xMAP Insights - Q1 2017

Connecting the xMAP Community to Innovative Applications & Resources

xMAP Insights ÂŽ

Q1 2017 Edition

Preparing for a Polio-Free World

Letter from the Editor

Welcome to xMAP® Insights A Newsletter for the Multiplexing Community Greetings, Welcome to xMAP® Insights. We’ve set out to create a quarterly newsletter that connects you to the multiplexing community by sharing innovative applications of xMAP technology and providing valuable resources to catalyze your next experiment.

Hilary Graham, MA Senior Manager, Scientific Marketing, Luminex Corporation

This issue focuses on assay development for the detection of infectious diseases. The following articles highlight the work of xMAP Innovators from across the globe: • The WHO Polio Research Unit | Novel tests to assess mucosal immunity against polio • Jan Bergervoet, Wageningen University | Custom plant pathogen assays • Margaret Ackerman, Dartmouth College | Tools for vaccine and therapeutic antibody development • Satoshi Kaneko, Nagasaki University | Surveillance of neglected tropical diseases You’ll also find new product features from our Partners (Bio-Rad, MilliporeSigma, Affymetrix, and R&D Systems) in the Resources Section.

We look forward to connecting with you in person at an upcoming conference, such as AACR, ECCMID, or Immunology 2017. See the Events Section for a full schedule.

Next quarter, xMAP Insights will focus on drug and vaccine development. If you have suggestions for future articles, please contact me directly at hgraham@luminexcorp.com. I’m grateful to have you as a reader and look forward to hearing from you. Also, if you ever find yourself in Austin, please reach out, as we’d be happy to host you on a tour of our campus.

Warm Regards,

Hilary Graham, MA Senior Manager, Scientific Marketing Luminex Corporation

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In This Issue 2 Letter from the Editor

Connections

Collaboration Community

Curiosity

5 Preparing for a

8 Enabling Plant Pathogen

20 Getting Started with

Polio- Free World

Detection 10 Demystifying the Immune Response 12 Bitten by the Tropical Medicine Bug

15 The Faces of Luminex: Anke Hoefnagels 17 Sharing Multiplex Solutions at xMAP Connect 2016

Luminex Technology 22 New Product Offerings 22 Bio-Rad 24 MilliporeSigma 25 Affymetrix 26 R&D Systems 27 Luminex IQ/OQ 28 Accelerate Your Success 29 Events

Connections

Connections

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Connections

Preparing for a Polio- Free World Supporting the development of new tests to assess mucosal immunity against polio

To date, smallpox is the only human infectious disease which has been eradicated, demonstrating the difficulty of eliminating a microbe from the environment.

Polio has existed for thousands of years - the first documented case of paralysis, likely from poliovirus, was engraved on an Egyptian tablet

dating back to 1580 BC.1 While the Salk inactivated injectable polio vaccine (IPV) and the Sabin live oral polio vaccine (OPV) became available in 1955 and 1961 respectively, a sharp decrease in the prevalence of polio wasn’t realized until the 1970s, when widespread immunization practices were established. Currently, wild polio is only circulating in parts of Afghanistan, Pakistan, and Nigeria, bringing global eradication within sight.

Harish Verma, along with colleagues at the World Health Organization (WHO) Polio Research unit, and collaborators across the globe are hard at work developing and deploying the most effective tools to stop the virus’s transmission.

Building a Better Assay

All photographs in this article are used with permission from the World Health Organization.

Mucosal immunity in the gut is critical for individual protection against polio and also to interrupt person-to-person spread of the virus. The gold standard to determine mucosal immunity is a challenge excretion study, where the gut is challenged with live, attenuated vaccine virus and levels of viral replication and shed virus are measured. The WHO Polio Research unit is always in search of

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Connections Unfortunately, there is a shortage of IPVs in the global market.

Researchers recently demonstrated that two fractional doses of IPV provide a greater protective effect than a single full dose.3 Needle adaptors have also been developed to control depth of intradermal injection and needle-free injectors have been developed to support the routine administration of the vaccines, as there are a limited number of trained vaccinators.4 Efforts around improving vaccine production are also being made, such as the development of safer production practices for Sabin IPV, as well as the manufacture of an IPV with non-infectious viral particles.

new technologies to detect and assess mucosal immunity that are faster, more reproducible, and require less skilled labor than conventional methods.

The Dartmouth xMAP-based IgA assay and mucosal neutralization development project is nearly underway, with a MAGPIX® instrument awaiting import to Cuba. Verma hopes that understanding how IPV primes the mucosal immune response will aid in the development of the next generation of polio vaccines to protect children in a postpolio world in the most effective manner possible.

Margaret Ackerman and Peter Wright from Dartmouth College, in collaboration with Sonia Resik, from the Laboratorio Nacional de Referencia de Poliovirus y otros Enterovirus in Cuba, submitted a proposal to the WHO to develop xMAP®-based polio-specific IgA and novel mucosal neutralization assays.2 In addition to the potential utility of the multiplex and neutralization assays to assess mucosal immunity, the WHO is also interested in supporting the project to build capacity in Resik’s lab beyond traditional serum-based assays, as she is a frequent collaborator on WHO polio projects as part of The Global Polio Laboratory Network.

Why Cuba?

Cuba provides a unique opportunity to study the development of mucosal polio immunity. In 1963, Cuba interrupted polio virus transmission. To prevent reemergence of the virus, Cubans are vaccinated twice yearly during distinct campaigns with OPV. Outside of the vaccination campaigns, there is no circulation of the vaccine virus in the population and the subsequent birth cohort is by definition immunologically naive. Without a continuous influx of vaccine virus into the population, in collaboration with the Dartmouth group, Resik will be able to determine the role of IPV in mucosal immunity sensitization.

Towards the Next Generation of Polio Vaccine OPV has long been the formulation of choice in areas with circulating polio as it provides mucosal immunity, hence enabling it to interrupt person-to-person spread of the virus, is less costly, and is easier to administer versus the inactive injectable version. Once polio has been eradicated, OPV will be phased out due to the small risk of vaccine-associated polio infection and paralysis. Once sufficient community immunity is reached, IPV will be used to maintain population immunity to prevent the occurrence of outbreaks.

