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3.
Clin Immunol ; 230: 108817, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34352391

RESUMO

Many studies have analyzed myelin-reactivity of T cells in multiple sclerosis (MS); however, with conflicting results. In this study we compare methods to determine myelin reactivity of T cells and aim to delineate the cause of inconsistency in the literature. Challenging T cells with myelin antigens we found a significant increase in antigen-reactivity of T cells from patients with MS using an ELISpot-assay, in contrast to a CFSE-dilution assay. Comparing the two assays showed that the myelin-reactive T cells detected in the ELISpot-assay originated primarily from effector memory T cells in contrast to the myelin-reactive T cells of the CFSE-assay representing a population of both naïve, central memory and effector memory T cells. This diversity in T cell populations activated in the two assays likely contribute to the discrepancy found in the literature and encourages thorough considerations when choosing an assay to determine antigen-specificity of T cells in future studies.


Assuntos
Imunoensaio/métodos , Esclerose Múltipla Recidivante-Remitente/imunologia , Proteínas da Mielina/imunologia , Linfócitos T/imunologia , Adulto , Autoantígenos/imunologia , Estudos de Casos e Controles , ELISPOT , Feminino , Fluoresceínas , Corantes Fluorescentes , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Proteína Básica da Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Succinimidas , Linfócitos T/classificação , Adulto Jovem
4.
Front Immunol ; 12: 696370, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386006

RESUMO

The COVID-19 pandemic is caused by SARS-CoV-2, a novel zoonotic coronavirus. Emerging evidence indicates that preexisting humoral immunity against other seasonal human coronaviruses (HCoVs) plays a critical role in the specific antibody response to SARS-CoV-2. However, current work to assess the effects of preexisting and cross-reactive anti-HCoVs antibodies has been limited. To address this issue, we have adapted our previously reported multiplex assay to simultaneously and quantitatively measure anti-HCoV antibodies. The full mPlex-CoV panel covers the spike (S) and nucleocapsid (N) proteins of three highly pathogenic HCoVs (SARS-CoV-1, SARS-CoV-2, MERS) and four human seasonal strains (OC43, HKU1, NL63, 229E). Combining this assay with volumetric absorptive microsampling (VAMS), we measured the anti-HCoV IgG, IgA, and IgM antibodies in fingerstick blood samples. The results demonstrate that the mPlex-CoV assay has high specificity and sensitivity. It can detect strain-specific anti-HCoV antibodies down to 0.1 ng/ml with 4 log assay range and with low intra- and inter-assay coefficients of variation (%CV). We also estimate multiple strain HCoVs IgG, IgA and IgM concentration in VAMS samples in three categories of subjects: pre-COVID-19 (n=21), post-COVID-19 convalescents (n=19), and COVID-19 vaccine recipients (n=14). Using metric multidimensional scaling (MDS) analysis, HCoVs IgG concentrations in fingerstick blood samples were well separated between the pre-COVID-19, post-COVID-19 convalescents, and COVID-19 vaccine recipients. In addition, we demonstrate how multi-dimensional scaling analysis can be used to visualize IgG mediated antibody immunity against multiple human coronaviruses. We conclude that the combination of VAMS and the mPlex-Cov assay is well suited to performing remote study sample collection under pandemic conditions to monitor HCoVs antibody responses in population studies.


Assuntos
Anticorpos Antivirais/sangue , Coronavirus/imunologia , Reações Cruzadas/imunologia , Imunoensaio/métodos , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , COVID-19/imunologia , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Coronavirus Humano OC43/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
5.
Front Biosci (Landmark Ed) ; 26(7): 198-206, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34340267

