xMAP Insights - 2018

xMAP Insights ÂŽ

Connecting the xMAPÂŽ Community to Innovative Applications & Resources

June 2018 Harnessing Immunogenicity for Vaccine Development

xMAP® Insights A Magazine for the Multiplexing Community Greetings, Welcome to xMAP® Insights — the magazine created to connect you to the multiplexing community by sharing innovative applications of xMAP® Technology, while also providing valuable resources to catalyze your next experiment.

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

This issue focuses on custom multiplex assay development for immunogenicity testing and vaccine development, including in-depth stories on: • The Serum Institute of India, which has produced more than 1.3 billion high-quality, low-cost vaccine doses • Ger Rijkers and the Pneumococcal Consortium, who are working to develop standardized multiplex assays for up to 93 antibodies of S. pneumonia • Siân Faustini, who developed serotype-specific assays that pinpoint threshold values missed by standard immune response tests • The clinical development of the human papillomavirus and Staphylococcus aureus vaccines • Brooke Gilliam, the lead MILLIPLEX® custom assay development scientist We are excited to announce that we are planning three xMAP® Connect regional user group meetings this year in Amsterdam, Boston, and China for multiplexers around the globe. We hope to see you there!

The next issue of xMAP Insights will focus on immuno-oncology. 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.

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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CONTENTS 04

Vaccines Aren’t a Luxury

08

Discriminating 93 Serotypes

10

Serotype-specific Assays

12

Assessing Immunogenicity

16

Meet Brooke Gilliam

18

5 Tips for Custom MILLIPLEX® Assays

20

Attend xMAP® Connect 2018

14

Meet Brooke Gilliam "While we have more than 600 validated antibody pairs, if you’re interested in an analyte not in our current commercial portfolio or need to tweak your assay format, just ask."

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Vaccines Aren’t a Luxury The Serum Institute of India has produced more than 1.3 billion high-quality, low-cost vaccine doses

Sunil Gairola, PhD, Director of Quality Control, and Manish Gautam, PhD, Senior Manager in the Analytical Development Group, at the Serum Institute of India share insights on the development of the next generation of vaccines. Since 1967, the Serum Institute of India has launched more than 20 vaccines, with five more vaccines in the pipeline specifically designed to target infectious diseases that are prevalent in developing countries. Gairola and Gautam rely on xMAP® Technology for the quality control and characterization of multivalent and combination vaccines.

Photo Above: Immunoasssay group at Serum Institute of India (group photo).

Bottom Row: Mr. Sachin Bhandare, Mr. Shrikanth Thorat, and Mr. Chetan Mandhane Top Row: Mr. Krishna Manoj, Mr. Krunal Patel, Manish Gautam, Prathamesh Kale and Laxmikant Kadam

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Luminex xMAP® Insights | June 2018

colorimetric and analytical separation techniques used for quantification of intermediate, final products, and detection of impurities.

2. Techniques used for measuring the biological activity, active concentration or confirmation of antigens. This group includes animal tests, cell-based assays, and immunochemical techniques.

The antigen concentration in the final lot is often relatively very low, which hampers the characterization of the final formulated vaccine.

Low antigen concentrations are a significant challenge, especially for combination and multivalent vaccines. To cater to such complex requirements, The Serum Institute has a specialized team and infrastructure centralized in the Analytical Development Group.

Tell me about the Analytical Development Group.

Pictured Above: Dr. Sunil Gairola

Talk about challenges in quality control for vaccine manufacturing.

Sunil Gairola (SG): Quality control of vaccines is challenging because vaccines are heterogeneous with respect to composition. Also, it’s often difficult to link a certain immunological effect to a specific product characteristic in regards to specific correlates of protection. Lastly, quality control assays aren’t one-size-fits-all, meaning each type of vaccine class requires a specific assay. For example:

Manish Gautam (MG): The Analytical Development (AD) Group develops, validates, and implements analytical technology solutions for quality control and characterization of multivalent and combination vaccines. We currently use xMAP®, Capillary Zone electrophoresis, HPSEC-MALS, and LC-MS technologies to test for quantity, potency, purity, and identity, as well as detailed structural characterization.

