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1.
Sheng Wu Gong Cheng Xue Bao ; 37(11): 4066-4074, 2021 Nov 25.
Article in Chinese | MEDLINE | ID: covidwho-1543003

ABSTRACT

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Chromatography, Affinity , Fluorescent Antibody Technique , Humans , Microspheres , Sensitivity and Specificity
3.
PLoS One ; 16(9): e0248444, 2021.
Article in English | MEDLINE | ID: covidwho-1394535

ABSTRACT

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


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing/methods , Immunoassay/methods , Immunoglobulin M/immunology , Microspheres , SARS-CoV-2/immunology , Antibodies, Viral/blood , High-Throughput Screening Assays/methods , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , ROC Curve , SARS-CoV-2/physiology
4.
Microbiol Spectr ; 9(1): e0013421, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1329041

ABSTRACT

Early in the pandemic when diagnostic testing was not widely available, serosurveys played an important role in estimating the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different populations. Dried blood spots (DBS), which can be collected in nonclinical settings, provide a minimally invasive alternative to serum for serosurveys. We developed a Luminex-based SARS-CoV-2 microsphere immunoassay (MIA) for DBS that detects IgG antibodies to nucleocapsid (N) and spike subunit 1 (S1) antigens. The assay uses a 384-well plate format and automated liquid handlers for high-throughput capacity. Specificity was assessed using a large collection of prepandemic DBS and well-characterized sera. Sensitivity was analyzed using serology data from New York State SARS-CoV-2 serosurvey testing and matched diagnostic test results. For DBS, the specificity was 99.5% for the individual N and S1 antigens. Median fluorescence intensity (MFI) values for DBS and paired sera showed a strong positive correlation for N (R2 = 0.91) and S1 (R2 = 0.93). Sensitivity, assessed from 1,134 DBS with prior laboratory-confirmed SARS-CoV-2 infection, ranged from 83% at 0 to 20 days to 95% at 61 to 90 days after a positive test. When stratified using coronavirus disease 2019 (COVID-19) symptom data, sensitivity ranged from 90 to 96% for symptomatic and 77 to 91% for asymptomatic individuals. For 8,367 health care workers reporting detailed symptom data, MFI values were significantly higher for all symptom categories. Our results indicate that the SARS-CoV-2 IgG DBS MIA is sensitive, specific, and well-suited for large population-based serosurveys. The ability to readily modify and multiplex antigens is important for ongoing assessment of SARS-CoV-2 antibody responses to emerging variants and vaccines. IMPORTANCE Testing for antibodies to SARS-CoV-2 has been used to estimate the prevalence of COVID-19 in different populations. Seroprevalence studies, or serosurveys, were especially useful during the early phase of the pandemic when diagnostic testing was not widely available, and the resulting seroprevalence estimates played an important role in public health decision making. To achieve meaningful results, antibody tests used for serosurveys should be accurate and accessible to diverse populations. We developed a test that detects antibodies to two different SARS-CoV-2 proteins in dried blood spots (DBS). DBS require only a simple fingerstick and can be collected in nonclinical settings. We conducted a robust validation study and have demonstrated that our test is both sensitive and specific. Furthermore, we demonstrated that our test is suitable for large-scale serosurveys by testing over 56,000 DBS collected in a variety of community-based venues in New York State during the spring of 2020.


Subject(s)
Antibodies, Viral/blood , COVID-19 Testing/methods , COVID-19/diagnosis , Immunoassay/methods , Immunoglobulin G/blood , Microspheres , SARS-CoV-2/isolation & purification , Female , Humans , Male , New York , Pandemics , Patient Care Team , Public Health , Sensitivity and Specificity , Seroepidemiologic Studies , Specimen Handling
5.
J Clin Microbiol ; 59(6)2021 05 19.
Article in English | MEDLINE | ID: covidwho-1255519

ABSTRACT

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R = 0.83, P < 0.0001 and R = 0.87, P < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R = 0.76, P = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (P < 0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.


Subject(s)
COVID-19 , Emergency Responders , Antibodies, Viral , COVID-19 Serological Testing , Colorado , Humans , Immunoassay , Microspheres , SARS-CoV-2 , Seroepidemiologic Studies
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.
PLoS Negl Trop Dis ; 14(10): e0008699, 2020 10.
Article in English | MEDLINE | ID: covidwho-932350

ABSTRACT

Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher binding to both EBOV NP and MARV NP coupled microspheres in the Guinea panel. Finally, the MMIA was successfully adapted to detect antibodies from bats that had been inoculated with EBOV- and MARV- virus-like particles, highlighting the versatility of this technique and potentially enabling the monitoring of wildlife as well as human populations with this assay. We were thus able to develop and validate a sensitive and broadly reactive high-throughput serological assay which could be used as a screening tool to detect antibodies against several highly pathogenic viruses.


