Enjoy the benefits of working with a single supplier for DNA isolation, protein purification, research, and diagnostics development and commercialization.
Diagnostic assays, kits, and devices all share one aim: to measure and observe aspects of a patient's health to help establish a diagnosis and guide treatment plans. Developing and commercializing new diagnostics that are more sensitive, robust, and selective are important to improve clinical decisions and ultimately, translate to better healthcare for patients.
But developing new diagnostic platforms is a highly involved and complex process, often requiring integration and validation of many components and processes. As such, diagnostic developers and manufacturers must find, validate, and coordinate the supplier for each component. Using a single supplier for multiple components across a diagnostic workflow can streamline these activities and ultimately, accelerate the development and commercialization of assays and kits.
To extend the single supplier concept even further, imagine the benefits of using that same supplier for your other research needs, too. Here, we explore examples that illustrate how using a broad range of products from Cytiva supports research – to develop a new diagnostic test, improve drug discovery, and potentially identify new biomarkers.
Developing point-of-care testing for food-borne pathogens
Human gnathostomiasis is a potentially fatal disease caused by several species of parasitic worm belonging to the genus Gnathostoma. While a definitive diagnosis can be made by recovery of Gnathostoma larvae from the patient, this type of direct detection can be extremely difficult. In practice, diagnosis is often made by evaluating the clinical symptoms (1).
While there are serological antibody-detection tests for human gnathostomiasis diagnosis, they are limited by their requirement for specialist operators and costly and sophisticated equipment (1). Therefore, a user-friendly, cost-effective, and rapid point of care (POC) diagnostic tool suitable for use in resource-limited areas would be beneficial to both patients and healthcare professionals.
The protein Gslic18 (rGslic 18) is a diagnostic antigen that shows high sensitivity and specificity for the detection of total IgG antibody in the serodiagnosis of human gnathostomiasis. Using a nickel-charged column with the ÄKTA chromatography system, Janwan et al. (2021) successfully separated and purified rGslic 18 for use in an immunochromatographic device (1).
The research group assembled the device, which included a conjugate pad composed of a Whatman™ glass microfiber filter sprayed with anti-human IgG4 conjugated with colloidal gold particles. There are several benefits to using Whatman™ conjugate pads in lateral flow devices:
- They do not require pre-treatment since they are inherently hydrophobic.
- The open structure of the material allows rapid penetration by both the conjugate and sample.
- They have high conjugate release levels that reduce both waste and reagent cost, and also increase the amount of conjugate reaching the capture line, in turn increasing capture line intensity for improved assay sensitivity.
The team evaluated the assembled diagnostic device for accuracy, sensitivity, specificity, and positive and negative predictive values. This culminated in a POC diagnostic device — the gnathostomiasis blood immunochromatographic test (GB-ICT) kit — with high sensitivity and specificity in simulated whole blood samples (WBS) for human gnathostomiasis.
In contrast to previously developed assays, the GB-ICT kit allows for finger-prick blood samples as input, circumventing the need for venous blood sampling and/or serum separation processes. Consequently, the authors concluded that the GB-ICT kit not only supports clinical diagnosis but is suitable for use in remote areas where sophisticated equipment or specialist personnel are not available.
Harnessing exosomes to improve drug screening
Binding kinetics of pharmaceuticals have been shown to be closely associated with drug efficacy and off-target toxicity (2). Consequently, the importance of binding kinetics in drug discovery has been broadly accepted over the past decade.
Designing simple molecular assays for determining binding kinetics can be a challenging task. Typically, membrane protein drug targets are integrated into a lipid bilayer to provide a testing platform. However, membrane proteins can denature easily, are difficult to produce, and don’t always retain their functional integrity in artificial lipid environments.
Exosomes are extracellular vesicles secreted by a wide range of cells. By carrying cell constituents – such as nucleic acids, proteins, lipids, and metabolites – between cells they play a crucial role in mediating intercellular communication. Since their plasma membrane proteins have the same outside-out orientation as at the surface of intact cells, they offer an attractive method to deliver proteins in their native conformation for molecular assays (3).
To harness the potential of exosomes, there is a need to find and refine a method for sorting proteins of interest to exosomes. To address this need, Desplantes et al. (2017) developed a novel approach that used a specific targeting peptide sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor (3). The research group achieved successful exosomal expression of five different membrane proteins, including two different BoNT/B receptors.