• Viral vaccines: we measure infectious particles and the level of impurities • Recombinant protein vaccines: we characterize the cell banks, perform protein sequence analysis, and measure the conformational stability of antigens • Toxoid based vaccines: we test in animals the potency and immunogenicity

Share your thoughts on technologies.

SG: In the last decade, the number of analytical techniques used for the characterization of antigens has increased substantially. Techniques can be broadly classified into two categories: 1. Techniques used for measuring the concentration and integrity of antigens: this category includes

Pictured Above: Dr. Manish Gautam

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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We also contribute to detailed immunogenicity assessments of vaccines with respect to the development of newer animal models and assays for characterizing the antigen-specific humoral and cell-mediated immunity responses in animal models. For example, the pneumococcal conjugate vaccine is a multi-serotype vaccine, and quality control testing involves more than 300 tests for release of one batch of vaccine.

How does The Serum Institute use xMAPÂŽ Technology?

MG: Safety and potency assessment of a candidate vaccine is a major stage-gate for vaccine development. In efficacy assessment, assays that measure antibody levels or any aspect of adaptive immune response such as antibody avidity, functional antibody assays, and cellular immune responses (T-cell proliferation and cytokine quantification) are required to understand the correlates of protection in animal models. Multiplexing is an advantageous technology because it enables simultaneous analysis of several antigens from very small sample volumes.

We use xMAP Technology in all phases of research and development, from toxicological studies to the final Phase 3 studies meant for licensure. We find the greatest value for multiplex technology with combination and multivalent vaccines, wherein higher throughput and reduced sample volumes help us to generate real-time data from animal models.

In addition to immunogenicity assays, we have also developed xMAP-based assays for routine testing of vaccines, including identity, potency, and dose confirmationbased assessments. We are also working on exploring applications of xMAP Technology in the genotypic characterization of strains used in the vaccines.

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The Serum Institute of India by the Numbers

#1

65%

140

India's No. 1 Biotechnicology company.

Estimated that about 65% of the children in the world receive at least one vaccine manufactured by The Serum Institute of India.

Supplies more than 140 countries.

1966

Founded in 1966 by Cyrus Poonawalla.

1967

Produced its first vaccine in 1967, which was Tetanus Toxoid.

1.3

The world's largest vaccine manufacturer by volume, having produced more than 1.3 Billion total vaccine doses.

Billion

The pneumococcal conjugate vaccine is a multi-serotype vaccine, and quality control testing involves more than 300 tests for release of one batch of vaccine.

What are specific challenges of coupling polysaccharides?

Can you share a few tips for fellow xMAP assay developers?

The specific challenge of coupling polysaccharides to the beads is the lack of a harmonized approach towards using a specific kind of conjugation chemistry for coupling. Many methods have been reported in the literature for coupling polysaccharides to the xMAP beads, each with advantages and limitations. While the scientific literature is rich with information on troubleshooting protein-coupling issues, polysaccharide-based applications are limited, so not much work has been reported regarding troubleshooting.

MG: Of course! Here are our top tips for multiplex assay development:

An additional issue is the availability of polysaccharides for coupling. We need to define the minimum acceptance criteria for polysaccharides to be used for coupling with respect to size, purity, and stability.

4. Choose the best curve-fitting model for optimal assay performance.

1. Invest up-front time to select the best assay format for your application.

2. Evaluate your assay outcomes with adequate assay controls.

3. Establish criteria cross-reactivity, specificity, and selectivity criteria in advance.

5. Validate your assay extensively and have a good monitoring plan.

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

Get 20 detailed protocols plus reagent lists for designing and optimizing your multiplex assay in the xMAPÂŽ Cookbook.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Discriminating 93 Serotypes

Ger Rijkers and the Pneumococcal Consortium are working to develop standardized multiplex assays for S. pneumoniae antibodies Ger Rijkers, PhD, Head of the Department of Science at University College Roosevelt, in Middelburg, the Netherlands, is developing a custom multiplex assay to efficiently identify the antibodies against specific serotypes of S. pneumoniae responsible for infection.

