Rethink Bioconjugation To Cut Costs and Improve Assay Performance

Bioconjugation enables the creation of novel biomolecules with enhanced or modified properties and has been the cornerstone of advancements in various fields like medicine, diagnostics, and materials science. It allows researchers to combine the desirable characteristics of different molecules together, such as the specificity of antibodies with the potency of drugs, detecting different pathogens with antibodies bound to particles in diagnostic settings, or the imaging capabilities of fluorescent dyes partnered with the precision detection of biomolecules. Typically, bioconjugation involves a biologically relevant macromolecule, such as a protein, carbohydrate, nucleic acid, or lipid, attached to a functional component, such as a therapeutic agent (drug molecule s), fluorescent probe, nanoparticle or other surfaces. Expands Functional Diversity of Biomolecules Bioconjugation allows for the chemical modification of biomolecules, expanding their functional capabilities beyond what is naturally available. By linking different molecules, researchers can create combined molecules (conjugates) with tailored properties for specific applications. For example, they can conjugate a fluorescent reporter probe to a protein or an antibody to enable visualisation of the protein - a commonly used technique in imaging disease. Applications in Industry: PHARMACEUTICALS • Targeted Drug Delivery: Bioconjugation plays a pivotal role in creating antibody-drug conjugates (ADCs), which link a therapeutic drug to an antibody. Bioconjugation is crucial in targeted drug delivery, allowing for the specific delivery of drugs to diseased cells or tissues, minimising off-target effects and improving efficacy. For example, in the treatment of cancer cells. In particular, the conjugation of proteins and peptides to nanoparticles and drugs helps to minimise off-target interactions, which remains one of the most insurmountable bottlenecks in therapeutics and diagnostics.2 Efficient targeting is not the only advantage. In some cases, bioconjugation serves several other purposes to improve drug efficacy. For instance, drug molecules can be coupled with biomolecules to keep them inactive until they are taken up by the target host cells. Thus, they protect drugs from enzymatic degradation, extending their half-life and reducing the required dosage. Furthermore, the attachment of hydrophilic biomolecules can increase the solubility of the drug to enhance the circulation and absorption of injectable or orally administered drugs.3 • Vaccine Development: Bioconjugation allows for the attachment of antigens to carrier proteins, enhancing immune response. This approach is commonly used in the creation of conjugate vaccines (e.g., the Haemophilus influenzae type b (Hib) vaccine). • Biomolecular Stability: Bioconjugating enzymes, proteins, or antibodies to stabilisers or carriers can increase the shelf life of biologics, ensuring higher stability during production and transport. BIOTECHNOLOGY • Biosensor Development: In biotechnology, bioconjugation helps in the design of biosensors that can detect specific biological markers. Enzymes or antibodies are conjugated to a sensing surface, allowing for high specificity and sensitivity in diagnostics. • Protein Engineering: Through bioconjugation, proteins can be modified to enhance functionality (e.g., improving enzyme activity, stability, or resistance to degradation) for industrial applications, such as biofuel production or in food processing. • Protein Purification: Conjugating affinity tags to proteins can simplify the purification process, as these conjugated proteins can be isolated using affinity chromatography. 2 https://www.sciencedirect.com/topics/immunology-and-microbiology/bioconjugation#:~:text=It%20is%20amazing%20that%20 the,purpose%20the%20imagination%20can%20conceive. 3 https://vectorlabs.com/blog/it-takes-two-to-tango-parwwt3-advantages-of-bioconjugation /?srsltid=AfmBOorjfnVqGOWxRo5oY_ZiZhEOjE4jb9bLT-DDdQBAmaBO7b9yxa6h AnteoBind™ Technology White Paper 3DIAGNOSTICS • Immunoassays and Diagnostic Kits: Bioconjugation is a critical process in the development of immunoassays (e.g., ELISA, lateral flow tests) where antibodies are conjugated to enzymes, fluorescent markers, or nanoparticles. These conjugates enhance detection sensitivity and specificity for diseases, infections, and biomarkers. This is where AnteoTech with AnteoBind™ and AnteoBind NXT™, is making an impact - offering fast, reliable, and scalable conjugation with enhanced surface control, FOOD AND BEVERAGES enabling superior reproducibility across assays and platforms. • In Vivo Imaging: In molecular imaging, bioconjugation links imaging agents (e.g., radioactive isotopes, fluorescent probes) to targeting molecules, allowing for noninvasive tracking of disease progression or therapy effects. • Fermentation Optimisation: Bioconjugation can improve the efficiency of fermentation processes by conjugating enzymes to stabilising agents or carriers, ensuring more effective enzyme activity and reducing costs in large-scale production. in food products, ensuring quality control and consumer safety. • Food