RESUMO

Background: High-throughput assays that can infer neutralizing activity against SARS-CoV-2 are of great importance for assessing the immunity induced by natural infection and COVID-19 vaccines. We aimed to evaluate the performance and degree of correlation of three fully automated anti-SARS-CoV-2 immunoassays with neutralization activity using a surrogate virus-neutralizing test (sVNT) from GenScript, targeting the receptor-binding domain. Methods: 110 sera collected from PCR-confirmed asymptomatic COVID-19 individuals were tested for neutralizing antibodies (nAbs) using the sVNT. Positive samples were tested on three automated immunoassays targeting different viral antigens: Mindray CL-900i®, Abbott Architect, and Ortho VITROS®. The diagnostic sensitivity, specificity, agreement, and correlation with the sVNT were assessed. Receiver operating characteristic (ROC) curve analysis was performed to determine optimal thresholds for predicting the presence of neutralizing activity by each assay. Results: All three assays showed 100% specificities. The highest sensitivity was 99.0%, demonstrated by VITROS®, followed by 94.3%, for CL-900i®, and 81.0%, for Architect. Both VITROS® and CL-900i® had the strongest correlation with the sVNT (ρ = 0.718 and ρ = 0.712, respectively), while Architect showed a moderate correlation (ρ = 0.618). ROC curve analysis indicated that the manufacturer's recommended cutoff values are adequate for predicting the presence of nAbs and providing a strong correlation with the sVNT. Conclusion: VITROS® and CL-900i® serological assays, which detect antibodies against SARS-CoV-2 spike protein, could serve as reliable assays to predict neutralization activity after infection or vaccination.


Assuntos
Anticorpos Neutralizantes/sangue , COVID-19/imunologia , Imunoensaio/métodos , SARS-CoV-2/imunologia , Automação , COVID-19/virologia , Humanos , Limite de Detecção
6.
ACS Appl Mater Interfaces ; 13(34): 40342-40353, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34412466

RESUMO

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the "hook effect." The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.


Assuntos
Antígenos Virais/análise , Técnicas Biossensoriais/métodos , Proteínas do Nucleocapsídeo de Coronavírus/análise , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/análise , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Fluorescência , Corantes Fluorescentes/química , Humanos , Imunoensaio/métodos , Limite de Detecção , Nanopartículas de Magnetita/química , Nasofaringe/virologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Pontos Quânticos/química , Saliva/virologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
7.
J Infect Dev Ctries ; 15(7): 904-909, 2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34343113

RESUMO

INTRODUCTION: As regard to all pandemics, the current COVID-19 pandemic, could also have been better managed with prudent use of preventive measures coupled with rapid diagnostic tools such as rapid antigen tests, but their efficacy is under question because of projected lower sensitivity as compared to Real Time Reverse Transcriptase Polymerase Chain Reaction, which although considered gold standard has its own limitations. METHODOLOGY: A prospective, single centre study was carried out to evaluate the performance of Standard Q COVID-19 Ag, a rapid immuno-chromatographic assay for antigen detection, against TrueNat, a chip-based, point-of-care, portable, Real-Time PCR analyzer for diagnosis of COVID-19; on 467 nasal swab samples from suspected subjects at a fever clinic in North India in month of July 2020. RESULTS: Of the 467 specimens tested, TrueNat showed positive result in 29 (6.2%), majority of whom were asymptomatic (72.4%) while 4/29 (13.9%) had influenza like illness and 2/29 (6.8%) presented with severe acute respiratory illness. Compared to TrueNat, Rapid antigen test gave concordance for 26 samples, while for 2 samples the result was false positive; giving an overall sensitivity of 89.7% (95% CI = 72.6- 97.8) and a specificity of 99.5%, indicating strong agreement between two methods. CONCLUSION: Community prevalence plays an important role is choosing the laboratory test and result interpretation. Rapid antigen detection tests definitely have a big role to play, especially in resource limited setting, for early diagnosis as well as for source control to halt the spread.


Assuntos
Teste Sorológico para COVID-19/métodos , Teste Sorológico para COVID-19/normas , COVID-19/diagnóstico , Imunoensaio/métodos , Imunoensaio/normas , SARS-CoV-2/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais , Infecções Assintomáticas , COVID-19/sangue , Teste de Ácido Nucleico para COVID-19/normas , Teste de Ácido Nucleico para COVID-19/estatística & dados numéricos , Criança , Pré-Escolar , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Nariz/virologia , Estudos Prospectivos , SARS-CoV-2/química , Sensibilidade e Especificidade , Adulto Jovem
8.
Viruses ; 13(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34452373

RESUMO

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.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , Vacinas contra COVID-19/imunologia , COVID-19/virologia , Imunoensaio/métodos , Medições Luminescentes/métodos , SARS-CoV-2/imunologia , COVID-19/sangue , COVID-19/imunologia , COVID-19/prevenção & controle , Teste Sorológico para COVID-19/instrumentação , Vacinas contra COVID-19/administração & dosagem , Humanos , SARS-CoV-2/genética , Vacinação
9.
J Clin Lab Anal ; 35(9): e23921, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34369009