Why does S. pneumoniae present a challenge?

Ger Rijkers (GR): S. pneumoniae comes in 93 different serotypes, i.e., there are 93 different polysaccharide capsules.

This also presents a challenge for vaccine development, and vaccine manufacturers to formulate a vaccine that covers the most common serotypes.

In the early days, before there was any multiplex immunoassay, the way to determine the serotype was by enzyme-linked immunosorbent assay (ELISA). Ideally, you would do 93 different ELISAs to detect all serotypes, which of course was not logistically possible. When xMAP® Technology launched, I immediately realized that we could use it to detect those multiple serotypes simultaneously, which would provide massive benefits to our laboratory.

Why is the measurement of serotype-specific antibodies important?

GR: There are some assays on the market that are nonspecific and measure a mixture of all the polysaccharides.

However, these give you limited information about the infection. The latest S. pneumoniae vaccine is made up of 13 serotypes. Discriminating between the serotype-specific antibodies enables you to identify whether the infection is due to a vaccine failure or a non-vaccine serotype.

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Tell us about the Pneumococcal Consortium.

GR: We started the Pneumococcal Consortium in 2013 because laboratories all over the world were struggling with the same problem of identifying the different S. pneumoniae serotypes, as there was no ready-to-use diagnostic kit available.

Since then, we’ve harmonized the protocols, agreed on common standards, and standardized results. We are working to publish a harmonized protocol so that other laboratories around the world can benefit from this technology. Our panel currently contains 25 serotypes, but our aim is to create an all-inclusive 93 serotype panel.

SS: What limits the number of serotypes you can detect at one time?

GR: Theoretically, the only limiting factor to the multiplex system would be the number of different color-coded beads available, but in reality, there are other hurdles. You can get unexpected cross-reactions, where one antibody can react with multiple polysaccharides or vice versa. When this happens, it is difficult to solve; you might have to go back to the drawing board and select completely different antibodies.

So, the higher the complexity of your multiplex system, the higher the risk that you’ll run into a problem with specificity.

We remain optimistic and hope that we’ll end up with the full 93 serotypes, but I would be satisfied if we could get 80 to 85.

How will pneumococcal research progress in the future?

GR: Our aim is to create an immunoassay that can be used in hospitals around the world to identify antibodies against all 93 serotypes of S. pneumoniae. Such an assay would aid effective patient treatment, inform vaccine development, and improve understanding of the disease.

Ideally, you would do 93 different ELISAs to detect all serotypes, which of course was not logistically possible. When xMAPÂŽ Technology launched, I immediately realized that we could use it to detect those multiple serotypes simultaneously, which would provide massive benefits to our laboratory.

2017 Pneumococcal Consortium Meeting

The Sixth Network Meeting for Users of Multiplex Immune Assays to Quantify Serotype-Specific IgG against Streptococcus pneumoniae polysaccharides was held at Chetham’s Library in the center of Manchester on Wednesday, May 16th and Thursday, May 17th, 2018.

Want to join the consortium?

Want to learn more about Rijkers' work?

Read his in-depth interview in SelectScience.

Contact Ger Rijkers at g.rijkers@ucr.nl or Jenna Gritzfeld at Jenna.Gritzfeld@phe.gov.uk.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Serotype-specific Assays Pinpoint Threshold Values Missed by Standard Tests xMAP® Assays reveal more about vaccine immune response than traditional ELISAs If you use ELISAs to test immunological responses, Siân Faustini from the University of Birmingham has this advice: “Serotype-specific assays are the way to go.” In her 20-minute webinar presentation from xMAP® Connect 2017, “Exploring immunological assays to assess responses to pneumococcal polysaccharide and conjugate vaccines in HIV,” Faustini shares data from a research study of pneumococcal vaccination response using both Luminex beadbased assays and traditional ELISAs.

