Advancing Immunodiagnostics With Magnetic Beads

Magneto sphere MAGBEAD MAGAZINE | ISSUE 2 | AUGUST 2024 Today’s trends shaping tomorrow’s healthcare P02 FOCUS ON IMMUNODIAGNOSTICS Membrane-based immunoassays – 10 top tips for lateral-flow assay (LFA) development P20 Magnetic bead-based protein purification and immunoassay development P08 The evolution of highsensitivity HIV diagnostic testing P12 In this issue 1 When designing your own lateral flow assay, there are several variables to consider: with the choice of detection reagents, assay materials, and manufacturing methods all influencing the accuracy and reliability of your test. Use this checklist to review ten essential considerations when developing your LFA, which will give you a deeper insight into lateral flow assays to streamline your development process. Know your target molecule The biology of your target molecule will be the crucial factor in developing the biology of your test. Is it a large or small molecule? What antigen or other ligand will bind specifically to your target but not other molecules? The kinetics of the binding reactions between your analyte and the antibodies used in the test will determine your assay’s optimal flow time, which will influence your choice of nitrocellulose (NC) membrane. A keen understanding of the biology of your target molecule will shape every choice in the design of your LFA. Know your sample type The biological source of your sample will influence the design of your test and help you determine which materials you should use in your assay. The type and volume of liquid samples (e.g., blood, urine, or saliva) are two parameters that will help you choose which sample and absorption pads are most appropriate for your test. Start developing your lateral flow immunoassay with our diagnostic services 1 Pick your test type The size of your target molecule will be a key factor in determining the type of LFA you choose. There are two approaches to lateral flow assay tests: sandwich and competitive. Sandwich assays rely on the binding of two separate antibodies to different regions of the same analyte molecule, making them well-suited for detecting high molecular weight analytes. Competitive assays can detect smaller molecules, such as mycotoxins or cortisol, and larger analytes, such as insulin. Fig 1. Comparison of sandwich and competitive lateral flow assay design. Materials matter! Choosing the right sample pad, conjugate release pad, and nitrocellulose membrane are critical to developing sensitive and reliable lateral flow assays. Sample pads are commonly made from either cotton or bound glass fiber. Cotton has a lower wicking rate and is suitable for low sample volumes. Bound glass is opriate for separating red blood cells (RBCs) from plasma. Using glass fibers tercanalso prevent hemolysis, the bursting of RBCs that turns sayreadouts. douchoose will store fthe10 top tips for lateral flow assay (LFA) development 2 Test line Control line 3 Result Sample pad Sandwich Conjugate pad Test line Control line Result Sample pad Competitive Conjugate pad Target Label-conjugate antibody Imm. captured antibody A note from Cytiva In this issue of Mangetosphere, we focus on immunodiagnostics, which is a field witnessing significant evolution. Characterized by augmented accuracy, speed, and simplicity, advancements in immunodiagnostics promise to streamline and enhance testing processes further than ever before. Read about some of these advancements in an excerpt from our whitepaper on page 2. It highlights some of today’s trends that are shaping tomorrow’s healthcare. As the world of immunodiagnostics evolves, emerging technologies such as microfluidics, nanotechnology, and artificial intelligence are bringing the next generation of immunoassays. Meanwhile, traditional technologies continue to develop. Find out how magnetic beads can help enhance ELISA on page 4. Today, immunoassays are an indispensable resource across numerous branches of medicine, including oncology, cardiology, infectious diseases, and more. They provide a rapid, sensitive, and cost-effective means of disease detection and monitoring. But their success relies heavily on the performance of the chosen antibodies. The article on page 16 talks about the importance of validating antibody specificity. We also understand that the lines between magnetic beadbased and membrane-based immunoassays are becoming more blurred, so for those considering supplementing or substituting your magnetic beads for membranes, check out the top tips for developing lateral-flow assays on page 20. We always appreciate your feedback, so please get in touch. We hope you enjoy this issue! Contents Magnetosphere | 01 02 Immunodiagnostics: Today’s trends shaping tomorrow’s healthcare 04 Enhancing ELISA with magnetic beads 08 Magnetic beadbased immunoassay development 16 Why validate antibody specificity? 12 The evolution of high-sensitivity HIV diagnostic testing 11 Chemistry: The science of success 20 From beads to branes: Top tips for lateral-flow assay (LFA) development Today’s trends shaping tomorrow’s healthcare Global demographic changes The global increase in life expectancy has led to a larger elderly population who are more susceptible to chronic and infectious diseases. An aging global population requires more frequent and comprehensive diagnostic testing to manage health conditions effectively. Increasing burden of infectious and chronic diseases The rising incidence of chronic diseases (such as cancer, diabetes, and cardiovascular disorders) and infectious diseases (like HIV/AIDS and COVID-19) has heightened the need for disease detection and monitoring tools. Increased emphasis on early detection Detecting diseases at an early stage is key to enhancing treatment effectiveness and minimizing healthcare costs. This increased need for early detection has driven the demand for highsensitivity immunoassays. Demand for personalized medicine There is a growing trend toward personalized medicine. In personalized medicine, treatments are tailored to the individual characteristics, needs, and preferences of a person. Immunodiagnostics play a crucial role in facilitating personalized medicine by enabling the sensitive identification of biomarkers that inform therapeutic strategies. Exploring immunodiagnostics market trends provides valuable insight into the emerging technologies and their applications. The immunodiagnostics market is rapidly expanding. This market growth is being fueled by several demand drivers, including: Immunodiagnostics market segments by application In the current immunodiagnostic market, infectious diseases represent the largest application. This emphasis is thanks to the increasing incidence of infectious disease globally, particularly following the COVID-19 pandemic. However, another rapidly growing application area in the immunodiagnostics space is in oncology. Immunodiagnostics are crucial for the screening, detection, prognosis, and monitoring of cancer treatments. Consequently, substantial expansion in the oncology segment is anticipated in the near future (2). Our whitepaper explores the rapidly evolving landscape of immunodiagnostics that has the potential to transform healthcare systems worldwide. Through investigating current and emerging technologies, market trends, and future prospects, we consider the opportunities and challenges that lie ahead. 