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1.
Biophys Physicobiol ; 18: 28-39, 2021.
Article in English | MEDLINE | ID: covidwho-1951733

ABSTRACT

PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.

2.
Front Med (Lausanne) ; 7: 603996, 2020.
Article in English | MEDLINE | ID: covidwho-1389195

ABSTRACT

Seroprevalence studies are crucial both for estimating the prevalence of SARS-CoV-2 exposure and to provide a measure for the efficiency of the confinement measures. Portuguese universities were closed on March 16th 2020, when Portugal only registered 62 SARS-CoV-2 infection cases per million. We have validated a SARS-CoV-2 ELISA assay to a stabilized full-length spike protein using 216 pre-pandemic and 19 molecularly diagnosed SARS-CoV-2 positive individual's samples. At NOVA University of Lisbon, presential work was partially resumed on May 25th with staggered schedules. From June 15th to 30th, 3-4 weeks after the easing of confinement measures, we screened 1,636 collaborators of NOVA university of Lisbon for the presence of SARS-CoV-2 spike specific IgA and IgG antibodies. We found that spike-specific IgG in 50 of 1,636 participants (3.0%), none of which had anti-spike IgA antibodies. As participants self-reported as asymptomatic or paucisymptomatic, our study also provides a measurement of the prevalence of asymptomatic/paucisymptomatic SARS-CoV-2 infections. Our study suggests that essential workers have a 2-fold increase in viral exposure, when compared to non-essential workers that observed confinement. Additional serological surveys in different population subgroups will paint a broader picture of the effect of the confinement measures in the broader community.

3.
Methods Mol Biol ; 2099: 89-97, 2020.
Article in English | MEDLINE | ID: covidwho-1292547

ABSTRACT

The Middle East respiratory syndrome (MERS) is the second novel zoonotic disease infecting humans caused by coronavirus (CoV) in this century. To date, more than 2200 laboratory-confirmed human cases have been identified in 27 countries, and more than 800 MERS-CoV associated deaths have been reported since its outbreak in 2012. Rapid laboratory diagnosis of MERS-CoV is the key to successful containment and prevention of the spread of infection. Though the gold standard for diagnosing MERS-CoV infection in humans is still nucleic acid amplification test (NAAT) of the up-E region, an antigen capture enzyme-linked immunosorbent assay (ELISA) could also be of use for early diagnosis in less developed locations. In the present method, a step-by-step guide to perform a MERS-CoV nucleocapsid protein (NP) capture ELISA using two NP-specific monoclonal antibodies is provided for readers to develop their in-house workflow or diagnostic kit for clinical use and for mass-screening project of animals (e.g., dromedaries and bats) to better understand the spread and evolution of the virus.


Subject(s)
Antigens, Viral/immunology , Coronavirus Infections/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Middle East Respiratory Syndrome Coronavirus/immunology , Nucleocapsid Proteins/immunology , Animals , Camelus/virology , Chiroptera/virology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Zoonoses
4.
Anal Chem ; 93(24): 8585-8594, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1253862

ABSTRACT

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has revealed the urgent need for accurate, rapid, and affordable diagnostic tests for epidemic understanding and management by monitoring the population worldwide. Though current diagnostic methods including real-time polymerase chain reaction (RT-PCR) provide sensitive detection of SARS-CoV-2, they require relatively long processing time, equipped laboratory facilities, and highly skilled personnel. Laser-scribed graphene (LSG)-based biosensing platforms have gained enormous attention as miniaturized electrochemical systems, holding an enormous potential as point-of-care (POC) diagnostic tools. We describe here a miniaturized LSG-based electrochemical sensing scheme for coronavirus disease 2019 (COVID-19) diagnosis combined with three-dimensional (3D) gold nanostructures. This electrode was modified with the SARS-CoV-2 spike protein antibody following the proper surface modifications proved by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) characterizations as well as electrochemical techniques. The system was integrated into a handheld POC detection system operated using a custom smartphone application, providing a user-friendly diagnostic platform due to its ease of operation, accessibility, and systematic data management. The analytical features of the electrochemical immunoassay were evaluated using the standard solution of S-protein in the range of 5.0-500 ng/mL with a detection limit of 2.9 ng/mL. A clinical study was carried out on 23 patient blood serum samples with successful COVID-19 diagnosis, compared to the commercial RT-PCR, antibody blood test, and enzyme-linked immunosorbent assay (ELISA) IgG and IgA test results. Our test provides faster results compared to commercial diagnostic tools and offers a promising alternative solution for next-generation POC applications.


