xMAP Insights - Q4 2016

Connecting the xMAP Community to Innovative Applications & Resources

xMAP Insights ®

INAUGURAL ISSUE!

Have you been to Papua, New Guinea?

MAGPIX® has.

In This Issue 3

Letter from the Editor

Connections 4 Have you been to Papua New Guinea? MAGPIX® has.

6

Shooting for the Moon – Proteogenomics – The Next Leap in Precision Cancer Care

Collaboration 7 DigiWest®: Reimaging Western Blotting on the Proteomic Scale 9

PiSCES: Reveals More Than Your Horoscope

11 PRISM: A Novel Bead-Based Biological Barcode Assay

Community 13 Get to know Stephanie Harris, Technical Applications Specialist at Luminex 14 Tips and Tricks from Luminex Tech Support 16 Become a Luminex Partner

Curiosity 17 Partner Product Offerings from Affymetrix, R&D Systems, EMD Millipore, and Luminex 20 Digital Learning: xMAP® Partner Webinars, Virtual Conferences, and How-to Videos 20 Upcoming In Person Trainings and Conferences

Letter from the Editor

Welcome to xMAP® Insights A Newsletter for the Multiplexing Community Greetings,

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

Welcome to the first issue of xMAP® Insights. We’ve set out to create a digital newsletter that is a collection of inspired and instructive articles that focus on you – our customer.

On a quarterly basis, we hope to capture a glimpse into the diverse applications of xMAP Technology and to spark ideas for your next experiment. Please take a moment to get to know the standing sections, as you might be more partial to some articles versus others.

• Connections – spotlights trending research and emerging opportunities • Collaboration – summarizes recent breakthrough applications and method publications • Community – profiles members of the multiplexing community • Curiosity – stimulates your imagination with resources, new product overviews, and upcoming learning events I’m grateful to have you as a reader and look forward to your feedback as well as suggestions for future articles. Please don’t hesitate to contact me directly at hgraham@luminexcorp.com. Also, if you ever find yourself in Austin, feel free to give me a shout, 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

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

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Not for use in diagnostic procedures.

3

Connections

Have you been to Papua, New Guinea?

MAGPIX® has.

The first commercially available MAGPIX instrument went to Peter Zimmerman’s field research site to fight malaria

Peter Zimmerman, Arsene Ratsimbasoa (Director, Madagascar National Malaria Control Program), and colleagues at the NMCP Headquarters in Antananarivo, capital of Madagascar.

Photograph used with permission from Peter Zimmerman.

Peter Zimmerman, PhD, Professor

choice for field use. This first MAGPIX ever sold is currently in

of International Health, Biology and

Zimmerman’s CWRU laboratory in Cleveland and stills sees

Genetics, was the first customer

constant use.

to receive the newly launched MAGPIX® instrument in 2010. While his lab is at Case Western Reserve University (CWRU) in Cleveland, Ohio, the instrument was installed at Zimmerman’s field research site in Papua New Guinea.

Zimmerman is currently working to elucidate how Plasmodium vivax, the protozoan parasite that is the most frequent cause of malaria, is infecting a historically resistant population of people in Madagascar.2 Duffy blood group negativity (Duffy (-)) was thought to provide resistance to P. vivax malaria infection, but this is no longer the case.

With the support of an NIAID grant, Zimmerman’s group is working

He is a longtime user of Luminex Technology, having developed

to uncover the newly evolved invasion mechanism of P. vivax and

a number of multiplex assays to decipher the impact of microbial

to define the current molecular susceptibility profile of Duffy (-)

genetic polymorphisms on the infection and pathogenesis of malaria

individuals. For this research project, he will be using a Bio-Plex 200®,

and HIV. Zimmerman compares developing multiplex assays for

provided to his collaboration with the Madagascar National Malaria

research applications to building with LEGOS - if you combine the

Control Program through the President’s Malaria Initiative.