Safety Testing: Conjugated antibodies are used in food safety kits to detect pathogens, allergens, or contaminants • Functional Ingredients: In food innovation, bioconjugation can be used to create functional food ingredients by linking bioactive molecules (e.g., peptides or polyphenols) to delivery systems that improve bioavailability and efficacy when consumed. Amongst the various bioconjugation strategies, covalent bonds are the most extensively utilised. Conventionally, bioconjugation often entails the reaction of specific functional groups, such as primary amines, thiols, or carboxylic acids, that are inherently present on biomolecules. These interact directly or via crosslinking agents to form durable covalent linkages (by sharing electron pairs), resulting in the integration of the two molecules into a unified, functional complex. Globally, the most widely used covalent bioconjugation approach in life sciences is the EDC/NHS (1-ethyl-3-(3dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) chemistry, which is particularly effective in conjugating carboxyl and primary amine groups. This method involves the formation of stable amide bonds and is commonly employed in protein conjugation protocols due to its reliability and biocompatibility. These traditional covalent chemistries, while strong and stable, have limited the flexibility and control in bioconjugation applications in the market: Non-Specific Reactions: Some bioconjugation chemistries lack specificity, which can result in off-target modifications and nonheterogeneous products. Loss of Functionality: The modifications resulting from the chemical reactions can alter the biological activity or structural integrity of proteins or other biomolecules, potentially reducing their robustness and, in turn efficacy, binding affinity and their functionality. Harsh Reaction Conditions: Certain reactions require harsh conditions (e.g., extreme pH, temperature, or solvents) that can denature or damage sensitive biomolecules making them ineffective. Complex Optimisation: Designing traditional bioconjugation strategies may require extensive optimisation of reaction conditions, linkers, and functional groups. Limited Control Over Stoichiometry: Traditional approaches can make it difficult to precisely control the number of molecules conjugated per target, impacting reproducibility and function. 4 AnteoBind™ Technology White Paper AnteoTech’s AnteoBind™ and AnteoBind NXT™ is changing all this. AnteoBind™ is AnteoTech’s proprietary metal-ion complex-based activation technology which utilises the principles of coordination chemistry to create stable ligand-metal complexes that can be bound to various surfaces. Coordination chemistry (compounds), as theorised by Alfred Werner (1866-1919), is considered the backbone of modern inorganic and bio–inorganic chemistry and the chemicals industry.4 AnteoBind™’s core competitive advantage lies in its affinity for “electron-donating” ligands, allowing stable binding with diverse biomolecules and surfaces while maintaining biomolecule functionality. The metal ions in AnteoBind™ carry a positive charge and can confer this property to the activated surface, in particles, enabling the tracking of the average particle size and their surface charge at each stage of the conjugation process, ensuring sensitivity, robustness and control. In the case of planar surfaces, a previously hydrophobic surface can become less hydrophobic or greatly hydrophilic depending on the AnteoBind™ formulation applied. What is Coordination chemistry? Coordination chemistry provides the foundation for using metal complexes such as AnteoBind™ as bioconjugation reagents. A coordinate bond is a form of covalent bond where metal ions (predominantly transition metals such as cobalt, nickel and zinc) bind to donor ligands that have lone pairs of electrons (e.g. functional groups rich in nitrogen, oxygen, or sulphur) that can form complexes. A common example of coordination chemistry found in biology is the binding and transport of oxygen in the blood by haemoglobin. One of the key uses of coordination chemistry is in immobilised metal ion affinity chromatography (IMAC), commonly used for protein purification in the life sciences industry and academia. It uses the histidine (His)-tag system, a short amino acid sequence that can be attached to a recombinant protein of interest that can form coordinate bonds with the metal ion (typically nickel or cobalt), allowing it to selectively bind to the solid support. Nonspecifically bound proteins are then washed away, and the His-tagged protein is eluted from the solid support using a chelating agent. AnteoBind™ Technology - How does it work? The AnteoBind™ system utilises the same principle as IMAC to immobilise biomolecules of interest to a surface. Indeed, significant opportunities exist for AnteoBind™ to be used in purification processes (affinity chromatography and related applications), and progression of this is being explored with potential strategic partners. Chemically, AnteoBind™ consists of a mixture of oligomeric metal complexes of various lengths, each centred around metal ions capable of forming multiple coordinate (dative) bonds. These oligomeric metal complexes are designed to interact with specific functional groups on biomolecules, facilitating the formation of strong and stable bonds - important requirements for the development of sensitive, reproducible diagnostic tests for medical use. When AnteoBind™ binds to a surface, it forms a multi-nanometre thin layer of the metal complex (around 2-15 nm thickness depending on available binding groups, e.g. carboxyl) and primes the surface for biomolecule attachment (activation). The AnteoBind™ activated surface will robustly bind with the electron-donating functional groups (e.g. carboxyl, hydroxyl, thiol) on another surface or biomolecule. 