Subject(s)
Biosensing Techniques , COVID-19 , Graphite , Point-of-Care Systems , Antibodies, Viral , COVID-19/diagnosis , COVID-19 Testing , Gold , Humans , Lasers , Nanostructures , SARS-CoV-2 , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus
5.
Int J Infect Dis ; 108: 330-332, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1253016

ABSTRACT

OBJECTIVES: Reverse-transcription PCR (RT-PCR) is considered the most sensitive method for the detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, this method is relatively resource- and time-consuming. This study was performed to compare SARS-CoV-2 nucleocapsid antigen (N-Ag) testing using an enzyme-linked immunosorbent assay (ELISA) with SARS-CoV-2 RNA detection. METHODS: Parallel SARS-CoV-2 RT-PCR and quantitative N-Ag ELISA analysis was executed on nasopharyngeal specimens obtained during SARS-CoV-2 screening in a cohort of pre-hospitalization patients. RESULTS: In total, 277 specimens were examined, including 182 (65.7%) RT-PCR-positive specimens, which demonstrated a median cycle threshold (Ct) value of 27 (interquartile range (IQR) 23-35). The SARS-CoV-2 N-Ag was detected in 164 of the 182 RT-PCR-positive specimens (overall sensitivity 90.1%). Among the 95 RT-PCR-negative specimens, 72 were N-Ag-negative (specificity 75.8%). SARS-CoV-2 RT-PCR and N-Ag ELISA results demonstrated a strong agreement (Cramer's V = 0.668; P < 0.001). N-Ag concentrations spanned from 5.4 to 296 000 pg/ml (median 901 pg/ml, IQR 43-1407 pg/ml) and were inversely correlated with Ct values (Spearman's r = -0.720; P < 0.001). CONCLUSIONS: SARS-CoV-2 N-Ag ELISA results were in close agreement with RT-PCR results, and N-Ag concentrations were proportional to viral loads. Thus, SARS-CoV-2 quantitative antigen testing could be an additional diagnostic instrument for SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nasopharynx , Nucleocapsid , RNA, Viral , Sensitivity and Specificity
6.
Anal Bioanal Chem ; 413(18): 4645-4654, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1245612

ABSTRACT

Nucleic acid detection technology based on polymerase chain reaction (PCR) and antibody detection based on immunochromatography still have many problems such as false negatives for the diagnosis of coronavirus disease 2019 (COVID-19). Therefore, it is of great importance to develop new techniques to improve the diagnostic accuracy of COVID-19. We herein developed an ultrasensitive, rapid, and duplex digital enzyme-linked immunosorbent assay (dELISA) for simultaneous detection of spike (S-RBD) and nucleocapsid (N) proteins of SARS-CoV-2 based on a single molecule array. This assay effectively combines magnetic bead encoding technology and the ultrasensitive detection capability of a single molecule array. The detection strategies of S-RBD protein and N-protein exhibited wide response ranges of 0.34-1065 pg/mL and 0.183-338 pg/mL with detection limits of 20.6 fg/mL and 69.8 fg/mL, respectively. It is a highly specific method for the simultaneous detection of S-RBD protein and N-protein and has minimal interference from other blood proteins. Moreover, the spike assay showed a satisfactory and reproducible recovery rate for the detection of S-RBD protein and N-protein in serum samples. Overall, this work provides a highly sensitive method for the simultaneous detection of S-RBD protein and N-protein, which shows ultrasensitivity and high signal-to-noise ratio and contributes to improve the diagnosis accuracy of COVID-19.