Assessing Individual Serotypes Among the most eye-opening parts of the presentation was Faustini’s finding that traditional vaccine response assays were not carefully assessing individual serotypes, which she had measured one by one using xMAP® Technology research reagents. “When our serotype data was compared with the whole PCP IgG assay, 36.4% of patients who were deemed protected by the whole PCP IgG assay had a significant number of serotypes that failed to reach threshold,” she reported. “What that’s saying is that the whole PCP IgG assay is actually misdiagnosing patients if you’re comparing it to the pneumococcal specific IgG assays.”

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Luminex xMAP® Insights | June 2018

Multiplexed Bead Approach To complete this work, Faustini developed serotype-specific assays with xMAP Technology and conjugated the Pneumovax-23 and Prevenar-13 vaccines to beads for a multiplexed approach. Luminex-based assays measured total antibodies for IgG, IgG1, IgG2, IgG3, and IgG4 from 50 HIV patient serum samples to 11 different serotypes. In the webinar, she reported, “The subclass analysis offers a further understanding of the IgG response to different vaccines and also shows the granularity of the response because it’s very different for each serotype.” Her analysis included both absolute values and fold changes, which showed different patterns.

In addition, Faustini presented “tips and tricks” for using Luminex-based multiplex assays.

She recommends daily calibration, sonicating the needle before and after use, and monthly validation of the machine and needles. For more data and advice, check out the full webinar.

The presentation is supplied by the presenter listed and is not endorsed by Luminex Corporation.

Want to learn more?

Visit the Luminex blog to watch more xMAP webinars.

Every other Wednesday, we post a new webinar recording that highlights the diverse work of the xMAP multiplexing community taken from presentations at xMAPÂŽ Connect users group meetings, as well as archived webinars by xMAP enthusiasts, Luminex Field Application Scientists, and Luminex Partners.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Assessing Immunogenicity A look at human papillomavirus and Staphylococcus aureus vaccine studies by the numbers.

As the majority of novel vaccines are now combination or multivalent, precise characterization of the individual components with minimal or no interference from other antigens is becoming increasingly challenging.

Accurately characterizing the polyclonal response to vaccination is critical, specifically monitoring typespecific neutralizing antibodies that mediate protective immunity. In contrast to running multiple singleplex tests, many vaccine developers have developed custom xMAP®-based immunoassays. Multiplex assays widely support epidemiologic studies and clinical trials investigating the efficacy, immunogenicity, and duration of investigational and on-market vaccines.

Highlighted are two case studies where multiplexed immunoassays supported the development of: • Merck’s Gardasil®, the original 4 and later 9 antigen human papillomavirus (HPV) vaccine, and

Both vaccine programs used competitive assays, where type-specific phycoerythrin (PE)-labeled, neutralizing monoclonal antibodies (mAbs) compete with an individual’s serum antibodies for binding to conformationally sensitive, neutralizing epitopes on the virus-like particles (VLPs). The fluorescent signals from bound type-specific mAbs-PE are inversely proportional to the patients' neutralizing antibody titers. The rationale for developing a multiplexed competitive immunoassay, rather than a capture assay, was that capture assays to VLPs measure a combination of antibodies to both neutralizing and non-neutralizing epitopes. Other advantages of the competitive assay format are that the serum sample does not need to be diluted and the assay measures antibodies specific to antigen type-specific neutralizing epitopes. These factors enabled the assay to demonstrate seroconversion and to link seroconversion to protective immunity in response to vaccination.

• Pfizer’s SA4Ag, a 4-antigen Staphylococcus aureus investigational vaccine which is currently being evaluated in phase 2b study.

As of March 2018, xMAP Technology has supported 143 clinical studies and 236 peer-reviewed publications investigating HPV vaccine immunogenicity and epidemiologic studies.

