: 113122, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1116328

ABSTRACT

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor's large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor's versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Biosensing Techniques/methods , COVID-19/diagnosis , Luminescent Measurements/methods , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , HEK293 Cells , Humans , Luciferases , Neutralization Tests , Virus Internalization
18.
Int J Mol Sci ; 22(3)2021 Jan 20.
Article in English | MEDLINE | ID: covidwho-1067753

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.


Subject(s)
COVID-19/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Proteins/genetics , Humans , Limit of Detection , Luminescent Measurements/methods , Polyproteins/genetics , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription
19.
Arch Virol ; 166(3): 715-731, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1046770

ABSTRACT

Coronaviruses (CoV) are a family of viral pathogens that infect both birds and mammals, including humans. Seven human coronaviruses (HCoV) have been recognized so far. HCoV-229E, -OC43, -NL63, and -HKU1 account for one-third of common colds with mild symptoms. The other three members are severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. These viruses are responsible for SARS, MERS, and CoV disease 2019 (COVID-19), respectively. A variety of diagnostic techniques, including chest X-rays, computer tomography (CT) scans, analysis of viral nucleic acids, proteins, or whole virions, and host antibody detection using serological assays have been developed for the detection of these viruses. In this review, we discuss conventional serological tests, such as enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunofluorescence assay (IFA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA), as well as biosensor-based assays that have been developed for diagnosing HCoV-associated diseases since 2003, with an in-depth focus on COVID-19.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Middle East Respiratory Syndrome Coronavirus/immunology , SARS-CoV-2/immunology , Severe Acute Respiratory Syndrome/diagnosis , Antibodies, Viral/immunology , Biosensing Techniques/methods , Blotting, Western/methods , COVID-19/virology , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Humans , Luminescent Measurements/methods , SARS Virus/immunology , Severe Acute Respiratory Syndrome/virology
20.
Dis Markers ; 2021: 8810196, 2021.
Article in English | MEDLINE | ID: covidwho-1039930

ABSTRACT

Several tests based on chemiluminescence immunoassay techniques have become available to test for SARS-CoV-2 antibodies. There is currently insufficient data on serology assay performance beyond 35 days after symptoms onset. We aimed to evaluate SARS-CoV-2 antibody tests on three widely used platforms. A chemiluminescent microparticle immunoassay (CMIA; Abbott Diagnostics, USA), a luminescence immunoassay (LIA; Diasorin, Italy), and an electrochemiluminescence immunoassay (ECLIA; Roche Diagnostics, Switzerland) were investigated. In a multigroup study, sensitivity was assessed in a group of participants with confirmed SARS-CoV-2 (n = 145), whereas specificity was determined in two groups of participants without evidence of COVID-19 (i.e., healthy blood donors, n = 191, and healthcare workers, n = 1002). Receiver operating characteristic (ROC) curves, multilevel likelihood ratios (LR), and positive (PPV) and negative (NPV) predictive values were characterized. Finally, analytical specificity was characterized in samples with evidence of the Epstein-Barr virus (EBV) (n = 9), cytomegalovirus (CMV) (n = 7), and endemic common-cold coronavirus infections (n = 12) taken prior to the current SARS-CoV-2 pandemic. The diagnostic accuracy was comparable in all three assays (AUC 0.98). Using the manufacturers' cut-offs, the sensitivities were 90%, 95% confidence interval [84,94] (LIA), 93% [88,96] (CMIA), and 96% [91,98] (ECLIA). The specificities were 99.5% [98.9,99.8] (CMIA), 99.7% [99.3,99.9] (LIA), and 99.9% [99.5,99.98] (ECLIA). The LR at half of the manufacturers' cut-offs were 60 (CMIA), 82 (LIA), and 575 (ECLIA) for positive and 0.043 (CMIA) and 0.035 (LIA, ECLIA) for negative results. ECLIA had higher PPV at low pretest probabilities than CMIA and LIA. No interference with EBV or CMV infection was observed, whereas endemic coronavirus in some cases provided signals in LIA and/or CMIA. Although the diagnostic accuracy of the three investigated assays is comparable, their performance in low-prevalence settings is different. Introducing gray zones at half of the manufacturers' cut-offs is suggested, especially for orthogonal testing approaches that use a second assay for confirmation.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Luminescent Measurements/methods , SARS-CoV-2/immunology , Adult , COVID-19 Testing , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Sensitivity and Specificity
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