4 https://ncert.nic.in/textbook/pdf/lech105.pdf Although a majority of the purified antibodies used in developing life sciences applications do not contain a His-tag, a histidine rich cluster is found in the Fc (fragment crystallisable) region of antibodies derived from many common hosts, including mice, humans and rabbits5. This histidine cluster in the Fc region allows for the coordinate complex-mediated orientation of the Fab (fragment, antigenbinding) regions away from the AnteoBind™ surface to freely bind available antigens (Figure 2). This ideal functional orientation of the antibody, achievable through AnteoBind™ use, facilitates increased sensitivity and helps reduce antibody usage by increasing the number of functional binding sites available for interactions with the analytes or antigen of interest. In other words, it creates a highly favourable orientation for attachment of molecules, a beacon! FIGURE 2: Ideal orientation of antibody on a surface to allow antigen binding. Typical antibodies are made of two regions (Fab and Fc), of which the Fab region is important for antigen recognition and binding. 5 Hale JE, Beidler DE. Purification of humanized murine and murine monoclonal antibodies using immobilized metal-affinity chromatography. Anal Biochem. 1994 Oct;222(1):29-33. doi: 10.1006/abio.1994.1449. PMID: 7856866.). 6 AnteoBind™ Technology White Paper Key advantages of AnteoBind™ technology over traditional products in the market Improved antibody orientation leading to better results An independent study by the Natural and Medical Sciences Institute (NMI) in Germany found that the deletion of lysine residues on the spike proteins due to certain mutations of SARS-CoV-2 led to a decrease in binding when using EDC/ NHS conjugations for Luminex-based multiplex assays ,whereas no decrease in performance was observed when using AnteoBind™ for the same assays.6 In other words, AnteoBind™ conjugates delivered better results for these specific mutants when compared to EDC/NHS conjugates in detecting the presence of these SARS-COV2 mutants. These differences are due to the variations in binding affinities of EDC/NHS and AnteoBindTM. Gao et al.7highlighted the differences in the distribution of lysine and histidine groups throughout a mouse IgG antibody. In their study it was determined that there was an even spread of lysine residues across the entirety of the antibody, meaning that there was a higher probability of attachment outside of the Fc region of the antibody, leading to non-optimal orientation. Conversely, the high concentration of histidine groups found in the Fc region and low occurrence elsewhere subsequently increases the probability of maintaining the ideal antibody orientation when using AnteoBind™, limiting the variability in orientation observed in EDC/NHS conjugations. Reproducibility The core of the AnteoBind™ technology lies in its affinity for “electron-donating” ligands (biomolecules) to form coordinate bonds. The oligomeric metal ions in AnteoBind™ confer a positive charge to the activated surface, enabling the tracking of particle size or contact angle and surface charge at each stage of the conjugation process. This allows for greater control over bioconjugation reproducibility, which is not possible with existing conjugation chemistries such as EDC/NHS. By way of an example application, an in-house study was undertaken to track the physicochemical changes throughout the bioconjugation process using 400nm blue latex particles. Following the standard instructions for use (IFU), the first step involved the activation of the 400nm latex particles, followed by the bioconjugation of a mouse monoclonal antibody to the activated 400nm blue latex particles and subsequent blocking using BSA. Table 1 shows size and charge data tracking the particles from their stock to conjugated state using Dynamic Light Scattering. The results show: • A progressive increase in particle size, confirming each successful binding layer. • A reversible shift in zeta potential, from negative to positive and back, clearly marking activation and antibody conjugation stages. TABLE 1: Physicochemical properties of particles during conjugation Stage Zeta-Size Poly-dispersity (d. nm) Stock bare blue latex 449.5 index (PdI) Zeta-Potential 0.027 (mV)- 50.8 AnteoBind™ NXT activated 460.1 0.004 + 58.1 Antibody conjugated particle 468.9 0.016- 51.3 6 Junker, D., Dulovic, A., Becker, M. et al. COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARSCoV-2 RBD mutants. Sci Rep 12, 7168 (2022). https://doi.org/10.1038/s41598-022-10987-2). 