Accelerate Antibody Pair-Based Immunoassay Success

How to: Save Time, Reduce Variability, and Optimize Assay Performance De-Risk and Streamline Antibody Pair-Based Immunoassay Development For Research Use Only. Not for Use in Diagnostic Procedures. $300 Billion Biopharma R&D investment in 2023 10%–15% of Drugs Go to market 75% of R&D Costs Attributed to drug development failures $28 Billion Annual spend on irreproducible pre-clinical research in the US $350 Million Annual spend on poor-performing antibodies in the US (of the $28B spend) 36% of Research Expenses Wasted Attributed to biological reagents in the US $10 Billion Estimated annual spend on wasted research expenses attributed to biological reagents in the US Introduction R&D investment in biopharma topped $300 billion1 global spend in 2023. Even so, bringing a new drug to market remains difficult, with only 10% to 15%2 of new candidates reaching the public. With nearly 75% of R&D costs attributed to drug development failures3, it’s critical to ensure accurate and reliable data is used to guide decisions at every stage of the drug development process. Prioritizing the development of robust and reproducible assays that leverage sensitive, specific antibodies can control costs and enable data quality. This ensures fast, accurate results that facilitate data-driven decision-making and minimize potential risks as projects continue to future stages. This how-to guide breaks down challenges associated with developing pair-based immunoassays and offers solutions to help de-risk drug development.2 cst-science.com/pair-based-assay For Research Use Only. Not for Use in Diagnostic Procedures. Solutions for Antibody Pair-Based Immunoassay Development Key Factors & Challenges in Antibody Pair-Based Immunoassay Development ELISA and ELISA-like immunoassays are staples in the drug discovery workflow, and are used during biomarker discovery, compound screening, and PK/PD studies. Utilizing antibody pairs to detect your protein of interest offers increased specificity, sensitivity, and accuracy compared to single antibody assays. Developing a robust pair-based assay to detect and quantify your target can be time-consuming, costly, and fraught with challenges that result in extended project timelines and wasted resources, due to the complexities around: Finding and identifying sensitive and specific immunoassays or antibodies. A significant amount of time can be wasted searching for and validating commercially available, off-the-shelf ELISA or ELISA-like assay kits, matched antibody pairs, or individual antibodies to create a custom assay. An off-the-shelf immunoassay kit must be evaluated to confirm that there is no compound binding site interference, that it meets specificity and sensitivity requirements, and/or whether further optimization is necessary. If a custom assay is being developed, all suitable combinations of commercial pairs or individual antibodies must be tested to find the ideal pair. However, many antibodies currently on the market lack the sensitivity, specificity, and consistency required, which can result in a time-intensive internal validation process. Verifying assay reproducibility and robustness. In the United States, an estimated $28 billion is spent annually on preclinical research that is not reproducible, with approximately $350 million attributed to subpar antibodies.4 Antibodies not properly validated across lots can lead to specificity or performance variations, resulting in assays that can become less reliable and reproducible over time. Additional variability may be introduced during antibody conjugation if the purity of the conjugate cannot be verified. Ensuring platform compatibility and supply. Many assay technology and instrumentation companies offer off-the-shelf assay kits, but may have a limited portfolio of targets. Even when a kit is available, it may lack the required sensitivity, may not be offered in the optimal format, or may not be available in sufficient quantities to support a project’s entire duration. Antibody vendors that do not own their manufacturing rely on third-party providers to source products and have limited control over supply and final formulation. They may have trouble accepting and filling bulk orders or offering custom formulations, causing unexpected delays and increased assay variability. Meeting accelerated development timelines. Preclinical timelines are typically fast-paced and require assay design, optimization, and validation efficiencies. However, every variable added at each stage can increase risk and delay project timelines. Managing cost constraints. Assay development is expensive, requiring organizations to balance cost-effectiveness with antibody quality and performance. 36% of research expenses wasted in the US are assigned to biological reagents, an estimated cost of $10 billion annually.5 References 1. Total global spending on pharmaceutical research and development from 2014 to 2030. Statista. June 20, 2025. July 7, 2025. https://www.statista.com/statistics/309466/ global-r-and-d-expenditure-for-pharmaceuticals/ 2. Sun D, Gao W, Hu H, Zhou S. Why 90% of clinical drug development fails and how to improve it?. Acta Pharm Sin B. 2022;12(7):3049-3062. doi:10.1016/j.apsb.2022.02.002 3. P. Corr, D. Williams. Conflict of Interest in Medical Research, Education, and Practice. E, The Pathway from Idea to Regulatory Approval: Examples for Drug Development. NIH National Library of Medicine. 