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Luminex xMAP® Insights | June 2018

GARDASIL TIMELINE

2003

HPV is the Most Common Sexually Transmitted Infection in the United States Opalka et al. developed a competitive Luminex immunoassay (cLIA) assay for HPV (6, 11, 16, 18) neutralizing antibodies:

2005

• Developed the first HPV-Luminex competitive immunoassay to measure titers of polyclonal antibodies in serum where phycoerythrin (PE)-labeled, neutralizing antibodies compete with patient serum antibodies for binding to conformationally sensitive, neutralizing epitopes on the VLPs.

Smith et. al evolved the 4-plex cLIA to enhance sensitivity and throughput and performed bridging studies: • Screening ELISAs with mAbs to ensure type and conformation specificity.

• Hemagglutination inhibition (HAI) and pseudovirus (PsV) neutralization assays.

• Competitive radioimmunoassay (RIA): This version of the cLIA has been widely used to monitor the immune response in Phase 2B and 3 clinical studies. It has also been used to monitor the duration of immunity, as well as efficacy, in expanded geographies and indications.

2008

2014

Dias et al. optimized & validated the 4-plex cLIA for use in epidemiology studies and vaccine clinical trials: • Determined the clinical serostatus cutoff as low titers of anti-HPV antibodies in the serum from naturally infected individuals are common.

• Switched antibodies to improve the analytical specificity of the assay.

• Conjugated R-phycoerythrin directly to the four mAbs to reduce the number of steps in the assay and reduce sources of error.

• Optimized concentrations of mAbs and VLPs. • Evaluated different sample matrices to improve the clinical sensitivity of the assay: ◆◆ Selected: ADHS. ◆◆ Alternative matrixes: PBS, PBS with 1% BSA, PBS with 1% rHA, NGS, DFP, and 3% nonfat milk in PBS. • Heat-inactivated freshly thawed samples to reduce variability due to pre-analytical factors.

• Automated the workflow with a TECAN Genesis Workstation to increase assay throughput, reproducibility, and operator safety.

Roberts et al. extended the original 4-plex cLIA to a 9-plex format to support Phase 3 studies of the investigational nine-valent HPV (HPV6/11/16/18/31/33/45/52/58) vaccine program: • Selected mAb pairs that are high affinity, type-specific, and recognized conformationdependent neutralizing epitopes.

• Assigned antibody concentrations to the HPV-9 cLIA reference standard to ensure comparability at the serostatus cutoffs.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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SA4Ag TIMELINE Begier et al.

1% of All U.S. Hospital Inpatient Admissions are Affected by S. aureus Disease

2015

• Used the SA4Ag cLIA to assess antigen-specific immunogenicity in a phase 1 clinical study evaluating 100 healthy adults.

2017

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Luminex xMAP® Insights | June 2018

• Developed a qualified 3+1 cLIA to indirectly measure the level of functional serum antibody titer to each of the four SA4Ag vaccine antigens (capsular polysaccharide (types 5 and 8), conjugates, clumping factor A (ClfA), and manganese transporter C (MntC)).

Frenck et al.

Creech et al.

• Used the qualified 4-plex cLIA to measure functional antibody responses in a phase 1/2 study of 284 healthy elderly adults.

The older cohort demonstrated similar rapidity and durability to the younger adult cohort, which implies that the vaccine antigens are capable of boosting low-level antibody titers generated by a lifetime of intermittent staphylococcal exposure or colonization.

Rozemeijer et al.

2017

2017

• Advanced 3+1 cLIA to a qualified 4-plex cLIA format to assess antigen-specific immunogenicity in a Phase 1/2 clinical study evaluating 454 healthy adults.

The original 3+1 cLIA was modified to a 4-plex format that used sera from human subjects vaccinated with SA4Ag as the reference standard.

This optimization eliminated potential matrix differences between the test samples and reference standard, as well as measured antibody specific to all four antigens in a single assay format.

A consortium of Canadian researchers validated the BioPlex® 2200 MMRV IgG assay for the quantitative detection of IgG antibodies to Measles, Mumps, Rubella and Varicella-zoster virus.