®

®

right pieces on the platform, it reliably works. Zimmerman still uses his 2006 Luminex-based assay for clinical research applications to diagnose malaria and detect single nucleotide polymorphisms (SNPs) associated drug resistance today.1

While there is a push to move malaria detection outside of the laboratory for clinical diagnosis, there is also the need to develop novel molecular tests to detect reservoirs of submicroscopic infections if malaria is to be eliminated. Researchers are presently

Before receiving the MAGPIX instrument he was using a Luminex®

working on techniques to amplify a nucleic acid signal – such as

200 system, but shipping sheath fluid to Papua New Guinea—

pooling mosquitos – because new strategies that improve detection

even when concentrated to 20x—is an expensive proposition. The

efficiency when testing entire populations of mosquitos are essential

reduced fluid consumption combined with the increased stability of

for success.

™

the CCD imager optics versus lasers, made the MAGPIX the obvious

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

4

Connections

Are you looking for a multiplexing instrument that is easy to learn, use, and maintain?

The MAGPIX instrument might be right for you.

MAGPIX®

Simple. Affordable. Reliable. Compact.

Countries with a MAGPIX® Presence ©2016 Luminex Corporation. All rights reserved. Luminex and xMAP are trademarks of Luminex Corporation, registered in the U.S. and other countries. GX1681.0916

xMAP Insights | 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

www.luminexcorp.com

5

Connections

Shooting for the Moon: Proteogenomics – the Next Leap in Precision Cancer Care APOLLO links proteomic and genomic analysis to develop more effective oncology treatment plans The Cancer Moonshot is a mission that all of us #CanServe according to Vice President Joe Biden.3 Announced at the State of the Union address in January 2016, the program aims to double the rate of progress toward a cure for cancer through increased collaboration and investment. Most recently the Applied Proteogenomics Organizational Learning and Outcomes (APOLLO) Network was established and will combine point-of-care genomic and proteomic analysis in hopes of matching a patient’s tumor types to targeted therapies. A cohort of 8,000 patients undergoing

Vice President Joe Biden Official White House Photo by David Lienemann.

In this issue of xMAP® Insights, the Collaboration section focuses on novel genomic and proteomic assays that utilize xMAP technology.

treatment for lung cancer as part of the Veterans Affairs (VA) and the Department of Defense (DoD) healthcare systems will be routinely screened at oncologist appointments with the goal of identifying treatment plans that are more effective based on each patient’s unique proteogenomic profile.

DigiWest enables a highly parallel analysis of protein expression and phospho-status by adapting the classical Western blot to a beadbased microarray platform.

The next step in the evolution of precision oncology is to identify targetable protein pathways and gene mutations that can serve as the basis for novel therapies. To advance research in the proteogenomic space, the NCI has gathered ideas from the public and will be releasing a new round of funding opportunity announcements in early 2017. 4

Do you have a peer-reviewed publication you’d like highlighted in the next issue of xMAP Insights?

Submit them to hgraham@luminexcorp.com

PRISM

(Profiling Relative Inhibition Simultaneously in Mixtures) employs 24 nucleotide long biological barcodes that are stably integrated into genetically distinct tumor cell lines, which allow for pooling and testing of multiple cell lines simultaneously against potential drug compounds enabling the accelerated the search for targeted therapies.

PiSCES

(Proteins in Shared Complexes Detected by Exposed Surface Epitopes) is an antibody-based multiplex method that is combined with mathematical analyses to construct protein-protein interaction networks to decipher differences in the relative abundances of particular protein complexes between healthy and diseased states to one-day facilitate personalized therapies.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

6

Collaboration

DigiWest®: Reimaging Western Blotting on the Proteomic Scale A novel high-throughput protein analysis approach utilizing a beadbased microarray platform

Inspired by the unparalleled size resolution of the classical Western blot, but frustrated by the lack of throughput, Markus Templin and his team set out to incorporate protein size information into their multiplex immunoassays. With DigiWest they’ve achieved an attractive alternative to currently available approaches, such as mass spectrometry, reverse-phase protein microarrays (RPPA), and next-generation Western blots based on: • Enhanced spatial resolution • Reduced sample requirement (as little as 50 μg) and quantitative phospho-status • Enhanced sensitivity – Able to detect 10 pg/μg with a 1,000 fold dynamic range • Increased throughput – Run 60 to 600 analytes per sample with 2 to 60 samples per study