2009. July 7, 2025. https://www.ncbi.nlm.nih.gov/books/NBK22930/ 4. M. Biddle, P. Stylianou, M. Rekas, A. Wright, J. Sousa, D. Ruddy, et al. Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge. MAbs. 2024;16(1):2323706. doi:10.1080/19420862.2024.2323706 5. Weller MG. Quality Issues of Research Antibodies. Analytical Chemistry Insights. 2016;11. doi:10.4137/ACI.S31614cst-science.com/pair-based-assay 3 Multiple Solutions to Meet Your Specific Assay Requirements There are many variables to consider and challenges to address when designing an immunoassay that produces accurate, reproducible results. How you achieve this may differ depending on the requirements of your project. It’s imperative to prioritize flexibility, reliability, and assay platform compatibility regardless of whether you use validated kits for ELISA or ELISA-like assays, antibodies for ELISA-like assay development, or custom antibody conjugation and formulations. For Research Use Only. Not For Use in Diagnostic Procedures. Solutions for Antibody Pair-Based Immunoassay Development Bulk Orders and Lot Reservations Customized and Carrier-Free Formulations Matched Antibody Pairs ELISA-Validated Antibodies Target Custom Conjugation and Labeling Services ELISA Kits Rapid, reproducible antibody pair-based assay development requires flexible solutions. Immunoassay Development Benefits Time Savings Reliable Supply Flexibility Reproducibility Product Performance4 cst-science.com/pair-based-assay Solutions for Antibody Pair-Based Immunoassay Development For Research Use Only. Not for Use in Diagnostic Procedures. Off-the-Shelf Solutions Commercially available, validated kits are an efficient way to start assay development as they decrease the time spent searching for the right pair of antibodies. These “off-the-shelf” solutions include a validated antibody pair and additional reagents for the preferred assay platform. Ideally, “off-the-shelf” commercial kits should also arrive quickly. Options that guarantee lot-to-lot consistency are vital to ensure reproducibility. Sandwich ELISA: Using Cell Signaling Technology (CST)® ELISA Kits Depending on the specific targets and sample types, PathScan®, PathScan® RP, or FastScan® ELISA kits may be suitable options. All kit types exhibit similar performance; however, PathScan RP and FastScan kits require less hands-on time and deliver results in 90 minutes. Multiple sizes are in stock for all kit types, so it’s easy to order 1, 5, 10, 20, or 50 kits from the same lot. There is an option to custom order kit quantities from the same lot beyond what is listed in the catalog; these can be shipped at a requested cadence. CST manufactures all kit components in house, which allows for further customization, such as more or less of a specific component, chemiluminescence detection, or requiring a 384-well format. All new kit lots are tested against previous lots to ensure lot-to-lot consistency. This includes testing with positive and negative controls, checking optical density signal thresholds, and checking inter-assay variance between the current and new lot. Follow-up quality control is performed at half the shelf life to ensure no signal degradation over time. ELISA-like Immunoassays: Using Platform Vendor Kits Check if the chosen assay platform vendor offers an off-the-shelf assay kit for the specific target and sample type. The kit will require testing and validation to deliver the required sensitivity and specificity. If successful, assess the number of kits needed to support a project and whether it fits within allotted budgets. Other considerations may include potential lot variations if the vendor does not guarantee lot-tolot consistency, and unexpected interruptions like order delays and/or kit discontinuations, as the antibodies in these kits are typically sourced from third parties. Custom Assays: Using CST® Matched Antibody Pairs and ELISA-Validated Antibodies If a commercial kit is unavailable or does not meet budgetary restrictions, using ready-to-ship matched antibody pairs and ELISA-validated antibodies to develop a custom ELISA-like assay may be the ideal solution. CST matched antibody pairs and ELISA-validated antibodies are tested using sandwich ELISA to identify and validate both capture and detection antibodies. They are carrier-free (BSA- and azide-free at ≥ 1 mg/mL in 100 µL), making them conjugation-ready for compatibility with a variety of ELISA-like immunoassays and platforms: • Meso Scale Discovery (MSD) • AlphaLISA, AlphaScreen • TR-FRET, HTRF, LANCE • Luminex xMAP • Quanterix Simoa Time Savingscst-science.com/pair-based-assay 5 An assay is only as good as the antibodies used. Antibodies can perform differently depending on the application, so it’s ideal to use ELISA-validated antibodies for your ELISA and ELISA-like immunoassay development. CST matched antibody pairs are selected by identifying “best-binders” on a combination of ELISA-type assays, including a classical sandwich ELISA. The assay specificity is confirmed using multiple positive cell lines, knock-out cell lines, when possible, and cell lines where the target is not expressed, or, in the case of a post-translational modification, not activated. After a potential pair