Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers

Prompt COVID-19 diagnosis is urgently required to support infection control measures. Currently available serological tests for measuring SARS-CoV-2 antibodies use different target antigens, although their sensitivity and specificity presents a challenge. We aimed to develop an “in-house” serological ELISA to measure antibodies against SARS-CoV-2 by combining different protein antigens. Sera (n = 44) from COVID-19-confirmed patients were evaluated against different SARS-CoV-2 protein antigens and all potential combinations using ELISA. Patients’ sera were also evaluated against commercially available ELISA diagnostic kits. The mixture containing RBD 2.5 μg/mL, S2 1 μg/mL and N 1.5 μg/mL was found to be the most potent. Plates were incubated with patients’ sera (1:100), and goat anti-human alkaline phosphatase-conjugated IgG, ΙgM and IgA antibody was added. The cut-off value for each assay was determined using the mean optical density plus two standard deviations of pre-pandemic controls. The “in-house” ELISA displayed 91% sensitivity and 97% specificity for IgG antibodies, whereas its sensitivity and specificity for IgM and IgA were 75% and 95% and 73% and 91%, respectively. The “in-house” ELISA developed here combined three SARS-CoV-2 antigens (RBD, S2 and N) as capture antigens and displayed comparable and even higher sensitivity and specificity than otherwise quite reliable commercially available ELISA diagnostic kits.

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Evaluation of the performance of three serological tests for diagnosis of Leishmania infantum infection in dogs using latent class analysis

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612020105 ◽

2020 ◽

Vol 29 (4) ◽

Author(s):

Asier Basurco ◽

Alda Natale ◽

Katia Capello ◽

Antonio Fernández ◽

María Teresa Verde ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Leishmania Infantum ◽

Latent Class ◽

Rapid Test ◽

Antibody Test ◽

Latent Class Models ◽

Canine Leishmaniasis ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests

Abstract Canine leishmaniasis (CanL) is a disease caused by Leishmania infantum. Serological methods are the most common diagnostic techniques used for the diagnosis of the CanL. The objective of our study was to estimate the sensitivity and specificity of one in-house ELISA kit (ELISA UNIZAR) and three commercially available serological tests (MEGACOR Diagnostik GmbH) including an immunochromatographic rapid test (FASTest LEISH®), an immunofluorescent antibody test (MegaFLUO LEISH®) and an enzyme-linked immunosorbent assay (MegaELISA LEISH®), using latent class models in a Bayesian analysis. Two hundred fifteen serum samples were included. The highest sensitivity was achieved for FASTest LEISH® (99.38%), ELISA UNIZAR (99.37%), MegaFLUO LEISH® (99.36%) followed by MegaELISA LEISH® (98.49%). The best specificity was obtained by FASTest LEISH® (98.43%), followed by ELISA UNIZAR (97.50%), whilst MegaFLUO LEISH® and MegaELISA LEISH® obtained the lower specificity (91.94% and 91.93%, respectively). The results of present study indicate that the immunochromatographic rapid test evaluated FASTest LEISH® show similar levels of sensitivity and specificity to the quantitative commercial tests. Among quantitative serological tests, sensitivity and specificity were similar considering ELISA or IFAT techniques.

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Diagnostic Accuracy of BD Phoenix CPO Detect for Carbapenemase Production in 190 Enterobacteriaceae Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.01043-18 ◽

2018 ◽

Vol 56 (12) ◽

Cited By ~ 11

Author(s):

Chiou Horng Ong ◽

Lasantha Ratnayake ◽

Michelle L. T. Ang ◽

Raymond Tzer Pin Lin ◽

Douglas Su Gin Chan

Keyword(s):