Subject(s)
Antibodies, Viral/blood , Immunoassay/methods , Microspheres , Nucleocapsid Proteins/immunology , Virus Diseases/veterinary , Animals , Chiroptera , Humans , Primates , Virus Diseases/diagnosis , Virus Diseases/virology
8.
J Clin Microbiol ; 59(6)2021 05 19.
Article in English | MEDLINE | ID: covidwho-1166353

ABSTRACT

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R = 0.83, P < 0.0001 and R = 0.87, P < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R = 0.76, P = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (P < 0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.


Subject(s)
COVID-19 , Emergency Responders , Antibodies, Viral , COVID-19 Serological Testing , Colorado , Humans , Immunoassay , Microspheres , SARS-CoV-2 , Seroepidemiologic Studies
9.
Int J Mol Sci ; 22(5)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1150749

ABSTRACT

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , COVID-19/blood , COVID-19/immunology , Neutralization Tests/methods , Neutralization Tests/standards , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cross Reactions , Flow Cytometry/methods , Fluorescence , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Microspheres , Receptors, Virus/chemistry , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
10.
Int J Mol Sci ; 22(5)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1134167

ABSTRACT

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , COVID-19/blood , COVID-19/immunology , Neutralization Tests/methods , Neutralization Tests/standards , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cross Reactions , Flow Cytometry/methods , Fluorescence , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Microspheres , Receptors, Virus/chemistry , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
11.
J Immunol Methods ; 490: 112952, 2021 03.
Article in English | MEDLINE | ID: covidwho-1065340

ABSTRACT

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Bacterial Proteins/metabolism , Biotin/analogs & derivatives , Biotin/metabolism , Cell Surface Display Techniques , Flow Cytometry , Fluorescent Dyes , HEK293 Cells , High-Throughput Screening Assays , Humans , Immunomagnetic Separation , Microspheres , Mutation/genetics , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
12.
J Infect Dis ; 222(12): 1965-1973, 2020 11 13.
Article in English | MEDLINE | ID: covidwho-1060647

ABSTRACT

We present a microsphere-based flow cytometry assay that quantifies the ability of plasma to inhibit the binding of spike protein to angiotensin-converting enzyme 2. Plasma from 22 patients who had recovered from mild coronavirus disease 2019 (COVID-19) and expressed anti-spike protein trimer immunoglobulin G inhibited angiotensin-converting enzyme 2-spike protein binding to a greater degree than controls. The degree of inhibition was correlated with anti-spike protein immunoglobulin G levels, neutralizing titers in a pseudotyped lentiviral assay, and the presence of fever during illness. This inhibition assay may be broadly useful to quantify the functional antibody response of patients recovered from COVID-19 or vaccine recipients in a cell-free assay system.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Serologic Tests/methods , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Binding Sites , Female , HEK293 Cells , Humans , Male , Microspheres , Middle Aged , Plasma/immunology , Protein Binding , SARS-CoV-2/immunology , Young Adult
13.
J Clin Microbiol ; 59(2)2021 01 21.
Article in English | MEDLINE | ID: covidwho-1041375

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 prepandemic samples. Sensitivity was measured and compared to that of the Abbott Architect SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned (n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% versus 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection (n = 534) of discarded sera was profiled from patients (n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7 [in days from symptom onset]), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled intensive care unit (ICU) patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taking the data together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Microspheres , SARS-CoV-2/isolation & purification , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/blood , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/immunology , Female , Fluoroimmunoassay , Humans , Immunoglobulin G/blood , Kinetics , Male , Middle Aged , Phosphoproteins/immunology , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
14.
Talanta ; 225: 122064, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1003087

ABSTRACT

Quantum dots (QDs) based fluorescent nanobeads are considered as promising materials for next generation point-of-care diagnosis systems. In this study, we carried out, for the first time, the synthesis of QDs nanobeads using polystyrene (PS) nanobead as the template. QDs loading on PS nanobead surface in this method can be readily achieved by the use of polyelectrolyte, avoiding the time-consuming and uncontrollable silane reagents-involved functionalization procedure that conventional synthesis of silica-based QDs nanobeads often suffer from. Notably, the application of QDs nanobeads in suspension microarray for H5N1 virus detection leads to a sensitivity lower than 25 PFU/mL. In addition, QDs nanobead was also incorporated into lateral flow assay for SARS-CoV-2 antibody detection, leading to more than one order of magnitude detection sensitivity as compared to that of commercial one based on colloid gold.