Are you developing an xMAP® based assay?

Share your story with the editor of xMAP Insights at hgraham@luminexcorp.com.

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Connections

Collaboration

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Collaboration

Prime Diagnostics: Custom Agricultural Assay Solutions Enabling plant pathogen detection to boost the global food supply Jan Bergervoet wants to maximize the global food supply. Not only has he developed numerous assays for the detection of plant pathogens, but Bergervoet has also pioneered statistical methods to set mathematically derived thresholds of sample positivity.

Bergervoet has been a member of the Plant Research International group at Wageningen University since 1980, and is responsible for the development and production of xMAP® assays for Prime Diagnostics, a Luminex Partner, since 2007.

Bergervoet was an early adopter of xMAP Technology, but struggled with assay development until the introduction of MagPlex® microspheres in 2006. He was one of the first to test the new magnetic beads and published his multiplex potato virus immunoassay in 2008.5 Since then, Bergervoet has developed custom xMAP-based assays to provide plant propagators and growers with elegant and efficient solutions.

Distinguishing the Living from the Dead Is a virus infectious or not when present in a plant sample? That was an important question for growers and propagators. The traditional molecular tests could detect viral RNA – however, no efficient assay was available to determine if the virus detected was infectious or not. Prime Diagnostics developed an xTAG®-based assay that reliably differentiated infectious from non-infectious cucumber green mottle mosaic virus, while the ELISA and TaqMan® assays always remained positive.6

Small but Destructive Pospiviroids are the smallest plant pathogens, with a genome of up to 400 nucleotides. These small RNA viruses can cause massive destruction in many food and feed crops. They have a tightly wound genetic structure that is very stable and infectious, and their genome doesn’t encode any proteins. In the lab, this brings distinct challenges: contamination of the lab must be carefully avoided and robust methods must be applied to ensure complete access to the RNA. While TaqMan PCR is often the go-to method for viral identification, viroid species often differ only by a single base – requiring sensitivity that TaqMan can’t offer.

To overcome the sensitivity challenge, colleagues from Wageningen and Australia recently developed a new data-driven, statistical method that establishes thresholds of positivity for individual assays within multiplexed arrays.6 While empirical thresholds can be developed for blood-based assays (human blood has little variation), plant samples vary between species and are also dependent on environmental conditions. While Bergervoet was ecstatic for a mathematicalderived threshold, the R:BASE solution was initially developed as an Excel® sheet, which had issues, as Excel varies across the globe (e.g., usage of periods versus commas). In 2017, a web tool will be available to remedy global Excel differences and make R:BASE ready files.

Pospiviroids. All photographs in this article are used with permission from Jan Bergervoet.

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Collaboration Combatting Globalized Pests A warming climate and global transportation of goods have enabled the whitefly-transmitted begomoviruses to infect food and fiber crops beyond their traditional geography, prompting researchers at Wageningen to develop a degenerative assay that detects virus from both plant and vector samples. This xTAG-based hierarchical set of arrays allows researchers to simultaneously detect multiple viral and fly species with applications in plant quarantine, biosecurity, and disease management programs.7

Interested in developing a commercial assay or offering testing services?

Assay Development Tips High quality antibodies are critical. Develop them yourself, buy them from reputable vendors, or borrow them from trusted collaborators.

Learn about the benefits of becoming a Luminex Partner.

Start testing your assay in a matrix with real samples as soon as possible. This way you’ll spend your time optimizing in your final assay format.

Dümmen Orange, a customer of Prime Diagnostics, won the 2016 FleuroStar Award for their new Begonia hybrid, Miss Malibu.

Test your negative and positive controls to ensure they are what you think they are. For example, if you’re developing an xTAG® assay, sequence your control nucleic acids. This way you’ll be sure of your signal, versus worrying about false cross-reactivity.

TaqMan is a trademark of Roche Molecular Systems, Inc. Excel is a trademark of Microsoft Corporation.

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Collaboration

Demystifying the Immune Response Developing tools to aid in the development of vaccines and therapeutic antibodies

Our understanding of the immune system has significantly progressed since Edward Jenner published his work in 1798 describing the development of the first vaccine.8 Jenner recognized that dairymaids previously infected with cowpox were immune to smallpox. To test his hypothesis, Jenner infected a young boy with a cowpox pustule from Margaret Ackerman, Assistant Professor in Thayer School the hand of a milkmaid. Jenner then of Engineering at Dartmouth College exposed the boy to smallpox, which he failed to contract. Jenner repeated the experiment on several children, including his son, finally concluding that the vaccination provided immunity.

Today’s vaccine developers are equipped with knowledge of the immune system workings as well as high throughput tools and computational technologies.

Tools of the Trade Since it was invented in 1971 to understand immune cell interactions, the ELISA has been the gold standard for protein detection.9,10 Today’s complex research questions require more sophisticated tools that can simultaneously detect multiple analytes, while conserving sample.

Instead of focusing on research questions that surround a single disease state, Margaret Ackerman, Assistant Professor in Thayer School of Engineering at Dartmouth College, has dedicated a portion of her efforts to building novel, high throughput tools to evaluate the antibody response for vaccine development. Her group is specifically interested in improving methods of profiling the Fc region of antibodies, because that region serves as the link between the adaptive and innate immune systems.

Ackerman’s foundational Fc array platform assesses the ability of antibodies to bind to a suite of antigenic targets (e.g., peptides, proteins, and whole virus) and simultaneously defines Fc characteristics using a custom suite of antibody receptors11 and reagents. In characterizing

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Collaboration the antigen specificities of clinical antibody samples (including collections of mucosal, blood, and stool products) and, in parallel, revealing the binding preferences of candidate Fc receptors, her Fc array provides a multi-dimensional profile of the humoral immune response. As these interactions are associated with known effector functions, these measurements can serve as a proxy for the functional capacity of an antibody response and, in turn, elucidate mechanisms of protection in vaccinated subjects. Ackerman’s group continues to expand the applications of the Fc array platform, including: • Identifying antibody specificities for many different variants of a single viral protein with panels containing dozens of variants from different clades/classes (e.g., HIV gp120, Influenza HA), or antibody specificities for different viral serotypes by coupling whole virus to beads (e.g., Poliovirus serotypes PV1-3) • Probing Fc interactions by determining antibody isotype, as well as interactions with receptors and complement proteins • Revealing receptor interactions that mediate immune system responses, such as ADCC, phagocytosis, complement-mediated destruction, neutrophil activation, dendritic cell uptake, and NK cell activation

From Discovery to Clinical Application While many academic discoveries don’t make it out of the lab – Ackerman’s Fc array is now used by numerous academic and corporate collaborators to support vaccine trials.