1. Data Bridge Market Research. Global Immunodiagnostics Market — Industry Trends and Forecast to 2030. Available from: https://www.databridgemarketresearch.com/reports/globalimmunodiagnosticsmarket/ 2. Skyquest. Global Immunodiagnostics Market Size, Share, Growth Analysis, By Type(Reagents & kits, instruments), By Application(Infectious diseases, oncology) — Industry Forecast 2023-2030. Available from: https://www.skyquestt.com/ 2030 $31.43 billion Predicted growth of immunodiagnostic market IMMUNODIAGNOSTICS 2 1 3 4 2022 $17.44 billion Read the full whitepaper here Magnetosphere 02 | Magnetosphere | 03 | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 E nzyme-linked immunosorbent assay (ELISA) is a widely adopted biochemical technique in immunology and molecular biology. It plays a pivotal role in various applications, including diagnostics, biomedical research, and drug development. While the capabilities of ELISA are well-established, recent innovations using magnetic beads are increasing its usefulness. However, intricacies of ELISA including the integration of magnetic beads, can be challenging to grasp for beginners and even experienced researchers. In this article, we aim to demystify ELISA, providing a clear understanding of its principles, types, and practical applications, with a special focus on the role of magnetic beads in advancing this essential technique. Understanding ELISA ELISA is a sensitive and specific technique used to detect and quantify the presence of specific molecules such as proteins, peptides, antibodies, and antigens. ELISAs use an enzymatic reaction between, for example, horseradish peroxidase (HRP) and luminol to generate a color change that is proportional to the concentration of antigen-bound antibody in the assay. ELISA is used in many different fields. In diagnostics, ELISA is extensively used in clinical laboratories for diagnosing diseases. It can detect specific antibodies or antigens associated with various diseases such as HIV, hepatitis, and autoimmune disorders. The technique can also be used to diagnose endocrine conditions such as thyroid disease, and a variation of it is used in home pregnancy tests. ELISA is also used in nonmedical fields. For environmental monitoring, it is used to screen environmental samples for contaminants such as pollutants, allergens, and pathogens. It can also be used for checking food and beverage safety and quality control. How magnetic bead-based ELISA works Magnetic bead-based ELISA, also known as magnetic bead ELISA or magnetic beadassisted ELISA uses magnetic beads to streamline the process. Magnetic beads, typically made of superparamagnetic materials, respond to an external magnetic field, making them particularly valuable in assay development. In a conventional ELISA, plates or wells coated with capture antibodies are used to immobilize target antigens from the sample. In magnetic bead-based ELISA, the capture 1 Multiplexing: Magnetic beadbased ELISA allows for simultaneous detection of multiple analytes, making it well-suited for high-throughput applications. 2 Point of care (POC) and portable devices: Compact and portable ELISA systems with magnetic bead integration could revolutionize POC testing, offering rapid diagnostics in remote or resourcelimited settings. 3 Customization for personalized medicine: Magnetic bead-based ELISA can be customized by selecting appropriate surface chemistries, coupling selected ligands to the beads, or using blocking agents. 4 Integration with emerging technologies: ELISA will continue to integrate with emerging technologies, such as microfluidics and nanomaterials, for improved sample handling and detection. 5 Data integration and bioinformatics: Advanced data analysis and bioinformatics will play a crucial role in extracting meaningful insights from magnetic bead-based ELISA experiments. The future of ELISA with magnetic beads Magnetic bead-based ELISA has been around for a while, but is likely to expand the use of ELISA in the following ways: “ ELISA is a valuable technique with a vast array of applications in research, diagnostics, and beyond Enhancing ELISA with magnetic beads By Laura Prescott, Scientific Marketing Writer, Cytiva Recent innovations are increasing the usefulness of ELISA as a biochemical technique. Find out more in our latest article. Magnetosphere 04 | Magnetosphere | 05 ELISA | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 antibodies are attached to magnetic beads instead. The process involves the following steps: • Antigen binding: Magnetic beads, precoated with capture antibodies specific to the target antigen, are mixed with the sample. The magnetic beads bind to the antigen in the sample. • Magnetic separation: A magnet is applied to the reaction mixture. The magnetic beads, along with the bound antigens are pulled to the side of the container, while unbound substances remain in solution. • Washing and detection: The unbound substances are removed by carefully aspirating the supernatant. Then, detection antibodies, typically labeled with enzymes or fluorescent markers, are added. These detection antibodies bind specifically to the antigens on the magnetic beads. • Signal amplification: As with traditional ELISA, an enzyme substrate is introduced, and a reaction occurs to produce a signal, which can be quantified. Magnetic bead-based ELISA has the advantage of signal amplification due to the close proximity of the magnetic beads, enhancing the assay’s sensitivity. Advantages of magnetic bead-based ELISA Using magnetic beads in your ELISA has many advantages. You’ll have enhanced sensitivity because magnetic beads allow “ Magnetic beads can also save the end user time. Assays are faster because magnetic bead-based ELISA often requires shorter incubation times for a higher binding capacity. This increased sensitivity is especially important when dealing with low-abundance antigens. Magnetic beads can also save the end user time. Assays are faster because magnetic bead-based ELISA often requires shorter incubation times. Magnetic beads are also automation-friendly, so assays using them are well-suited for high-throughput applications. These advantages could lead to faster and earlier diagnoses for patients allowing them to receive more timely treatment. Magnetic bead-based ELISA also has applications beyond diagnostics. Researchers use this technique in pharmaceutical research for drug discovery, pharmacokinetics, and pharmacodynamics studies. It is also valuable in protein-protein interaction studies and in studying binding affinities. And genomic and proteomic researchers use magnetic bead-based ELISA for DNA and protein purification and analysis. ELISA is a valuable technique with a vast array of applications in research, diagnostics, and beyond. Understanding the principles and types of ELISA is key to obtaining reliable and meaningful results. The technique is evolving to meet the demands of modern research and healthcare. Automation, miniaturization, and magnetic bead-based ELISA represent a promising future, providing new avenues for innovation with an eye toward efficiency, sensitivity, and customization. n 06 | Magnetosphere 10 cytiva.com Magnetosphere magazine Article Choose the right magnetic beads for your needs Download our decision tree for a hassle-free way to choose the right magnetic beads for your application! Download now Simplify your magnetic bead selection process today! Download now N ot only are magnetic beads available in different shapes and sizes, they also come with different magnetic properties, chemical coatings, and functional groups. It is important to know these properties before beginning to work with the bead so that you can choose a magnetic bead that suits the purpose of your experiment. Be clear on what you wish to achieve and know the attributes of your chosen target to enhance the binding, sensitivity, and specificity of an experiment. Each experiment is different and needs to be empirically optimized. Protein purification with magnetic beads Magnetic beads are also a convenient tool for protein purification. They are used to purify single proteins, large protein complexes, and antibodies and are used in high-throughput purifications. “ Small bead size provides a greater surface area for binding and can also be used for large molecules to maximize binding capacity per bead Magnetosphere 08 | Magnetosphere | 09 concentration of the blocker and incorporate the same blocker into the wash buffer to prevent nonspecific binding. We also recommended that you first “clean” the solution containing your target by mixing it with the beads in the absence of the antibody to reduce nonspecific binding. Antibody selection is a very important step in immunoassays. Optimize the antibody using standard immunoblotting to identify lack of specificity if any. Selecting a specific antibody and a high-affinity bead can greatly enhance the sensitivity of an experiment. Optimize your antigen with different tags and antibodies and select the one that provides excellent results. Sometimes all you need is to standardize what works in a good way for your needs. The excess antibody can remain unbound and result in background signals, while insufficient concentrations will not coat the beads uniformly and can reduce target binding capacity. Contaminations from light and heavy chains of antibodies can be eliminated using magnetic bead-antibody cross-linking and elution in a low pH and nonreducing buffer. Antigen-antibody binding is determined by the level of antigen expression. This critical step needs to be standardized for different antigens against specific antibody concentrations. • For scarce antigens, you can increase the binding time (which can also increase nonspecific binding) and concentrate the samples. • For abundant targets, you can increase antibody concentration or bead surface area. • Incorporate detergent in the binding buffer to reduce nonspecific binding. • Although binding at room temperature for 10 min is sufficient, slow binding at 4°C for an optimal time of 4 h might help in certain cases. Magnetic bead-based immunoassay development By Chike Ejiofor, Product Manager, Cytiva Magnetic beads are convenient tools for protein purification and immunoassay development, here’s how and why they can be used effectively. MAGNETIC BEADS To begin, you must adjust the volume of beads to protein in the starting material. Insufficient affinity material will reduce binding capacity, and excess binding sites can amplify nonspecific signals when sites are saturated. If your biomolecule of interest is small, more will bind per bead than will bind on larger biomolecules. Therefore, when the size of the antibody, tag, or antigen is large, the volume of beads used can be increased to provide a sufficient binding surface. Small bead size provides a greater surface area for binding and can also be used for large molecules to maximize binding capacity per bead. For diluted samples like cell supernatants or low-abundance proteins, either larger volumes of sample need to be applied to relatively smaller bead volumes or the samples need to be concentrated. Alternatively, bead sample incubation times can be increased. Glycerol applied to the samples might reduce nonspecific binding. Select high-affinity tags (commonly used ones include GST, histidine, and streptavidin) while ensuring they specifically express only in your protein of interest. Elute in a buffer having ligands with higher affinity for the beads than the specific protein or with a buffer that has a higher pH. Magnetic beads for immunoassays Magnetic beads provide better efficacy for target detection, pull-down, protein-protein, and protein-DNA interaction studies. Magnetic beads are often blocked to remove unwanted nonspecific background by the addition of bovine serum albumin (BSA), skimmed milk, sperm DNA, gelatin, polyethylene glycol (PEG), or sera, but ready-to-use streptavidin-blocked beads are available that do not require these additions. If a blocker is used, equilibrate the beads with lysis buffer and a low | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 The science of success