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1.
PLoS One ; 16(2): e0246346, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33529223

RESUMEN

BACKGROUND: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. AIM: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples. METHODS: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined. RESULTS: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases. CONCLUSION: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.


Asunto(s)
Anticuerpos Antivirales/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , /aislamiento & purificación , /diagnóstico , Ensayo de Inmunoadsorción Enzimática/instrumentación , Humanos , Inmunoglobulina G/análisis , Fosfoproteínas/análisis , Sensibilidad y Especificidad
2.
mSphere ; 6(1)2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536325

RESUMEN

Reported coronavirus disease 2019 (COVID-19) case counts likely underestimate the true prevalence because mild or asymptomatic cases often go untested. Here, we use a sero-survey to estimate the seroprevalence of IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the St. Louis, MO, metropolitan area in a symptom-independent manner. Five hundred three adult and 555 pediatric serum/plasma samples were collected from patients presenting to Barnes-Jewish Hospital or St. Louis Children's Hospital between 14 April 2020 and 12 May 2020. We developed protocols for in-house enzyme-linked immunosorbent assays (ELISAs) using spike and nucleoprotein and used the assays to estimate a seroprevalence rate based on our samples. Overall IgG seropositivity was estimated to be 1.71% (95% credible interval [CI], 0.04% to 3.38%) in pediatric samples and 3.11% (95% CI, 0.92% to 5.32%) in adult samples. Seropositivity was significantly lower in children under 5 years of age than in adults, but rates between adults and children aged 5 or older were similar. Of the 176 samples tested from children under 4 years of age, none were positive.IMPORTANCE This study determined the percentages of both children and adult samples from the greater St. Louis metropolitan area who had antibodies to SARS-CoV-2 in late April to early May 2020. Approximately 1.7 to 3.1% of the tested individuals had antibodies, indicating that they had previously been infected by SARS-CoV-2. These results demonstrate that the extent of infection was about 10 times greater than the number of confirmed cases at that time. Furthermore, it demonstrated that by 5 years of age, children were infected to an extent similar to that of adults.


Asunto(s)
Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , /inmunología , /inmunología , Adolescente , Adulto , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Lactante , Masculino , Persona de Mediana Edad , Missouri/epidemiología , Estudios Seroepidemiológicos , Adulto Joven
3.
Medicine (Baltimore) ; 100(3): e23961, 2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33545977

RESUMEN

INTRODUCTION: This protocol is for a meta analysis that aims to systematically review the diagnostic value of anti-hepatitis B virus in serum tested by the enzyme linked immunosorbent assay in patients with hepatitis B. METHODS AND ANALYSIS: The following electronic databases will be searched from inception to Mar 2021: PubMed, Web of Science, ScienceDirect, EMBASE, MEDLINE, Springer, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, VIP Chinese Science and Technology Periodical Database, and Wanfang Database. All study about enzyme linked immunosorbent assay reagents have been published at home and abroad to diagnose hepatitis B virus will be included. MetaDisc 1.4 soft will used to calculate pooled effect size in sensitivity, specifi city, likelihood ratio, diagnostic odds ratio and summary receiver operating characteristic curve, and area under the curve as well. ETHICS AND DISSEMINATION: Formal ethical approval is not required, as the data are not individualized. The findings of this systematic review will be disseminated in a peer-reviewed publication and/or presented at relevant conferences. REGISTRATION NUMBER: INPLASY2020100051.


Asunto(s)
Protocolos Clínicos , Virus de la Hepatitis B/inmunología , Hepatitis B/enzimología , Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Hepatitis B/patogenicidad , Humanos , Metaanálisis como Asunto , Revisiones Sistemáticas como Asunto
4.
Mem Inst Oswaldo Cruz ; 115: e200287, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33533869

RESUMEN

BACKGROUND: The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES: To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS: Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS: Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS: Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.


Asunto(s)
Antígenos Virales/sangre , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Inmunológicas/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas no Estructurales Virales/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Dengue/sangre , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/inmunología , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/genética
5.
Nat Commun ; 12(1): 113, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397956

RESUMEN

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.