In less than 2 years, Ackerman and Joshua Weiner established a GCLP (Good Clinical Laboratory Practices) facility and developed a qualified version of the Fc array to process clinical samples.

The Dartmouth GCLP Antibody Laboratory (dAbL) is a true, high throughput facility equipped with two FLEXMAP 3D® instruments, an automated liquid handling workstation to facilitate work with 384-well plates, and three dedicated staff members – often running hundreds of samples and generating up to 100,000 antibody assessments each day.

To date, the dAbL has performed analysis of more than 30 primate and human studies in collaboration with private and public sectors and used this platform to characterize antibody responses to HIV, influenza, polio, rotavirus, and emerging pathogens in response to infection or vaccination. dAbL is just getting started. They plan to leverage their expertise for evaluating Fc binding interactions and the capabilities of the dAbL to develop additional assays to determine safety and efficacy of biologic therapeutics.

All photographs in this article are used with permission from Margaret Ackerman.

High throughput techniques generate large data sets that can be challenging to manage.

When designing your high throughput assay, be sure to plan for data analysis and storage. dAbL collaborated with scientists from the Computer Science Department to build a database complete with a web interface.

Multiplex Assay Development Tips from the Dartmouth GCLP Antibody Laboratory (dAbL)

Automation of the steps that lead up to analysis greatly increases consistency and reduces technician hands-on time. dAbL selected the Eppendorf epMotion® for their liquid handling and they also use liquid plate washers from BioTek.

Cultivate relationships.

Collaborators can often provide access to high quality reagents not available for purchase.

Good reagents are the foundation of a great assay.

Whether you develop your reagents yourself or buy them from a trusted vendor, don’t assume they will work well in your assay format. Be sure to test each reagent when developing your assay to ensure high quality data and reproducibility.

Luminex xMAP Insights | Q1 2017 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Consider investing in an instrument service contract. A devoted service technician will understand you and your assay best, ensuring minimal downtime.

Eppendorf and epMotion are trademarks of Eppendorf AG.

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Collaboration

Bitten by the Tropical Medicine Bug Developing multiplex assays and surveillance systems for neglected tropical diseases All photographs in this article are used with permission from Satoshi Kaneko.

In 1995, Satoshi Kaneko was a practicing surgeon in Tokyo until he was invited by a colleague to join the Japan International Cooperation Agency in Guatemala to support a Chagas disease project. While he was not bitten by the kissing bug that transmits Chagas, he was infected with a passion for disease surveillance.

Since joining the Institute of Tropical Medicine at Nagasaki University in 2005, Kaneko’s work has focused on neglected tropical diseases (NTDs). The World Health Organization (WHO)12 lists 17 NTDs of viral, bacterial, and parasitic origins that occur in tropical regions and principally affect the poorest populations.

As an epidemiologist, Kaneko seeks to provide a community diagnosis —not individual—to understand which areas have disease, what the prevalence of disease is, and to determine the course of mass drug administration efforts (start, stop, or refine). The results of Kaneko’s assays are provided to local governmental authorities, which are responsible for the treatment of individuals.16

Passionate to understand and curtail the spread of NTDs, Kaneko was in search of a tool that would enable the simultaneous diagnosis of multiple pathogens and a colleague suggested he consider multiplexing. Kaneko partnered with Yoshito Fujii, a biochemist also at Institute of Tropical Medicine, and they developed a multiplexed seroprevalence detection system against eight antigens from six pathogens, and confirmed its utility in nearly 3,500 Kenyan blood samples.13 Since their 2014 proof of concept publication in PLoS Neglected Tropical Diseases, Kaneko and Fujii have gone on to develop two more multiplex assays for the species-specific detection for schistosomiasis14 and for human African trypanosomiasis.15

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Collaboration While his seroprevalence assay provided the most comprehensive and cost-effective epidemiological status of NTDs to date, Kaneko continues to innovate. His current efforts are focused on increasing the menu of pathogens in his multiplex assays and expanding the geographic reach of adoption from Kenya to also include Nigeria, the Democratic Republic of Congo, and Egypt.

Interested in developing your own assay, but don’t know where to start?

Check out the xMAP Cookbook for detailed instructions on how to design, optimize, and validate your xMAP assay.

Kaneko’s team of four researchers in Japan, and three researchers in Kenya, face both common and unique challenges when developing assays for NTDs. Difficulties fixing the protein antigen to the bead.

To remedy the problem, Fujii used recombinant antigen with a polyhistidine tag that allowed for easy monitoring of binding efficiency.

High background binding.

When Fujii was developing the assay, nearly half of binding was non-specific. To remedy this problem, he treated with SDS, reduced the amount of antigen and switched to magnetic beads – all tips he found in the xMAP® Cookbook. Antibodies from parasitic infections are difficult to detect in the blood, so stool or saliva samples often provide better results.

Lack of tools.

Reagents, such as antigen and antibody libraries, as well as animal models are hard to come by for NTDs.

Difficulty obtaining samples.

Kaneko relied on dried blood spots versus more invasive blood sampling techniques to boost participation.

Instrument installation and maintenance.

Kaneko has a Luminex® 200™ and MAGPIX® in both his research and field laboratories. He's been pleased with the dependable and responsive service for his instruments, regardless of location.

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Connections

Community

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Community

The Faces of Luminex: Anke Hoefnagels, Field Applications Specialist All photographs in this article are used with permission from Anke Hoefnagels.

