Advanced Western Blotting Solutions for SARS-CoV-2 Research and Serology Testing

Simple Western systems are automated, hands-free western blotting platforms that can be used for antigen-down serology assays for SARS-CoV-2. Jess, can process 24 samples in 3 hours and offers the ability to use viral antigen ladders to measure serum antibodies reactive against multiple SARS-CoV-2 antigens simultaneously and provide information-rich views into differences in antibody binding patterns across patients. This approach also has promise as an orthogonal confirmatory test to validate results derived from lateral flow, ELISA or other immunoassay serology techniques.

Traditional Western blots are commonly used as a confirmation for antigen-down serological assays due to several technical advantages:

• Antibody binding to multiple viral antigens can be visualized simultaneously
• Antigen molecular weight information can be used to confirm specificity of antibody binding

However, traditional Western blots have many drawbacks in a fast-moving research or clinical setting including being slow, labor intensive and lacking reproducibility. Jess overcomes the drawbacks of traditional western blots by providing:

How do SARS-CoV-2 Serological Assays on Simple Western work?

 

Simple Western IgG SARS-CoV-2 serology assay using ladder of recombinant viral antigens as capture antigens

The SARS-CoV-2 Multi-Antigen Serology Module for Jess/Wes is an antigen down serology assay capable of detecting IgG serum antibodies reactive against 5 key viral antigens. Recombinant viral antigens are mixed and separated by size to create a ladder for capture of reactive serum antibodies. The module delivers the flexibility to utilize multiple viral antigens including the addition of proprietary vaccine antigens, providing more information about antibody binding profiles in each patient. Multiple patient samples can be processed in each 3-hour run, enabling scientists to characterize variability in the immune response to SARS-CoV-2.

The SARS-CoV-2 Multi-Antigen Serology Module provides all the antigens, controls, and diluents needed to develop your serological assay to detect serum antibodies reactive against recombinant Nucleocapsid protein (N), S1 Receptor Binding Domain protein (RBD), S1 subunit full length, S2 subunit full length, and Spike (S1+S2) viral antigens.

 

Figure 2:  Five-antigen SARS-CoV-2 Simple Western serology assay simultaneously detects N, S1 RBD, S1 full length, S2 full length, and Spike (S1+S2) protein peaks in a COVID-19 ladder using a Jess automated western blot system. The specific viral antigens used are shown in the table below. The histidine-tagged viral proteins were detected using an anti-His primary antibody.

 

Figure 3:  Simple Western SARS-CoV-2 serology assay demonstrates differential patient reactivity to N, S1 RBD, S1 full length, S2 full length, and Spike (S1+S2) COVID-19 viral antigens using a Jess automated western blot system. A viral antigen ladder was used and tested with different PCR+ SARS-CoV-2 patient serum samples. SARS-CoV-2 positive serum samples were loaded onto a Jess system and human primary antibodies specific to each viral antigen were detected using an Anti-Human IgG HRP-conjugated secondary antibody (ProteinSimple DM-005). The data is represented in both lane view and an electropherogram format, with quantitative analysis of relative peak areas shown in the accompanying chart.

 

Simple Western SARS-CoV-2 IgG serology assay using whole viral lysates as capture antigens

Viral lysates can also be used as capture antigens to present the most biologically relevant epitopes for antibody capture. Further, viral lysates include all viral antigens which can provide rich views into multi-analyte antibody binding profiles in each run. This can be important in confirmatory testing and validation of other orthogonal SARS-CoV-2 serology tests.

Figure 4:  Detection of human serum antibodies reactive to nucleocapsid (N) and other SARS-CoV-2 proteins found in SARS-CoV-2 viral lysate using a Jess automated western blot system. SARS-CoV-2 viral lysate was separated, immobilized and then incubated with different PCR+ SARS-CoV-2 patient serum samples. Serum IgG detection was done using an Anti-Human IgG HRP-conjugated secondary antibody (ProteinSimple DM-005). A faint N protein band was observed in the PCR negative serum sample, which is consistent with known cross-reactivity of this protein with other coronaviruses.

 

 

Simple Westerns are not new to coronavirus research and can be used to measure host susceptibility and study how to block or reduce virus entry and virus replication. Check out a recent Nature Communications paper on how Simple Western was used to study the Middle Eastern Respiratory Syndrome (MERS) infection mechanism.

 

Highly Sensitive Detection and Characterization of ACE2 and TMPRSS2

Simple Western assays allow for highly sensitive and quantitative measurement of Angiotensin-Converting Enzyme2 (ACE2) and TMPRSS2 in tissue and cell lysate samples. ACE2 is a receptor for SARS-CoV-2 that binds the S protein and acts as the entry receptor. Cellular serine protease TMPRSS2 cleaves the Spike receptor at S1/S2 and S2’ sites to enable S2 mediated cell fusion and infection. Some COVID-19 therapeutic approaches are targeting AC2 or TMPRSS2.

 

 

 

Simple Westerns are used routinely in vaccine development including in the following application areas:

1. Serological assays to characterize immune response to a vaccine
2. Characterizing vaccine proteins
3. Monitoring vaccine component expression over time
4. Quantitating vaccine manufacturing process intermediate contamination

Serological assays to assess immune response to a vaccine

Antigen-down serological assays on Simple Western can be used to assess antibody levels and characterize the immune response during vaccine development. Time course studies can be used to quantify durability of response. Using combinations of viral antigens for antibody capture provide robust measurements of antibody binding profiles and information about relative immunogenicity of viral antigens. Further, a western blotting-based serology test is often an important datapoint for confirming lateral flow, ELISA or other immunoassay serology techniques.

In this Nature publication, Simple Western was used for the development of a vaccine candidate for SARS-CoV-2. Simple western assay was performed for the Immunoblot analysis of SARS-CoV-2 Spike (S1/2, S0 and S1) expression after transduction of BHK-21 cells with different YF-S vaccine candidates.

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Characterize vaccine proteins and monitor vaccine component expression over time

Manufacture of vaccines requires rigorous analytical characterization of vaccine components to assess identity and stability. Enjoy the benefits that Simple Western assays offer over traditional approaches like ELISA and manual western blotting including speed, automation, reproducibility. Simple Westerns enable vaccine researchers to quantify vaccine proteins using a standard curve, identify protein fragments, and track protein expression over time. Learn how scientists from Merck Research Laboratories used Simple Western assays to quantitate specific components in vaccine candidates with high reproducibility.

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Quantitate vaccine manufacturing process intermediate contamination

When manufacturing a vaccine, Simple Western can be used to determine the amount of residual BSA in samples collected throughout the vaccine manufacturing and purification process. In Loughney et al, 2014, ELISAs were not accurate enough for residual BSA quantitation. Traditional SDS PAGE could not be used due to the size of the vaccine proteins which were too close in molecular weight to BSA. In this publication, Simple Western assay was utilized to measure BSA in various process intermediates in their vaccine manufacturing process, enabling the design of a robust and scalable manufacturing process.

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