Immunoprecipitation • Sera-Mag™ speedbeads protein A/G magnetic particles • Protein A mag Sepharose™ magnetic beads • Protein G mag Sepharose magnetic beads Chemiluminescence • Sera-Mag carboxylate beads and speedbeads • Sera-Mag streptavidin coated magnetic beads and speedbeads • Sera-Mag speedbeads amine-blocked particles • Sera-Mag speedbeads streptavidin-blocked magnetic particles Colorimetric • Sera-Mag carboxylate beads and speedbeads • Sera-Mag streptavidin coated magnetic beads and speedbeads • Sera-Mag speedbeads amine-blocked particles A guide to magnetic bead chemistries optimized for immunodiagnostic applications. Easily connect our magnetic bead chemistries with your chosen applications for a perfect match. CHEMISTRY ELISA • Sera-Mag carboxylate beads and speedbeads • Sera-Mag streptavidin coated magnetic beads and speedbeads • Sera-Mag speedbeads amine-blocked particles • Sera-Mag speedbeads streptavidin-blocked magnetic particles Fluorescence • Sera-Mag carboxylate beads and speedbeads • Sera-Mag streptavidin coated magnetic beads and speedbeads • Sera-Mag speedbeads amine-blocked particles • Sera-Mag speedbeads streptavidin-blocked magnetic particles View the full range of magnetic beads Our commitment to sustainability Thriving people, thriving planet, a resilient business. For Cytiva, these three aims are deeply connected. Achieving them is our ambition and our responsibility. That’s why we commit to integrating respect for people and care of the environment in our decisions in ways that have a lasting impact on society, our customers, patients, and Cytiva associates. Our impact to the United Nations Sustainable Development Goals “ For sandwich immunoassays, donor and acceptor beads need to be designed carefully so they do not bind to each other • Unless stated otherwise, all steps of the experiment can be performed on ice to reduce lack of specificity. • Elute at 70°C for 10 min instead of 95°C to prevent the antibody from being released from the beads. For sandwich immunoassays, donor and acceptor beads need to be designed carefully so they do not bind to each other. Select beads that distinctly differ in their excitation and emission wavelengths, binding specificity for the respective antigen, and are not influenced by sample or buffer-specific interferences. High biomolecular concentrations should be avoided because a high concentration overloads the beads and reduces donoracceptor signals. View our magnetic bead troubleshooting guide Our commitment to sustainability Thriving people, thriving planet, a resilient business. For Cytiva, these three aims are deeply connected. Achieving them is our ambition and our responsibility. That’s why we commit to integrating respect for people and care of the environment in our decisions in ways that have a lasting impact on society, our customers, patients, and Cytiva associates. Want to learn more? cytiva.com/sustainability Sciencebased targets + Net Zero Our impact to the United Nations Sustainable Development Goals This statement of our commitment to sustainability reflects our support of the UN Global Compact and its 10 principles on human and labor rights, environmental protection, and anti-corruption. It is echoed in relevant policies, procedures and strategic priorities across Cytiva. Our commitment to sustainability Thriving people, thriving planet, a resilient business. For Cytiva, these three aims are deeply connected. Achieving them is our ambition and our responsibility. That’s why we commit to integrating respect for people and care of the environment in our decisions in ways that have a lasting impact on society, our customers, patients, and Cytiva associates. Want to learn more? cytiva.com/sustainability Sciencebased targets + Net Zero Our impact to the United Nations Sustainable Development Goals This statement of our commitment to sustainability reflects our support of the UN Global Compact and its 10 principles on human and labor rights, environmental protection, and anti-corruption. It is echoed in relevant policies, procedures and strategic priorities across Cytiva. 10 | Magnetosphere Optimize and standardize your protocols to achieve exceptional results Magnetic beads are versatile and often provide a faster and easier solution than more complex and conventional methods. The points discussed here should help enhance binding capacity and specific binding across different experiments when selecting and using magnetic beads. Certain protocols might require more optimization, depending on the characteristics of the components and requirements of an experiment. Optimizing and standardizing your protocols will yield the most consistent, reliable results with exceptional efficiency. n Magnetosphere | 11 | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 I t was 1981. Around the globe, music fans were recovering from John Lennon’s murder and focused on the attempt on Ronald Reagan’s life. Raiders of the Lost Ark was big at the box office, and the world was watching the royal wedding of Prince Charles and Lady Diana. It was also the year that five previously healthy young men presented with Pneumocystis jirovecii (formerly P. carinii) pneumonia, a rare and deadly fungal infection (1). Later that year, the New York Times ran the article “Rare Cancer Seen in 41 Homosexuals” (2), which described the increase in Kaposi sarcoma among young gay men—not its usual target population (3). It wasn’t long thereafter that Pneumocystis jirovecii pneumonia and Kaposi sarcoma started showing up in some people who received blood transfusions and blood products, prompting the global search for an etiologic agent for the disorder. We now know that these individuals were infected with the human immunodeficiency virus (HIV), and this was the beginning of the acquired immunodeficiency syndrome (AIDS) epidemic. But it was not until 1984 that the causal relationship between HIV and AIDS was determined so that tests could be developed (4). Since those first infections were reported in 1981, HIV has infected ~ 85.6 million people and killed over 40 million people (5). But earlier and more accurate detection is helping to slow the spread and is assisting in getting treatments to those who need them faster. Improving HIV diagnostic testing sensitivity The first generation of HIV antibody tests could not detect the virus. They only detected the presence of IgG antibodies against the virus, which typically appear 6 to 12 weeks post infection. These tests were designed not for screening patients, but