Who are the people behind Luminex? Anke Hoefnagels is a Field Applications Specialist supporting Global Partner Operations who celebrated her second anniversary with Luminex on January 1st. When Anke isn’t working out of the Den Bosch office in The Netherlands, you’ll find her traveling or improving her musicality.

How did you come to work at Luminex?

For my masters, I developed a ChIP-seq assay to elucidate the role of the HNF1B transcription factor in regulating magnesium levels in the kidney. Once the assay was optimized, I looked for additional research questions that the method could be used to address. My mentor pointed out to me that most scientists ask research questions and then find the appropriate technique – but I operated in the opposite manner. At that moment, I came to understand that I enjoyed the methodical troubleshooting required for assay development.

What’s a typical day for you?

For the past two years, I’ve worked as a Technical Support Associate.

Technical Support at Luminex operates in a follow-the-sun model, meaning my colleagues from Asia, The Netherlands, and Austin all work as a single team to provide 24-hour support to customers.

As each European country speaks a different language, we tend to communicate in English as a common language. For this reason, the majority of our customer cases are submitted via email versus phone calls. The benefit of primarily receiving email cases is that customers often attach the Support Utility files, enabling us to have a better understanding of the issue at hand.

On January 1st, I started a new position as a Field Applications Specialist (FAS) and I support Luminex Partners located in Europe.

As an FAS, I help new customers to adopt, and existing customers to optimize, their Luminex assays.

Tell us something about science or medicine in The Netherlands.

A hot topic in The Netherlands at the moment is the use of antibiotics. To prevent antibiotic-resistant bacteria, The Netherlands has a very strict policy on antibiotic use. I recently had an ear infection and had to visit the doctor multiple times before an antibiotic was prescribed. While this was a personal inconvenience, I think antibiotic stewardship is important, given the increasing emergence of resistant bacteria.

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Community When you’re not working, how do you spend your time?

Traveling! This year I went to Thailand and earned my diving certificate. I also went on a night safari in the jungle and saw amazing snakes. Last year, I went to Indonesia and climbed down into the crater of the Ijen Volcano on Java. The trek up was specifically at night to be able to see the sulfur gas burning as blue fire – it was quite an adventure!

I’m currently taking a musical orientation course where I will play approximately 20 instruments. Growing up I could never choose an instrument, so my goal of this course is to select an instrument and learn how to play it. So far I’ve played: piano, accordion, clarinet, saxophone, hobo, flute, trumpet, guitar, and drums!

Need help resolving an issue?

Anke Hoefnagels is here to help

Anke Hoefnagels is a Field Applications Specialist based in Europe. She loves assay optimization and assisting customers with resolving their problems. Anke shares a few tips we hope you find helpful.

TIP #1

Are you getting an error in xPONENT®?

If you have an error, check the log file or the CAL/VER report (for errors during calibration and verification) for a description of what might be wrong.

In the sentence with the error code, it often mentions what to do (such as reboot the instrument) or what the problem is (such as low bead counts).

In some cases, the error code is a general code without a description. In these cases, please contact Luminex Tech Support for assistance.

TIP #2

Are you having low bead counts?

Low bead counts can be caused by several factors, but the most common causes are incorrect probe height, a clogged probe, or a small clog in the instrument. Samples can become ‘sticky’ – especially for protein-based assays. As sample flows through the probe and small tubing, it is important to have a proper cleaning procedure set up for the instrument to avoid clogging. Sonication of the probe, followed by resetting the probe height, may resolve the issue. Make a habit to run a cleaning procedure before shutting down the instrument or when the instrument will be idle to avoid future clogs.

TIP #3

Unsure how to resolve a problem?

Luminex Tech Support is always available to help. When contacting Technical Support, consider providing the Support Utility file. This file contains data from the instrument, which is helpful in determining what happened and how it can be resolved.

If the problem might be assay-related, adding a batch is also helpful. Adding a batch provides the raw data of the run, which allows Tech Support to upload the data into their version of xPONENT®. With this information, they can determine if the mapping was correct, what the bead counts are, and if the MFI values are in range.

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Community

Sharing Multiplex Solutions at xMAP® Connect 2016 Learn about the diverse applications of xMAP Technology and spark inspiration for your next experiment

All photographs in this article are used with permission from Hilary Graham.

Most conferences focus on research advances and the development of therapies for specific diseases. xMAP® Connect is different.

• Improving protein and peptide-multiplexing technology for the development of tumor autoantibody based diagnostics

Whether you’re a multiplexing expert eager to share your work or just getting started – xMAP Connect is the premier event for networking with fellow xMAP enthusiasts and sharing best practices. In 2016, more than 200 researchers from 36 countries in Europe, the Middle East, India, Africa, and Asia attended the annual xMAP users’ meeting held in Amsterdam.

• Characterizing circulating tumor cells using a Luminex®-based liquid biopsy

The open architecture of xMAP Technology enables endless applications, which is evident in the diversity of the presentations.

• Identifying markers of exposure and immunity in antibody response profiles from individuals living in malaria endemic settings

Day 1 focused on biomarkers, immunology, and multiplex screening developments:

• Determining biomarkers of tumoral infiltration in central nervous systems of patients with Lymphoma

• Identifying biomarkers for asthma phenotype analysis

• Mapping the presence, abundance, and location of the proteins across the human body with affinity proteomics of dried blood spots

• Designing phenotypic drug screening assays and hit validation tools using cytokine profiling

• Developing a new tool for the detection and quantification of microRNA

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Community

Day 2 was devoted to clinical infectious disease research and public health solutions: • Tracking the longitudinal humoral response to reveal virulence factors involved in osteomyelitis • Discriminating up to 51 mucosotropic HPV types for epidemiological investigation • Distinguishing bacterial from non-bacterial pediatric clinical pneumonia using 56 biomarkers • Monitoring of S. pneumoniae vaccine response • Preventing the feeding of processed animal proteins through the identification of species-specific and tissue-specific molecules • Development of public health surveillance tools for the detection of: Salmonella Typhimurium, fungi in indoor air, and genetically modified foods • Identifying predictive S. aureus antigens for the development of diagnostic tools In addition to the invited presentations, 25 researchers also shared their recent work during the poster sessions. The Protagen Diagnostics team was recognized for the most innovative poster: “Discovery of Novel Autoantigens in Sjögren's Syndrome with Potential Subgrouping of Disease.” They screened more than 2,000 serum samples from patients with autoimmune diseases and more than 1,000 healthy individuals with the goal of confirming known, and discovering new, autoantibodies. They discovered a small set of novel autoantibodies appearing with a prevalence of 8 to >20% in Primary Sjögren’s syndrome patients.