Confidence Interval ◽

Infection Control ◽

Diagnostic Accuracy ◽

Positive Result ◽

Sensitivity And Specificity ◽

Patient Management ◽

Susceptibility Testing ◽

Control Measures ◽

Content Type ◽

Infection Control Measures

ABSTRACT The rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) is necessary for patient management and infection control measures. We compared the performance of the BD Phoenix CPO Detect with that of a homemade Carba NP assay and a modified carbapenem inactivation method (mCIM) by challenging all 3 assays with 190 isolates of Enterobacteriaceae with meropenem MICs of >0.125 mg/liter. A total of 160 isolates produced KPC-, IMI-1-, NDM-, IMP-, and OXA-type carbapenemases, while 30 isolates were negative for carbapenemase production. The sensitivity and specificity were 90.6% (95% confidence interval [CI], 85.0% to 94.7%) and 100.0% (95% CI, 88.4% to 100.0%), respectively, for the Carba NP; 100.0% (95% CI, 97.7% to 100.0%) and 96.7% (95% CI, 82.7% to 99.9%), respectively, for the mCIM; and 89.4% (95% CI, 83.5% to 93.7%) and 66.7% (95% CI, 47.2% to 82.7%), respectively, for the BD Phoenix CPO Detect. In particular, the BD CPO Detect failed to detect a significant number of CPE with IMI-1. While the BD Phoenix CPO Detect is able to classify carbapenemases and is built into routine susceptibility testing with the potential to reduce the time to CPE detection, its low specificity means that a positive result will need confirmatory testing by another method.

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Performance of an Immunochromatographic Test (ICT) in Comparison to Some Commonly Used Serological Tests for the Diagnosis of Brucellosis in Dromedary Camels (Camelus dromedarius)

Microorganisms ◽

10.3390/microorganisms7120591 ◽

2019 ◽

Vol 7 (12) ◽

pp. 591

Author(s):

Serhan ◽

Khan ◽

Gasim ◽

Alketbi ◽

De Massis ◽

...

Keyword(s):

Small Ruminants ◽

Control Measures ◽

Camelus Dromedarius ◽

Enzyme Linked Immunosorbent Assay ◽

Lower Sensitivity ◽

Immunochromatographic Test ◽

Dromedary Camels ◽

Serological Tests ◽

Significant Difference ◽

Fluorescence Polarization Assay

Serological tests may represent an essential tool for the diagnosis of camel brucellosis; however, concerns arise in the scientific community regarding the direct transposition from cattle and small ruminants without adequate validation. The present study was made to compare four serological tests for the diagnosis of brucellosis in dromedary camels (Camelus dromedarius). In terms of sensitivity, our results show that the Immunochromatographic Test (ICT) shows the higher value of sensitivity, 98.67% (95% Confidence Level (C.L): 94.36%–99.99%), followed by the Fluorescence Polarization Assay (FPA) with 95.05% (95% C.L: 88.23%–99.51%), then the Competitive Enzyme-Linked Immunosorbent Assay (c-ELISA) with 94.94% (95% C.L: 88.25%–99.45%) and, finally, the Rose Bengal Test (RBT) with 68.95% (95% C.L: 56.55%–80.69%), which is the only test showing a significantly lower sensitivity compared to the others. On the other hand, our study revealed no significant difference in terms of specificity between all the tests under study, with a range from 99.06% (95% C.L: 98.34%–99.64%) for the ICT to 99.92% (95% C.L: 99.64%–100%) for the RBT. The ICT was found to be comparable in terms of sensitivity and specificity with the most commonly used tests for camel brucellosis. The results of the present study are of paramount importance for designing surveillance and control measures for brucellosis in camel populations.

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Serological Diagnosis of Brucella Infections in Odontocetes

Clinical and Vaccine Immunology ◽

10.1128/cvi.00413-08 ◽

2009 ◽

Vol 16 (6) ◽

pp. 906-915 ◽

Cited By ~ 11

Author(s):

Gabriela Hernández-Mora ◽

Charles A. Manire ◽

Rocío González-Barrientos ◽

Elías Barquero-Calvo ◽

Caterina Guzmán-Verri ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Brucella Melitensis ◽

Enzyme Linked Immunosorbent Assay ◽

Dot Blot ◽

Serum Samples ◽

Igg Antibody ◽

Serological Tests ◽

Diagnostic Potential ◽

Control Serum ◽

History Of

ABSTRACT Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a “gold” standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (R i/g 2 = 0.65 and κi/g = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (R i/c 2 = 0.46, R g/c 2 = 0.37 and κi/c = 0.62, κg/c = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.