Subject(s)
COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Single Molecule Imaging/methods , Spike Glycoprotein, Coronavirus/isolation & purification , Antibodies, Viral/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay/standards , Humans , Immunoassay/methods , Magnetics , Microspheres , Phosphoproteins/genetics , Phosphoproteins/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics
7.
J Thromb Haemost ; 19(8): 2007-2013, 2021 08.
Article in English | MEDLINE | ID: covidwho-1223529

ABSTRACT

INTRODUCTION: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following ChAdOx1 nCOV-19 vaccine has been described, associated with unusual site thrombosis, thrombocytopenia, raised D-dimer, and high-titer immunoglobulin-G (IgG) class anti-platelet factor 4 (PF4) antibodies. Enzyme-linked immunosorbent assays (ELISA) have been shown to detect anti-PF4 in patients with VITT, but chemiluminescence assays do not reliably detect them. ELISA assays are not widely available in diagnostic laboratories, and, globally, very few laboratories perform platelet activation assays. METHODS: Assays that are commercially available in the United Kingdom were evaluated for their ability to identify anti-PF4 antibodies in samples from patients with suspected VITT. Four IgG-specific ELISAs, two polyspecific ELISAs, and four rapid assays were performed on samples from 43 patients with suspected VITT from across the United Kingdom. Cases were identified after referral to the UK Expert Haematology Panel multidisciplinary team and categorized into unlikely, possible, or probable VITT. RESULTS AND DISCUSSION: We demonstrated that the HemosIL AcuStar HIT-IgG, HemosIL HIT-Ab, Diamed PaGIA gel, and STic Expert assays have poor sensitivity for VITT in comparison to ELISA. Where these assays are used for heparin-induced thrombocytopenia (HIT) diagnosis, laboratories should ensure that requests for suspected VITT are clearly identified so that an ELISA is performed. No superiority of IgG-ELISAs over polyspecific ELISAs in sensitivity to VITT could be demonstrated. No single ELISA method detected all possible/probable VITT cases; if a single ELISA test is negative, a second ELISA or a platelet activation assay should be considered where there is strong clinical suspicion.


Subject(s)
Laboratories , Platelet Factor 4 , COVID-19 Vaccines , ChAdOx1 nCoV-19 , Enzyme-Linked Immunosorbent Assay , Heparin , Humans , Immunoglobulin G , United Kingdom , Vaccination
8.
ACS Appl Mater Interfaces ; 13(19): 22262-22270, 2021 May 19.
Article in English | MEDLINE | ID: covidwho-1221187

ABSTRACT

The coronavirus disease 2019 (COVID-19) can present a similar syndrome to an influenza infection, which may complicate diagnosis and clinical management of these two important respiratory infectious diseases, especially during the peak season of influenza. A rapid and convenient point-of-care test (POCT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus is of great importance for prompt and efficient control of these respiratory epidemics. Herein, a multichannel electrochemical immunoassay (MEIA) platform was developed based on a disposable screen-printed carbon electrode (SPCE) array for the on-site detection of SARS-CoV-2 and A(H1N1). The developed MEIA was constructed with eight channels and allowed rapid detection on a single array. On the SPCE surface, monoclonal antibodies against influenza A(H1N1) hemagglutinin (HA) protein or SARS-CoV-2 spike protein were coated to capture the target antigens, which then interacted with a horseradish peroxidase (HRP)-labeled detection antibody to form an immuno-sandwich complex. The results showed that the MEIA exhibited a broader linear range than ELISA and comparable sensitivity for A(H1N1) HA and SARS-CoV-2 spike protein. The detection results on 79 clinical samples for A(H1N1) suggested that the proposed MEIA platform showed comparable results with ELISA in sensitivity (with a positive rate of 100% for positive samples) but higher specificity, with a false-positive rate of 5.4% for negative samples versus that of 40.5% with ELISA. Thus, it offers great potential for the on-the-spot differential diagnosis of infected patients, which would significantly benefit the efficient control and prevent the spread of these infectious diseases in communities or resource-limited regions in the future.


Subject(s)
Biosensing Techniques/methods , COVID-19/diagnosis , Electrochemical Techniques/methods , Immunoassay/methods , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/diagnosis , SARS-CoV-2/isolation & purification , Humans , Point-of-Care Testing , Sensitivity and Specificity
9.
Clin Infect Dis ; 71(15): 778-785, 2020 07 28.
Article in English | MEDLINE | ID: covidwho-1217823