We hope to see you at xMAP® Connect 2017!

Sign up for notifications of upcoming events where you can connect with the xMAP user community.

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Connections

Curiosity

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Select your instrument

I have a research question

Getting Started with Luminex Technology

MAGPIX®

LUMINEX® 200™

FLEXMAP 3D®

Portable

Gold Standard

Advanced

All xMAP® Microspheres

# targets I have ____

96-well plate in ≤ 60 min (up to 4,800 tests/hour)

96-well plate in ≤ 45 min (up to 12,800 tests/hour)

96-well plate in ~20 min (up to 144,000 tests/hour) 384-well plate in ~75 min (up to 153,600 tests/hour)

More than

Less than

500

3 to 500

3

Identify reagents and equipment

What method is right for me?

Antibodies/ Probes

Sample

Low Plex Methods Protein Western blot ELISA Immunohistochemistry Flow cytometry Electrochemiluminescence

Buffers

Thermocycler

Shaker

High Plex Methods

xMAP®-based Multiplexing

Protein Protein chip Mass spectrometry NMR

Open architecture technology enables the develop of protein and genomic assays

Nucleic Acid Microarray Mass spectrometry Next generation sequencing

e your ass z i a tim

y

Op

Nucleic Acid Northern blot Southern blot RT-PCR

Microspheres

method is right for me?

Buy aWhat xMAP kit or design a custom assay Buy

Build

a ready-made kit with Kit Finder a ready-made kit with ourour xMAP Kit Finder

a custom solution from one of our partners

a custom assay with the xMAP Cookbook

a li

V

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Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. © 2017 Luminex Corporation. All Rights Reserved. Luminex , xMAP, MAGPIX, and FLEXMAP 3D are trademarks of Luminex Corporation, registered in the U.S. and other countries. 200 is a trademark of Luminex Corporation. GX1757

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Buy

Get more answers while conserving sample, time, and reagents with multiplexing

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Investigating Immune Response Profiles Using Bio-Plex® Multiplex Assays One of the body’s first detection mechanisms when encountering a pathogen or immunization is the innate immune system.

Once activated, the innate branch of the immune system communicates through cytokines and chemokines, as well as cell surface bound molecules. This communication triggers an adaptive immune response, leading to the generation of specific antibodies and cytotoxic cells targeting the particular etiological agent. Ideally, this response also leads to the production of memory cells that are able to rapidly respond to future infections.

Depending on the type of pathogen, different pathways can be activated to generate this specific and long-lasting immunity.

Viral and intracellular bacterial antigens can be presented via MHC 1 on the surface of infected cells to CD8 cytotoxic T-cells, whereas extracellular bacterial or parasitic antigens will be bound

by MHC II and presented to CD4 helper T cells. The former will trigger a cell-mediated immune response and the latter leads to an antibody-mediated immune response.17 The long-lasting success of vaccines hinges on the ability of the innate immune system to effectively stimulate an adaptive immune response. Understanding the concentrations and ratios of important cytokines over the course of vaccination and by individual, for example, is critical when evaluating overall vaccine performance and efficacy.

pediatric vaccines, termed low vaccine responders (LVRs).

Peripheral blood mononuclear cells (PBMCs) were collected from LVRs, as well as infants with normal vaccine responses, and were subjected to in vitro stimulation with anti-viral compound R848. Both sample types were interrogated using a subset of the Bio-Plex Pro™ Human Cytokine 27-plex Assay (#M500KCAF0Y).

The LVR group showed higher baseline levels of RANTES and higher post-stimulation levels than normal responders.

RANTES expression can be induced by respiratory infections.

In addition, the stimulated LVR PBMCs secreted significantly less IL-12p70 than normal responders, as well as lower levels of pro-inflammatory IL-1β, suggesting an overall poorer capacity to respond to viral and bacterial infections.18 Understanding the diversity of immune responses is critically important to vaccine efficacy. Variations in patient populations should be taken into consideration not only during vaccine development, but also when structuring clinical trials, and during post-market surveillance and reporting.

Multiplex immunoassay technology is a powerful tool that can help researchers and vaccine developers evaluate the immune response to vaccination, as well as long-term vaccine efficacy, by allowing the simple and rapid measurement of many targets from a single sample.

Surendran et. al., describe a population of approximately 10% of infants who display a sub-optimal innate response to routine

Assay Notes • Format: 1 x 96-well kit

• Magnetic beads for simplified plate processing

• This multiplex assay detects: FGF basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF

• All-in-one, space-saving complete kit • Premixed standards for generation of curves over a broad dynamic range for each target • For multiplexing capabilities with other cytokines, refer to Bulletin 5927

Bio-Plex and the Bio-Rad logo are trademarks of Bio-Rad Laboratories, Inc.

Luminex xMAP Insights | Q1 2017 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

22

Bio-Plex® Pro Mouse Chemokine and Cytokine Panel—Available Q1 2017!

The Bio-Plex® Pro Mouse Chemokine and Cytokine Panel enables you to detect and quantify 33 biologically-relevant cytokines and chemokines from mouse serum or plasma samples, requiring minimal input volumes, thereby conserving precious sample. Whether you are studying oncology, immunology, autoimmunity, neurology, infectious diseases, or engaging in biomarker discovery, the Bio-Plex Pro Mouse Chemokine Panel provides a robust, efficient tool to screen even low-level analytes in a high-throughput, multiplex assay.

Assay Notes: Increased Sensitivity

Pre-mixed, Ready to Use Reagents

Gain confidence in your results with a high-performing assay

Streamline your workflow with pre-mixed reagents and a simplified magnetic bead-based protocol

Excellent Signal-to-Noise Ratio Detect more target analytes with optimized chemistry and a broad dynamic range

Broad Panel Coverage Obtain data for a large number of biologically-relevant targets from a single experiment

Bio-Plex and the Bio-Rad logo are trademarks of Bio-Rad Laboratories, Inc.