Subject(s)
Biosensing Techniques/methods , COVID-19/diagnosis , Influenza, Human/diagnosis , Microspheres , Nanostructures/chemistry , Polystyrenes/chemistry , Quantum Dots , Antibodies, Viral/immunology , COVID-19/virology , Fluorescent Dyes/chemistry , Humans , Influenza A Virus, H5N1 Subtype/physiology , Influenza, Human/virology , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Nanostructures/ultrastructure , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Sensitivity and Specificity , Silicon Dioxide/chemistry
15.
Talanta ; 225: 121977, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1003086

ABSTRACT

SARS-COV-2 is a novel coronavirus discovered in Wuhan in December 30, 2019, and is a family of SARS-COV (severe acute respiratory syndrome coronavirus), that is, coronavirus family. After infection with SARS-COV-2, patients often experience fever, cough, gas prostration, dyspnea and other symptoms, which can lead to severe acute respiratory syndrome (SARS), kidney failure and even death. The SARS-COV-2 virus is particularly infectious and has led to a global infection crisis, with an explosion in the number of infections. Therefore, rapid and accurate detection of the virus plays a vital role. At present, many detection methods are limited in their wide application due to their defects such as high preparation cost, poor stability and complex operation process. Moreover, some methods need to be operated by professional medical staff, which can easily lead to infection. In order to overcome these problems, a Surface molecular imprinting technology (SM-MIT) is proposed for the first time to detect SARS-COV-2 virus. For this SM-MIT method, this review provides detailed detection principles and steps. In addition, this method not only has the advantages of low cost, high stability and good specificity, but also can detect whether it is infected at designated points. Therefore, we think SM-MIT may have great potential in the detection of SARS-COV-2 virus.


Subject(s)
COVID-19/diagnosis , Magnetite Nanoparticles/chemistry , Molecular Imprinting , Polymers/chemistry , SARS-CoV-2/metabolism , Viral Proteins/metabolism , COVID-19/virology , Humans , Microspheres , Ovalbumin/chemistry , Ovalbumin/metabolism , SARS-CoV-2/physiology , Sensitivity and Specificity , Viral Proteins/chemistry
16.
Front Cell Infect Microbiol ; 10: 553837, 2020.
Article in English | MEDLINE | ID: covidwho-983733

ABSTRACT

Purpose: To develop a rapid detection reagent for SARS-CoV-2 antigen for the auxiliary diagnosis of new coronary pneumonia (COVID-19), and perform the methodological evaluation and clinical evaluation of the reagent. Method: SARS-CoV-2 N-protein test strip was created by combining fluorescent microsphere labeling technology and immunochromatographic technology, based on the principle of double antibody sandwich. Then we evaluated the analytical capability and clinical application of the strips. Result: The limit of detection of the strips for recombinant N protein was 100 ng/ml and for activated SARS -CoV-2 virus was 1 × 103 TCID50/ml. The strips also have high analytical specificity and anti-interference capability. According to the predetermined cut-off value, the specificity of the test strip in healthy controls and patients with other respiratory disease was 100.00 and 97.29%, the sensitivity in COVID-19 cases at progress stage and cured stage was 67.15 and 7.02%. The positive percentage agreement and negative percentage agreement of antigen strip to RNA test were 83.16 and 94.45%. Conclusion: SARS-CoV-2 fluorescence immunochromatographic test strip can achieve fast, sensitive and accurate detection, which can meet the clinical requirements for rapid detection of viruses on the spot.


Subject(s)
Antigens, Viral/immunology , COVID-19 Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, Viral/analysis , Child , Child, Preschool , Chromatography, Affinity/methods , Coronavirus Nucleocapsid Proteins/analysis , Female , Fluorescent Dyes , Humans , Limit of Detection , Male , Microspheres , Middle Aged , Sensitivity and Specificity , Young Adult
17.
PLoS One ; 15(12): e0238010, 2020.
Article in English | MEDLINE | ID: covidwho-961459

ABSTRACT

Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.


Subject(s)
Cell Separation/methods , Immunoglobulin G/immunology , Immunomagnetic Separation/methods , Antigens, Protozoan/immunology , Child , Child, Preschool , Female , Humans , Magnetic Phenomena , Malaria/immunology , Malaria, Vivax/immunology , Male , Microspheres , Papua New Guinea/epidemiology , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Technology
18.
Paediatr Anaesth ; 30(11): 1269-1274, 2020 11.
Article in English | MEDLINE | ID: covidwho-955522