RESUMO

BACKGROUND: SARS-CoV-2 pandemic is currently ongoing, meanwhile vaccinations are rapidly underway in some countries. The quantitative immunoassays detecting antibodies against spike antigen of SARS-CoV-2 have been developed based on the findings that they have a better correlation with the neutralizing antibody. METHODS: The performances of the Abbott Architect SARS-CoV-2 IgG II Quant, DiaSorin LIAISON SARS-CoV-2 TrimericS IgG, and Roche Elecsys anti-SARS-CoV-2 S were evaluated on 173 sera from 126 SARS-CoV-2 patients and 151 pre-pandemic sera. Their correlations with GenScript cPass SARS-CoV-2 Neutralization Antibody Detection Kit were also analyzed on 173 sera from 126 SARS-CoV-2 patients. RESULTS: Architect SARS-CoV-2 IgG II Quant and Elecsys anti-SARS-CoV-2 S showed the highest overall sensitivity (96.0%), followed by LIAISON SARS-CoV-2 TrimericS IgG (93.6%). The specificities of Elecsys anti-SARS-CoV-2 S and LIAISON SARS-CoV-2 TrimericS IgG were 100.0%, followed by Architect SARS-CoV-2 IgG II Quant (99.3%). Regarding the correlation with cPass neutralization antibody assay, LIAISON SARS-CoV-2 TrimericS IgG showed the best correlation (Spearman rho = 0.88), followed by Architect SARS-CoV-2 IgG II Quant and Elecsys anti-SARS-CoV-2 S (all rho = 0.87). CONCLUSIONS: The three automated quantitative immunoassays showed good diagnostic performance and strong correlations with neutralization antibodies. These assays will be useful in diagnostic assistance, evaluating the response to vaccination, and the assessment of herd immunity in the future.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/virologia , Imunoensaio/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Neutralizantes/sangue , Teste Sorológico para COVID-19/instrumentação , Humanos , Imunoglobulina G/sangue , Testes de Neutralização , Curva ROC , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes Sorológicos
10.
Microbiol Spectr ; 9(1): e0013421, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34319133

RESUMO

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.


Assuntos
Anticorpos Antivirais/sangue , Teste para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Microesferas , SARS-CoV-2/isolamento & purificação , Feminino , Humanos , Masculino , New York , Pandemias , Equipe de Assistência ao Paciente , Saúde Pública , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Manejo de Espécimes
11.
Adv Clin Chem ; 103: 215-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34229851

RESUMO

The measurement of cardiac troponin (cTn) is recommended by all guidelines as the gold standard for the differential diagnosis of Acute Coronary Syndromes. The aim of this review is to discuss in details some key issues regarding both analytical and clinical characteristics of the high-sensitivity methods for cTn (hs-cTn), which are still considered controversial or unresolved. In particular, the major clinical concern regarding hs-cTn methods is the difficulty to differentiate the pathophysiological mechanism responsible for biomarker release from cardiomyocytes after reversible or irreversible injury, respectively. Indeed, recent experimental and clinical studies have demonstrated that different circulating forms of cTnI and cTnT can be respectively measured in plasma samples of patients with reversible or irreversible myocardial injury. Accordingly, a new generation of hs-Tn methods should be set up, based on immunometric immunoassays or chromatographic techniques, specific for circulating peptide forms more characteristics for reversible or irreversible myocardial injury. It is conceivable that this new generation of hs-cTn methods will complete the mission regarding the laboratory tests for specific cardiac biomarkers, started more than 20 years ago, which has already revolutionized the diagnosis, prognosis and management of patients with cardiac diseases.


