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

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.

Download Full-text

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

10.1101/2020.11.26.20238733 ◽

2020 ◽

Author(s):

S. Edouard ◽

R. Jaafar ◽

N. Orain ◽

P. Parola ◽

P. 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 Jess™ Simple Western system, an automated capillary-based assay was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as antigen, and IgT 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 exposition of people to HCoVs including SARS-CoV-2.

Download Full-text

Diagnostic Efficiency of Three Fully Automated Serology Assays and Their Correlation with a Novel Surrogate Virus Neutralization Test in Symptomatic and Asymptomatic SARS-COV-2 Individuals

Microorganisms ◽

10.3390/microorganisms9020245 ◽

2021 ◽

Vol 9 (2) ◽

pp. 245

Author(s):

Salma Younes ◽

Hadeel Al-Jighefee ◽

Farah Shurrab ◽

Duaa W. Al-Sadeq ◽

Nadin Younes ◽

...

Keyword(s):

Diagnostic Performance ◽

Neutralization Test ◽

Cross Reactivity ◽

Diagnostic Efficiency ◽

Receptor Binding Domain ◽

Rt Pcr ◽

Viral Antibodies ◽

Asymptomatic Patients ◽

Intended Use ◽

Control Samples

To support the deployment of serology assays for population screening during the COVID-19 pandemic, we compared the performance of three fully automated SARS-CoV-2 IgG assays: Mindray CL-900i® (target: spike [S] and nucleocapsid [N]), BioMérieux VIDAS®3 (target: receptor-binding domain [RBD]) and Diasorin LIAISON®XL (target: S1 and S2 subunits). A total of 111 SARS-CoV-2 RT-PCR- positive samples collected at ≥ 21 days post symptom onset, and 127 pre-pandemic control samples were included. Diagnostic performance was assessed in correlation to RT-PCR and a surrogate virus-neutralizing test (sVNT). Moreover, cross-reactivity with other viral antibodies was investigated. Compared to RT-PCR, LIAISON®XL showed the highest overall specificity (100%), followed by VIDAS®3 (98.4%) and CL-900i® (95.3%). The highest sensitivity was demonstrated by CL-900i® (90.1%), followed by VIDAS®3 (88.3%) and LIAISON®XL (85.6%). The sensitivity of all assays was higher in symptomatic patients (91.1–98.2%) compared to asymptomatic patients (78.4–80.4%). In correlation to sVNT, all assays showed excellent sensitivities (92.2–96.1%). In addition, VIDAS®3 demonstrated the best correlation (r = 0.75) with the sVNT. The present study provides insights on the performance of three fully automated assays, which could help diagnostic laboratories in the choice of a particular assay according to the intended use.

Download Full-text

Developing a Paper-Based Antigen Assay to Differentiate Between Coronaviruses and SARS-CoV-2 Spike Variants

10.26434/chemrxiv.13476738.v1 ◽

2020 ◽

Author(s):

Delyan Hristov ◽

Hom Rijal ◽

Jose Gomez-Marquez ◽

Kimberly Hamad

Keyword(s):

Test Line ◽

Human Saliva ◽

Cross Reactivity ◽

Spike Protein ◽

Signal Quality ◽

Binding Pattern ◽

Rt Pcr ◽

Global Pandemic ◽

Antigen Assay ◽

Assay Design

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location dependent, time consuming and relatively expensive. Thus, there is a need for a more flexible method which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here we present the first steps towards a paper-based test which can differentiate between different between the Spike protein of various coronaviruses, SARS-CoV-1, SARS-CoV-2 and CoV-HKU1 with negligible cross reactivity for HCoV-OC43 and HCoV-229E in a single assay which takes less than 30 minutes. Furthermore, our test can distinguish between fractions of the same Spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration and antigen interference is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality. <br>

Download Full-text

Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18189630 ◽

2021 ◽

Vol 18 (18) ◽

pp. 9630

Author(s):

Pierre Nsele Mutantu ◽

Mya Myat Ngwe Tun ◽

Takeshi Nabeshima ◽

Fuxun Yu ◽

Patrick Kakoni Mukadi ◽

...

Keyword(s):

Immunoglobulin G ◽

Expression System ◽

Negative Control ◽

Spike Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

N Protein ◽

E Coli ◽

System A ◽

Recombinant Nucleocapsid Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay’s performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.

