Harnessing Magnetic Beads in Immunodiagnostics

ebook Harnessing magnetic beads in immunodiagnostics How magnetic beads can be used to streamline protein purification and enhance immunoassays Immunodiagnostics has been a game changer for healthcare. It’s at the heart of disease detection, therapeutic monitoring, and biomarker identification. In the last few years, immunodiagnostics has advanced even further with the help of magnetic beads. These humble, yet powerful tools assist in protein purification and immunoassays, enhancing both assay sensitivity and speed. In immunoassays, magnetic beads can separate, detect, and quantify a whole range of molecules including target proteins, antibodies, and antigens. Essentially, they offer an easier and more effective alternative to traditional protein separation methods. Magnetic beads are particularly beneficial in complex diagnostic tests such as multiplexed assays. When coated with specific antibodies, magnetic beads selectively bind to target proteins and antigens. This binding creates high-purity samples that are easy to isolate through a magnetic field. With these broad benefits in mind, this e-book will explore the use of magnetic beads in mmunodiagnostics, focusing on protein purification and immunoassay applications. Delve into the key benefits and challenges of magnetic beads across these applications and find protocol tips and magnetic bead recommendations. Fig 1. The biotinylated primary antibody is incubated with the beads and binds to them. This target binds to the antibody. 2 Optimizing protein purification with magnetic beads Protein purification without antibodies Magnetic beads are revolutionizing protein purification processes. They can efficiently isolate and purify target proteins (or protein complexes) from a wide range of samples and volumes. The adaptability of magnetic bead surfaces for different functions provides unparalleled flexibility when purifying proteins. For example, magnetic beads such as His Mag Sepharose™ Ni beads can be used to purify Histagged proteins, enhancing standard nickel ion affinity chromatography. There are many additional magnetic bead conjugation options available. For instance, they can come either preconjugated with surface ligands such as antigens, enzymes, or peptides, or you can select coated magnetic beads with specific surface chemistry to conjugate with your specific ligands. Magnetic beads are widely used across many different protein purification applications. For example, you can get magnetic beads conjugated with protein A or protein G, such as Protein A Mag Sepharose Xtra beads. These beads (see Fig 2) are commonly used for purifying IgG antibodies but can also be used to purify polyclonal and monoclonal antibodies. In a recent 2024 study, researchers used Protein A magnetic beads to develop a one-step protocol, purifying two monoclonal antibodies directly from nonclarified, crude plant extracts (1). The study showcases how magnetic beads can significantly save on time and resources and can improve protein quality and biological activity via efficient purification. Magnetic beads with functional carboxylic acid groups are also useful for protein purification applications. For example, these beads have been conjugated with an aptamer designed to capture the human anticoagulant serine protease, activated protein C (APC). Using these conjugated carboxylic acid magnetic beads, highly pure APC was eluted in its active form, which is highly useful for downstream therapeutic developments to prevent blood clotting (2). Another key magnetic bead type for this application is streptavidin-coated magnetic beads. These beads can be used to purify biotinylated antibodies and proteins such as glycol-engineered antibodies. These antibodies are widely used for the treatment of many diseases, including cancers, autoimmune disorders, and inflammatory and infectious diseases (3). Antibody-free approaches are useful for high-throughput workflows or when multiple protein purifications are needed, eliminating time-consuming and costly antibody production. Magnetic beads make protein purification without antibodies even faster—and because they’re adaptable to small samples or viscous solutions—they’re ideal for modern lab workflows. Fig 2. An illustration of the immunoprecipitation steps using magnetic beads conjugated with protein A or protein G. 3 Challenges with magnetic beads in protein purification When using magnetic beads for protein purification, one of the main challenges is the risk of nonspecific binding. Nonspecific binding can occur when using a suboptimal bead size for the desired target protein or a suboptimal ratio of sample-to-bead concentration. For example, with a low sample concentration, an excess of available binding sites on the beads can lead to increased nonspecific binding. Conversely, for larger proteins, there may be insufficient surface area on the magnetic beads for effective binding. Also, when using antibodies, not prechecking protein specificity can also lead to nonspecific binding. To mitigate these challenges, you need to optimize protein concentration, carefully select appropriate magnetic beads, and verify the specificity of antibodies prior to use. Protein purification with antibodies The use of antibodies immobilized to magnetic beads for protein purification is termed “magnetic immunoprecipitation.” For this process, an antibody is conjugated to the surface of the magnetic bead, which is then mixed with the sample containing the antigen. Alternatively, a secondary antibody could be conjugated to the magnetic beads, which is then mixed with the sample containing the primary antibody that is bound to the antigen. Another common method is using proteins like protein A or protein G conjugated to magnetic beads. These proteins capture the primary antibody that is bound to the target antigen. Antibody-coated magnetic beads can provide exceptional specificity for the capture of the target protein and have been used across a broad range of clinical applications. For example, they have been used to purify diabetes-associated mutant insulin from plasma samples (4). More recently, they have been used to purify age-distinct insulin secretory granules for downstream proteomics and lipidomics analyses (5). Discover more options for modifying the surface of magnetic beads to suit your protein purification needs = Antibody Protein A/G Fig 3. Protein A/G magnetic beads are used for antibody isolation applications, including affinity purification and pull-down, as well as immunoprecipitation. This is due to their surface chemistry which binds lgA and lgG proteins, features a coating based on lgA/lgG-fusion proteins and offers broad binding capabilities. 