The Western blot has been a staple in the molecular biology tool kit for nearly 40 years.5 While at Stanford, George Stark was the first to describe the transfer of proteins by capillary action from a gel onto diazobenzyloxymethyl-paper, submitting his work to the Proceedings of the National Academy of Sciences (PNAS) in April 1979.6

• Automated workflows and digital data output Challenges are inherent to developing a new method, but Templin and his team weren’t deterred with DigiWest. To develop and verify it against Western blotting, they established a panel of over 1,000 commercial antibodies.

Simultaneously, Harry Towbin was working to determine the specificity of antibodies against proteins in complex macromolecular structures and unknowingly developed the same gel electrophoresis method, but with a transfer onto nitrocellulose. Towbin submitted his

Sounds too good to be true? Templin’s group validated the novel method with a series of four experiments:9 1. DigiWest exhibited comparable sensitivity and linearity

version of the method to PNAS in June 1979.7 Only when both articles were published in the September issue of PNAS did the authors realize they had developed the same method.

But it was a third man, W. Neal Burnette, who was the first to coin the term Western blot – in homage to the Southern and Northern blots –

during a spike-in experiment but required only 1/100th of the initial sample as compared to a traditional Western blot. Additionally, the intra-assay variation (CV of replicate measurements from the same bead-set) was below 5%.

2. Highly similar expression patterns were observed as

noted at the end of the introduction section in his 1981 publication in

compared to a mass spectrometry approach that used

the journal of Analytical Biochemistry.8

Kinobeads to identify a set of 24 kinases, which showed

Flash forward to today and Western blot analysis is practiced in the same analog format and remains as laborious and resource-intensive as it was back in 1979.

significant differences between Lapatinib-resistant and the H292 parental cell line.

3. Major changes in the expression levels of Ephrin-receptors were revealed, indicating rewiring of Ephrin signaling during

Markus Templin and colleagues from the NMI Natural and Medical

the development of resistance. Additionally, 15 out of the 37

Sciences Institute at the University of Tübingen, have developed the

differentially expressed proteins were associated with p53.

next generation of the Western blot. Known as DigiWest , it enables ®

a highly parallel analysis of protein expression and phospho-status by adapting the classical Western blot to a bead-based microarray platform. This novel protein profiling immunoassay is described in Nature Communications and is also available in a fee-for-service model through NMI TT Pharmaservices.9

4. DigiWest protein profiling detected expression differences for approximately 200 proteins from mammary carcinoma tissue that was collected via laser capture microdissection.

Additionally, Her2 signal intensity detected via histology was reproduced by the DigiWest.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

7

Collaboration Now that Templin’s group has demonstrated proof of concept with DigiWest, they are working to broaden its applicability. While the

Want to learn more?

primary application of their method thus far has been biomarker

Read the Nature Communications article.

Visit the DigiWest website.

discovery and lead compound profiling in oncology research, DigiWest’s ability to simultaneously analyze up to 600 proteins, including phospho-status, would likely also be of interest to preclinical

Interested in developing your own commercial assay?

and clinical scientists in other fields. They have also been working to expand their antibody panels from human and mouse to also include rat, dog, and mini pig as well.

Contact the Luminex Business Development team.

DigiWest is a trademark of NMI registered in Germany.

DigiWest

®

A HIGH-THROUGHPUT PROTEIN ANALYSIS APPROACH UTILIZING A BEAD-BASED MICROARRAY PLATFORM

1

3

Proteins are size separated via SDS-PAGE.

4

Sample lanes are cut into 96 strips to generate 96 molecular weight fractions immobilized on membrane.

2 Proteins are transferred to a blotting membrane.

Proteins are eluted into 96-well plates.

5 Biotinylated proteins are immobilized on the surface Neutravidin-coated Luminex beads. One distinct bead set is added to each of the 96 wells, which results in a collection of distinguishable protein-loaded bead-sets from a single sample.