Asunto(s)
Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , /inmunología , Anticuerpos Antivirales/inmunología , /epidemiología , /métodos , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Pandemias , Estándares de Referencia , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/inmunología
6.
Sensors (Basel) ; 21(2)2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33429915

RESUMEN

The outbreak of the coronavirus disease (COVID-19) pandemic caused by the novel coronavirus (SARS-CoV-2) has been declared an international public health crisis. It is essential to develop diagnostic tests that can quickly identify infected individuals to limit the spread of the virus and assign treatment options. Herein, we report a proof-of-concept label-free electrochemical immunoassay for the rapid detection of SARS-CoV-2 virus via the spike surface protein. The assay consists of a graphene working electrode functionalized with anti-spike antibodies. The concept of the immunosensor is to detect the signal perturbation obtained from ferri/ferrocyanide measurements after binding of the antigen during 45 min of incubation with a sample. The absolute change in the [Fe(CN)6]3-/4- current upon increasing antigen concentrations on the immunosensor surface was used to determine the detection range of the spike protein. The sensor was able to detect a specific signal above 260 nM (20 µg/mL) of subunit 1 of recombinant spike protein. Additionally, it was able to detect SARS-CoV-2 at a concentration of 5.5 × 105 PFU/mL, which is within the physiologically relevant concentration range. The novel immunosensor has a significantly faster analysis time than the standard qPCR and is operated by a portable device which can enable on-site diagnosis of infection.


Asunto(s)
Técnicas Biosensibles/instrumentación , /diagnóstico , Pruebas en el Punto de Atención , Glicoproteína de la Espiga del Coronavirus/análisis , Antígenos Virales/análisis , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Ensayo de Inmunoadsorción Enzimática/instrumentación , Ensayo de Inmunoadsorción Enzimática/métodos , Diseño de Equipo , Grafito , Humanos , Límite de Detección , Pandemias , Prueba de Estudio Conceptual , Subunidades de Proteína , Imagen Individual de Molécula/instrumentación , Imagen Individual de Molécula/métodos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Factores de Tiempo
7.
Arch Virol ; 166(3): 715-731, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33492524

RESUMEN

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


Asunto(s)
Anticuerpos Antivirales/sangre , /diagnóstico , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Síndrome Respiratorio Agudo Grave/diagnóstico , Anticuerpos Antivirales/inmunología , Técnicas Biosensibles/métodos , Western Blotting/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Mediciones Luminiscentes/métodos , Virus del SRAS/inmunología , Síndrome Respiratorio Agudo Grave/virología
8.
BMC Vet Res ; 17(1): 51, 2021 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-33494765

RESUMEN

BACKGROUND: Infectious bronchitis virus (IBV), a coronavirus, is one of the most important poultry pathogens worldwide due to its multiple serotypes and poor cross-protection. Vaccination plays a vital role in controlling the disease. The efficacy of vaccination in chicken flocks can be evaluated by detecting neutralizing antibodies with the neutralization test. However there are no simple and rapid methods for detecting the neutralizing antibodies. RESULTS: In this study, a peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine was developed. The pELISA could indirect evaluate neutralizing antibody titers against different types of IBV in all tested sera. The titers measured with the pELISA had a coefficient of 0.83 for neutralizing antibody titers. CONCLUSIONS: The pELISA could detect antibodies against different types of IBV in all tested sera. The pELISA has the potential to evaluate samples for IBV-specific neutralizing antibodies and surveillance the infection of IBV.