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Not for use in diagnostic procedures.

23

MILLIPLEX® MAP 384-Well High Sensitivity Human Cytokine Magnetic Bead Panel Low levels of inflammation are involved in many clinical and sub-clinical disease states, such as autoimmune disease, cardiovascular disease, diabetes, neurological disorders, and cancer.

Measuring picogram levels of cytokines, therefore, is critical for understanding the pathogenesis of these diseases.

The MILLIPLEX® MAP 384-Well High Sensitivity Human Cytokine Magnetic Bead Panel enables you to focus on the therapeutic potential of cytokines, as well as the modulation of even low levels of cytokine expression. MILLIPLEX® MAP offers you the ability to select a 21-plex pre-mixed kit or to choose any combination of 21 analytes to design a custom kit that better meets your needs. The 384-well Human High Sensitivity T Cell Panel is the 384-well version of the current 96-well Human High Sensitivity T Cell Panel (HSTCMAG-28SK).

Data obtained from the High Sensitivity T Cell Panel correlates with data for the respective cytokines in the Human Cytokine/ Chemokine Panels I, II, and III, furthering your ability to measure specific cytokine response in both normal and disease states.

Additionally, the MILLIPLEX MAP 384-well Human High Sensitivity T Cell Panel is analytically validated to deliver the quality research results you expect from the MILLIPLEX MAP brand. Extensive testing for standard curve range, sensitivity, precision, cross-reactivity, and recovery shows that the 384-well format produces comparable, if not superior, results to the 96-well format kit.

Assay Notes • This kit may be used for the analysis of all or any combination of the 384-well Human High Sensitivity T Cell panel analytes in serum, plasma, or tissue/cell culture supernatant.

• Available analytes: Fractalkine/CX3CL1, GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12 (p70), IL-13, IL-17A/CTLA8, IL-21, IL-23, ITAC/CXCL11, MIP1α/CCL3, MIP1β/CCL4, MIP-3α/CCL20, TNFα.

• This assay uses 20 µL of neat samples.

• Serum or plasma samples with high analyte values may be further diluted in serum matrix prior to the addition of 20 µL to the sample wells.

• Of note, all of the validation work for this panel was done with the BioTek® Multiflo™ FX Automated Washer (Cat. No. 40-099). Hand washing is not recommended, since this assay works with such small volumes.

Available formats of the MILLIPLEX® MAP 384-well Human High Sensitivity T Cell Panel • Cat. No. HSTC384-28K (configurable analyte selection, up to 21 analytes) • Cat. No. HSTCMAG384-PX21 (21-plex premixed kit) • Panel-Space Saver (Bulk) Packaging, Cat. No. HSTCMAG384PX21BK (21-plex bulk premixed kit)

Milliplex and the Millipore Sigma logo are trademarks of Merck KGaA. BioTek and Multiflo are trademarks of BioTek Instruments, Inc.

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Not for use in diagnostic procedures.

24

Accelerate your biomarker research by analyzing 65 immune targets simultaneously The ProcartaPlex® Human Immune Monitoring Panel19 enables you to analyze 65 soluble protein targets in parallel in a single well. The panel contains predominately key cytokines, chemokines, and growth factors, offering the broadest Luminex panel available (find a complete analyte list below). While multiplexed immunoassays are a proven tool, this is the first ready-made xMAP®-based kit to exceed 50-plex for immune network analysis.

Accelerate your biomarker research by analyzing 65 immune targets simultaneously.

Interested to learn more about the development of this 65-plex panel? Check out this poster presented at ID Week 2015 of a 63-plex pilot study20 in patients with and without syphilis.

Inflammation Fibroblast liver

TNF-α IL-1β TGF-β PDGF IL-6

Acute phase proteins

e

in

am

st Hematopoiesis

IL-1, IL-6, IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, TNF-α, MCP-1, MIP-1α

Neutrophil elastase Neutrophil Respiratory burst Reactive oxygen species

IL-18 IL-3, IL-4, IL-10, IL-13, IFN-γ, TNF-β

Resting B cell

Resting T cell

IL-3, IL-6, IL-7, GM-CSF IL-3, IL-4, IL-10

IL-10, IL-14, IL-4, IL-6

Mast cell

Allergy anaphylaxis

IL-12, IL-15 Innate immunity

IL-3, IL-4, IL-5

Activated LFA-1, VLA-4, T cell Mac-1

IFN-γ

Eotaxin

IL-2

IL-6

Activated B cell

Lung epithelium

Eosinophil

IL-2 Viral infection, tumor surveillance

IL-23

NK cell

TGF-β, IL-2, IL-4,IL-5, IL-13, IFN-γ

Plasma cell

Th17/22 Th1/Th2

Humoral immunity

Allergy anaphylaxis

IL-2, IFN-γ, IL-4 Viral infection, tumor surveillance

• Be careful not to invert the 96-Well Flat Bottom Plate or allow contents from one well to mix with another well.

• Use a multi-channel pipette and reagent reservoirs whenever possible to achieve optimal assay precision.

CTL

Th9

IgE IgM/G/A

• This assay can be analyzed with the Luminex® 200™ or FLEXMAP 3D® instrument only. Please contact tech@eBioscience.com for more information if you are a MAGPIX® user.

• During the incubation steps, cover the 96-Well Flat Bottom Plate with the Black Microplate Lid provided in the kit to minimize exposure of the beads to light.

Cardiovascular pathology

M-CSF, TNF-α, GM-CSF, G-CSF, IL-1, IL-6, IL-11, IL-12

Macrophage

IL-8, TNF-α

Endothelial cell

Hi

Fever

TNF, IL-1β

• Analyte parameters: APRIL, BAFF, BLC, CD30, CD40L, ENA-78, Eotaxin, Eotaxin-2, Eotaxin-3, FGF-2, Fractalkine, G-CSF, GM-CSF, Gro a, HGF, IFN-a, IFN-g, IL-10, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-18, IL-1a,IL-1b, IL-2, IL-20, IL-21, IL-22, IL-23, IL-27, IL-2R, IL-3, IL-31, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, I-TAC, LIF, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIF, MIG, MIP1a, MIP-1b, MIP-3a, MMP-1,NGF beta, SCF, SDF-1a, TNF b, TNF-a, TNF-R2, TRAIL, TSLP, TWEAK, VEGF-A.