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rSodC is a potential antigen to diagnose Corynebacterium pseudotuberculosis by enzyme-linked immunoassay

AMB Express ◽

10.1186/s13568-020-01125-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Antonio Pedro Fróes de Farias ◽

José Tadeu Raynal Rocha Filho ◽

Silvana Beutinger Marchioro ◽

Luan Santana Moreira ◽

Andressa Souza Marques ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Corynebacterium Pseudotuberculosis ◽

Semiarid Region ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Control Programs ◽

Epidemiological Surveys ◽

Purified Antigen

Abstract Caseous lymphadenitis (CL) is a chronic infectious disease that affects sheep and goats. Many serological tests have been developed to detect the disease; one of the most widely used is the enzyme-linked immunosorbent assay (ELISA), due to its advantages, which include acceptable cost-effectiveness, applicability, sensitivity and specificity. ELISA formulations using recombinant proteins can exhibit significant sensitivity and specificity when using a single purified antigen. DTxR, Trx, TrxR, LexA, SodC, SpaC, NanH, and PknG recombinant proteins can be considered target proteins for ELISA development due to its extracellular or on the cell surface location, which allows a better recognition by the immune system. Therefore, the objectives of this study were to evaluate the antigenic reactivity of Corynebacterium pseudotuberculosis recombinant proteins in goat and sheep serum. Of eight proteins evaluated, rSodC was selected for validation assays with small ruminant serum samples from the semiarid region of the state of Bahia, Brazil. Validation assays with goat serum samples showed that ELISA-rSodC presented sensitivity and specificity of 96% and 94%, respectively. Validation assays with sheep serum showed that ELISA-rSodC exhibited sensitivity and specificity of 95% and 98%, respectively. Analysis of 756 field serum samples showed that rSodC identified 95 positive samples (23%) in goats and 75 positive samples (21%) in sheep. The ELISA with recombinant SodC protein developed in this study discriminated positive and negative serum samples with high levels of sensitivity and specificity. This formulation is promising for epidemiological surveys and CL control programs. Trial registration AEC No 4958051018. 12/18/2018, retrospectively registered

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rSodC is a potential antigen to diagnose Corynebacterium pseudotuberculosis by enzyme-linked immunoassay

10.21203/rs.3.rs-54005/v2 ◽

2020 ◽

Author(s):

Antonio Pedro Fróes de Farias ◽

José Tadeu Raynal Rocha Filho ◽

Silvana Beutinger Marchioro ◽

Luan Santana Moreira ◽

Andressa Souza Marques ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Corynebacterium Pseudotuberculosis ◽

Semiarid Region ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Control Programs ◽

Epidemiological Surveys ◽

Purified Antigen

Abstract Caseous lymphadenitis (CL) is a chronic infectious disease that affects sheep and goats. Many serological tests have been developed to detect the disease; one of the most widely used is the enzyme-linked immunosorbent assay (ELISA), due to its advantages. ELISA formulations using recombinant proteins can exhibit significant sensitivity and specificity when using a single purified antigen. DTxR, Trx, TrxR, LexA, SodC, SpaC, NanH, and PknG recombinant proteins can be considered target proteins for ELISA development. Therefore, the objectives of this study were to evaluate the antigenic reactivity of Corynebacterium pseudotuberculosis recombinant proteins in goat and sheep serum. Of eight proteins evaluated, rSodC was selected for validation assays with small ruminant serum samples from the semiarid region of the state of Bahia, Brazil. Validation assays with goat serum samples showed that rSodC presented sensitivity and specificity of 96% and 94%, respectively. Validation assays with sheep serum showed that recombinant SodC exhibited sensitivity and specificity of 95% and 98%, respectively. Analysis of 756 field serum samples showed that rSodC identified 95 positive samples (23%) in goats and 75 positive samples (21%) in sheep. The ELISA with recombinant SodC protein developed in this study discriminated positive and negative serum samples with high levels of sensitivity and specificity. This formulation is promising for epidemiological surveys and CL control programs.