Download Full-text

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

PLoS ONE ◽

10.1371/journal.pone.0257351 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257351

Author(s):

Carolina de la Guardia ◽

Giselle Rangel ◽

Alcibiades Villarreal ◽

Amador Goodridge ◽

Patricia L. Fernández ◽

...

Keyword(s):

Area Under The Curve ◽

High Sensitivity ◽

Cross Reactivity ◽

Spike Protein ◽

Close Relative ◽

Rt Pcr ◽

Characteristic Analysis ◽

Serum Samples ◽

Health Authorities ◽

Significant Difference

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.

Download Full-text

Developing a Paper-Based Antigen Assay to Differentiate Between Coronaviruses and SARS-CoV-2 Spike Variants

10.26434/chemrxiv.13476738 ◽

2020 ◽

Author(s):

Delyan Hristov ◽

Hom Rijal ◽

Jose Gomez-Marquez ◽

Kimberly Hamad

Keyword(s):

Test Line ◽

Human Saliva ◽

Cross Reactivity ◽

Spike Protein ◽

Signal Quality ◽

Binding Pattern ◽

Rt Pcr ◽

Global Pandemic ◽

Antigen Assay ◽

Assay Design

Download Full-text

Thrombotic Thrombocytopenia after COVID-19 Vaccination: In Search of the Underlying Mechanism

Vaccines ◽

10.3390/vaccines9060559 ◽

2021 ◽

Vol 9 (6) ◽

pp. 559

Author(s):

Piotr Rzymski ◽

Bartłomiej Perek ◽

Robert Flisiak

Keyword(s):

Direct Interaction ◽

Underlying Mechanism ◽

The United States ◽

Cross Reactivity ◽

Spike Protein ◽

Vaccine Hesitancy ◽

Future Research ◽

Platelet Factor ◽

Precautionary Measures ◽

Adenoviral Proteins

The rollout of COVID-19 vaccines brings hope for successful pandemic mitigation and getting the transmission of SARS-CoV-2 under control. The vaccines authorized in Europe displayed a good safety profile in the clinical trials. However, during their post-authorization use, unusual thrombotic events associated with thrombocytopenia have rarely been reported for vector vaccines. This led to the temporary suspension of the AZD1222 vaccine (Oxford/AstraZeneca) in various European countries and the Ad26.COV2 vaccine (Janssen/Johnson&Johnson) in the United States, with regulatory bodies launching investigations into potential causal associations. The thromboembolic reactions were also rarely reported after mRNA vaccines. The exact cause of these adverse effects remains to be elucidated. The present paper outlines the hypotheses on the mechanisms behind the very rare thrombotic thrombocytopenia reported after the COVID-19 vaccination, along with currently existing evidence and future research prospects. The following are discussed: (i) the role of antibodies against platelet factor 4 (PF4), (ii) the direct interaction between adenoviral vector and platelets, (iii) the cross-reactivity of antibodies against SARS-CoV-2 spike protein with PF4, (iv) cross-reactivity of anti-adenovirus antibodies and PF4, (v) interaction between spike protein and platelets, (vi) the platelet expression of spike protein and subsequent immune response, and (vii) the platelet expression of other adenoviral proteins and subsequent reactions. It is also plausible that thrombotic thrombocytopenia after the COVID-19 vaccine is multifactorial. The elucidation of the causes of these adverse events is pivotal in taking precautionary measures and managing vaccine hesitancy. It needs to be stressed, however, that the reported cases are currently sporadic and that the benefits of COVID-19 vaccines vastly outweigh their potential risks.

Download Full-text

SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy

Virology Journal ◽

10.1186/s12985-021-01620-1 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Brian Mondeja ◽

Odalys Valdes ◽

Sonia Resik ◽

Ananayla Vizcaino ◽

Emilio Acosta ◽

...

Keyword(s):

High Resolution ◽

Control Sample ◽

Negative Control ◽

High Rate ◽

Apical Region ◽

The Novel ◽

Rt Pcr ◽

Crown Shape ◽

Novel Coronavirus ◽

High Resolution Microscopy

Abstract Background The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. Methods Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. Results The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. Conclusions The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Download Full-text

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.

Download Full-text

Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692831 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bochao Liu ◽

Ze Wu ◽

Chaolan Liang ◽

Jinhui Lu ◽

Jinfeng Li ◽

...

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Primary Screening ◽

Global Pandemic ◽

Novel Coronavirus ◽

Acid Test ◽

Control Samples

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors’ control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

Download Full-text