Asunto(s)
Infecciones por Coronavirus/prevención & control , Ensayo de Inmunoadsorción Enzimática , Virus de la Bronquitis Infecciosa/inmunología , Pruebas de Neutralización , Enfermedades de las Aves de Corral/prevención & control , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Pollos/inmunología , Pollos/virología , Infecciones por Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas de Neutralización/métodos , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
9.
Commun Biol ; 4(1): 129, 2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33514825

RESUMEN

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , /química , Glicoproteína de la Espiga del Coronavirus/química , Adulto , /inmunología , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , /virología , Convalecencia , /metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Sueros Inmunes/química , Inmunidad Humoral , Lentivirus/genética , Lentivirus/inmunología , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Fosfoproteínas/química , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Unión Proteica , Receptores Virales/química , Receptores Virales/inmunología , Receptores Virales/metabolismo , /patogenicidad , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Análisis de Supervivencia
10.
PLoS Pathog ; 17(1): e1009161, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33444413

RESUMEN

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs.


Asunto(s)
/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Argentina/epidemiología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pandemias , Estudios Seroepidemiológicos
11.
Food Chem ; 337: 127617, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-32799156

RESUMEN

In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a broad-spectrum monoclonal antibody for tropane alkaloids (TAs) was established for the rapid screening of atropine, scopolamine, homatropine, apoatropine, anisodamine, anisodine and L-hyoscyamine residues in pig urine, pork and cereal flour samples through a simple sample preparation procedure. The half inhibitory concentrations of atropine, homatropine, L-hyoscyamine, apoatropine, scopolamine, anisodamine and anisodine were 0.05, 0.07, 0.14, 0.14, 0.24, 5.30 and 10.15 ng mL-1, respectivelyThe detection and quantitative limits of this method for TAs in samples were 0.18-73.18 and 0.44-74.77 µg kg-1. The spiked recoveries ranged from 69.88% to 147.93%, and the coefficient of variations were less than 14%. Good correlation (R2 = 0.9929) between the results of the ic-ELISA and the high performance liquid chromatography-tandem mass spectrometry support the reliability of the developed ic-ELISA method.


Asunto(s)
Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/métodos , Harina/análisis , Carne de Cerdo/análisis , Tropanos/análisis , Animales , Anticuerpos Monoclonales/inmunología , Atropina/análisis , Atropina/orina , Cromatografía Líquida de Alta Presión/métodos , Femenino , Análisis de los Alimentos/métodos , Ratones Endogámicos BALB C , Reproducibilidad de los Resultados , Escopolamina/análisis , Escopolamina/orina , Alcaloides Solanáceos/análisis , Alcaloides Solanáceos/orina , Porcinos , Espectrometría de Masas en Tándem , Tropanos/inmunología , Tropanos/orina
12.
Food Chem ; 335: 127600, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-32736155

RESUMEN

Toosendanin (TSN), as an important Chinese traditional insecticide, has been registered and commercialized in China. In this report, the residual analytical methods, residue dynamics and final residues of TSN in tobacco, cabbage and soil under field condition were studied by IC-ELISA and HPLC. The sensitivity, precision and repeatability of IC-ELISA method were more suitable in comparison with HPLC for the demand of TSN residue analysis. Using IC-ELISA, the half-lives (t1/2) of TSN were found to be 1.30 days in cabbage, 1.70 days in tabacco and 0.71 days in soil, respectively. At the recommended dose, the final residues of TSN detection by IC-ELISA was 0.009 mg·kg-1 in cabbage and 0.043 mg·kg-1 in tobacco, as well as was not detected in soil. Therefore, TSN is easily degradable, and IC-ELISA could be a convenient and supplemental analytical tool for monitoring TSN residue in crops and environment.


Asunto(s)
Brassica/metabolismo , Medicamentos Herbarios Chinos/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Contaminantes del Suelo/metabolismo , Suelo/química , China , Cromatografía Líquida de Alta Presión/métodos , Semivida , Cinética , Residuos de Plaguicidas/análisis , Tabaco
13.
Int J Nanomedicine ; 15: 9975-9985, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33363367