Clinical Studies Using Commercial Kits Researchers at Sanofi used the MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel to assess the cytokine profile of recipients of the recombinant yellow fever-17D–dengue virus, liveattenuated, tetravalent dengue vaccine (CYD-TDV) candidate in two Phase III vaccine efficacy trials that demonstrated significant protection against dengue disease during the two years of active surveillance.

To assess other potential predictors of Zostavax® responses, Stanford researchers quantified serum cytokine concentrations before and after vaccination with varicella-zoster virus vaccine using a 51-plex ProcartaPlex fluorescent-bead assay from ThermoFisher Scientific.

A consortium of Canadian researchers validated the BioPlex® 2200 MMRV IgG assay for the quantitative detection of IgG antibodies to Measles, Mumps, Rubella and Varicella-zoster virus. The team evolved the commercial qualitative kit to a quantitative format using the anti-measles WHO standard to generate a calibration curve that correlated relative fluorescence intensity to quantitative antibody titer. This change allowed for comparison of the calculated measles antibody titer from the BioPlex 2200 MMRV IgG assay titer to the plaque reduction neutralization test (PRNT) reference method.

MILLIPLEX is a registered trademark of Merck KGaA. Gardasil and Zostavax are registered trademarks of Merck Sharp & Dohme Corp ProcartaPlex is a registered trademark of Affymetrix, Inc., which is part of Thermo Fisher Scientific. BioPlex is a trademark of Bio-Rad Laboratories, Inc.

Curious how other vaccine development programs are leveraging xMAP® Technology?

Check out the Luminex Research Publications Database, which has more than 32,000 curated publications that you can search and sort.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Meet Brooke Gilliam Gilliam helps researchers with custom MILLIPLEX® assay solutions 16

Luminex xMAP® Insights | June 2018

What do you do when commercially available multiplex kits are not optimally configured to meet your experimental needs? Custom immunoassay development might be the right solution for you if you want to combine analytes across existing panels, include off-menu analytes, or conjugate a specific antibody to xMAP® beads with robust analytical validation. Meet Brooke Gilliam, MS, who has 15 years of immunoassay development experience, and learn more about the custom assay development process.

What do you do at MilliporeSigma?

Brooke Gilliam (BG): I’m the lead MILLIPLEX® custom assay development (www.emdmillipore.com/customassay) scientist at MilliporeSigma. I spend my time consulting with customers on new assay feasibility, as well as overseeing the scientific development process, which includes:

While we have more than 600 validated antibody pairs, if you’re interested in an analyte not in our current commercial portfolio or need to tweak your assay format, just ask.

• Developing new analytes • Combining currently available analytes into custom panels • Considering cross-reactivity and sample dilution for analyte compatibility • Generating work plans and conducting project milestone calls with customers and our project management team • Reviewing customer data and making suggestions • Keeping customers informed throughout the project

What aspect of your job do you enjoy most?

BG: I like consulting with customers and providing solutions to their unique challenges. I have the pleasure of directly interacting with customers to collaboratively develop and implement assay solutions. It’s gratifying to see the assays that I helped to develop working well for customers in their labs, as many of my customers come to us because they previously struggled with lot-to-lot variation, sensitivity, or assay validation issues with commercially available kits.

Not sure if your targets are available in a commercial kit?

Check out the xMAP Kit Finder to browse 1,300 commercially available kits. ®

Tell me something about MilliporeSigma most people don’t know.

BG: While we have more than 600 validated antibody pairs, if you’re interested in an analyte not in our current commercial portfolio, just ask. We publish new analytes in our Analyte Quarterly brochure, but additional analytes may also be in development or available. We often include new analytes into our development pipeline based on customer demand.

If you’re interested in a specific analyte, the best way to get started is to contact our custom assay team directly via our website at emdmillipore.com/customassay.

If you could solve any scientific challenge or human health crisis, what would it be?

BG: Before I started at MilliporeSigma, I was a researcher working in academic labs that focused on autoimmune inflammatory disorders, specifically rheumatoid arthritis and Lupus. I was moved when writing the patient demographics section for a manuscript of a large Lupus study; many of the patient participants were young children, so I would hope for a cure or even a targeted treatment for Lupus.