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Rapid and Simple Diagnosis of Chlamydophila pneumoniae Pneumonia by an Immunochromatographic Test for Detection of Immunoglobulin M Antibodies

Clinical and Vaccine Immunology ◽

10.1128/cvi.00085-08 ◽

2008 ◽

Vol 15 (7) ◽

pp. 1128-1131 ◽

Cited By ~ 5

Author(s):

Naoyuki Miyashita ◽

Kazunobu Ouchi ◽

Fumio Kishi ◽

Mitsuaki Tabuchi ◽

Naoki Tsumura ◽

...

Keyword(s):

Antibiotic Therapy ◽

Sensitivity And Specificity ◽

Chlamydophila Pneumoniae ◽

Immunoglobulin M ◽

Immunochromatographic Test ◽

Serological Tests ◽

Specific Immunoglobulin ◽

Selection Of

ABSTRACT To evaluate a newly developed immunochromatographic test (the MySet test) for the detection of Chlamydophila pneumoniae-specific immunoglobulin M antibodies, the results obtained by the MySet test were compared with those obtained by two serological tests. The sensitivity and specificity of the MySet test were 100% and 92.9%, respectively. The MySet test is rapid and simple to use and is thought to be a useful tool for the selection of appropriate antibiotic therapy.

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rSodC is a potential antigen to diagnose Corynebacterium pseudotuberculosis by enzyme-linked immunoassay

10.21203/rs.3.rs-54005/v3 ◽

2020 ◽

Author(s):

Antonio Pedro Fróes de Farias ◽

José Tadeu Raynal Rocha Filho ◽

Silvana Beutinger Marchioro ◽

Luan Santana Moreira ◽

Andressa Souza Marques ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Corynebacterium Pseudotuberculosis ◽

Semiarid Region ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Control Programs ◽

Epidemiological Surveys ◽

Purified Antigen

Abstract Caseous lymphadenitis (CL) is a chronic infectious disease that affects sheep and goats. Many serological tests have been developed to detect the disease; one of the most widely used is the enzyme-linked immunosorbent assay (ELISA), due to its advantages. ELISA formulations using recombinant proteins can exhibit significant sensitivity and specificity when using a single purified antigen. DTxR, Trx, TrxR, LexA, SodC, SpaC, NanH, and PknG recombinant proteins can be considered target proteins for ELISA development. Therefore, the objectives of this study were to evaluate the antigenic reactivity of Corynebacterium pseudotuberculosis recombinant proteins in goat and sheep serum. Of eight proteins evaluated, rSodC was selected for validation assays with small ruminant serum samples from the semiarid region of the state of Bahia, Brazil. Validation assays with goat serum samples showed that rSodC presented sensitivity and specificity of 96% and 94%, respectively. Validation assays with sheep serum showed that recombinant SodC exhibited sensitivity and specificity of 95% and 98%, respectively. Analysis of 756 field serum samples showed that rSodC identified 95 positive samples (23%) in goats and 75 positive samples (21%) in sheep. The ELISA with recombinant SodC protein developed in this study discriminated positive and negative serum samples with high levels of sensitivity and specificity. This formulation is promising for epidemiological surveys and CL control programs.

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rSodC is a potential antigen to diagnose Corynebacterium pseudotuberculosis by enzyme-linked immunoassay

10.21203/rs.3.rs-54005/v4 ◽

2020 ◽

Author(s):

Antonio Pedro Fróes de Farias ◽

José Tadeu Raynal Rocha Filho ◽

Silvana Beutinger Marchioro ◽

Luan Santana Moreira ◽

Andressa Souza Marques ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Corynebacterium Pseudotuberculosis ◽

Semiarid Region ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Control Programs ◽

Epidemiological Surveys ◽

Purified Antigen

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A new ELISA and western blot technique based on recombinant TES antigen and/or larval antigen for the detection of toxocariasis in humans

Parasitology ◽

10.1017/s0031182020002085 ◽

2020 ◽

pp. 1-8

Author(s):

Marie-Kristin Raulf ◽

Daniela Jordan ◽

Herbert Auer ◽

Jens M. Warnecke ◽

Bernd Lepenies ◽

...

Keyword(s):

Sensitivity And Specificity ◽

High Reliability ◽

High Sensitivity ◽

Cross Reactivity ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serological Tests ◽

Western Blots ◽

Blot Technique ◽

Human Sera

Abstract Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.

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