RESUMEN

Background: As two important tumor markers, vascular endothelial growth factor (VEGF) and carcinoembryonic antigen (CEA) have a great value for clinical application in the early diagnosis of cancer. Due to the complex composition of biological samples, the results from combined detection of CEA and VEGF are often taken as a comprehensive indicator in order to make an accurate judgment on a disease. However, most of the current methods can only be used to detect the content of one biomarker. Therefore, it is necessary to explore a simple, rapid, low-cost, and highly sensitive method for the simultaneous detection of CEA and VEGF. Methods: Based on specific aptamers and magnetic separation, a time-resolved chemiluminescence enzyme-linked aptamer assay was developed for the simultaneous detections of CEA and VEGF in serum samples. Results: Under the optimal conditions, the linear range of the calibration curve for VEGF was from 0.5 to 80 ng mL-1, and the limit of detection was 0.1 ng mL-1. The linear range of the calibration curve for CEA was 0.5 to 160 ng mL-1, and the limit of detection was 0.1 ng mL-1. The established method was applied to detect VEGF and CEA in serum samples. The results were consistent with those of commercial kits. Conclusion: The method has high sensitivity and can quickly obtain accurate results, which could greatly improve the measurement efficiency, reduce the cost, and also reduce the volume of sample consumed. It can be seen that the method established in this study has important application value and broad application prospect in clinical diagnosis.


Asunto(s)
Aptámeros de Péptidos/metabolismo , Antígeno Carcinoembrionario/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Mediciones Luminiscentes , Factor A de Crecimiento Endotelial Vascular/sangre , Fosfatasa Alcalina/metabolismo , Biocatálisis , Calibración , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Cinética , Mediciones Luminiscentes/métodos , Fenómenos Magnéticos , Factores de Tiempo
14.
Ann Clin Lab Sci ; 50(6): 852-854, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33334805

RESUMEN

Due to the COVID-19 pandemic, the Food and Drug Administration issued an Emergency Use Authorization to permit developers of certain serological tests to market their product prior to a comprehensive review. Nonetheless, the reliability of these assays is of great importance in order to be useful as a tool in estimating the relative proportions of different populations that have been exposed to SARS-CoV-2. We provide a sampling of 145 individuals from an ambulatory setting simultaneously tested with a qualitative point of care rapid finger prick Lateral Wave® IgM and IgG assay and a sample for the Mayo Clinic enzyme linked immunosorbent assay (ELISA) IgM/IgG antibody assay. Significant discrepancies did exist between the purported antibody responses as demonstrated by each assay.


Asunto(s)
Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Sistemas de Atención de Punto/normas , /aislamiento & purificación , Bioensayo , /virología , Humanos , Pruebas Serológicas/métodos
15.
J Insect Sci ; 20(6)2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-33347589

RESUMEN

A molecular gut analysis technique is described to identify predators of Lygus hesperus (Knight), a significant pest of many crops. The technique is unique because it can pinpoint which life stage of the pest was consumed. Sentinel egg masses designed to mimic the endophytic egg-laying behavior of L. hesperus were marked with rabbit serum, while third instar and adult L. hesperus were marked with chicken and rat sera, respectively. Then, the variously labeled L. hesperus life stages were introduced into field cages that enclosed the native arthropod population inhabiting an individual cotton plant. After a 6-h exposure period, the predator assemblage, including the introduced and native L. hesperus population, in each cage were counted and had their gut contents examined for the presence of the variously marked L. hesperus life stages by a suite of serum-specific enzyme-linked immunosorbent assays (ELISA). The whole-plant sampling scheme revealed that Geocoris punticpes (Say) and Geocoris pallens Stal (Hemiptera: Geocoridae) and members of the spider complex were the numerically dominant predator taxa in the cotton field. The gut content analyses also showed that these two taxa appeared to be the most prolific predators of the L. hesperus nymph stage. Other key findings include that Collops vittatus (Say) (Coleoptera: Melyridae) and Solenopsis xyloni McCook (Hymenoptera: Formicidae) appear to be adept at finding and feeding on the cryptic L. hesperus egg stage, and that L. hesperus, albeit at low frequencies, engaged in cannibalism. The methods described here could be adapted for studying life stage-specific feeding preferences for a wide variety of arthropod taxa.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Hemípteros/inmunología , Conducta Predatoria , Animales , Hormigas , Escarabajos , Huevos , Conducta Alimentaria , Ninfa/inmunología , Arañas
16.
Zhonghua Nan Ke Xue ; 26(6): 518-521, 2020 Jun.
Artículo en Chino | MEDLINE | ID: mdl-33356040

