Identifying Novel Neurodegenerative Disease Biomarkers with Western Blotting and Multiplex Workflows
Another study, published in Alzheimer’s & Dementia by a multi-institutional team led out of Sapienza University of Rome in Italy, investigated how brain insulin resistance, derived from altered insulin signaling in the brain, may affect synaptic plasticity and increase the risk of early-onset Alzheimer’s disease–like neurodegeneration in individuals with Down syndrome (DS). The study examined the role of mTOR, a “master regulator” which, in other studies, was found to be responsible for maintaining synaptic and neuronal health, among other anabolic and catabolic cellular processes. The insulin/mTOR pathway has previously been found to be altered early in life in people with DS. Here, Bio-Rad western blotting reagents and instruments and its ChemiDoc MP Imaging System were used to assess the purity of neuronal-derived extracellular vesicles by confirming the expression of neuronal markers. Subsequently, the Bio-Rad Bio-Plex™ Pro Cell Signaling Akt Panel (8-plex) was used to evaluate the expression of biomarkers involved in the mTOR pathway in these vesicles (2).
The use of total protein normalization and highly validated reagents and panels enabled researchers to obtain reliable and reproducible results. Their findings contributed to an increased understanding of neurodegeneration associated with DS and helped identify novel exosomal biomarkers that could serve as therapeutic targets.
Genomic and Proteomic Tools to Uncover SARS-CoV-2 Omicron Variant Pathogenicity
The persistence of the COVID-19 pandemic has seen the emergence of SARS-CoV-2 variants and accompanying risks of pathogenic changes, leading to immune response evasion and reduced vaccine effectiveness. In a 2022 study published in Nature, a team of researchers led by the Cambridge Institute of Therapeutic Immunology and Infectious Disease looked to identify key differences in the pathogenic mechanisms between Omicron and Delta variants. Using a selection of Bio-Rad tools, including the ZE5 Cell Analyzer for flow cytometry analysis of spike protein expression at the cell surface, CFX Connect Real-Time PCR Detection System for assessment of viral RNA copy number following infection, and ChemiDoc MP Imaging System for protein expression analysis of infected cells, the study concluded that the Omicron spike protein confers substantial evasion of vaccine-elicited neutralizing antibodies, which could be offset by booster doses of an mRNA vaccine. Additionally, the Omicron variant showed increased ACE2 binding affinity as well as reduced ability to fuse with or infect cells expressing TMPRSS2 (3).
In this study, tools from Bio-Rad helped advance the field’s understanding of the phenotypic impact of this emerging variant, both in terms of vaccine strategy and the implications on cellular entry patterns that could play a role in viral pathogenesis and disease progression.
Optimizing SARS-CoV-2 Spike Protein Purification Utilizing Cation Exchange Chromatography
Process chromatography resins from Bio-Rad also supported COVID-19 vaccine development. Published in Scientific Reports, a team from the National Institute of Allergy and Infectious Diseases in Maryland, U.S., used the small-particle-size, high-resolution cation exchange resin, Nuvia HR-S Resin, designed for intermediate and final polishing purification steps, to demonstrate that commercially available resins are suitable for automated, high yield purification of the SARS-CoV-2 spike protein. The spike protein had previously only been purified using low-yield, expensive, and difficult-to-scale methods, such as affinity and size-exclusion chromatography (4).
Using a Bio-Rad cation exchange resin, this proof-of-concept study revealed a highly scalable process for spike protein purification — an essential stepping stone to mass spike protein production, facilitating further research into the virus and its pathogenicity.
Accelerating SARS-CoV-2 Diagnostics Development Using Droplet Digital™ PCR (ddPCR™)
The COVID-19 pandemic placed unprecedented strain on the scientific world to rapidly develop accurate SARS-CoV-2 diagnostic and detection tools. In a paper published in Scientific Reports, researchers from the University of California San Francisco used the Bio-Rad QX200™ Droplet Digital PCR System for absolute quantification of viral load in both purified nucleic acid and crude lysate from patient samples (5). Unlike real-time quantitative PCR, which is used extensively for SARS-CoV-2 detection, Droplet Digital PCR does not rely on a standard curve or perfect amplification conditions.
Using ddPCR technology from Bio-Rad, the team developed a streamlined SARS-CoV-2 detection process directly from patient crude lysate, requiring no purification steps, to allow for faster, cost-effective, and more accurate testing.
With the goal of supporting researchers across a spectrum of translational research disciplines, Bio-Rad continues to be a trusted partner throughout discovery and development. The breakthrough papers summarized here have helped to advance our understanding of diagnostics, underlying disease mechanisms, and treatments, consequently shaping the way we seek to address unmet medical needs.
| Research Objectives | Bio-Rad Tools and Reagents | Solution or Technique |
|---|---|---|
| Antibody–drug conjugate purification (1) | CHT Ceramic Hydroxyapatite Media | CHT Media used for the purification of high ratio drug-to-antibody therapeutics during the manufacturing process |
| Disease biomarker identification (2) |
|
Separation and quantification of neuronal markers |
|
Simultaneous evaluation of biomarkers involved in signaling pathways crosstalk | |
| Viral pathogenesis (3) |
|
Flow cytometry analysis of viral protein expression at the cell surface |
|
Quantification of viral copy number following infection | |
|
Chemiluminescent Western Blotting of virus-infected cells | |
| Viral component purification (4) |
|
Purification of viral spike surface proteins, for vaccine development and pathogenicity research |
| Viral detection and quantification (5) |
|
Quantification of viral load from patient crude lysates without nucleic acid purification |
References
- Kumar A et al. (2020). Design and validation of linkers for site-specific preparation of antibody–drug conjugates carrying multiple drug copies per cysteine conjugation site. Int Journ Mol Sci 21, 6882.
- Perluigi M et al. (2021). Aberrant crosstalk between insulin signaling and mTOR in young Down syndrome individuals revealed by neuronal‐derived extracellular vesicles. Alzheimer’s Dement.
- Meng, B et al. (2022). Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity. Nature 603, 706–714.
- Cibelli N et al. (2022). Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods. Scientific Reports 12.
- Vasudevan H et al. (2021). Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification. Scientific Reports 11.
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