is identified, additional tests are run to rule out non-specific binding and to determine species specificity. Most pairs that make it this far are then conjugated to confirm the pair works in conjugated form for compatibility with most ELISA-like immunoassays. What to keep in mind when identifying the optimal matched antibody pair: Select ELISA-validated antibodies. ELISA-validated antibodies enhance the rate of success, ultimately saving time and resources. If ELISA-validated antibodies are unavailable, look for antibodies against the target of interest and validated for multiple applications. Antibodies validated across several applications have a greater chance of success of working as a matched pair, compared to antibodies validated for a single application. Choose antibodies that target non-overlapping epitopes. Use antibodies that bind to non-overlapping epitopes to avoid competitive binding between capture and detection antibodies. Also, confirm that no compound binding site interference exists between the tested compound and either antibody. Check antibody species reactivity. Confirm the antibodies will recognize the target protein in your sample and avoid non-specific binding that can increase background noise or cause false positives. Ensure the assay sensitivity and specificity meet the requirements. Perform titration and standard curve experiments, keeping an eye on signal-to-noise levels, to establish the Limit of Detection (LoD) and/or Lower Limit of Quantification (LLQ). This ensures an appropriate dynamic range and assay sensitivity. Use positive and negative control samples or perform blocking or competitive inhibition experiments to determine the assay specificity. Address lot-to-lot consistency. Confirm the antibody vendor provides reagents that will perform the same on your project’s first and last day. CST eliminates lot-to-lot variability by comparing data from new antibody and kit lots across multiple applications with previous lots to ensure equivalent performance. Once a matched antibody pair is identified, customize formulations to meet specific assay requirements. Solutions for Antibody Pair-Based Immunoassay Development For Research Use Only. Not For Use in Diagnostic Procedures. Antibodies Shouldn’t Be a Variable In Your Immunoassay Development Product Flexibility Performance6 cst-science.com/pair-based-assay Solutions for Antibody Pair-Based Immunoassay Development For Research Use Only. Not for Use in Diagnostic Procedures. Do-It-Yourself Conjugation—or Not In most ELISA-like immunoassay platforms, at least one antibody in the pair must be conjugated to a specific detection label. However, creating high-quality, reliable conjugated antibodies is challenging. Specific chemistries tailored for different labels are required to achieve a stable conjugation and to prevent post-conjugation antibody aggregation. Precise ratios of antibody-to-label and robust purification are critical for optimizing labeling, maximizing recovery of functional product, and maintaining specificity and sensitivity for the intended application. Do-it-yourself (DIY) conjugation kits are popular for their convenience and ease of use, but performance can vary between vendors and may not meet the specific needs of your assay. Lot-to-lot variability, even from the same vendor, can lead to inconsistent antibody conjugation and compromised assay performance. These kits often lack the flexibility to adjust the critical antibody-to-label ratio level or optimize the conjugation process, as they rely on a single conjugation chemistry, provide a fixed amount of materials, and require specific antibody quantities and starting material formulations. Kit protocols often fail to include purification and characterization steps—key processes that remove free dye and unreacted antibody to improve assay performance, as their presence can artificially increase or decrease the assay signal and compromise the accuracy of your results. Scaling up conjugation reactions to support larger projects is also challenging, as most kits are designed for small-scale use and typically accommodate 1 mg of antibody or less. This limitation often necessitates preparing multiple batches, which can introduce inconsistencies in the conjugated antibody over the course of the project. Finally, if an antibody conjugate prepared using a DIY conjugation kit fails, identifying the root cause can be difficult, as the issue may stem from the antibody, the conjugate, or the kit components. These challenges subsequently require more time and resources to resolve. CST Antibody Conjugation vs DIY Antibody Conjugation Flexibility Product Performance Analysis of FGF Receptor 1 (D8E4) XP® Rabbit mAb #9740 conjugated using CST methods compared to DIY kits from two other vendors via Ultra-High-Performance Liquid Chromatography (UHPLC). The optimization of bio-conjugation chemistries and stringent purification to remove free dye and antibody performed by CST results in a higher quality product, compared to other vendors’ products that contain higher levels of free dye and debris, which can interfere with assay results. Utilizing premium conjugation and purification methods paired with rigorous quality control standards can circumvent DIY pitfalls and ensure the quality, performance, and lot-to-lot reproducibility of conjugates. This results in consistently high-quality conjugates. CST Conjugate Competitor C Conjugate Competitor B Conjugate Competitor C free dye Competitor B free dye and debris CST free dye Absorbance [mAU] 30.0 25.0 20.0 15.0 10.0 5.0 0.0 0.01 -5.