4 Workflow and bead selection considerations 1 Optimize bead size, volume, and incubation times for the target protein To optimize binding capacity, adjust the bead volume according to the size and concentration of your target protein. For large target molecules, increase bead volume to maximize binding sites. Alternatively, you can decrease the bead size to create more surface area per mass for binding. In workflows using His-tagged proteins, His Mag Sepharose Ni beads are ideal due to their high surface area, maximizing binding capacity while reducing nonspecific binding. When working with low-concentration samples, increasing bead incubation time can provide more binding opportunities. 2 Adjust elution buffers for recovery and integrity Optimized elution conditions are essential for recovering pure proteins and preventing degradation. For protein-specific release, use high-affinity ligands in your elution buffer, or adjust the pH accordingly. For example, acidic conditions release proteins from protein A or protein G beads, while imidazole elution is suited for His-tagged proteins. 3 Carefully prepare buffers to minimize nonspecific binding Nonspecific interactions can also be reduced with careful buffer preparation. Blocking agents like BSA, PEG, or glycerol decrease nonspecific binding. Beads should be equilibrated with a lysis buffer that includes a low concentration of the blocker, and the same blocker should be added to the wash buffer. To further reduce background signals, you can preincubate beads in the target solution without antibodies. You can also perform a pre-step during which the sample is exposed to beads without the capture molecule. The pre-step will remove components in the sample that nonspecifically bind to the beads. The beads without the capture molecule can then be removed and the beads with the capture molecule can be added to the sample to isolate the protein of interest. 4 Control temperatures for protein integrity Conduct binding steps at optimal temperatures to maintain protein stability and bead integrity. Avoid excessive heating in elution stages, as this can cause protein denaturation and can lower your binding efficiency. For some workflows, slower binding at 4°C provides better specificity without compromising protein integrity. 5 Magnetic immunoassays: enhancing sensitivity and speed While magnetic beads can be used to purify and concentrate proteins prior to downstream analytical methods, they can also be used directly in magnetic immunoassays. Benefits of using magnetic beads in immunoassays include improved assay sensitivity, faster results, and higher specificity, due to the increased surface area per volume for antigen-antibody interactions. Magnetic beads are used in various immunoassays, like enzyme-linked immunosorbent assays (ELISAs), chemiluminescent immunoassays (CLIAs), fluorescent immunoassays (FIAs), lateral-flow assays (LFAs), and multiplex immunoassays. Their integration allows for quicker and more efficient antigen capture, which is crucial in both clinical and research settings. See how a magnetic immunoassay works to provide a quick detectable signal Applications in ELISAs Magnetic beads are frequently used in ELISAs, including those to detect infectious diseases. In a 2021 study, nickel ion-coated magnetic beads immobilized SARSCoV-2 antigens containing a His-tag. The antigen-bound magnetic beads were then incubated with an anti-human IgG antibody conjugated to horseradish peroxidase. This process enabled the amount of antigen captured to be analyzed. The result was a chromogenic magnetic immunoassay for SARS-CoV-2 that was both easy and quick to visually interpret. The detection range is comparable to gold standard immunoassays (such as traditional ELISA and Luminex) but requires significantly less time and resources, making it suitable for point-of-care (POC) use (6). Magnetic beads are also used in more advanced types of ELISAs such as chemiluminescent ELISAs. These assays offer greater sensitivity and specificity, especially when detecting low-abundance proteins (7). Magnetic beads coated with antibodies or protein A/G improve binding kinetics in chemiluminescent assays, leading to faster protein capture and more accurate signal detection. To achieve assays with high sensitivity, select assay components, such as Sera-Mag™ SpeedBeads in conjugation with the capture molecule, that enable high assay specificity while minimizing background noise. Multiplex immunoassays with magnetic beads Magnetic beads are invaluable in multiplex immunoassays, which require the simultaneous detection of multiple biomarkers. By using fluorescent beads with distinct excitation and emission wavelengths, cross-reactivity can be minimized and signal clarity improved, even when handling complex samples. Streptavidin-coated magnetic beads are advantageous for multiplex assays due to their rapid and highly specific binding to biotinylated antibodies. Commonly used in multiplex cancer assays, these beads isolate tumor-specific proteins, enabling researchers to track cancer progression and gain insights into the disease’s development and treatment response. Non-fluorescently labelled beads can be used and then fluorescently labelled detection molecules can be used to enable the multiplexing. Fig 4. Streptavidin-blocked magnetic beads are ideal for high-specificity binding applications, like immunoand molecular diagnostics, as well as NGS library preparation. 