7 Shipeng Gao, Francisco Rojas-Vega, Javier Rocha-Martin, José M. Guisán, Oriented immobilization of antibodies through different surface regions containing amino groups: Selective immobilization through the bottom of the Fc region, International Journal of Biological Macromolecules, Volume 177, 2021, Pages 19-28, ISSN 0141-8130, AnteoBind™ Technology White Paper 7FIGURE 3: Average particle diameter during key conjugation steps. Particle size was measured for the stock particles (Bare-COOH particles- in red), particles incubated with AnteoBindTM (AnteoBindTM activated particles - in green), and activated particles conjugated with a mouse monoclonal antibody (Antibody Conjugate -in blue), using a Malvern Zetasizer Nano ZS instrument. As shown in Table 1, the addition of AnteoBindTM (green) and subsequently the antibody (blue) changes the size of the particles as expected. FIGURE 4: Surface charge during key conjugation steps. Particle charge was measured for the stock particles (Bare-COOH particles- in red), particles incubated with AnteoBindTM (AnteoBindTM activated particles- in green) and activated particles conjugated with a mouse monoclonal antibody (Antibody Conjugated Particles-in blue) using a Malvern Zetasizer Nano ZS instrument. As shown in Table 1, the addition of AnteoBindTM (Green) moved the particles to a positive charge on the right, and once the antibody is added to these particles the charge moves back to negative (Blue). These results are visualised in Figures 3 and 4. In Figure 3, a gradual shift to the right is observed as the particle becomes larger with each additional layer, and in Figure 4, there is a jump from negative to positive charge (measured using a Zetasizer instrument) once activated with AnteoBind™, and back to negative when the activated particles are conjugated with the antibody and blocked with an appropriate molecule. These real-time, traceable physicochemical changes offer additional opportunities for quality assurance, something traditional chemistries simply can’t provide. AnteoBind™ provides a simple, comprehensive in-process quality check throughout the conjugation process, whereas with conventional methods, this can only occur at the end of the process. This feature has the potential to save time and resources in the conjugation workflow and ensures greater batch-to-batch reproducibility features that are important for decision-making during large scale production. 8 AnteoBind™ Technology White Paper Stability While the AnteoBind™ reagents themselves are stable for up to two years at room temperature, surfaces treated with AnteoBindTM reagents have been shown to maintain their ability to reproducibly attach biomolecules for longer periods than traditional chemistries. In-house real time stability studies were conducted on AnteoTech’s newest product, AnteoBind™ NXT, over a 9-week period using a lateral-flow format in triplicates. The results (Figure 5) demonstrate that the AnteoBind™ NXT activated particles can be effectively used for conjugation and subsequent assays for up to nine weeks. These studies are ongoing, and the results will be updated as they become available. FIGURE 5: AnteoBind™ NXT activated particles can be used for conjugation for nine consecutive weeks. The graph shows average RFU for three replicates of lateral flow strips using streptavidin conjugated particles. Similar stability studies have been performed with particles activated using other AnteoBind™ products achieving over one-year real time stability and up to six years equivalent under accelerated conditions. This significant benefit is not available with EDC/NHS, where best practice requires the bioconjugation to be completed within hours of initiating the chemical reaction. The flexibility to perform bioconjugations at any time after initial activation not only improves workflow, but also enables the bulk activation of particles for subsequent use, improving reproducibility and reducing the costs associated with multiple activations. AnteoBind™ Technology White Paper 9AnteoBind™ compatibility with a wide range of particles and surfaces Table 2 below shows a selection of particles activated using AnteoBind™ with minimal optimisation of the standard methods. These include a wide range of particle sizes (40 nm-5.6µm), functional groups (-COOH/-OH/-silica), and surface materials (polystyrene, silica, polyvinyl alcohol, gold). AnteoBind™ clearly delivers considerable flexibility regarding surface functionalisation AnteoBind™ with optimal performs best overall when coupled with a carboxylated surface. Table 2: Selection of micro- and nanoparticles assessed inhouse Particle Functional Group Size (diameter) Material Magnetic or other Ademtech Carboxyl-Adembeads -COOH 200 nm 500 nm Polystyrene Magnetic Asahi Kasei NanoAct™8-COOH 300nm Cellulose Non-Magnetic Agilent LodeStars9 Carboxyl-COOH 2.7 µm Polystyrene Magnetic Bangs ProMag10 (PMC1N)-COOH 1 µm Polystyrene Magnetic Bangs ProMag (PMC3N)-COOH 2.8 µm Polystyrene Magnetic Bangs Silica Microspheres11-silica/OH 1, 5 µm Silica Non-Magnetic BBI Gold Colloids12-Au 40nm Gold Non-Magnetic JSR Micro Magnosphere (MX100/Carboxyl)-COOH 1.1 µm Hydrophobic polymer