8 The sample is detected on a Luminex FLEXMAP 3D® instrument.

6 The beads are pooled to reconstruct the initial sample lane, where a defined color code correlates with each molecular weight of the immobilized proteins.

9 DigiWest profile of protein analytes.

7

E

Immunoassay: a small aliquot of the bead pool is incubated with Western blot antibodies overnight before Phycoerythrin-labeled secondary antibodies are added for signal generation. www.digiwest.de

© 2016 Luminex Corporation. All rights reserved. Luminex and FLEXMAP 3D are trademarks of Luminex Corporation, registered in the U.S. and other countries. DigiWest is a trademark of NMI registered in Germany.

GX1668.0816

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

www.luminexcorp.com

Not for use in diagnostic procedures.

8

Collaboration

PiSCES: Reveals More Than Your Horoscope Novel multiplex matrix network analysis method for the elucidation of protein-protein interaction signatures While the genome is the molecular recipe, the proteome provides a glimpse into what is happening in real time. Like letters that join

Top row: *Steven Neier, Alex Ferrer, *Tessa Davis, *Adam Schrum, *Stephen E.P. Smith, *Brendan Reed.

Bottom row: Kyoo-A Lee, Katelynn Wilton, *Diana Gil, Robert Stiles, Michele Hoffmann. * for co-authors on the paper Photograph used with permission from Adam Schrum.

together to compose ‘words’, individual proteins can bind to form distinctive protein complexes, where the unique composition of each protein complex signals to the cell a specific message. The human genome was sequenced in 2003, but the different combinations that proteins can form remain largely uncharted due to technological limitations.

Although detecting the subtle changes in the proteome is no easy

analysis of specific protein signatures. Twenty proteins in a pairwise combination matrix resulted in the detection of 210 unique pairs of PiSCES. They found a preliminary signature that distinguishes control from disease, and the protein complexes involved point toward an unforeseen hypothesis about which signaling activities might drive disease. In the future,

task, the lab of Adam Schrum was up for the challenge. Schrum

heterogeneity patterns identified across groups could

and colleagues recently published an article in Science Signaling

be used to determine personalized treatments based on

detailing the development of a new method, PiSCES (Proteins in

known patterns associated with treatment responsiveness.

Shared Complexes Detected by Exposed Surface Epitopes), capable of deciphering the molecular signatures unique to healthy and aberrant signaling networks associated with disease.10

When asked what inspired PiSCES, Shrum noted that “the prospect of obtaining network-level information about protein complexes driving biological signal transduction was quite enticing, but there really

T cell antigen receptor (TCR) is capable of transmitting multiple

wasn’t a good technical approach that could provide rich information

messages, and in autoimmune diseases the message can be patho-

from clinical patient samples, mainly because they are so small. We

logic. Therefore, the protein complexes that form a signalosome

needed something that could do this like an ELISA assay would, with

around TCR were examined from 4 mm skin punch biopsies from the

that kind of sensitivity, and possibility for broad survey. The multiplex

scalp of controls or patients with alopecia areata, an autoimmune

microsphere platform seemed very promising, like it could fit the bill,

disorder where T cells attack hair follicles. One of the strengths of this

and it turned out to provide an informative protein interaction signa-

method is the ability to detect patterns even with limited biomate-

ture like we had hoped.”

rial—a common limitation when working with patient samples.

Moving forward, Schrum hopes to translate his method from the lab to

His team was looking for unique combinations of protein associations

patients. Once concise, clinically relevant bio-signatures are identified,

that they could interpret via a matrix approach to generate a network

this subset could serve as the basis of a molecular diagnostic assay.

Want to hear directly from the authors?

Want to read more?

Download the Science Signaling podcast.