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Time [min] 1 2 3cst-science.com/pair-based-assay 7 Solutions for Antibody Pair-Based Immunoassay Development For Research Use Only. Not For Use in Diagnostic Procedures. Conjugation/Label Type Popular Labels Platforms & Techniques Fluorophores Type I Alexa Fluor® 647 • TR-FRET Fluorophores Type II Allophycocyanin (APC) Lanthinides trFluor™ Europium Cryptate Haptens Biotin • Meso Scale Discovery (MSD) • AlphaLISA, AlphaScreen • Luminex xMAP • Quanterix Simoa Digoxigenin (DIG) • Bio-Techne Ella Visit CST Custom Antibody Conjugation & Labeling Services for a comprehensive list of offerings and applications. Avoid Assay Variability with Bulk Orders and Lot Reservations Nobody likes the extra work needed if a bridging study is required after a new antibody or kit lot is introduced mid-project. Ordering the correct ELISA kit, matched antibody pair, or single ELISA-validated antibody quantities eliminates the additional work and helps avoid product supply delays. CST provides custom bulk orders from the same lot and with the option to reserve an entire lot of ELISA kits, matched antibody pairs, or ELISA-validated antibodies, ensuring assay consistency for the duration of your project. Bulk orders can be shipped based on a preferred schedule, pre-aliquoted, and packaged to fit into project workflows seamlessly. Reproducibility Reliable Supply Partner with CST scientists to get optimized, reproducible conjugated antibodies to keep your projects on track. Custom Conjugation & Labeling Services offer a broad range of conjugations and labels that can be tailored to match needs and budgets. CST labels for antibody pair-based immunoassays:Cell Signaling Technology (CST) is a different kind of life sciences company—one founded, owned, and led by active research scientists, with the highest standards of product and service quality, technological innovation, and scientific rigor for over 25 years. We consistently provide fellow scientists around the globe with best-in-class products and services to fuel their quests for discovery. Helping researchers find new solutions is our main mission every day—but it’s not our only mission. We’re also dedicated to helping identify solutions to other problems facing our world. We believe that all businesses must be responsible and work in partnership with local communities, while seeking to minimize their environmental impact. That’s why we joined 1% for the Planet as its first life sciences member, and have committed to achieving net-zero emissions by 2029. At CST, we believe in the power and promise of science to solve the challenges we face as a global community. We’re a company of caring people driven by a devotion to facilitating good science—a company committed to doing the right thing for our customers, our communities, and our planet. 25-PLC-64150 For Research Use Only. Not for Use in Diagnostic Procedures. © 2025 Cell Signaling Technology, Inc. All rights reserved. Cell Signaling Technology, CST, XP, IntraSeq, PTMScan, and SignalStar are trademarks, or registered marks of Cell Signaling Technology, Inc. 10x Genomics is a registered trademark of 10x Genomics, Inc. Simple Western is a registered trademark of Bio-Techne. All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information. CST Antibody Performance Guarantee: CST antibodies are guaranteed to work—the first time, and every time. To learn more, please visit: www.cellsignal.com/abguarantee cellsignal.com Doing good science is important. So is just doing good. Solutions for Antibody Pair-Based Immunoassay Development CST Solutions for Immunoassay Development Benefits Time Savings — An extensive collection of kits, pairs, and antibodies makes it easy to quickly find the optimal products for your immunoassay development and deliver timely results. Reliable Supply — Get started fast with ready-to-ship kits, pairs, and antibodies that arrive quickly. Avoid project delays and interruptions through bulk ordering and lot reservation. Flexibility — Choose from a continuously growing menu of products purpose-built for immunoassay development. If you can’t find what you’re looking for, custom order antibody pairs and ELISA-validated antibodies, or work with us on a custom antibody conjugation. Product Performance — Rigorously validated ELISA kits, pairs, and antibodies deliver reliable results with guaranteed functionality, specificity, and sensitivity. Reproducibility — Lot-to-lot and assay-to-assay consistency throughout the length of your project ensures consistent and accurate results. Visit the CST Solutions for Antibody Pair-Based Immunoassay Development webpage for more information on how to jump-start your assay development. Rapid, Reproducible Antibody Pair-Based Assay Development Starts with CST The flexibility and reliability you want to support your assay development for the entire duration of your project will allow you to focus on delivering reproducible and accurate results. From ready-to-use to fully custom, CST solutions support developing, scaling, and delivering reproducible immunoassays quickly.