Magnetic JSR Micro Magnosphere (MS160/Carboxyl)-COOH 1.5 µm Hydrophilic polymer Magnetic JSR Micro Magnosphere (MS300/Carboxyl)-COOH 3 µm Polystyrene Magnetic Luminex® 13 Microspheres - MagPlex or MicroPlex-COOH 5.6 µm Polystyrene Fluorescent Merck Millipore Estapor®14 (EM1-100/40)-COOH 1 µm Polystyrene Magnetic Merck Millipore Estapor® (M1-200/20)-COOH 2 µm Polystyrene Magnetic Merck Millipore Estapor® (M1-020/50)-COOH 200 nm Polystyrene Magnetic Merck Millipore Estapor® Europium Carboxyl-COOH 300 nm Polystyrene Fluorescent Microparticles Polystyrene (PS-MAG-COOH-S1978)-COOH 350 nm Polystyrene Magnetic nanoComposix Bioready Gold Nanoshells15-COOH 150nm Gold Non-Magnetic Revvity Chemagen M-PVA (C21)-COOH 1 µm Poly vinyl alcohol Magnetic Thermo Fisher Colored Latex (DR/DB1040CA)-COOH 400 nm Polystyrene Non-Magnetic Thermo Fisher Dynabeads16 MyOne/Carboxyl-COOH 1 µm Polystyrene Magnetic Thermo Fisher Dynabeads M-270/carboxyl-COOH 2.6 µm Polystyrene Magnetic Thermo Fisher Fluoro-Max™ 17 Europium particles-COOH 100 nm, 200 nm, 300 nm Polystyrene Fluorescent In addition, AnteoBind™ has been used to activate a selection of other non-particle-based surfaces including glass, biosensor chips, cyclic olefin copolymer (COC) and polystyrene 96- and 384-well plates.8 8 https://www.asahi-kasei.co.jp/fibers/en/cnb/about/index.html 9 https://www.agilent.com/en/product/biomolecules-conjugation-synthesis-purification/resins-latex-magneticparticles/magnetic-particles/lodestars-carboxyl 10 https://www.bangslabs.com/products/magnetic-microspheres-particles/promag-particle-lines 11 https://bangslabs.com/product-category/silica-microspheres/plain-silica/ 12 https://shop.bbisolutions.com/product/gold-colloid-40nm-10ml/01tTt000004aSLFIA2 13 https://www.luminexcorp.com/xmap-technology/#microspheres 14 https://www.merckmillipore.com/AU/en/product/Estapor-Magnetic-Microspheres,MM_NF-C121726 15 https://nanocomposix.com/products/150-nm-bioready-gold-nanoshells-for-covalent-conjugation 16 https://www.thermofisher.com/au/en/home/brands/product-brand/dynal.html 17 https://www.thermofisher.com/order/catalog/product/93470350010150 10 AnteoBind™ Technology White Paper Superior Versatility EDC/NHS conjugation, the most used covalent chemistry in bioconjugation, is dependent on the presence of lysine residues on proteins, as they contain a primary amine group (ε-amino group) that readily reacts with the NHS ester to form a stable amide bond. However, the quantity and distribution of lysine groups varies across different antibodies. This variability affects the performance and reproducibility of the end conjugate, usually resulting in inconsistent performance and greater variability between batches of conjugates. Unlike EDC/NHS, AnteoBind™ bonds with a wide range of functional groups on surfaces of biomolecules through the formation of stable ligand-metal ion complexes. Both independent19 and internal studies have shown that the reliance on coordinate bonds increases the compatibility of AnteoBindTM. It is compatible with a wide range of biomolecules such as those listed below, making it a superior choice in the market. • Antibodies IgG, IgM): • Viral particles (e.g., HAV) • Virus like particles (e.g., Flavivirus) • Antigens (e.g., COVID-19 spike proteins) • Binding proteins (e.g., Streptavidin, Protein G/A) • Serum proteins (e.g., BSA) • Nucleic acids (e.g., single-strain DNA, poly-Ts) • Polysaccharides (e.g., Polysialic acid) • Synthetic polymers (e.g., Polyacrylic acid) • Particle to particle (e.g., Qdots to nanoparticles Comparative Summary of AnteoBind™ NXT vs. EDC/NHS EDC/NHS chemistry has long been acknowledged as the industry standard for covalently linking biomolecules, namely through its amide bond formation between carboxyl and amine groups. Its widespread use is attributed to its effectiveness in various applications, including protein labelling, nanoparticle functionalisation, and antibody conjugation. Two in-house comparative studies have been completed to evaluate the AnteoBindTM technology (AnteoBindTM NXT was used for these studies) against EDC/NHS, focusing on: 1. A direct comparison of AnteoBindTM NXT performance with standardised EDC/NHS protocols. 2. AnteoBindTM NXT compatibility with particles of different materials and sizes. 3. AnteoBindTM NXT compatibility and advantages across multiple assay platforms. 4. Potential antibody savings using AnteoBindTM NXT. Appendix 1 contains these case studies which set out the excellent results that AnteoBindTM has shown when compared to EDS/NHS across multiple assays. Table 3 lists some of the key operational differentiators between EDC/NHS and AnteoBind™. To highlight a few, once opened, the stock EDC/NHS chemicals must be processed quickly before aliquots are disposed of or the bulk returned back to the freezer. In contrast, AnteoBind™ reagents can be stored at room temperature or refrigerated for up to two years. The hygroscopic nature of EDC makes it difficult to re-use EDC aliquots and can lead to substantial reagent wastage, whereas AnteoBindTM reagents are supplied as a water-soluble, ready-to-use solution at room temperature, avoiding these common pitfalls. TABLE 3: Operational differences between AnteoBindTM