Visit Dr Schrum's faculty webpage for article access.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

9

Collaboration

PiSCES

A MULTIPROTEIN MATRIX ANALYSIS SYSTEM FOR PROTEIN-PROTEIN INTERACTIONS

1

MULTIPLEX IMMUNOPRECIPITATION PANEL

3

BIOSTATISTICAL EVALUATION FOR THE VISUALIZATION OF NETWORK ACTIVITY Unsupervised hierarchical clustering of the normalized fold changes in median fluorescence intensity

Principal component analysis of log2 median fluorescence intensity

Protein complexes were captured from cell lysates by immunoprecipitation antibodies covalently coupled to a panel of Luminex beads (red circle). Co-associated proteins were identified with fluorochrome-labeled antibodies (yellow star).

2

DETECTION OF PROTEIN COMPLEXES

ANC (Adaptive Nonparametric with empirical Cutoff) analysis identified high-confidence, statistically significant PiSCES that were consistently identified as hits in at least 70% of experiments

WCNA (Weighted Correlation Network Analysis) identifies subgroups of PiSCES whose expression changes are correlated

Common hits compose high-confidence co-regulated PiSCES biosignatures, which are able to distinguished the control from disease state.

PiSCES are visualized via a protein-protein interaction web, which includes indirect and direct protein interactions.

Reproduced with permission, courtesy of BioRad

A Bio-Plex 200 instrument was customized for the analysis of protein complexes. Specifically, an insulated plexiglass divider enabled samples to be refrigerated at 4° C during data acquisition, while the cytometer atop the plexiglass remained at room temperature.

Š2016 Luminex Corporation. All rights reserved. Luminex and xMAP are trademarks of Luminex Corporation, registered in the U.S. and other countries. GX1660.0816

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

www.luminexcorp.com

Not for use in diagnostic procedures.

10

Collaboration

PRISM: A Novel Bead-Based Biological Barcode Assay Enabling large-scale screening of small-molecule libraries in pooled cancer cell lines to discover genotype-specific vulnerabilities

Golub’s team validated the PRISM assay in vitro and in vivo and found that it:13 • Performs as intended distinguishing genotype-specific drug responses in lung cancer cell lines • Detects the vast majority of cell lines from a single xenograft • Accurately assesses drug sensitivity in wild-type and mutant cell lines • Is suitable for drug discovery, specifically library screening applications • Generates hypotheses about the mechanism of action and molecular target of unknown candidate compounds based on activity pattern across multiple cell lines compared to reference libraries of compounds Now that the Golub lab has demonstrated proof of concept with PRISM, Mader explains, “we are working to industrialize the process to

Barcoding is no longer confined to the black stripes of the ubiquitous

enable cell profiling at the earliest stage of drug discovery in an effort

Universal Product Code (UPC) label found on nearly every product

to rapidly find new opportunities for cancer therapeutics.”

we purchase. The conservationist Paul Hebert was the first to demonstrate the feasibility and utility of DNA barcoding as a way to identify species in 2003.11 Applications outside the research lab include the detection of food fraud, where lower quality fish is substituted for a more desirable species. A recent study by OCEANA found that 39 percent of 142 seafood samples collected and DNA tested from grocery stores, restaurants and sushi venues in New York City were mislabeled.12 While DNA barcoding has already empowered single-cell analysis,

Interested in developing your own multiplex assay?

Download the xMAP® Cookbook to get started.

Todd Golub’s team at the Broad Institute developed a new method to speed oncology drug discovery.13 PRISM, Profiling Relative Inhibition Simultaneously in Mixtures, recently reported in the journal Nature Biotechnology, relies on 24 nucleotide long biological barcodes that are stably integrated into genetically distinct tumor cell lines.14 “We wanted an approach to screen cancer cell lines that both recognized their complexity and allowed us to scale our studies,” said Chris Mader, Senior Group Leader, PRISM Project.

