Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase–quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98–0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24–0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92–0.99), specificity (range 0.93–0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87–0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.

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EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION

10.1101/2020.10.09.20210039 ◽

2020 ◽

Author(s):

Beatriz Araujo Oliveira ◽

Lea Campos de Oliveira ◽

Franciane Mendes de Oliveira ◽

Geovana Maria Pereira ◽

Regina Maia de Souza ◽

...

Keyword(s):

Sensitivity And Specificity ◽

High Sensitivity ◽

Diagnostic Techniques ◽

Cross Reactivity ◽

Cross Reaction ◽

List Type ◽

Rt Pcr ◽

Serum Samples ◽

Igm Antibodies ◽

Good Agreement

AbstractBackgroundCOVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population.MethodsHere we evaluate the sensitivity and specificity of eleven different immunochromatographic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests.ResultsOur results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%.ConclusionsWe found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.HighlightsImmunochromatographic assays demonstrated high sensitivity and specificity and good agreement with the gold-standard RT-PCR;Increase in sensitivity and specificity of assays using samples collected after the 10th day of symptoms;Cross-reaction with Dengue serology in evaluation of IgM.

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Detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in Henan Province, China, using real-time reverse transcription PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.049577-0 ◽

2013 ◽

Vol 62 (7) ◽

pp. 1060-1064 ◽

Cited By ~ 8

Author(s):

Xueyong Huang ◽

Licheng Liu ◽

Yanhua Du ◽

Hongxia Ma ◽

Yujiao Mu ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

High Sensitivity ◽

Cross Reactivity ◽

Henan Province ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Serum Samples ◽

Reverse Transcription Pcr

A novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome (FTLS) was discovered in Henan Province, China. Here, we report the development of an assay for this novel bunyavirus based on real-time reverse transcription PCR (RT-PCR). The assay exhibited high sensitivity and specificity without cross-reactivity towards 13 other viruses that cause similar symptoms. To evaluate the performance of this assay in detecting clinical samples, we analysed 261 serum samples from patients in Henan Province between 2007 and 2010. Of these samples, 91.95 % were bunyavirus positive. Compared with serological assays, the real-time PCR assay was much more sensitive in identifying infected patients 1 to 7 days after the onset of symptoms.

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Automated Western immunoblotting detection of anti-SARS-CoV-2 serum antibodies

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04203-8 ◽

2021 ◽

Author(s):

Sophie Edouard ◽

Rita Jaafar ◽

Nicolas Orain ◽

Philippe Parola ◽

Philippe Colson ◽

...

Keyword(s):

Negative Control ◽

Cross Reactivity ◽

Spike Protein ◽

Rt Pcr ◽

Substantial Agreement ◽

Western Immunoblotting ◽

Elisa Kit ◽

Line Test ◽

Serologic Assay ◽

Control Samples

AbstractELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary serologic assay for COVID-19. The JessTM Simple Western system, an automated capillary-based assay, was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as the source of antigen, and total immunoglobulins (IgG, IgM, IgA) detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen’s Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2.

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Novel circulating protein biomarkers for thyroid cancer determined through data-independent acquisition mass spectrometry

PeerJ ◽

10.7717/peerj.9507 ◽

2020 ◽

Vol 8 ◽

pp. e9507

Author(s):

Dandan Li ◽

Jie Wu ◽

Zhongjuan Liu ◽

Ling Qiu ◽

Yimin Zhang

Keyword(s):

Mass Spectrometry ◽

Area Under The Curve ◽

High Sensitivity ◽

Factor H ◽

Complement Factor ◽

Protein Biomarkers ◽

Serum Samples ◽

Data Independent Acquisition ◽

Median Serum ◽

And Control

Background Distinguishing between different types of thyroid cancers (TC) remains challenging in clinical laboratories. As different tumor types require different clinical interventions, it is necessary to establish new methods for accurate diagnosis of TC. Methods Proteomic analysis of the human serum was performed through data-independent acquisition mass spectrometry for 29 patients with TC (stages I–IV): 13 cases of papillary TC (PTC), 10 cases of medullary TC (MTC), and six cases follicular TC (FTC). In addition, 15 patients with benign thyroid nodules (TNs) and 10 healthy controls (HCs) were included in this study. Subsequently, 17 differentially expressed proteins were identified in 291 patients with TC, including 247 with PTC, 38 with MTC, and six with FTC, and 69 patients with benign TNs and 176 with HC, using enzyme-linked immunosorbent assays. Results In total, 517 proteins were detected in the serum samples using an Orbitrap Q-Exactive-plus mass spectrometer. The amyloid beta A4 protein, apolipoprotein A-IV, gelsolin, contactin-1, gamma-glutamyl hydrolase, and complement factor H-related protein 1 (CFHR1) were selected for further analysis. The median serum CFHR1 levels were significantly higher in the MTC and FTC groups than in the PTC and control groups (P < 0.001). CFHR1 exhibited higher diagnostic performance in distinguishing patients with MTC from those with PTC (P < 0.001), with a sensitivity of 100.0%, specificity of 85.08%, area under the curve of 0.93, and detection cut-off of 0.92 ng/mL. Conclusion CFHR1 may serve as a novel biomarker to distinguish PTC from MTC with high sensitivity and specificity.