Assuntos
Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/metabolismo , Imunoensaio/métodos , Troponina/sangue , Troponina/metabolismo , Biomarcadores/sangue , Humanos , Sensibilidade e Especificidade
12.
PLoS One ; 16(7): e0254486, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34283860

RESUMO

The coronavirus disease (COVID-19) is the global public health challenge currently persisting at a grand scale. A method that meets the rapid quantitative detection of antibodies to assess the body's immune response from natural COVID-19 illness or vaccines' effects is urgently needed. In the present study, an attempt was made to integrate a newly designed spectrometer to the COVID-19 test strip procedure; this augmentation provides the quantitative capacity to a lateral flow immunoassay (LFIA). Optical interpretation of results by quantitative α index, rather than visual qualification, can be done quickly, in 5-10 minutes. The developed product was compared with several other serological IgM/IgG antibody reagents on the market by recruiting 111 participants suspected of having COVID-19 infection from March to May 2020 in a hospital. Taking RT-PCR as the diagnostic gold standard, the quantitative spectral LIFA platform could correctly detect all 12 COVID-19 patients. Concerning RT-PCR negative patients, all three antibody testing methods found positive cases. The optical-based platform exhibited the ability of early detection of immunoglobulins of RT-PCR negative patients. There was an apparent trend that elevation of IgM levels in the acute phase of infection; then IgG levels rose later. It exhibited the risk of a false-negative diagnosis of RT-PCR in COVID-19 testing. The significant detection ability of this new optical-based platform demonstrated clinical potential.


Assuntos
COVID-19/imunologia , Imunoensaio/métodos , Isotipos de Imunoglobulinas/análise , Humanos , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Curr Issues Mol Biol ; 43(2): 728-748, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34287238

RESUMO

The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a severe threat to human health and the global economy and has resulted in overwhelming stress on health care systems worldwide. Despite the global health catastrophe, especially in the number of infections and fatalities, the COVID-19 pandemic has also revolutionized research and discovery with remarkable success in diagnostics, treatments, and vaccine development. The use of many diagnostic methods has helped establish public health guidelines to mitigate the spread of COVID-19. However, limited information has been shared about these methods, and there is a need for the scientific community to learn about these technologies, in addition to their sensitivity, specificity, and limitations. This review article is focused on providing insights into the major methods used for SARS-CoV-2 detection. We describe in detail the core principle of each method, including molecular and serological approaches, along with reported claims about the rates of false negatives and false positives, the types of specimens needed, and the level of technology and the time required to perform each test. Although this study will not rank or prioritize these methods, the information will help in the development of guidelines and diagnostic protocols in clinical settings and reference laboratories.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ensaio de Imunoadsorção Enzimática/métodos , Coloide de Ouro , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoensaio/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação
15.
Sci Rep ; 11(1): 15321, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321523

RESUMO

The spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose-response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/imunologia , Teste em Amostras de Sangue Seco/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Bloqueadores , Anticorpos Antivirais/imunologia , COVID-19/sangue , Humanos , Imunoensaio/métodos , Testes de Neutralização/métodos , Reprodutibilidade dos Testes , Testes Sorológicos
16.
Artigo em Inglês | MEDLINE | ID: mdl-34224962

RESUMO

Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC-MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4-1500 pmol/L for E1 and 4-2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC-MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC-MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.


Assuntos
Cromatografia Líquida/métodos , Estradiol/sangue , Estrona/sangue , Imunoensaio/métodos , Espectrometria de Massas em Tandem/métodos , Adolescente , Adulto , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Menopausa , Pessoa de Meia-Idade , Valores de Referência , Reprodutibilidade dos Testes , Adulto Jovem
17.
J Immunol ; 207(4): 1211-1221, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34312257

RESUMO

Long half-life of therapeutic Abs and Fc fusion proteins is crucial to their efficacy and is, in part, regulated by their interaction with neonatal Fc receptor (FcRn). However, the current methods (e.g., surface plasmon resonance and biolayer interferometry) for measurement of interaction between IgG and FcRn (IgG/FcRn) require either FcRn or IgG to be immobilized on the surface, which is known to introduce experimental artifacts and have led to conflicting data. To study IgG/FcRn interactions in solution, without a need for surface immobilization, we developed a novel (to our knowledge), solution-based homogeneous binding immunoassay based on NanoBiT luminescent protein complementation technology. We optimized the assay (NanoBiT FcRn assay) for human FcRn, mouse FcRn, rat FcRn, and cynomolgus FcRn and used them to determine the binding affinities of a panel of eight Abs. Assays could successfully capture the modulation in IgG/FcRn binding based on changes in Fc fragment of the Abs. We also looked at the individual contribution of Fc and F(ab)2 on the IgG/FcRn interaction and found that Fc is the main driver for the interaction at pH 6. Our work highlights the importance of using orthogonal methods to validate affinity data generated using biosensor platforms. Moreover, the simple add-and-read format of the NanoBiT FcRn assay is amenable for high-throughput screening during early Ab discovery phase.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoensaio/métodos , Medições Luminescentes/métodos , Receptores Fc/imunologia , Sequência de Aminoácidos , Animais , Técnicas Biossensoriais/métodos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Testes Imunológicos/métodos , Camundongos , Ligação Proteica/imunologia , Ratos
18.
Mikrochim Acta ; 188(8): 261, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34278534