and EDC/NHS AnteoBindTM Shipping Ambient EDC/NHS Dry Ice / Cold Storage Room Temperature- 80°C Reagent stability (opened) Up to 24 months 3 to 6 months Reagent wastage Nil Up to 90%* Optimal period for conjugation 12 months Immediate IPQC of Activation Yes No Single step co-conjugation Yes No Antibody usage Low High Total processing time 295 minutes 490 minutes^ * Based on customer feedback regarding using an SOP during research and development activities ^ Time based on a particle manufacturer’s EDC/NHS procedure The findings from the case studies indicate that AnteoBind™ possesses significant benefits over EDC/NHS depending on the biomolecule of interest and the assay format being used. These benefits may include significant antibody savings to achieve similar levels of sensitivity. In combination with improved workflow benefits, compatibility with a wider range of biomolecules and the flexibility to activate multiple surface types/chemistries. 18 https://anteotech.com/life-sciences/papers-publications/ AnteoBind™ Technology White Paper 11The AnteoBind™ Product Offering AnteoBind™ is a ONE-STEP ACTIVATION SOLUTION, available in ready to use kits and stand-alone reagents. The AnteoBindTM product range and what these products are used for is set out in Table 4 below and includes: TABLE 4: AnteobindTM Product Range along with most appropriate applications Products Usage AnteoBind™ NXT For nano- & microparticle based assay development (lateral flow, chemiluminescence, turbidimetric etc.) & bioseparations. AnteoBind™ Micro Activation of carboxylated particles >1μm in diameter for protein coupling AnteoBind™ Sub-Micron Activation of carboxylated particles from 0.2 to 1μm in diameter for protein coupling AnteoBind™ Biosensor Activation of planar surfaces (glass slides, 96 well plates, COC plastics etc.) for protein coupling AnteoBind™ Nano (for <0.5µm particles) Nanoparticle-based kit for lateral flow assay development & bioseparations AnteoBind™ Micro (for >0.5µm particles) AnteoBind™ Activation Kit Multiplex Microspheres Microparticle-based kit for assay development & bioseparations For developing Multiplex assays using low carboxyl load microparticles like the Luminex® Magplex particles. These reagent and kits are compatible with microspheres, nanospheres, ranging from 40 nm to6 microns and several planar surfaces. Supported particles include magnetic, coloured and fluorescent latex nanoparticles, Luminex microspheres, and gold colloids/nanoshells. 12 AnteoBind™ Technology White Paper Summary AnteoBind™ is redefining bioconjugation for the diagnostics and life sciences industry. As a smart surface activation technology, it enables fast, reliable, and highly reproducible conjugation of biomolecule, giving developers more control, better performance, and faster time to market. Unlike conventional chemistries like EDC/NHS, AnteoBind™ allows users to track each stage of the conjugation process through measurable changes in particle size and charge, ensuring unmatched consistency and quality. The result is lower reagent consumption, enhanced assay sensitivity, and reduced development costs. Whether you’re scaling a diagnostic platform or optimising R&D, AnteoBind™ delivers the precision, efficiency, and confidence needed to stay competitive in a rapidly evolving market. AnteoBind™ Technology White Paper 13 AnteoBind™ Technology White Paper 13Appendix 1 Case Study 1: A comparison of NXT with EDC/NHS in a nanoparticle-based, lateral flow format This study compared two different particle types (latex and gold nanoshells) commonly used in lateral flow tests. EDC/NHS chemistry or AnteoBind™ NXT were used to conjugate the protein of interest to the particles and subsequently detected in a lateral flow assay. To enable unbiased comparisons and quantify the differences between the two chemistries, a colorimetric strip reader was used to measure visual intensities. 400nm Blue latex particles Coloured carboxylated particles are widely used in lateral flow assays due to their enhanced chemical and physical stability across various storage conditions but also their ability to multiplex in a lateral flow device. Similar to gold, the results can be read visually and do not require special equipment (a reader) to interpret the results. In these experiments a half-strip lateral flow test to detect recombinant Influenza A nucleoprotein was developed. The conjugates were prepared using either EDC/NHS or AnteoBind™ NXT using the standard protocols. In order to demonstrate the advantages in antibody savings, three different antibody concentrations were conjugated to the particles (10, 25 and 100 microgram (µg) antibody per milligram (mg) of particles) for both the EDC/NHS or NXT mediated activations. These particles were then used to detect recombinant Influenza A nucleoprotein in a buffer-based assay. Key Findings: 1. Less antibody required: NXT activated conjugates prepared at 10µg of antibody/mg of particles displayed visible bands in all antigen samples while presenting no line in the blank sample. This band at the lowest antigen concentration was not visible in any of the EDC/NHS conjugates respective of the antibody loading. This example demonstrates a 90% savings in antibody usage to obtain similar or better visual results. 