for screening blood products used in transfusions (6). When finally adopted for use in screening patients, the early tests were fraught with false positives, and confirmatory testing (Western blot) was needed (6). First- and second-generation tests measured only anti-HIV IgG antibodies (IgGsensitive tests). It was not until the thirdgeneration tests that IgM antibodies were able to be detected (IgM/IgG-sensitive tests), providing a shorter window for detection. Newer fourth-generation HIV tests measure the HIV-1 p24 antigen in addition to IgM and IgG antibodies. The p24 protein is the most abundant viral capsid protein (7), meaning that the presence of HIV virions can be detected prior to activation of adaptive immunity. The fourth-generation tests also more precisely measure antibodies, which is important as p24 levels rise and fall with HIV viral load (8). The evolution of high-sensitivity HIV diagnostic testing By Dr. Lindsay Sun, MD, MS, Medical Direct, Beckman Diagnostics Magnetosphere 12 | Magnetosphere | 13 In 2014, the Joint United Nations Programme on HIV/ AIDS (UNAIDS) set a goal to have 90% of those living with HIV diagnosed, on treatment, and virally suppressed (23). Increased sensitivity of fourth-generation HIV antigen/antibody combination immunoassays will continue to shorten the window for HIV detection to enable earlier diagnosis of HIV infection. They will allow improved assessment for individuals who have been exposed to HIV (24, 25). The accuracy of an HIV diagnostic test is dependent on its analytical sensitivity for identifying the p24 antigen early after HIV exposure as well as its ability to detect the diversity in HIV strains (26). The advent of highsensitivity, fourth-generation HIV diagnostic tests allows for earlier testing and faster time to antiretroviral treatment, ultimately improving patient outcomes (27). High-sensitivity diagnostic immunoassay testing is vital for meeting the increased HIV testing demands and enabling earlier detection and treatment, which may curb viral transmissions. Here’s why! HIV diagnostic testing Advancements in HIV diagnostic testing for early detection and treatment HIV has infected around 85.6 Million people And killed over 40 Million people Since 1981 | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 Over the past four decades, increases in test specificity and sensitivity have improved the positive predictive values of HIV diagnostic testing and shortened the time from HIV exposure to positive test result. While no HIV test can detect viral presence immediately after exposure, earlier, more sensitive immunoassay testing and results may lead to alterations in behavior that reduce disease spread. Earlier results also give the opportunity to start antiretroviral therapy sooner and begin monitoring HIV-associated comorbidities such as cardiovascular (9) and kidney (10) diseases. Earlier diagnosis saves both cost and lives, especially in high-prevalence areas (11, 12) despite the increased cost of treatment (13). Testing sensitivity and specificity Advancements in HIV diagnostics and therapeutics have enabled healthcare providers to quickly identify, diagnose, and monitor HIV infection earlier, helping to slow the spread of the virus. High-sensitivity immunoassay testing is central to meeting heightened expectations of diagnostics for improved patient care. Currently, HIV diagnostic tests can be performed at a clinic or at home with a salivaor blood-based test kit. While most HIV diagnostic tests are highly accurate when performed correctly, a false-positive HIV diagnosis may lead to high levels of stress and unnecessary initiation of treatment References: 1. Sencer DJ. 1. The AIDS epidemic in the United States, 1981-early 1990s. Centers for Disease Control and Prevention. March 26, 2021. Accessed March 22, 2024. https://www.cdc. gov/museum/online/story-of-cdc/aids/index.html 2. 1. Altman LK. RARE CANCER SEEN IN 41 HOMOSEXUALS. The New York Times. July 3, 1981:20. 3. Akasbi Y, Awada A, Arifi S, Mellas N, El Mesbahi O. Non-HIV Kaposi’s sarcoma: a review and therapeutic perspectives. Bull Cancer. 2012;99(10):92-99. doi:10.1684/bdc.2012.1636 4. Gallo RC, Montagnier L. The discovery of HIV as the cause of AIDS. N Engl J Med. 2003;349(24):2283-2285. doi:10.1056/ NEJMp038194 5. WHO. HIV. THE GLOBAL HEALTH OBSERVATORY. 2024. Accessed March 22, 2024. https://www.who.int/data/gho/data/ themes/hiv-aids 6. Alexander TS. Human immunodeficiency virus diagnostic testing: 30 years of evolution. Clin Vaccine Immunol. 2016;23(4):249-253. doi:10.1128/CVI.00053-16 7. Spach DH, Wood BR. HIV Diagnostic Testing. National HIV Curriculum. October 7, 2023. 8. Burrell CJ, Howard CR, Murphy FA. Retroviruses. In: Fenner and White’s Medical Virology. Elsevier; 2017:317-344. doi:10.1016/ B978-0-12-375156-0.00023-0 9. Triant VA. HIV infection and coronary heart disease: an intersection of epidemics. J Infect Dis. 2012;205 Suppl 3:S355- 61. doi:10.1093/infdis/jis195 10. Wyatt CM. Kidney disease and HIV infection. Top Antivir Med. 2017;25(1):13-16. 11. Zah V, Toumi M. Economic and health implications from earlier detection of HIV infection in the United Kingdom. HIV AIDS (Auckl). 2016;8:67-74. doi:10.2147/HIV.S96713 12. Halperin J, Katz M, Pathmanathan I, et al. Early HIV diagnosis leads to significantly decreased costs in the first 2 years of HIV care in an urban charity hospital in new orleans. J Int Assoc Provid AIDS Care. 2017;16(6):527-530. doi:10.1177/2325957417737381 13. Baggaley RF, Irvine MA, Leber W, et al. Cost-effectiveness of screening for HIV in primary care: a health economics modelling analysis. Lancet HIV. 2017;4(10):e465-e474. doi:10.1016/S2352-3018(17)30123-6 14. Johnson C, Fonner V, Sands A, et al. A report on the misdiagnosis of HIV status. In: Consolidated Guidelines on HIV Testing Services: 5Cs: Consent, Confidentiality, Counselling, Correct Results and Connection 2015. Geneva: World Health Organization; 2015 Jul. ANNEX 14. Available from: https://www.ncbi.nlm.nih.gov/books/NBK316023/ 15. HIV.gov. HIV Testing Overview. September 15, 2023. Accessed March 24, 2024. https://www.hiv.gov/hiv-basics/hiv-testing/ learn-about-hiv-testing/hiv-testing-overview 16. NHS. HIV and AIDS - Diagnosis. April 22, 2021. Accessed March 25, 2024. https://www.nhs.uk/conditions/hiv-and-aids/ diagnosis/ 17. UNAIDS. Siezing the Moment: Tackling Entrenched Inequalities to End Epidemics. UNAIDS; 2020. 