RESUMO

The ongoing global pandemic of SARS-CoV-2 has promoted to develop novel serological testing technologies since they can be effectively complementary to RT-PCR. Here, a new all-fiber Fresnel reflection microfluidic biosensor (FRMB) was constructed through combining all-fiber optical system, microfluidic chip, and multimode fiber bio-probe. The transmission of the incident light and the collection and transmission of Fresnel reflection light are achieved using a single-multi-mode fiber optic coupler (SMFC) without any other optical separation elements. This compact design greatly simplifies the whole system structure and improves light transmission efficiency, which makes it suitable for the label-free, sensitive, and easy-to-use point-of-care testing (POCT) of targets in nanoliter samples. Based on Fresnel reflection mechanism and immunoassay principle, both the SARS-CoV-2 IgM and IgG antibodies against the SARS-CoV-2 spike protein could be sensitively quantified in 7 min using the secondary antibodies-modified multimode fiber bio-probe. The FRMB performs in one-step, is accurate, label-free, and sensitive in situ/on-site detection of SARS-CoV-2 IgM or IgG in serum with simple dilution only. The limits of detection of SARS-CoV-2 IgM and SARS-CoV-2 IgG were 0.82 ng/mL and 0.45 ng/mL, respectively. Based on our proposed theory, the affinity constants of SARS-CoV-2 IgM or IgG antibody and their respective secondary antibodies were also determined. The FRMB can be readily extended as a universal platform for the label-free, rapid, and sensitive in situ/on-site measurement of other biomarkers and the investigation of biomolecular interaction.


Assuntos
Anticorpos Antivirais/sangue , Técnicas Biossensoriais/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas Analíticas Microfluídicas/métodos , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Humanos , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Limite de Detecção , SARS-CoV-2/química , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
19.
EBioMedicine ; 69: 103465, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34229274

RESUMO

BACKGROUND: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. METHODS: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. FINDINGS: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. INTERPRETATION: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. FUNDING: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.


Assuntos
Teste Sorológico para COVID-19/métodos , Imunoensaio/métodos , Espectrometria de Massas/métodos , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Automação Laboratorial/métodos , Automação Laboratorial/normas , Teste Sorológico para COVID-19/normas , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Imunoensaio/normas , Aprendizado de Máquina , Espectrometria de Massas/normas , Fosfoproteínas/química , Fosfoproteínas/imunologia , Sensibilidade e Especificidade
20.
Sci Rep ; 11(1): 14926, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290350

RESUMO

Infection with SARS-CoV-2, the Betacoronavirus, caused a pandemic that affected the globe negatively. The gold method, RT-PCR, can detect SARS-CoV-2 but it is time-consuming and needs sophisticated equipment and professional personnel. On the other hand, rapid tests offer fast results and can detect anti-SARS-CoV-2 antibodies (Abs). The aim of this study is to develop a new rapid and cost-effective method for the detection of anti-SARS-CoV-2 IgG/IgM Abs. A new top-loading detection device was developed and composed of a small piece of plastic (25 × 25 × 0.5 mm) with an opening in the center, a piece of nitrocellulose (NC) membrane enough to block the opening from one side and adhesive tape to affix the NC to the plastic piece. The NC is blotted with anti-human IgG/IgM and rabbit serum. The device was evaluated against a commercially available IgG/IgM ELISA detection kit using normal, Covid-19-positive, HCV, HBV, and Cytomegalovirus-positive sera. Outcomes demonstrated simplicity, reproducibility, and accuracy of the new device and results can be obtained in less than 5 min. We anticipate our developed assay method to be used widely in point of care before deciding on the use of expensive nucleic acid assays.


Assuntos
COVID-19/diagnóstico , Imunoensaio/métodos , SARS-CoV-2/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/imunologia , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Testes Imediatos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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