• When working with samples and standards, change the pipette tips after every transfer and avoid creating bubbles when pipetting.

PECAM-1, VCAM-1, ICAM-1, P-selectin, E-selectin

Bacterial products (LPS, peptidoglycans, etc.)

Assay Notes

Fibrosis

IL-17A, IL-17F, IL-22, IL-6, GM-CSF

Autoimmunity Tissue repair, protozoan immunity

Procartaplex and the Affymetrix logo are trademarks of Affymetrix, Inc.

Luminex xMAP Insights | Q1 2017 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

25

Custom Luminex® Assays by R&D Systems® Interested in building a custom assay – don’t want to do it yourself? R&D Systems® has more than 30 years of experience in immunoassay development. Currently offering more than 100 analytes – designing your multiplex panel is as easy as visitingUnited States the sign in or register Quick Order Items in Cart (0) R&D website. The R&D Systems Luminex High Performance Assays provide similar performance to the Quantikine® ELISA kits.

Search keyword, molecule name, target,the catalog number, or product type to obtain Additionally, the R&D Systems platform provides easy assay correlation between two formats, as well as the ability comparable data across platforms. The R&D Systems Luminex High Performance Assays are suitable for biomarker discovery, validation, and detection.

Want to learn more?

Watch this informational video from R&D Systems.

PRODUCTS SERVICES PATHWAYS RESOURCES R&D SYSTEMS QUALITY ABOUT US Home » Resources » Technical Information » Luminex Assay Principle

Luminex Assay Principle

The sample is added to a mixture of color-

Biotinylated detection antibodies specific to the

The sample is added to a mixture of color­ Biotinylated detection antibodies specific to the coded beads, pre-coated with analyteanalytes of interest are added and form an antibodycoded beads, pre­coated with analyte­ analytes of interest are added and form an specific capture antibodies. The antibodies antigen sandwich. Phycoerythrin (PE)-conjugated specific capture antibodies. The antibodies antibody­antigen sandwich. Phycoerythrin (PE)­ bind to the analytes of interest. streptavidin is added. It binds to the biotinylated bind to the analytes of interest. conjugated streptavidin is added. It binds to detection antibodies. the biotinylated detection antibodies.

Polystyrene beads are read on a dual-laser Polystyrene beads are read on a dual­ flow-based detection instrument, such as laser flow­based detection instrument, ® the Luminex® 100™, Luminex 200™ or such as the Luminex 100™ , Luminex ™ ® ® FLEXMAP 3D®. One Bio­Plex laser classifies the bead 200 or Bio­Rad analyzer.

One laser classifies the bead and and determines the analyte that is being determines the analyte that is being detected.

The second laser determines the detected. The second laser determines the magnitude of the PE-derived signal, which is inmagnitude of the PE­derived signal, which direct proportion to the amount of analyte is in direct proportion to the amount of bound. analyte bound.

In addition to the Luminex 100, Luminex 200, or In addition to the Luminex 100, Luminex 200 or FLEXMAP 3D®, flow-based analyzers, magnetic beads Bio­Rad Bio­Plex dual­laser, flow­based can be read using the Luminex MAGPIX® Analyzer. A analyzers, magnetic beads can be read using magnet in the MAGPIX®analyzer captures and holds the Luminex MAGPIX Analyzer. A magnet in the MAGPIX analyzer captures and holds the the magnetic beads in a monolayer, while two specmagnetic beads in a monolayer, while two trally distinct light-emitting diodes (LEDs) illuminate spectrally distinct light­emitting diodes (LEDs) the beads. One LED identifies the analyte that is being illuminate the beads. One LED identifies the detected, and the second LED determines the magnianalyte that is being detected and, the second tude of the PE-derived signal. Each well is imaged with LED determines the magnitude of the PE­ a CCD camera. derived signal. Each well is imaged with a CCD camera.

Related Information R&D Systems, the R&D Systems logo, and Quantikine are trademarks of Bio-Techne Corporation.

Luminex Assays Luminex Assays and High Performance Assays Luminex High Performance Assays Luminex Instrumentation Luminex xMAP Insights | Q1 2017 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

26

Do you need Installation Qualification and Operational Qualification Services?

Installation Qualification and Operational Qualification (IQ/OQ) are an integral part of any validation program for newly installed or relocated instruments. Additional OQ services can also be individually ordered on a yearly basis. Luminex IQ/OQ protocols provide the documented evidence laboratories need to prove that their Luminex instrument was installed correctly and is operating according to specifications. These documents can be used to fulfill a broad range of internal and external quality management requirements. Consider purchasing IQ/OQ services from Luminex if any of the below apply to you.

• Are you working in a regulated industry?

• Have you moved your instrument to a new location or laboratory?

• Have you had major (hardware or software) repairs or upgrades to your instrument?

• Would you like added assurance that your instrument is functioning properly to produce the highest quality data?

Contact your Luminex Representative for more information or to place an order.

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Not for use in diagnostic procedures.

27

Curiosity

Accelerate Your Success:

Become a Luminex Partner Proven Technology

Efficiency

Flexibility

Unmatched in level of end-user adoption and validation

1 to 500 analytes

Allows for both

nucleic acid and protein applications

>4,000

>32,000 scientific publications cite Luminex technology and/or our partners’ products

simultaneously tested from one sample,

in one well

Meets a wide range of multiplexing and throughput needs with

analytes

can be accessed

3 instrument options

on one platform

>65 510(k) clearances

510 (k)

Open architecture systems enables use of commercial kits and internally developed assays

Partner Facts

>70 Luminex Partner Companies

A wide install base - with

FDA

CLEARED

1 IVD assay

st The using xMAP Technology was FDA cleared in 2001—

>12,500

by a Partner

xMAP® Instruments Sold Worldwide

Are you interested in commercializing a multiplex assay?