18. Peabody R. How accurate is self-testing (home testing) for HIV? aidsmap October 2023. Accessed March 25, 2024. https://www.aidsmap.com/about-hiv/how-accurate-selftesting-home-testing-hiv 19. Saunders J, Brima N, Orzol M, et al. Prospective observational study to evaluate the performance of the BioSure HIV Self-Test in the hands of lay users. Sex Transm Infect. 2018;94(3):169-173. doi:10.1136/sextrans-2017-053231 20. Figueroa C, Johnson C, Ford N, et al. Reliability of HIV rapid diagnostic tests for self-testing compared with testing by health-care workers: a systematic review and meta-analysis. Lancet HIV. 2018;5(6):e277-e290. doi:10.1016/S2352- 3018(18)30044-4 21. Ganguli I, Bassett IV, Dong KL, Walensky RP. Home testing for HIV infection in resource-limited settings. Curr HIV/AIDS Rep. 2009;6(4):217-223. doi:10.1007/s11904-009-0029-5 22. Wray TB, Chan PA, Simpanen EM. Longitudinal effects of home-based HIV self-testing on well-being and health empowerment among men who have sex with men (MSM) in the United States. AIDS Care. 2020;32(2):148-154. doi:10.108 0/09540121.2019.1622636 23. UNAIDS. 90-90-90 An Ambitious Treatment Target to Help End the AIDS Epidemic. UNAIDS; 2014. 24. Panlilio AL, Cardo DM, Grohskopf LA, Heneine W, Ross CS, U.S. Public Health Service. Updated U.S. Public Health Service guidelines for the management of occupational exposures to HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep. 2005;54(RR-9):1-17. 25. Marrazzo JM, del Rio C, Holtgrave DR, et al. HIV prevention in clinical care settings: 2014 recommendations of the International Antiviral Society-USA Panel. JAMA. 2014;312(4):390-409. doi:10.1001/jama.2014.7999 26. Abdullah, Din M, Waris A, et al. The contemporary immunoassays for HIV diagnosis: a concise overview. Asian Biomed (Res Rev News). 2023;17(1):3-12. doi:10.2478/abm2023-0038 27. NIH. Early HIV diagnosis and treatment important for better long-term health outcomes. National Institutes of Health. October 12, 2022. Accessed March 28, 2024. https://www. nih.gov/news-events/news-releases/early-hiv-diagnosis- “ High-sensitivity immunoassay testing is central to meeting the increased demand and heightened expectations of diagnostics for improved patient care Magnetosphere | 15 and open the patient to discrimination and damaged relationships. Conversely, a false-negative HIV diagnosis fails to link patients to prevention, treatment, and care services. Both these outcomes highlight the importance of high sensitivity and specificity (14). The proper testing window must also be considered when determining the accuracy of any test type. Nucleic acid testing may identify a positive result 10 to 33 days following exposure. In the laboratory, fourth-generation antigen/antibody HIV diagnostic testing may identify a positive result 18 to 45 days post-exposure, but fourth-generation at-home HIV diagnostic tests may take up to 90 days. Finally, firstor second-generation antibody testing may identify a positive result 23 to 90 days following exposure (15, 16). While HIV at-home self-testing has the advantage of improving access to testing and empowering people to choose when and where they test (17), at-home tests are subject to user error (18, 19) primarily from incorrect specimen collection (20) Rapid, home HIV diagnostic tests also do not come with the care of a physician, which can lead to poor psychological outcomes (21). And prescription of antiretroviral therapies relies on the patient being willing and able to access medical care (22). Beckman Coulter’s commitment to diagnostic testing Beckman Coulter and its proven highsensitivity immunoassay capabilities are empowering healthcare providers, research organizations, and laboratories to address emerging diagnostic challenges across diverse specialties, from neurology to infectious diseases. We are revolutionizing the next generation of specialized diagnostics through high-sensitivity immunoassays, driving innovation, advancing medical insights, and enhancing diagnostic availability and adaptability to better serve patients in a variety of disease states. n Learn how to bring highsensitivity immunoassay testing solutions to your laboratory. 14 | Magnetosphere The information in this blog is for educational purposes only and should not be taken as medical advice. If you have been exposed to HIV or would like more information about HIV diagnostic testing, please contact your healthcare provider. The Future of Diagnostics: Advancements, Challenges and Opportunities: • Identify key external factors impacting the diagnostics industry positively and negatively over the next five years. • Examine major challenges and opportunities for the diagnostics industry in the next five years. Broaden your applications with magnetic bead conjugation: • Magnetic bead chemistry technology. • Understand the various surface chemistries and sizes for different applications. • Review how customizing magnetic beads can streamline genomics workflows and expand diagnostic applications. Check out these on-demand webinars Watch now Watch now | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 cell lines in peer-reviewed papers and online protein databases, including: • Gene Cards • Broad Institute • NCBI • Protein Atlas • Uniport However, there are a few limitations to defining protein expression profiles using online databases: • Expression data may not be complete in some cases “ An antibody that recognizes the protein only in its native form should not be used on samples using denaturing conditions such as western blot Why validate antibody specificity? By Abcam Antibody validation is critical to success and is very much application and experiment specific. Here are some tips on efficiently completing the validation process. Magnetosphere 16 | Magnetosphere | 17 • It can be challenging to find negative cell lines or tissues, particularly for essential housekeeping genes. • RNA levels are often very unreliable in determining the quantity of proteins. Furthermore, antibodies may or may not recognize the protein in its native or denatured state. It is therefore essential to prepare the test samples accordingly. For example, an antibody that recognizes the protein only in its native form should not be used on samples using denaturing conditions such as western blot. Validation Benefits Limitations Mass spectrometry or immunoprecipitationmass spectrometry (IP-MS) Protein complexes are first immunoprecipitated from a cell lysate and then analyzed with mass spectrometry Amenable to a high-throughput format Potential to estimate abundance of target protein bound to the antibody of interest using