ABSTRACT

BACKGROUND: During the coronavirus pandemic, preventing contamination of the anesthesia machine, critical to avoid cross-contamination between patients, has proven challenging when treating premature infants and neonates. While attaching a HEPA filter to the endotracheal tube will protect the anesthesia machine and the gas sampling line from contamination, this contribution to the dead space makes ventilation of these small patients challenging. Direct filtration of the gas sampling line eliminates this problem; however, appropriate filters are not readily available. AIMS: Identify a small filter capable of filtering out particles of a size similar to the SARS-CoV-2 virus for the gas sampling line. METHODS: We used fluorescence microspheres suspended in a solution for a challenge test to determine the filtration efficiency of various filters. The microspheres varied in diameter (0.02 µm, 0.042 µm, 0.109 µm, and 0.989 µm). A fluorescence plate reader was used to evaluate the degree of fluorescence intensity in the flow-through from various filters and referenced to the fluorescence intensity of the input. RESULTS: AHEPA filter, as recommended as an anti-viral filter, effectively filtered all the particles tested. The B. Braun PERIFIX Flat Epidural Filter was the second most effective filter, filtering particles larger than 0.042 µm. Other filters tested did not filter fluorescence microspheres equivalent in size to a single coronavirus particle (0.07 µm). CONCLUSIONS: Although the Food and Drug Administration (FDA) has not approved the Flat Epidural Filter for use as an anesthesia machine gas filter, our simple challenge test suggests that it could be used to effectively filter the anesthesia gas sampling line.


Subject(s)
Anesthesia, Endotracheal/instrumentation , COVID-19/prevention & control , Equipment Contamination/prevention & control , Filtration/instrumentation , Microspheres , SARS-CoV-2/isolation & purification , Fluorescence , Humans , Infant , Infant, Newborn
19.
J Biophotonics ; 14(3): e202000338, 2021 03.
Article in English | MEDLINE | ID: covidwho-908749

ABSTRACT

The appearance of antibodies in blood is a critical signal to suggest the infection. A rapid and accurate detection method for the antibody is significant to the disease diagnosis, especially for the epidemic. To this end, a highly sensitive whispering-gallery-mode (WGM) optical testing kit is designed and fabricated for detecting the specific immunoglobulin antibodies. The key component of the kit is a silica self-assembled microsphere decorated with the nucleocapsid proteins (N-proteins) of the SARS-CoV-2 virus. After the N-protein antibody immunoglobulin G (N-IgG) and immunoglobulin M (N-IgM) solutions being injected into the kit, the WGM red-shifts due to the antigen-antibody reaction. The wavelength displacement rates are proportional to the concentrations of these two antibodies from 1 to 100 µg/mL. A good specificity of the kit is demonstrated by the nonspecific human immunoglobulin G (H-IgG) and immunoglobulin M (H-IgM).


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing , COVID-19/diagnosis , Microspheres , Silicon Dioxide/chemistry , Antigens/immunology , Biosensing Techniques , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Epidemics , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Optics and Photonics , Phosphoproteins/immunology , Polymethyl Methacrylate/chemistry , SARS-CoV-2 , Silanes
20.
Int J Mol Sci ; 21(18)2020 Sep 09.
Article in English | MEDLINE | ID: covidwho-760933

ABSTRACT

Currently available COVID-19 antibody tests using enzyme immunoassay (EIA) or immunochromatographic assay have variable sensitivity and specificity. Here, we developed and evaluated a novel microsphere-based antibody assay (MBA) for detecting immunoglobulin G (IgG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (NP) and spike protein receptor binding domain (RBD). The seropositive cutoff value was set using a cohort of 294 anonymous serum specimens collected in 2018. The specificity was assessed using serum specimens collected from organ donors or influenza patients before 2020. Seropositive rate was determined among COVID-19 patients. Time-to-seropositivity and signal-to-cutoff (S/CO) ratio were compared between MBA and EIA. MBA had a specificity of 100% (93/93; 95% confidence interval (CI), 96-100%) for anti-NP IgG, 98.9% (92/93; 95% CI 94.2-100%) for anti-RBD IgG. The MBA seropositive rate for convalescent COVID-19 patients was 89.8% (35/39) for anti-NP IgG and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than EIA. MBA could better differentiate between COVID-19 patients and negative controls with higher S/CO ratio for COVID-19 patients, lower S/CO ratio with negative controls and fewer specimens in the equivocal range. MBA is robust, simple and is suitable for clinical microbiology laboratory for the accurate determination of anti-SARS-CoV-2 antibodies for diagnosis, serosurveillance, and vaccine trials.


Subject(s)
Antibodies, Viral/blood , Coronavirus Infections/blood , Nucleocapsid Proteins/immunology , Pneumonia, Viral/blood , Serologic Tests/methods , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , COVID-19 , Child , Child, Preschool , Coronavirus Infections/diagnosis , Coronavirus Nucleocapsid Proteins , Female , Humans , Infant , Male , Microspheres , Middle Aged , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Sensitivity and Specificity , Serologic Tests/standards
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