6 Challenges and key considerations in magnetic bead immunoassays 1 Nonspecific binding and signal optimization Nonspecific binding is a common challenge in immunoassays. In the case of magnetic immunoassays, magnetic beads can capture unwanted components alongside the target protein. Solving this issue is essential to achieve high assay specificity. Optimizing bead surface chemistry and incorporating blocking agents during washes can prevent unintended interactions and improve target capture rates. Also, to avoid signal bleed, ensure each biomarker is tagged with beads that do not interfere with other markers in the assay. 2 Bead-to-antibody ratios and incubation times Optimizing the bead-to-antibody ratio is crucial for the specificity and sensitivity of immunoassays. Insufficient antibody concentration can lead to incomplete coverage of the capture beads, while excess antibodies can produce background noise. The ratio can be optimized based on assay conditions and sample characteristics, such as protein size and abundance. 3 Selecting high-affinity and specificity beads and antibodies To maximize assay performance, select high-affinity beads such as Sera-Mag Protein A/G, Protein A Mag Sepharose, Protein A Mag Sepharose Xtra, Protein G Mag Sepharose, and Protein G Mag Sepharose Xtra magnetic beads. These beads bind selectively to IgG antibodies, allowing target proteins to be captured from complex samples with minimal off-target interactions. Choosing antibodies with high specificity also improves binding precision. For high-sensitivity applications, streptavidin-coated magnetic beads offer strong binding to biotinylated antibodies, which is particularly useful in multiplex assays. 4 Washing steps for contaminant removal Washing is a critical step in magnetic immunoassays. Gentle centrifugation or magnetic washing with buffers containing mild detergents helps remove any unbound proteins without impacting bead stability or target integrity. A common protocol involves three washes with phosphate-buffered saline with Tween-20 (PBS-T), which removes unbound proteins while preserving the bead’s magnetic properties. Avoid excessive washes to reduce the risk of target molecule loss. 5 Handling and storage of magnetic beads Proper handling techniques should be followed to preserve magnetic bead functionality and enhance assay performance. Avoid extreme temperatures and use manufacturer-recommended storage buffers. For example, the magnetic beads from Cytiva are temperature-stable, but should still be stored in optimal conditions for assay consistency. 7 What does the future hold for magnetic beads in immunodiagnostics? Magnetic beads are streamlining protein purification steps and are enhancing the sensitivity and reliability of immunoassays. These powerful tools can also be used across other immunodiagnostic workflow steps, including targeting nucleic acids and cell surface proteins for cell enrichment and other clinical diagnostics purposes. For example, magnetic beads can be used for: • Increased automation to enhance throughput and consistency of diagnostic assays • Precision biomarker detection, which can tailor treatments to individual patients needs • POC testing to detect a wider array of biomarkers including emerging diseases • Systems that use artificial intelligence and data analytics to improve diagnostic accuracy and provide deeper insight to disease mechanisms As precision diagnostics evolve, the use of magnetic beads in immunodiagnostics promises to further streamline detection processes, improve assay sensitivity, and reduce turnaround time. This advancement paints a bright future for healthcare, with its potential to save resources, lower healthcare costs, and provide patients with faster and more accurate diagnoses. Ready to streamline your immunodiagnostic workflows? Browse Cytiva’s full range of magnetic beads for proteins and immunoassays 8 References 1. Faye L, Grünwald-Gruber C, Vezina LP, Gomord V, Morel B. A fast and easy onestep purification strategy for plant-made antibodies using Protein A magnetic beads. Front Plant Sci. 2023;14:1276148. doi:10.3389/FPLS.2023.1276148/BIBTEX 2. Hamedani NS, Happich FL, Klein EM, et al. Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C. Scientific Reports 2022 12:1. 2022;12(1):1-11. doi:10.1038/s41598-022-11198-5 3. Chuang HY, Huang CC, Hung TC, et al. Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies. Bioorg Chem. 2021;112:104863. doi:10.1016/J.BIOORG.2021.104863 4. Støy J, Olsen J, Park SY, Gregersen S, Hjørringgaard CU, Bell GI. In vivo measurement and biological characterisation of the diabetes-associated mutant insulin p.R46Q (GlnB22-insulin). Diabetologia. 2017;60(8):1423. doi:10.1007/ S00125-017-4295-2 5. Neukam M, Sala P, Brunner AD, et al. Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging. Cell Rep. 2024;43(3). doi:10.1016/J.CELREP.2024.113836/ASSET/37AC6EE1-2D42-4696- 8CE2-84FD0060EAC9/MAIN.ASSETS/GR2.JPG 6. Huergo LF, Selim KA, Conzentino MS, et al. Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies. ACS Sens. 2021;6(3):703-708. doi:10.1021/ACSSENSORS.0C02544/ SUPPL_FILE/SE0C02544_SI_002.MP4 7. Liu H, Shi Y, Ye J, et al. An automated magnetic beads-based chemiluminescence immunoassay system for simultaneous quantification of multi-mycotoxins in agricultural products. Sens Actuators B Chem. 2024;420:136424. doi:10.1016/J. SNB.2024.136424 9 cytiva.com Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva. Sepharose and Sera-Mag are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. 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