RESUMEN

Objective: To compare two ELISA methods for the detection of the prostatic exosomal protein (PSEP) in the urine. METHODS: Using the double-antibody sandwich (DAS) method and the indirect method of ELISA, we detected PSEP in the urine samples from 100 IIIA chronic prostatitis (CP) patients and another 100 normal healthy males. Meanwhile, we examined 30 clinical urine samples using the diluent (0.1 mol/L PBS buffer) and the urine matrix standard curves to verify the consistency of the standard diluent with the sample collected. Result: The sensitivities of DAS and indirect ELISA were 89% versus 87% and their specificities 91% versus 90%, with total consistency rates of 90% versus 88.5%, with no statistically significant difference in between. The scatter plot for the results of the PBS diluent and the urine matrix standard curves showed a good linearity (R2 = 0.999). No significant difference was found in the results of detection of the clinical urine samples in different matrices. CONCLUSIONS: Considering the characteristics of PSEP, the indirect ELISA method is more practical and feasible for the clinical detection of PSEP in the urine samples of prostatitis patients.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Exosomas , Prostatitis , Proteinuria/diagnóstico , Enfermedad Crónica , Humanos , Masculino , Prostatitis/diagnóstico , Proteínas/análisis
17.
Biomed Res Int ; 2020: 9878453, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33224987

RESUMEN

Knowledge of the sensitivities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests beyond 35 days after the clinical onset of COVID-19 is insufficient. We aimed to describe positivity rate of SARS-CoV-2 assays employing three different measurement principles over a prolonged period. Two hundred sixty-eight samples from 180 symptomatic patients with COVID-19 and a reverse transcription polymerase chain reaction (RT-PCR) test followed by serological investigation of SARS-CoV-2 antibodies were included. We conducted three chemiluminescence (including electrochemiluminescence assay (ECLIA)), four enzyme-linked immunosorbent assay (ELISA), and one lateral flow immunoassay (LFIA) test formats. Positivity rates, as well as positive (PPVs) and negative predictive values (NPVs), were calculated for each week after the first clinical presentation for COVID-19. Furthermore, combinations of tests were assessed within an orthogonal testing approach employing two independent assays and predictive values were calculated. Heat maps were constructed to graphically illustrate operational test characteristics. During a follow-up period of more than 9 weeks, chemiluminescence assays and one ELISA IgG test showed stable positivity rates after the third week. With the exception of ECLIA, the PPVs of the other chemiluminescence assays were ≥95% for COVID-19 only after the second week. ELISA and LFIA had somewhat lower PPVs. IgM exhibited insufficient predictive characteristics. An orthogonal testing approach provided PPVs ≥ 95% for patients with a moderate pretest probability (e.g., symptomatic patients), even for tests with a low single test performance. After the second week, NPVs of all but IgM assays were ≥95% for patients with low to moderate pretest probability. The confirmation of negative results using an orthogonal algorithm with another assay provided lower NPVs than the single assays. When interpreting results from SARS-CoV-2 tests, the pretest probability, time of blood draw, and assay characteristics must be carefully considered. An orthogonal testing approach increases the accuracy of positive, but not negative, predictions.