PRISM provides a practical solution for screening drug candidate compounds against a large number of cancer cell lines. While cancer is inherently genetically diverse, methods limitations have historically limited the number of cell lines that researchers use to develop new drugs. This breakthrough method allows for the simultaneous interrogation of multiple cell lines. In brief, cells are barcoded, pooled, and then treated with a drug. Genomic DNA is isolated from surviving cells, hybridized to Luminex® microspheres and then identified and quantified with a FLEXMAP 3D® instrument.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

11

Collaboration

PRISM

A NOVEL METHOD FOR PROFILING DRUG CANDIDATES ACROSS A LARGE NUMBER OF POOLED CELL LINES

1

BARCODING AND MIXING CANCER CELL LINES

3

24-base-pair DNA barcodes are stably integrated into individual tumor cell lines

PROCESSING TO EXTRACT GENOMIC DNA & PCR TO AMPLIFY BARCODES ISOLATE GENOMIC DNA FROM VIABLE CELLS

4

BEAD HYBRIDIZATION PE

PE

PE

PE

PE

PE

Luminex microspheres of distinct colors (each coupled to a different anti-barcode oligonucleotide) are hybridized to the amplicons and stained with phycoerythrin-streptavidin

5

SAMPLE DETECTION

Barcoded cell lines are individually frozen and later thawed to generate mixtures of equal numbers of barcoded cell lines.

2

SMALL MOLECULE TREATMENT

CONTROL

TREATMENT

Quantify hybridization events on a Luminex FLEXMAP 3D®. The bead color indicates the barcode identity and the hycoerythrin intensity corresponds to barcode abundance, a direct reflection of cell number

© 2016 Luminex Corporation. All rights reserved. Luminex and FLEXMAP 3D are trademarks of Luminex Corporation, registered in the U.S. and other countries. GX1650.0816

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

www.luminexcorp.com

Not for use in diagnostic procedures.

12

Community

Get to Know Stephanie Harris Technical Applications Specialist at Luminex Q: What do you do at Luminex?

I’m a Technical Applications Specialist providing customers with troubleshooting support. I’m the protein specialist in our group, so I use this expertise to work on internal development projects as well as provide solutions to researchers’ queries. I generally receive four significant cases per day plus a couple of straightforward queries.

Stephanie Harris, Technical Applications Specialist

The longest call I’ve ever had was a little over two hours, and the customer was great – she was willing to try everything I suggested

Customers might not know that the MAGPIX RUO has three types

to resolve her issue.

of error codes: sample delivery, optics module, and calibration and

When I started at Luminex two years ago, this was the first customer facing role I’ve had. I began by answering emails and submissions from the web form, which we respond to in less

verification, which they can tell apart by the four digit code inside of the bracket following the hardware code 2091 (e.g., 2091[4113] where 4113 pertains to the filter wheel of the optics module).

than an hour, but now I am fully confident when fielding complex

Luminex 200 and FLEXMAP 3D® provide software warning and

customer calls in real time. When talking with customers, I gener-

error codes. There are four log types that show the category of the

ally stand in front of the instrument, as this helps to bring all my

error: batch, maintenance, security, and system.

knowledge front-of-mind.

I feel that I found my niche with this position, as I’m able to combine

Q: How can customers speed up the trouble shooting process?

When contacting Tech Support, it’s helpful if the customer provides

my research immunoassay development expertise with my experi-

the instrument serial number, has generated the Calibration and

ence from using these instruments for many years – both of which

Verification report (or Performance Verification report), and is near

are needed to support the resolution of customer cases. My favorite

the instrument for troubleshooting. Additionally, we get the best

calls are from customers who are developing novel research assays

issue resolution results when the lab technician who is experiencing

or pushing their instrument to/beyond its capability. The most

the problem contacts us directly.

diverse calls definitely come from MAGPIX® users because the instrument is so versatile in research applications, which is reflected

Q: Where were you before Luminex?

in the customer base and their applications.

I spent the previous eight years at EMD Millipore (now a part

Q: Tell us a recent project that you’re especially proud of.

development. In my first role, I manufactured subsequent lots of

of Millipore Sigma) doing mostly antibody and immunoassay

I recently created four error code and solutions internal help

beads and detection antibody cocktails for Luminex-based multi-

documents that are used by my colleagues to resolve customers’

plex kits. When I was promoted to R&D, my main responsibility

instrumentation issues. These documents save my colleagues and

was making the first lots of beads for all novel multiplex research

our customers’ time when troubleshooting, as we can now provide

immunoassays. I had gained so much bead coupling experience

on-point solutions when provided error codes from the Luminex®

that my colleagues starting calling me “The Bead Master”. Later, I

200™ and MAGPIX RUO instruments. To develop these documents,

moved on to antibody purifications for subsequent lots of kits and

I started keeping a listing of error codes and then checked them

developed new antibody pairs for novel multiplex immunoassays. I

against closed cases to determine how the issue was resolved.