ABSTRACT

BACKGROUND: The emergence of coronavirus disease 2019 (COVID-19) is a major healthcare threat. The current method of detection involves a quantitative polymerase chain reaction (qPCR)-based technique, which identifies the viral nucleic acids when present in sufficient quantity. False-negative results can be achieved and failure to quarantine the infected patient would be a major setback in containing the viral transmission. We aim to describe the time kinetics of various antibodies produced against the 2019 novel coronavirus (SARS-CoV-2) and evaluate the potential of antibody testing to diagnose COVID-19. METHODS: The host humoral response against SARS-CoV-2, including IgA, IgM, and IgG response, was examined by using an ELISA-based assay on the recombinant viral nucleocapsid protein. 208 plasma samples were collected from 82 confirmed and 58 probable cases (qPCR negative but with typical manifestation). The diagnostic value of IgM was evaluated in this cohort. RESULTS: The median duration of IgM and IgA antibody detection was 5 (IQR, 3-6) days, while IgG was detected 14 (IQR, 10-18) days after symptom onset, with a positive rate of 85.4%, 92.7%, and 77.9%, respectively. In confirmed and probable cases, the positive rates of IgM antibodies were 75.6% and 93.1%, respectively. The detection efficiency by IgM ELISA is higher than that of qPCR after 5.5 days of symptom onset. The positive detection rate is significantly increased (98.6%) when combining IgM ELISA assay with PCR for each patient compared with a single qPCR test (51.9%). CONCLUSIONS: The humoral response to SARS-CoV-2 can aid in the diagnosis of COVID-19, including subclinical cases.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Immunity, Humoral/immunology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Adult , Amino Acid Sequence , Antibodies, Viral/immunology , COVID-19 , Child , Child, Preschool , Coronavirus Infections/virology , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , SARS-CoV-2
10.
J Med Virol ; 93(3): 1732-1738, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196496

ABSTRACT

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400. ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Inactivated/immunology , Animals , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/genetics , Humans , Mice , Mice, Inbred BALB C , Phosphoproteins/genetics , Phosphoproteins/immunology , Rabbits , SARS-CoV-2/genetics , Sequence Deletion/genetics , Sequence Deletion/immunology , Spike Glycoprotein, Coronavirus/genetics
11.
ACS Omega ; 6(14): 9667-9671, 2021 Apr 13.
Article in English | MEDLINE | ID: covidwho-1191080

ABSTRACT

SARS-CoV-2 is the etiologic agent of COVID-19, which has led to a dramatic loss of human life and presents an unprecedented challenge to public health worldwide. The gold standard assay for SARS-CoV-2 identification is real-time polymerase chain reaction; however, this assay depends on highly trained personnel and sophisticated equipment and may suffer from false results. Thus, a serological antibody test is a supplement to the diagnosis or screening of SARS-CoV-2. Here, we develop and evaluate the diagnostic performance of an IgM/IgG indirect ELISA method for antibodies against SARS-CoV-2 in COVID-19. The ELISA was constructed by coating with a recombinant nucleocapsid protein of SARS-CoV-2 on an enzyme immunoassay plate, and its sensitivity and specificity for clinical diagnosis of SARS-CoV-2 infection was assessed by detecting the SARS-CoV-2-specific IgM and IgG antibodies in COVID-19 patient's sera or healthy person's sera. The SARS-CoV-2 positive serum samples (n = 168) were collected from confirmed COVID-19 patients. A commercial nucleocapsid protein-based chemiluminescent immunoassay (CLIA) kit and a colloidal gold immunochromatography kit were compared with those of the ELISA assay. The specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of IgM were 100, 95.24, 100, and 91.84%, whereas those of IgG were 100, 97.02, 100, and 94.74%, respectively. We developed a highly sensitive and specific SARS-CoV-2 nucleocapsid protein-based ELISA method for the diagnosis and epidemiologic investigation of COVID-19 by SARS-CoV-2 IgM and IgG antibody detection.