normalization techniques Can recognize all protein isoforms to which the antibody binds Can identify post-translational modifications, interacting partners, and complexes Confirms specificity based upon digested protein fragments Too many washes of your IP could remove weak or moderate binders Not all antibodies are suitable for IP It can be challenging to distinguish partner proteins pulled down in a complex from offtarget binding Interpreting the data can be tricky as the highest enrichment score does not always mean that this is the target the antibody preferentially binds to. For example, off-target binding can be difficult to demonstrate. An isotype control Ab is required, but there may still be discrepancies between IPs. Western blot Protein is detected in a sample via initial size separation and then blotting onto a membrane to be visualized by an antibody Useful for determining antibody specificity against target protein based on molecular weight Time-consuming assay Not easily automatable compared to some of the other applications Immunocytochemistry (ICC) Proteins in cells are detected via specific antibodies and reporter molecules Validates whether an antibody recognizes the correct protein based on cellular localization Specificity is confirmed in cells that either do or do not express the target protein Qualitative assay Unable to determine if an antibody recognizes other proteins nonspecifically with identical cellular localization Immunohistochemistry (IHC) Proteins in tissues are detected via specific antibodies and reporter molecules Validates whether an antibody recognizes the correct protein based on localization within the tissue Specificity is confirmed in tissue samples that either do or do not express the target protein Qualitative assay Unable to determine if an antibody recognizes other proteins nonspecifically with identical tissue localization Protein or peptide array Antibody binding events are detected by first spotting arrays with the proteins or peptides and then adding the antibody (similar to ELISA) Allows screening for antibody binding with numerous different proteins or peptides High-throughput screening process Protein array only: unable to screen for posttranslationally modified proteins if the arrays are made of E. coli-synthesized proteins Arrays made by peptides and denatured proteins only present linear epitopes for interrogation Note that the two following methods can’t be considered exhaustive tests of antibody specificity; therefore, we do not recommend using them alone: • Blocking with an immunizing peptide can confirm that an antibody binds its immunogen. However, the immunizing peptide will block both specific and nonspecific antibodies, so it can’t serve as a comprehensive method to confirm antibody specificity. • Omitting a primary antibody can evaluate the tissue or secondary detection reagents but not the primary antibody specificity. Table 1. Benefits and limitations of various validation methods A ntibody validation revolves around proving three key aspects: • Specificity and functionality: an antibody can differentiate between various antigens in the intended application • Affinity: the strength of binding between antibody and epitope • Reproducibility: your validation data can be reproduced in any lab Here we will focus on how you can validate antibody specificity in your experimental setup to obtain accurate and consistent results. Although manufacturers usually test an antibody in several applications and species, it’s impossible to account for numerous protocols and reagents with which researchers may use the antibody. Therefore, your antibody validation steps are essential because they are specific to your setup. Validating antibody specificity in various applications You can use several different methods to validate antibodies. Table 1 outlines some popular applications, their benefits, and their limitations. Key points to consider when validating antibody specificity 1. Choice and preparation of positive and negative controls Identifying and using appropriate positive and negative controls is essential for successful antibody validation. • A positive control is a relevant cell line or tissue sample strongly expressing the target protein of interest that can be used to confirm the selective binding of your antibody. • A negative control is a cell line or tissue sample that does not express the target protein and, therefore, can provide data on the nonselective binding properties of your antibody. When a true negative control is not available, a sample expressing low levels of the target proteins can work as an acceptable alternative. It’s often challenging to determine cell or tissue types that do or do not express the target protein. You can find information about target protein expression in different tissues or ANTIBODIES | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 Incubation times can vary dramatically from a minimum of 1 hour to overnight at 4°C 2. Protocols Ensure you use an optimized protocol to give the antibody the best chance of passing the validation process. For instance, incubation times can vary dramatically from a minimum of 1 hour to overnight at 4°C, so you’ll need to determine the optimal incubation period for each antibody. If the incubation period is too short, you may encounter sensitivity issues. Conversely, prolonged incubation time may lead to background staining. You will also need to optimize other factors, such as working dilutions, blocking conditions, and the use of native vs denatured conditions. See our protocol library. 