2. Better sensitivity: Using a strip reader the NXT conjugates showed better Signal:Blank (S:B) ratios at all concentrations of the Influenza A antigen compared to the corresponding EDC/NHS conjugated particles (Table 1). These differences in the detection limits were also confirmed in the visual band intensities seen in Figure 1. Figure 1: Lateral flow strip images demonstrating conjugates using either NXT or EDC/NHS. Each panel in the figure represents a set of strips that were incubated with decreasing concentrations of the antigen in triplicate, with the leftmost set of strips in each panel representing a blank condition. In the strips, the top line represents a control line, and a visible signal at the top indicates a valid test. The bottom line represents the test line, and a visual signal represents the presence of the antigen. As the concentration of an antigen increases in a sample, the visual signal intensity also increases in a lateral flow test. The red boxes show the best conditions for EDC/NHS conjugated particles compared with the NXT conjugated particles using the least amount of antibody. NXT - 100µg NXT - 25µg NXT - 10µg EDC - 100µg EDC - 25µg EDC - 10µg FIGURE 1: Lateral flow strip images demonstrating conjugates using either NXT or EDC/NHS. Each panel in the figure represents a set of strips that were incubated with decreasing concentrations of the antigen in triplicate, with the leftmost set of strips in each panel representing a blank condition. In the strips, the top line represents a control line, and a visible signal at the top indicates a valid test. The bottom line represents the test line, and a visual signal represents the presence of the antigen. As the concentration of an antigen increases in a sample, the visual signal intensity also increases in a lateral flow test. The red boxes show the best conditions for EDC/NHS conjugated particles compared with the NXT conjugated particles using the least amount of antibody. 14 AnteoBind™ Technology White Paper TABLE 1: Shows the Signal:Blank ratios observed for high, medium and low concentrations of the Influenza A antigen when detected with either NXT or EDC/NHS conjugated particles (conjugated with 100, 25 or 10 µg of the antibody) in a lateral flow assay. Conjugates Signal:Blank High Med Low NXT-100µg Ab 27.8 16.2 2.7 NXT-25µg Ab 32.2 19.3 4.1 NXT-10µg Ab 28.8 18.3 3.8 EDC-100µg Ab 12.5 3.7 1.4 EDC-25µg Ab 19.7 3.6 1 EDC-10µg Ab 11.4 4.1 1.1 150nm gold nanoshells Gold nanoshells (GNS) are another set of particles that are extensively used for lateral flow applications. The GNS used in the following experiment were 150 nm in size and had carboxylated surface groups to allow for EDC/NHS mediated conjugation. The GNS were conjugated with anti-Influenza A antibodies using the respective supplier protocols for EDC/NHS and AnteoBind™ NXT conjugation. The standard procedure for conjugating GNS for lateral flow use suggested a starting concentration of 30µg antibody/mg of particles, though the antibody was titrated down to 1µg antibody/mg particles due to observed incompatibilities of high antibody loading with the AnteoBind™ chemistry in the GNS (low specific signal and high non-specific signals observed). Although this is not an optimal concentration to test the EDC/NHS chemistry, this experiment highlights the differences in ideal antibody load. The AnteoBindTM NXT or EDC/NHS Influenza A conjugated GNS particles were used in a lateral flow format to detect two different concentrations of the recombinant Influenza A nucleoproteins (the same panel used in the Blue latex particles). The signals obtained from these strips were also quantitatively measured using a reader and compared between the two activation methods. Key Findings1. AnteoBindTM NXT delivers better antibody functionality: Although the background non-specific signals for both AnteoBindTM NXT and EDC/NHS conjugated particles were similar (Table 2), AnteoBindTM NXT conjugated particles comparatively produced a much higher specific signal for both antigen concentrations tested when 1ug of antibody was used for the conjugation. The 30µg EDC/NHS conjugate particles displayed both higher specific and non-specific signals leading to a similar level of detection, albeit using 30x more antibody. TABLE 2: Absolute Signals of AnteoBindTM NXT and EDC/NHS Conjugated GNS for high, medium and blank concentrations of the Influenza A antigen panel. Conjugates High Medium Blank NXT- 1µg Ab 8870 3257 137 EDC-1µgAb 1430 300 136 EDC- 30µgAb 10874 5473 208 2. AnteoBindTM NXT affords antibody savings with higher sensitivity - The Signal:Blank ratios for strips using AnteoBindTM NXT conjugated particles were similar compared to EDC-30µg particles. This can also be attributed to the lower background (blank) signals observed in the NXT-1µg particles. TABLE 3: Signal:Blank ratios of AnteoBindTM NXT and EDC/NHS conjugated GNS for the High and Medium concentrations of the Influenza A antigen panel. Conjugates High Medium NXT-1µg Ab 64.7 23.8 EDC-1µg Ab 10.5 2.2 EDC-30µg Ab 52.3 26.3 AnteoBind™ Technology White Paper 15Case Study 2: A comparison of AnteoBind™ NXT with EDC/NHS