12.
J King Saud Univ Sci ; 33(4): 101439, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1185114

ABSTRACT

By the end of year 2019, the new virus SARS-CoV-2 appeared, causing the Coronavirus Disease 2019 (COVID-19), and spread very fast globally. A continuing need for diagnostic tools is a must to contain its spread. Till now, the gold standard method, the reverse transcription polymerase chain reaction (RT-PCR), is the precise procedure to detect the virus. However, SARS-CoV-2 may escape RT-PCR detection for several reasons. The development of well-designed, specific and sensitive serological test like enzyme immunoassay (EIA) is needed. This EIA can stand alone or work side by side with RT-PCR. In this study, we developed several EIAs including plates that are coated with either specially designed SARS-CoV-2 nucleocapsid or surface recombinant proteins. Each protein type can separately detect anti-SARS-CoV-2 IgM or IgG antibodies. For each EIAs, the cut-off value, specificity and sensitivity were determined utilizing RT-PCR confirmed Covid-19 and pre-pandemic healthy and other viruses-infected sera. Also, the receiver operator characteristic (ROC) analysis was performed to define the specificities and sensitivities of the optimized assay. The in-house EIAs were validated by comparing against commercial EIA kits. All in-house EIAs showed high specificity (98-99%) and sensitivity (97.8-98.9%) for the detection of IgG/IgM against RBD and N proteins of SARS-CoV-2. From these results, the developed Anti-RBD and anti-N IgG and IgM antibodies EIAs can be used as a specific and sensitive tool to detect SARS-CoV-2 infection, calculate the burden of disease and case fatality rates.

13.
MAbs ; 13(1): 1905978, 2021.
Article in English | MEDLINE | ID: covidwho-1177234

ABSTRACT

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Epitopes/immunology , Nucleocapsid Proteins/immunology , SARS-CoV-2/enzymology , Animals , Humans
14.
Folia Microbiol (Praha) ; 66(4): 579-586, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1171477

ABSTRACT

From 7 to 8 days after the onset of symptoms in COVID-19 infection, the sensitivity of serological tests was found to be higher than that of nucleic acid tests. The aims of this study were to investigate antibody levels in patients with SARS-CoV-2 infection, to examine the relationship between antibody levels and virus load, and to evaluate the performance of 2 different commercial kits. A total of 103 patients with confirmed SARS-CoV-2 infection were included in the study. Antibodies against SARS-CoV-2 in serum samples taken from patients were investigated simultaneously with anti-SARS-CoV-2 IgG and IgA ELISAs (Euroimmun) and COVID-19 (SARS-CoV-2) IgG/IgM (Deep Blue) kits. No positivity was detected with any of the test kits in 18 (17.4%) of the 103 samples. In symptomatic patients, 100% of IgM and IgA tests were found to be positive in the group sampled after 10 days, while 100% of IgG tests were found positive after 20 days. The sensitivity of the Deep Blue COVID-19 IgG antibody kit was calculated as 81.48% and the specificity was 97.96%. While there was no statistically significant difference between the PCR CT and ELISA OD values, a positive correlation was found between the ELISA OD values and the days since the date of symptom initiation. The sensitivity and specificity of the rapid antibody test used in this study were found to be quite high. In conditions where ELISA tests cannot be applied, it is thought that it can give an idea in terms of the presence of antibodies as a simple and fast test. Although ELISA tests are valuable in the diagnosis of COVID-19 during the acute period, they are tests that can be used safely in the diagnosis of previous infections and seroepidemiological studies.


Subject(s)
Antibodies, Viral/blood , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing/methods , COVID-19/immunology , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , COVID-19/diagnosis , Female , Humans , Male
15.
Expert Rev Clin Immunol ; 17(6): 573-599, 2021 06.
Article in English | MEDLINE | ID: covidwho-1160272

ABSTRACT

Introduction: The gold standard for diagnosis of coronavirus disease 2019 (COVID-19) is detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription polymerase chain reaction (RT-PCR), which is expensive, time-consuming and may result in false-negative results. Serological tests can be employed for RT-PCR negative patients, contact tracing, determining the probability of protection against re-infection, and seroepidemiological studies.Areas covered: The main methodologies of serology-based tests for the detection of SARS-CoV-2 including enzyme-linked immunosorbent assays (ELISAs), chemiluminescent immunoassays (CLIAs) and lateral flow immunoassays (LFIAs) were reviewed and their diagnostic performances were compared. Herein, a literature review on the databases of PubMed, Scopus and Google Scholar between January 1, 2020 and June 30, 2020 based on the main serological methods for COVID-19 detection with the focus on comparative experiments was performed. The review was updated on December 31, 2020.Expert opinion: Serology testing could be considered as a part of diagnostic panel two-week post symptom onset. Higher sensitivity for serology-based tests could be achieved by determining combined IgG/IgM titers. Furthermore, higher sensitive serological test detecting neutralization antibody could be developed by targeting spike (S) antigen. It was also demonstrated that the sensitivity of ELISA/CLIA-based methods are higher than LFIA devices.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing , COVID-19/diagnosis , Immunoglobulin G/blood , Immunoglobulin M/blood , SARS-CoV-2/immunology , Biomarkers/blood , COVID-19/immunology , COVID-19/virology , Enzyme-Linked Immunosorbent Assay , Host-Pathogen Interactions , Humans , Luminescent Measurements , Predictive Value of Tests , Reproducibility of Results
16.
Diagnostics (Basel) ; 11(3)2021 Mar 09.
Article in English | MEDLINE | ID: covidwho-1158367