Contact the Luminex Business Development team at businessdevelopment@luminexcorp.com to discuss your assay application and the benefits of partnering with Luminex.

www.luminexcorp.com © 2016-2017 Luminex Corporation. All rights reserved. Luminex and xMAP are trademarks of Luminex Corporation, registered in the U.S. and other countries. INFG20904

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Curiosity

Connect with Luminex at These Upcoming Conferences

AACR Annual Meeting

April 1-5

Washington DC

ECCMID

April 22-25

Vienna, Austria

Immunology 2017

May 12-16

Washington DC

Biotechnica

May 16-18

Hannover, Germany

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29

References 1.

Polio Today - History of Polio. Polio Global Eradication Initiative (Internet). Cited 2017 Jan. Available from: http://polioeradication.org/polio-today/ history-of-polio/.

2. Wright PF, Connor RI, Wieland-Alter WF, et. al. Vaccine-induced mucosal immunity to poliovirus: analysis of cohorts from an open-label, randomised controlled trial in Latin American infants. The Lancet Infectious Diseases, 2016;16(12),1377-84.

3.

Schipper P, van der Maaden K, Romeijn S, et. al. Repeated fractional intradermal dosing of an inactivated polio vaccine by a single hollow microneedle leads to superior immune responses. Journal of Controlled Release, 2016;242,141-7.

4. Resik S, Tejeda A, Mach O, et. al. Needle-free jet injector intradermal delivery of fractional dose inactivated poliovirus vaccine: Association between injection quality and immunogenicity. Vaccine, 2015;33(43),5873-7.

5.

Bergervoet JH, Peters J, van Beckhoven JR, et. al. Multiplex microsphere immuno-detection of potato virus Y, X and PLRV. J Virol Methods, 2008 Apr;149(1), 63-8.

6. van Brunschot SL, Bergervoet JH, Pagendam DE, et. al. Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens. PloS One, 2014;9(1), e84743.

7.

van Brunschot SL, Bergervoet JHW, Pagendam DE, et. al. A bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors. J Virol Methods,2014;198, 86-94.

8. An inquiry into the causes and effects of the variolae vaccinae: a disease discovered in some of the western counties of England, particularly Gloucestershire, and known by the name of the cow pox. U.S. National Library of Medicine (Internet). Cited 2017 Jan. Available from: https://collections.nlm.nih.gov/catalog/ nlm:nlmuid-2559001R-bk.

9. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8:871-874.

10. Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical chemistry, 2005;51(12), 2415-2418.

11. Boesch AW, Brown EP, Cheng HD, et. al. (2014 July). Highly parallel characterization of IgG Fc binding interactions. In MAbs (Vol. 6, No. 4, pp. 915-927).

Taylor & Francis.

12. Neglected tropical diseases. World Health Organization (WHO): Africa (Internet). Cited 2017 Jan. Available from: http://www.afro.who.int/en/neglectedtropical-diseases.html.

13. Fujii Y, Kaneko S, Nzou SM, et. al. Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models. PLoS Negl Trop Dis, 2014; 8(7), e3040.

14. Tanigawa C, Fujii Y, Miura M, et. al. Species-specific serological detection for schistosomiasis by serine protease inhibitor (SERPIN) in multiplex assay. PLoS Negl Trop Dis, 2015;9(8), e0004021.

15. Nzou SM, Fujii Y, Miura M, et. al. Development of multiplex serological assay for the detection of human African trypanosomiasis. Parasitology, 2016;65(2), 121-127.

16. Development of a simultaneous diagnosis method for multiple infections of neglected tropical diseases (NTDs) in Africa: (Multiplex Project). Institute of Tropical Medicine Nagasaki University (Internet). Cited 2017 Jan. Available from: http://www.tm.nagasaki-u.ac.jp/multiplex/phase1/index_eng.html.

17. Clem AS. Fundamentals of vaccine immunology. J Glob Infect Dis, 2011;3(1), 73.

18. Surendran N, Nicolosi T, & Pichichero M. Infants with low vaccine antibody responses have altered innate cytokine response. Vaccine, 2016;34(47), 5700-5703.

19. ProcartaPlex Human Immune Monitoring Panel (65 plex). Affymetrix (Internet). Cited 2017 Jan. Available from: http://www.ebioscience.com/humanimmune-monitoring-panel-65-plex-procartaplex-multiplex-kit.htm.

20. A Pilot Study of 63 Sera Cytokines in Patients with and without Syphilis. ID Week Poster. Cited 2017 Jan. Available from: https://idsa.confex.com/idsa/2015/ webprogram/Handout/id3295/POSTER134_883.pdf.

21. Simple Plex webinars. Protein Simple (Internet). Cited 2017 Jan. Available from: http://www.proteinsimple.com/webinar_simple_plex.html?mkt _qWTNOVGRrTTJVNCIsInQiOiIxYUVQVk15SHFVbEpqM3JMZlN 6dzZ1aXpRRG5OM2Y1XC9PN2xwYzVoaDlzSTkzelwvYXRFU zNsVW9B dU9mTDlESkFcL2 F0Z2crTVlvczBqSFwvenpSdHh4Tnc9PSJ9#sp5.

22. Simple Plex: The Hands-Free, Low-Volume, Microfluidic ELISA Alternative. Application Note. Cited 2017 Jan. Available from: http://www.proteinsimple.com/ documents/App_Note_SimplePlex_ELISA_Alternative.pdf.

23. Bioanalytical qualification of clinical biomarker assays in plasma using a novel multi-analyte Simple Plex™ platform. Future Science (Internet). Cited 2017 Jan. Available from: http://www.future-science.com/doi/abs/10.4155/bio-2016-0196?src=recsys&journalCode=bio.

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Not for use in diagnostic procedures.

30

www.luminexcorp.com © 2017 Luminex Corporation. All Rights Reserved. Luminex, xMAP, MAGPIX, FLEXMAP 3D, xTAG, and xPONENT are all trademarks of Luminex Corporation, registered in the U.S. and other countries. Luminex 200 is a trademark of Luminex Corporation. NEWS1783