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A rapid, sensitive enzyme-linked immunoassay for human thyrotropin.

Clinical Chemistry ◽

10.1093/clinchem/31.7.1131 ◽

1985 ◽

Vol 31 (7) ◽

pp. 1131-1134 ◽

Cited By ~ 11

Author(s):

Y C Tseng ◽

K D Burman ◽

J R Baker ◽

L Wartofsky

Keyword(s):

Color Change ◽

Clinical Laboratory ◽

High Sensitivity ◽

Normal Subjects ◽

Turnaround Time ◽

Cross Reactivity ◽

Serum Samples ◽

Assay Sensitivity ◽

Nitrophenyl Phosphate ◽

Set Up

Abstract In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.

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Measuring the palpable pulsatility length as a physical examination test in defining the severity of inflow stenosis for hemodialysis fistulas

BMC Nephrology ◽

10.1186/s12882-019-1536-2 ◽

2019 ◽

Vol 20 (1) ◽

Author(s):

Matt Chiung-Yu Chen ◽

Mei-Jui Weng ◽

Misoso Yi-Wen Wu ◽

Yi-Chun Liu ◽

Wen-Che Chi

Keyword(s):

Operating Characteristic ◽

Area Under The Curve ◽

High Sensitivity ◽

Characteristic Analysis ◽

Main Trunk ◽

Arteriovenous Fistulas ◽

Cannulation Site ◽

Before And After ◽

Physical Examinations ◽

Hemodialysis Fistulas

Abstract Background Pulsatility is an important property of hemodialysis arteriovenous fistulas (AVF) and can be perceived by the fingers as a gradual decrease in strength downstream from the anastomosis along the main trunk of the fistula. The distance from the point at which the pulse becomes imperceptible to the anastomosis is termed the palpable pulsatility length (PPL); we considered this length may play a role in assessing the severity of inflow stenosis for hemodialysis fistulas. Methods This study was performed by retrospective analysis of routinely collected data. Physical examinations and fistula measurements were performed in a selected population of 76 hemodialysis patients with mature fistulas during half a year. Fistula measurements included the PPL before and after treatment and the distance between the anastomosis and the arterial cannulation site (aPump length). The aPump index (API) was calculated by dividing the PPL by the aPump length. Angiograms were reviewed to determine the location and severity of stenosis. PPL and API were used to detect the critical inflow stenosis, which indicates severe inflow stenosis of an AVF. Results Receiver operating characteristic analysis showed that the area under the curve was 0.895 for API and 0.878 for PPL. A cutoff value of API < 1.29 and PPL < 11.0 cm were selected to detect the critical inflow stenosis. The sensitivity was 96.0% versus 80.0% and specificity was 84.31% versus 84.31% for API and PPL, respectively. Conclusions PPL and API are useful tools in defining the severity of pure inflow stenosis for mature AVFs in the hands of trained examiners with high sensitivity and specificity.

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Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.2.374-377.2002 ◽

2002 ◽

Vol 9 (2) ◽

pp. 374-377 ◽

Cited By ~ 6

Author(s):

Daniela A. Cunha ◽

Roseli M. Zancopé-Oliveira ◽

M. Sueli ◽

S. Felipe ◽

Silvia M. Salem-Izacc ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Paracoccidioides Brasiliensis ◽

High Sensitivity ◽

Cross Reactivity ◽

Antibody Responses ◽

Healthy Individuals ◽

Immunological Reactivity ◽

Serum Samples ◽

Recombinant Antigens

ABSTRACT The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

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Parasite-derived circulating microRNAs as biomarkers for the detection of human Schistosoma japonicum infection

Parasitology ◽

10.1017/s0031182019001690 ◽

2019 ◽

Vol 147 (8) ◽

pp. 889-896 ◽

Cited By ~ 1

Author(s):

Yi Mu ◽

Pengfei Cai ◽

Remigio M. Olveda ◽

Allen G. Ross ◽

David U. Olveda ◽

...