ABSTRACT

SARS-CoV-2 is a zoonotic betacoronavirus associated with worldwide transmission of COVID-19 disease. By the beginning of March, WHO reported about 113,820,000 confirmed cases including more than 2,527,000 deaths all over the world. However, the true extent of virus circulation or its real infection/fatality ratio is not well-estimated due to the huge portion of asymptomatic infections. In this observational study, we have estimated the prevalence of specific immunoglobulin M and G directed towards SARS-CoV-2 antigen in a cohort of 1383 adult volunteers aged over 65 years old, living in the district of Benevento, in the South of Italy. Serological screening was carried out on capillary blood in September 2020, seven months after pandemic outbreak in Italy, to evaluate virus circulation and antibody response among elderly adults, in which severe symptoms due to viral infection are more common. The overall seroprevalence of anti-SARS-CoV-2 antibodies was 4.70% (CI 3.70%-5.95%) with no statistically significant differences between sexes. Among these, 69.69% (CI 55.61%-77.80%) tested positive to IgM, 23.08% (CI 14.51%-34.64%) to IgG and 9.23% (CI 4.30%-18.71%) was positive for both. All patients that were positive to IgM underwent molecular testing through RT-qPCR on oral-rhino pharyngeal swabs and only one specimen was positive for SARS-CoV-2 RNA detection. Instead, the presence of IgG from screened volunteers was confirmed by re-testing serum samples using both an ELISA assay validated for in vitro diagnostic use (IVD) and a recently published synthetic peptide-based ELISA assay. In conclusion, our report suggests that (1) early restrictions were successful in limiting COVID-19 diffusion in the district of Benevento; (2) rapid serological analysis is an ideal testing for both determining real seroprevalence and massive screening, whereas detection of viral RNA remains a gold standard for identification of infected patients; (3) even among people without COVID-19 related symptoms, the antibody response against SARS-CoV-2 antigens has individual features.

17.
J Immunol Methods ; 494: 113046, 2021 07.
Article in English | MEDLINE | ID: covidwho-1155530

ABSTRACT

BACKGROUND: Frequently SARS-CoV-2 results in mild or moderate disease with potentially lower concentrations of antibodies compared to those that are hospitalised. Here, we validated an ELISA using SARS-CoV-2 trimeric spike glycoprotein, with targeted detection of IgG, IgA and IgM (IgGAM) using serum and dried blood spots (DBS) from adults with mild or moderate disease. METHODS: Targeting the SARS-CoV-2 trimeric spike, a combined anti-IgG, IgA and IgM serology ELISA assay was developed using 62 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 624 COVID-19 negative samples. The assay was validated using 73 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 359 COVID-19 negative serum samples with an additional 81 DBSs. The assay was further validated in 226 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 426 COVID-19 negative clinical samples. RESULTS: A sensitivity and specificity of 98.6% (95% CI, 92.6-100.0), 98.3% (95% CI, 96.4-99.4), respectively, was observed following validation of the SARS-CoV-2 ELISA. No cross-reactivities with endemic coronaviruses or other human viruses were observed, and no change in results were recorded for interfering substances. The assay was stable at temperature extremes and components were stable for 15 days once opened. A matrix comparison showed DBS to correlate with serum results. Clinical validation of the assay reported a sensitivity of 94.7% (95% CI, 90.9-97.2%) and a specificity of 98.4% (95% CI, 96.6-99.3%). CONCLUSIONS: The human anti-IgGAM SARS-CoV-2 ELISA provides accurate and sensitive detection of SARS-CoV-2 antibodies in non-hospitalised adults with mild or moderate disease. The use of dried blood spots makes the assay accessible to the wider community.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing , COVID-19 , SARS-CoV-2/metabolism , Adult , COVID-19/blood , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Middle Aged
18.
Anal Bioanal Chem ; 413(13): 3501-3510, 2021 May.
Article in English | MEDLINE | ID: covidwho-1151992