performed all of my antibody pair screening assays on the MAGPIX

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

13

Community and validated them using the Luminex 200. This is how I became

Q: Outside of work, how do you spend your time?

quite familiar with Luminex-based assays and instruments before

I’m pursuing a Master’s in Biotechnology Management from the

formally joining the Luminex family.

University of Maryland University College, so I’m currently taking one course at a time for that and expect to finish the program in 2018.

Q: What science are you currently interested in?

I’m intrigued by all things GE/GMO. I’ve recently read about

For fun, I do course exercises on Codecademy to learn programming

research that would allow us to be vaccinated while eating our

languages such as Python and Java. I’m also learning more about

vegetables, on the development of artificial wombs, and generating

SQL. I’m learning to speak Spanish with the Duolingo app and love

spider silk through goats’ milk that is used for the manufacture of

playing Mortal Kombat via PS3, PS4, and Wii.

bulletproof vests.

Tips and Tricks

TIP #3

from Luminex Tech Support

Looking for proteins to develop your own assay?

In addition to the xMAP® Kit Finder and the xMAP Cookbook, Linscott’s Directory is a great resource with

TIP #1

over 2,100,000 unique antibody listings and 1,000,000

Frustrated by verification failure?

other immunological and biological reagents.

Some MAGPIX verification failures are the result of ®

debris on the chamber or clogs in the sample probe and in the sample lines. As the composition of the de-

bris is generally unknown, running the Enhanced Startup routine twice - once with 0.1N NaOH for the Sanitize and Clean commands, and again with 20% bleach – will

TIP #4

generally clear up minor debris and clogs. Still not fixed?

Does your quantitative protein assay lack sensitivity?

Try increasing the primary incubation. Primary incubation times to test are 1 hour, 2 hours, and

The degree of clog can be determined by reviewing the

overnight. One and 2 hour incubations can be performed

Calibration/Verification Report and Support Utility.

at room temp while shaking. Overnight incubations should be performed while refrigerated and shaking.

TIP #2

Getting an error code?

The most common instrument code is 800C9580.

This is a general system code that accompanies both MAGPIX and LXR (Luminex 200 and FLEXMAP 3D ) ®

®

error codes and messages. MAGPIX error codes resemble

TIP #5

Want stellar protein coupling?

Before coupling proteins (including antibodies) to beads, verify the protein concentration via NanoDrop™, bicinchoninic acid (BCA) assay, or Bradford

2091[XXXX] and 2XXX; LXR codes will begin with “0x-“.

assay. While we don’t suggest a specific concentration

If you receive this code check the System Log or contact

it’s always best to verify the protein concentration that’s

Luminex Technical Support with the support utility file to

on the label or in the certification of analysis because

determine the actual error.

they aren’t always correct.

NanoDrop is a trademark of Thermo Fisher Scientific.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

14

Community

xMAP Insights | 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

15

Community

Accelerate Your Success Become a Luminex Partner

EFFICIENCY

1 to 500

PROVEN TECHNOLOGY

FLEXIBILITY

Unmatched in level of end-user adoption and validation

Allows for both

nucleic acid and protein applications

analytes

>28,000

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

>4,000 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

>12,500

FDA

CLEARED

xMAP instruments

1st IVD assay using The

xMAP Technology was FDA cleared in 2001—

SOLD

by a partner

by Luminex and our partners 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.

© 2016 Luminex Corporation. All rights reserved. Luminex and xMAP are trademarks of Luminex Corporation, registered in the U.S. and other countries. GX1644.0716

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

www.luminexcorp.com

Not for use in diagnostic procedures.