microparticles in four chemiluminescence assays This study compared the conjugation of antibodies (using either EDC/NHS or AnteoBindTM NXT) to 2.8µm carboxylated magnetic microparticles for the detection of four different analytes (Tumor Necrosis factor alpha (TNF-α), Interleukin-6 (IL-6), Interferon gamma (IFN-γ) and Granulocyte Macrophage Colony Stimulation Factor (GM-CSF)) in a chemiluminescence immunoassay (CLIA). All the ligand antibody conjugations were prepared in accordance with either the AnteoBind™ NXT instructions for use (IFU) or the EDC two-step protocol with NHS for magnetic particles. The microparticle antibody loading for each of the four antibodies was titrated incrementally and the performance was assessed in a CLIA format under buffered conditions. The two best antibody loading conditions (according to signal to blank ratios [S:B]) from each activation method were identified and directly assayed in the same plate to eliminate differences in inter-assay variability. TNF-α Table 4: Average signals obtained from the TNF-α CLIA at two antibody loadings TNF-α antigen NXT - 10μg Ab 2ng/mL 2,219,728 EDC - 10μg Ab 1,475,136 NXT-5 μg Ab 1,687,998 EDC-5ug 829,253 0.5ng/mL 528,244 346,025 419,705 232,356 0.125ng/mL 115,227 103,774 107,029 60,054 0ng/mL 11,471 9,328 11,338 10,393 FIGURE 2: TNF-α CLIA - Signal:Blank ratios of the respective activation methods against two antibody loadings In the TNF-α CLIA AnteoBind™ NXT (NXT) displayed consistently higher signals and slightly higher blank values leading to better performance compared to the EDC control when loading 10µg of antibody per mg of particles. At 5µg Ab/mg the NXT conjugates maintained very similar performance to the EDC conjugates at 10 µg Ab/mg loading whereas the EDC conjugates at 5µg Ab/mg had ~ 50% reduction in specific signal at all antigen points, leading to a noticeable drop off in performance. IL-6 TABLE 5: Average signals obtained from the IL-6 CLIA at two antibody loadings IL-6 antigen NXT - 10μg Ab EDC - 10μg Ab 2ng/mL 1,100,173 2,197,334 NXT - 1.25μg Ab EDC - 1.25μg Ab 2,375,590 148,376 0.5ng/mL 311,185 556,499 423,669 43,357 0.125ng/mL 91,427 146,369 88,046 7,470 0ng/mL 11,922 5,473 5,313 3,322 16 AnteoBind™ Technology White Paper FIGURE 3: IL-6 CLIA - Signal:Blank ratios of the respective activation methods against two antibody loadings The NXT conjugates significantly underperformed at the 10µg Ab/mg antibody loading due to low specific signals and high blanks compared with the equivalent EDC conjugates. However, decreasing the antibody amount by 8x improved the performance of the NXT-conjugated particles, whereas the EDC/NHS particles did not perform well under these conditions. IFN-γ TABLE 6: Average signals obtained from the IFN-γ CLIA at two antibody loadings IFN-γ antigen NXT - 10μg Ab EDC - 10μg Ab 2ng/mL 1,395,474 1,279,653 NXT - 2.5μg Ab 715,722 EDC - 2.5μg Ab 705,100 0.5ng/mL 293,762 259,124 177,829 145,322 0.125ng/mL 86,193 63,769 50,910 39,410 0ng/mL 11,294 6,329 7,089 6,449 FIGURE 4: IFN-γ CLIA - Signal:Blank ratios of the respective activation methods against two antibody loadings At higher concentrations of the antibody (10µg/mg) the EDC conjugated particles performed much better compared to the NXT particles, however at lower concentrations (5µg/mg) both NXT and EDC conjugated particles resulted in similar results, though only becoming closer in performance as antigen levels were reduced. AnteoBind™ Technology White Paper 17GM-CSF TABLE 7: Average signals obtained from the GM-CSF CLIA at two antibody loadings GM-CSF antigen NXT - 20μg Ab EDC - 20μg Ab 2ng/mL 0.5ng/mL 0.125ng/mL 0ng/mL 2,995,800 704,803 177,563 13,091 5,514,129 1,488,314 366,752 26,008 NXT - 10μg Ab 2,895,370 691,503 173,249 EDC - 10μg Ab 2,928,340 853,021 226,599 9,519 27,002 FIGURE 5: GM-CSF CLIA - Signal:Blank ratios of the respective activation methods against two antibody loadings Both the antibody concentrations tested for this analyte resulted in significantly higher blank (non-specific signals) for the EDC conditions compared to the NXT conditions. Despite the higher blank values both the EDC and NXT conditions performed similarly at the 20µg Ab/mg loading, while the NXT condition at half the antibody loading was seen to outperform the EDC conjugates at both concentrations of antibody loading. Key Findings: 1. NXT allows significant antibody savings: The NXT activation achieved comparable performance to EDC using 50% less antibody in the TNF-α CLIA, and 87.75% less in the IL-6 CLIA. 2. NXT conjugation may not work at traditional antibody concentrations - Notably, the optimal IL-6 antibody loading for the EDC method (10 µg Ab/mg) resulted in an almost non-functional conjugate when used with NXT. This underscores both a fundamental difference between the two technologies and the critical importance of optimising antibody loading when evaluating the AnteoBind™ platform. To discuss opportunities of how AnteoBind can best support your product development and research please contact us at support@anteotech.com 18 AnteoBind™ Technology White Paper To discuss opportunities of how AnteoBind can best support your product development and research please contact us at support@anteotech.com.