ABSTRACT

Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false positive results. In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. Schematic representation of sample collection and analysis. The figure was created using BioRender.com.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/analysis , Enzyme-Linked Immunosorbent Assay/methods , Humans , Phosphoproteins/analysis , Sensitivity and Specificity , Specimen Handling
19.
J Virol Methods ; 292: 114122, 2021 06.
Article in English | MEDLINE | ID: covidwho-1120398

ABSTRACT

INTRODUCTION: Reliable methods for the detection of SARS-CoV-2 neutralising antibodies (NAbs) are essential for the evaluation of vaccine candidates and for the selection of convalescent plasma donors. Virus neutralisation tests (NTs) are the gold standard for the detection and quantification of NAbs, but they are complex and require BSL3 facilities. In contrast, surrogate enzyme-linked immunosorbent assays (sELISA) offer the possibility of high-throughput testing under standard laboratory safety conditions. In this study, we investigated two commercial sELISA kits (GenScript, AdipoGen) designed for the detection of SARS-CoV-2 NAbs. METHODS: 276 plasma samples were screened using commercial IgG-ELISA and NAbs titres were determined by micro-neutralisation test (micro-NT). In addition, all samples were tested in both sELISA. Sensitivity and specificity for both sELISA were determined in comparison to the micro-NT results. RESULTS: 57 % of the samples were SARS-CoV-2 NAb positive in micro-NT, while 43 % tested negative. Comparison with micro-NT results showed a sensitivity of 98.2 % and a specificity of 69.5 % for the GenScript ELISA. The AdipoGen ELISA had a sensitivity of 83.5 % and a specificity of 97.8 %. False negative results were obtained mainly on samples with low NAbs titres. CONCLUSION: Both sELISA were able to qualitatively detect NAbs in plasma samples. Sensitivity and specificity differed between sELISA with GenScript superior in sensitivity and AdipoGen superior in specificity. Both sELISA were unable to quantify NAbs, thus neither of them can completely replace conventional NTs. However, in a two-step diagnostic algorithm, AdipoGen could potentially replace NT as a subsequent confirmatory test due to its high specificity but only in settings where no exact NAbs quantification is needed.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , Humans , Immunoglobulin G/blood , Neutralization Tests
20.
Am J Trop Med Hyg ; 104(4): 1513-1515, 2021 Feb 25.
Article in English | MEDLINE | ID: covidwho-1102598

ABSTRACT

Laboratory diagnosis of the COVID-19 relies on RT-PCR to amplify specific fragments of SARS-CoV-2 genome. However, serological tests are required to determine the immune response elicited after infection. Here, we analyzed convalescent sera collected from positive individuals by RT-PCR to SARS-CoV-2 (n = 78), Zika (n = 20), dengue (n = 20), chikungunya (n = 54), intestinal parasites (n = 11), and HIV (n = 1), from different areas of Ecuador, with an in-house ELISA using a SARS-CoV-2 receptor binding domain recombinant (rRBD) antigen to detect IgG antibodies elicited by SARS-CoV-2 infection. Of the 78 samples positive for SARS-CoV-2 by RT-PCR, 73 showed high absorbance value compared with the cutoff and five were negative. All tested sera from other infections showed no reactivity. Sensitivity, specificity, positive predictive value, and negative predictive value were 93.6%, 100%, 100%, and 95.4%, respectively. This in-house anti-IgG rRBD ELISA offers an economic and simple alternative to determine IgG immune responses after SARS-CoV-2 infection.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , COVID-19/epidemiology , COVID-19 Serological Testing/economics , Ecuador/epidemiology , Enzyme-Linked Immunosorbent Assay/economics , Humans , Immunoglobulin G/blood , Protein Binding , Protein Domains , Recombinant Proteins/immunology , Spike Glycoprotein, Coronavirus/chemistry
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