3. The choice of buffers Most antibody assays will use two buffer types: PBS or TBS. You will need to determine the optimal buffer for your experiment, considering parameters that can influence buffer performance such as pH. See our buffer recommendations for optimal western blot results and antibody storage. Models for designing positive and negative controls Cell lines or tissues that endogenously express or lack the target protein can serve as positive or negative controls, respectively. You can use several various cell lines with different protein expression levels to provide a range of controls. Alternatively, appropriate positive and negative controls can be designed using multiple methods including knock-out models, siRNA knockdown, and cell treatment. n 4°C Magnetosphere 18 | Magnetosphere | 19 eLearning Get to know: developing diagnostic assays • Top considerations for developing a diagnostic assay • Critical questions for identifying suitable collaborators • Steps of the component and service design process eLearning Get to know: working with magnetic beads • Why different bead types and surface chemistries suit different applications • Key considerations for selecting a suitable bead for your application • How to improve results for common molecular biology applications eLearning Get started: optimizing rapid POC immunoassays • Different types of POC immunoassays • How to develop your lateral-flow assay (LFA) • Optimizing and troubleshooting your LFA development • Using data to help you select the right reagent Enroll now Free interactive courses Enroll now Enroll now Subscribe Watch this | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 Know your target molecule: 1 The biology of your target molecule will be the crucial factor in developing the biology of your test. Is it a large or small molecule? What antigen or other ligand will bind specifically to your target but not other molecules? The kinetics of the binding reactions between your analyte and the antibodies used in the test will determine your assay’s optimal flow time, which will influence your choice of nitrocellulose (NC) membrane. A keen understanding of the biology of your target molecule will shape every choice in the design of your LFA. Know your sample type: 2 The biological source of your sample will influence the design of your test and help you determine which materials you should use in your assay. The type and volume of liquid samples (e.g., blood, urine, or saliva) are two parameters that will help you choose which sample and absorption pads are most appropriate for your test. Pick your test type: 3 The size of your target molecule will be a key factor in determining the type of LFA you choose. There are two approaches to lateral-flow assay tests: sandwich and competitive. Sandwich assays rely on the binding of two separate antibodies to different regions of the same analyte molecule, making sandwich assays well-suited for detecting high molecular weight analytes. Competitive assays can detect smaller molecules, such as mycotoxins or cortisol, and larger analytes, such as insulin. Materials matter: 4 Choosing the right sample pad, conjugate release pad, and nitrocellulose membrane are critical to developing sensitive and reliable lateral-flow assays. Sample pads are commonly made from either cotton or bound glass fiber. Cotton has a lower wicking rate and is suitable for low sample volumes. Bound glass is appropriate for separating red blood cells (RBCs) from plasma. Using glass fibers with a higher diameter can also prevent hemolysis, the bursting of RBCs that turns plasma red and prevents accurate assay readouts. It’s important to determine that the conjugate release pad you choose will store the detection reagents without damage or aggregation over the shelf life of the test strip. Choosing hydrophilic materials with an open structure can support efficient liquid flow through your immunoassay. Test membrane flow rates: 5 Nitrocellulose (NC) membranes are one of the most critical components of a lateral-flow assay; their interactions with your sample will directly affect test performance. To help ensure a good balance between assay speed and sensitivity, try testing the flow rate of your sample with a range of different NC membranes. The membrane you choose should allow adequate time for the interaction of analyte and reagents at the test and control lines but also provide results within your chosen timeframe. n Download the full checklist here “ Sandwich assays rely on the binding of two separate antibodies to different regions of the same analyte molecule, making sandwich assays well-suited for detecting high molecular weight analytes From beads to branes Top tips for lateral-flow assay (LFA) development Magnetosphere | 21 Use this checklist to review essential considerations when developing your LFA, which will give you a deeper insight into lateral-flow assays to streamline your development process. 1 When designing your own lateral flow assay, there are several variables to consider: with the choice of detection reagents, assay materials, and manufacturing methods all influencing the accuracy and reliability of your test. Use this checklist to review ten essential considerations when developing your LFA, which will give you a deeper insight into lateral flow assays to streamline your development process. Know your target molecule The biology of your target molecule will be the crucial factor in developing the biology of your test. Is it a large or small molecule? What antigen or other ligand will bind specifically to your target but not other molecules? The kinetics of the binding reactions between your analyte and the antibodies used in the test will determine your assay’s optimal flow time, which will influence your choice of nitrocellulose (NC) membrane. A keen understanding of the biology of your target molecule will shape every choice in the design of your LFA. Know your sample type The biological source of your sample will influence the design of your test and help you determine which materials you should use in your assay. The type and volume of liquid samples (e.g., blood, urine, or saliva) are two parameters that will help you choose which sample and absorption pads are most appropriate for your test. Start developing your lateral flow immunoassay with our diagnostic services 1 Pick your test type The size of your target molecule will be a key factor in determining the type of LFA you choose. There are two approaches to lateral flow assay tests: sandwich and competitive. Sandwich assays rely on the binding of two separate antibodies to different regions of the same analyte molecule, making them well-suited for detecting high molecular weight analytes. Competitive assays can detect smaller molecules, such as mycotoxins or cortisol, and larger analytes, such as insulin. tralflow assay design. 10 top tips for lateral flow assay (LFA) development 2 Test line Control line 3 Result Sample pad Sandwich Conjugate pad Test line Control line Result Sample pad Competitive Conjugate pad belconjugate antibody Imm. captured antibody 1 20 | Magnetosphere Request samples | FOCUS ON IMMUNODIAGNOSTICS | ISSUE #2 Sample pad/ blood separator Conjugate release Nitrocellulose membrane Absorption pad Test line Control line 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. © 2024 Cytiva For local office contact information, visit cytiva.com/contact CY code CY44664-09Aug24-EB