Keyword(s):

Schistosoma Japonicum ◽

Diagnostic Performance ◽

Area Under The Curve ◽

Infection Intensity ◽

The Philippines ◽

Pcr Analysis ◽

Rt Pcr ◽

Serum Samples ◽

Sensitivity Specificity ◽

Pcr Targeting

AbstractNovel tools for early diagnosis and monitoring of schistosomiasis are urgently needed. This study aimed to validate parasite-derived miRNAs as potential novel biomarkers for the detection of human Schistosoma japonicum infection. A total of 21 miRNAs were initially validated by real-time-polymerase chain reaction (RT-PCR) using serum samples of S. japonicum-infected BALB/c mice. Of these, 6 miRNAs were further validated with a human cohort of individuals from a schistosomiasis-endemic area of the Philippines. RT-PCR analysis showed that two parasite-derived miRNAs (sja-miR-2b-5p and sja-miR-2c-5p) could detect infected individuals with low infection intensity with moderate sensitivity/specificity values of 66%/68% and 55%/80%, respectively. Analysis of the combined data for the two parasite miRNAs revealed a specificity of 77.4% and a sensitivity of 60.0% with an area under the curve (AUC) value of 0.6906 (P = 0.0069); however, a duplex RT-PCR targeting both sja-miR-2b-5p and sja-miR-2c-5p did not result in an increased diagnostic performance compared with the singleplex assays. Furthermore, the serum level of sja-miR-2c-5p correlated significantly with faecal egg counts, whereas the other five miRNAs did not. Targeting S. japonicum-derived miRNAs in serum resulted in a moderate diagnostic performance when applied to a low schistosome infection intensity setting.

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Correlation Between Chest CT Findings and Clinical Features of 211 COVID-19 Suspected Patients in Wuhan, China

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa171 ◽

2020 ◽

Vol 7 (6) ◽

Cited By ~ 2

Author(s):

Songlin Song ◽

Feihong Wu ◽

Yiming Liu ◽

Hongwei Jiang ◽

Fu Xiong ◽

...

Keyword(s):

Clinical Features ◽

Area Under The Curve ◽

High Sensitivity ◽

Pulmonary Involvement ◽

Chest Ct ◽

Chain Reaction ◽

Basic Model ◽

Group 4 ◽

Significant Difference ◽

Polymerase Chain

Abstract Background Chest computed tomography (CT) has been widely used to assess pulmonary involvement in COVID-19. We aimed to investigate the correlation between chest CT and clinical features in COVID-19 suspected patients with or without fever. Methods We retrospectively enrolled 211 COVID-19 suspected patients who underwent both chest CT and reverse transcription polymerase chain reaction in Wuhan, China. The performance of CT in patients with relevant onset of symptoms, with fever (n = 141) and without fever (n = 70), was assessed respectively. Results The sensitivity of CT for COVID-19 was 97.3%, with area under the curve (AUC) of 0.71 (95% confidence interval [CI], 0.66–0.76). There were 141 suspected patients with fever and 70 without fever. In the fever group, 4 variables were screened to establish the basic model: age, monocyte, red blood cell, and hypertension. The AUC of the basic model was 0.72 (95% CI, 0.63–0.81), while the AUC of the CT-aided model was 0.77 (95% CI, 0.68–0.85), a significant difference (P < .05). In the nonfever group, only dry cough was screened out to establish the basic model. The AUC was 0.76 (95% CI, 0.64–0.88), which was not significantly different than the CT-aided model (P = .08). Conclusions Chest CT has a high sensitivity in patients with COVID-19, and it can improve diagnostic accuracy for COVID-19 suspected patients with fever during the initial screen, whereas its value for nonfever patients remains questionable.

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Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

Clinical and Vaccine Immunology ◽

10.1128/cvi.00295-09 ◽

2009 ◽

Vol 16 (12) ◽

pp. 1774-1780 ◽

Cited By ~ 25

Author(s):

Eduardo A. F. Coelho ◽

Laura Ramírez ◽

Mariana A. F. Costa ◽

Vinicio T. S. Coelho ◽

Vivian T. Martins ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Ribosomal Proteins ◽

High Sensitivity ◽

Leishmania Species ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Canine Visceral Leishmaniasis ◽

Leishmania Chagasi ◽

Serum Samples ◽

Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

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