How do you spend your time away from work?

BG: I chauffeur my kids to various sports and activities.

Outside the organized activities, I enjoy spending time together with my family at home.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Tips for Custom

MILLIPLEX速 Assays Brooke Gilliam is the lead custom multiplex assay development scientist at MilliporeSigma and is based in downtown St. Louis, Missouri. Gilliam shares a few best practices based on her 15 years of experience developing immunoassays.

1

What information is required to confirm the feasibility of a custom MILLIPLEX速 assay?

To get started, we need to know your species and sample type and whether or not the customer would like QC ranges established for their assay.

2

What parameters do you typically consider for assessing the feasibility of a custom assay?

We assess all of the following up front (as required or known): sample dilution, sample preparation, serum matrix solution compatibility (for dilution of standards in serum/ plasma samples), bead regions, type of Luminex instrument and analysis software, analyte cross-reactivity, and desired assay format.

3

Can you give me some examples of customers who may require a custom multiplex assay?

Our most popular request is for combining analytes between panels, or for validating an assay on a new sample type or species. We also do quite a bit of development work for new analytes or proprietary customer targets. With the biomarker detection field rapidly evolving, it is not always possible to have every analyte available, and this is the rationale behind our development offerings. Finally, some customers require enhanced verification/validation depending on their assay needs or developing an assay in a new format. We do all that as well. Lastly, we do offer services to assist our customers. Examples include our Core Lab MapMate program, bead conjugations, biotinylation, iodination, and data analysis.

MILLIPLEX速 is a registered trademark of Merck KGaA.

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Luminex xMAP速 Insights | June 2018

4

What is one important tip that all customers should know?

5

What types of assay validation do you offer in your custom assays?

Every customer’s sample is different; not all serum/plasma samples are created equally.

For example, I have seen proteins at off the chart levels in some diseased patients or mouse samples. We encourage customers to send us a subset of their samples to use when developing a custom assay to ensure we hit the range they need to measure all of their analytes. This will avoid customers running (and wasting) a kit and then having to adjust the dilution factor because their analyte is abnormally high. If that’s not possible, we will send a test kit for customers to run in-house and can modify the assay depending on the customer’s results before we send the final kitted assay. This two-way partnership is essential and is one way that we deliver the highest quality assay possible.

I can’t stress enough the importance of the assay quality. At a bare minimum, we always measure analyte cross-reactivity, determine a minimum required dilution for the customer’s sample type, establish an assay range to ensure samples read in the linear portion of the curve, and supply an optimal serum matrix solution for dilution of the standards (serum/ plasma samples). We also consider lot #1 to be our gold standard reference lot and match the performance of subsequent assays back to our gold standard. Beyond this, we offer a menu of validation services depending on the nature of your work or your specific validation requirements. An example could include spike-recovery in samples.

Every customer’s sample is different; not all serum/ plasma samples are created equally.

Ready to get started?

Connect with a custom assay scientist by visiting:

www.emdmillipore.com/customassay

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Attend xMAP® Connect 2018

Users’ group meetings in Amsterdam, Boston, and China

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

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. xMAP Connect is structured to grow your network and to enable you to discover the endless applications of xMAP Technology. During the program, you will hear from multiplex experts about developing cutting-edge methods and innovative projects from the research and clinical spaces, as well as have the opportunity to connect with fellow researchers, Luminex partners, and Luminex specialists on-site.

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Luminex xMAP® Insights | June 2018

Interesting in attending xMAP Connect 2018?

Sign up to receive updates about attending the events and presenting your work.

Amsterdam

Boston

China

Want to learn more about xMAP Connect?

Watch this video to see what past attendees enjoyed about the meeting.

View archives from the 2017 events in Amsterdam, Boston, and China.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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www.luminexcorp.com Š 2018 Luminex Corporation. All Rights Reserved. Luminex and xMAP are trademarks of Luminex Corporation, registered in the U.S. and other countries.

Luminex xMAP Insights | June 2018 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.