Asunto(s)
Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Anticuerpos Antivirales/sangre , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunoensayo/métodos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Pruebas Serológicas/métodos
18.
Viruses ; 12(11)2020 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-33171590

RESUMEN

Knowledge of the antibody-mediated immune response to SARS-CoV-2 is crucial to understand virus immunogenicity, establish seroprevalence, and determine whether subjects or recovered patients are at risk for infection/reinfection and would therefore benefit from vaccination. Here, we describe a novel and simple cell-ELISA specifically designed to measure viral spike S1-specific IgG produced in vitro by B cells in peripheral blood mononuclear cell (PBMC) cultures from a cohort of 45 asymptomatic (n = 24) and symptomatic (n = 21) individuals, with age ranging from 8 to 99 years. All subjects underwent ELISA serological screening twice, at the same time as the cell-ELISA (T2) as well as 35-60 days earlier (T1). Cryopreserved PBMCs of healthy donors obtained years before the COVID-19 pandemic were also included in the analysis. The preliminary results presented here show that out of 45 tested subjects, 16 individuals (35.5%) were positive to the cell-ELISA, 11 (24.5%) were concomitantly positive in the serological screening (T1 and/or T2), and only one person was exclusively positive in ELISA (T1) and negative in cell-ELISA, though values were close to the cutoff. Of note, five individuals (11.2%) tested negative in ELISA but positive in cell-ELISA and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum, which challenges the negative results of the serological screening. The relative level of in vitro secreted IgG was measurable in positive subjects, ranging from 7 to 50 ng/well. Accordingly, all anti-SARS-CoV-2 antibody-positive subjects previously reported moderate to severe symptoms attributable to COVID-19, even though the RT-PCR data were rarely available to confirm viral infection. Overall, the described cell-ELISA might be an effective method for detecting subjects who encountered the virus in the past, and thus helpful to improve serological ELISA tests in the case of undetectable/equivocal circulating IgG levels, and a suitable and improved tool to better evaluate SARS-CoV-2-specific humoral immunity in the COVID-19 pandemic.


Asunto(s)
Anticuerpos Antivirales/sangre , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Neumonía Viral/diagnóstico , Glicoproteína de la Espiga del Coronavirus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Betacoronavirus/inmunología , Niño , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Pandemias , Pruebas Serológicas , Adulto Joven
20.
BMC Infect Dis ; 20(1): 790, 2020 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-33096994

RESUMEN

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, mainly among adults. JCV is widely distributed in North America and the number of JCV cases in the U.S. has increased in recent years. Therefore, the central nervous system disease caused by JCV can be considered a potentially re-emerging viral disease. However, the seroprevalence of JCV is unknown in Japan. The purpose of this study is to evaluate the seroprevalence of JCV in the Japanese population. METHODS: We used an IgG enzyme-linked immunosorbent assay (IgG-ELISA) with JCV-infected cell-lysates and/or a neutralizing (NT) antibody assay. The cut-off value of IgG-ELISA was determined using IgG-ELISA to analyze serum specimens from 37 healthy Japanese donors. IgG-ELISA was validated by assessing its sensitivity and specificity, using 38 human serum samples previously tested for the presence or absence of antibodies against JCV and snowshoe hare virus (SSHV), in an in-house NT antibody assay conducted by the Public Health Agency of Canada. The seroepidemiological study was performed using IgG-ELISA and NT antibody assay to analyze 246 human serum samples from the serum bank of the National Institute of Infectious Diseases (NIID) in Japan. RESULTS: The cut-off value of IgG-ELISA was determined at 0.20, based on the mean (- 0.075) and standard deviation (0.092) values using Japanese donors' sera. The sensitivity and the specificity of IgG-ELISA determined using 25 JCV-positive and 4 JCV-negative serum samples were 96 and 100%, respectively. Analysis of the 246 Japanese serum samples revealed that no specimen showed a higher value than the cut-off value of IgG-ELISA, and no sample tested positive by the NT antibody assay. CONCLUSIONS: Our results showed that JCV is not circulating significantly in Japan. To the best of our knowledge, this is the first report to demonstrate the seroprevalence of JCV in the general population in Japan.


Asunto(s)
Anticuerpos Antivirales/inmunología , Virus de la Encefalitis de California/inmunología , Encefalitis de California/epidemiología , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas de Neutralización/métodos , Adolescente , Adulto , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Niño , Preescolar , Culicidae/virología , Encefalitis de California/virología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Lactante , Recién Nacido , Japón/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Adulto Joven
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