The researchers assessed the binding properties of ligands to the membrane protein-expressing exosomes using various techniques including 125I-dendrotoxin binding assays and surface plasmon resonance (SPR) analysis. Radiolabeled 125I-dendrotoxin was incubated with membrane protein-expressing exosomes and rapidly filtered through Whatman™ glass microfiber filters, Grade GF/B to separate free and bound toxin. The results demonstrated proper assembly and/or conformation of the membrane proteins at the surface of exosomal membranes.
Surface Plasmon Resonance (SPR) analysis of the immunocaptured exosomes, using the Biacore™ 3000 or Biacore™ T200 system, enabled the researchers to evaluate expression levels, analyze ligand binding potential and, ultimately, establish a simplified automated assay to measure precise binding properties of ligands to membrane receptors.
Analyzing transcriptional drivers of oncogenesis
MEF2B is a gene that encodes a transcriptional factor (myocyte-specific enhancer factor 2B) that is mutated in approximately 8%-18% of diffuse large B cell lymphomas (DLBCLs; 4). While these mutations are thought to affect its transcriptional activity and ultimately promote lymphomagenesis, its genome-wide impacts on gene expression haven’t been thoroughly explored.
One research group conducted a comprehensive study to understand the impacts of MEF2B mutations in DLBCL. It involved a combination of expression microarray analysis and mRNA sequencing to identify MEF2B target genes and evaluate their differential expression in mutant and wildtype cell lines (4).
The group also used ChIP sequencing to identify the areas in the genome bound by myocyte-specific enhancer factor 2B (MEF2B). To construct ChIP libraries, they enriched DNA sequences bound by MEF2B, size separated ChIPed DNA using polyacrylamide gel electrophoresis, and purified the DNA using SeraMag™ SpeedBeads.
Finally, the team performed gel-shift assays to determine whether mutations in MEF2B affect DNA binding. The ÄKTA™ chromatography system allowed the researchers to purify wildtype and mutated forms of his-tagged MEF2B using a 1 mL HisTrap™ FF column. The purified transcription factor proteins were allowed to bind with radioactively-labelled DNA probes and loaded onto a gel. Subsequently, the gel was exposed to a phosphor screen overnight and scanned using a Typhoon™ imager.
Ultimately, the researchers were able to identify numerous candidate MEF2B target genes – both direct and indirect – that hadn’t been identified previously. Moreover, the results show that MEF2B increases cell migration and the expression of genes involved in epithelial–mesenchymal transition (EMT).
EMT has a critical role in cancer, enabling carcinoma cells to suppress their epithelial features and acquire mesenchymal characteristics. As a result, these cells can gain mobility and the capacity to migrate from the primary site.
The results also showed that certain MEF2B mutations associated with cancer decrease DNA binding and likely affect its ability to activate transcription. Overall, the research significantly contributes to our understanding of the role of MEF2B mutations in cancer and their potential as diagnostic biomarkers.
The benefits of working with a single supplier
In these examples, we have described how research groups used multiple Cytiva products in their work to discover novel diagnostic approaches across several applications, and how their work can potentially translate to the diagnostic laboratory.
Ensuring projects and processes are as efficient and streamlined as possible is a common theme across research, development, and commercialization. So, it can be advantageous to use suppliers that can satisfy multiple requirements, as well as meet scalability demands as diagnostic tools transition from research to manufacturing.
Through collaborative services and access to a wide selection of high-performance, customizable components and solutions, Cytiva is committed to helping you accelerate your immunoassay and molecular diagnostic process. And we can put you in touch with other team members who can help you with your other research needs.Contact us to discuss your diagnostics needs
- Janwan P, Intapan PM, Sadaow L et al. Development of immunochromatographic test kit for rapid detection of specific IgG4 antibody in whole-blood samples for diagnosis of human gnathostomiasis. Diagnostics (Basel). 2021;11(5):862. doi:10.3390/diagnostics11050862
- Meyer-Almes FJ. Kinetic binding assays for the analysis of protein-ligand interactions. Drug Discov. Today Technol. 2015;17:1-8. doi: 10.1016/j.ddtec.2015.08.004.
- Desplantes R, Lévêque C, Muller B et al. Affinity biosensors using recombinant native membrane proteins displayed on exosomes: Application to botulinum neurotoxin B receptor. Sci. Rep. 2017;7:1-11.
- Pon JR, Wong J, Saberi S et al. MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation. Nat. Commun. 2015; 6:7953. doi: 10.1038/ncomms8953.