16

Curiosity

Partner Product Offerings Factors Involved in the Cancer Immunity Cycle Step 3: T cell priming & activation

The Immuno-Oncology Checkpoint Marker Panel is the first commercially available, multiplex immunoassay system that allows the simultaneous detection of the soluble forms of the following 14 checkpoint modulators: BTLA, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2,

Step 3

Lymph node

Tumor mic roenvi ronment

de no Lym ph

Step 2

Step 2: Step 1 Cancer antigen presentation TNF alpha IL-1 IFN alpha IL-10 IL-4 IL-13 Step 1: Release of cancer cell antigens Antigens

are also available as Simplex assays and can be added to custom Mix&Match assays to create a custom assay that allows for the sample (25 μl for serum or plasma samples; 50 μl for cell culture

Step 5: Infiltration of T cells into tumors ICAM-1 VEGF Selectins

Step 5

Dendritic cell

TIM-3, CD28, CD80, 4-1BB, CD27, and CTLA-4. These assays

simultaneous quantification of up to 80 analytes from a single small

Step 4 Blood vessel

Active T cell

el ess

ProcartaPlex® Immuno-Oncology Checkpoint Molecules Panel (14 plex)

Step 4: Trafficking T cells to tumors Fractalkine IP-10 Rantes

v od Blo

Affymetrix: Checkmate on Cancer— get the full view soluble immune checkpoint molecules

CD28 PD-L2 CD152/CTLA-4 CD27 CD80 CD137/4-1BB IL-2 IL-12 HVEM GlTR

Active T cell

Step 6

Step 7

Tumor cell

Step 6: Recognition of cancer cells by T cells

Step 7: Killing of cancer cells PD-L1 LAG-3 PD-1 TIM-3 B7.1 MIC IDO TGF beta BTLA

Reproduced with permission, courtesy of Affymetrix®.

supernatant).

QuantiGene® Plex Assay

The Immuno-Oncology Checkpoint Marker Panel is an ideal

plexed gene expression and DNA copy number variation analysis,

RUO solution:

allowing researchers to measure 3 to 80 genes in every well of a

QuantiGene Plex assay is the highest throughput solution for multi-

• to identify responder profiles prior to treatment stratification via biomarker • to provide complementary information to the data obtained by pathologists from tumor tissues; • to monitor disease progression in longitudinal studies • to associate biomarker levels with checkpoint blockade therapy response To learn more download the Immuno-Oncology Panel Tech Note, product flyer15 or visit the product webpage.16

96-well plate.19 Over 15,000 genes can be mixed to create custom pathway- and disease-themed panels or purchased in an off the shelf panel. Eighty-plex off the shelf RUO kits are available for the following pathways: • Angiogenesis

• Inflammation (human)

(human & mouse)

• Immunology (human & mouse)

• Apoptosis (human & mouse)

• Jak-Stat (human & mouse)

• Breast cancer-ER (human)

• Kinase (human & mouse)

• Cancer (human)

• p53 (human)

• Cell cycle (human & mouse)

• Stem cell (human & mouse)

Not finding what you’re looking for in a readymade panel?

• Cytokine (human & mouse)

• Toxicity (human)

Add a ProcartaPlex Simplex Bead Set to include additional individual

• Drug Metabolism

• Wnt signaling (human)

®

analytes to panels or build your own panel by combining multiple ProcartaPlex Simplex bead sets ProcartaPlex Basic Kit.17 Don’t want to build your own custom assay? Affymetrix can build a custom immunoassay for you in as little as 2 weeks.

(human & mouse) Learn more about the benefits and capabilities of QuantiGene Plex Assay in the resource literature.

18

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

17

Curiosity

EMD Millipore: MILLIPLEX® MAP Human Neuroscience Magnetic Bead Panel 1

R&D Systems, a Bio-Techne Brand: Want a custom biomarker assay?

Don’t want to build it yourself?

Researching Parkinson’s Disease, Alzheimer’s Disease, or Traumatic Brain Injury?

The Luminex® Assay, by R&D Systems, offers 347 biomarkers to select from enabling you to configure the exact assay you need. This

The MILLIPLEX MAP Human Neuroscience Magnetic Bead Panel 1

near custom assays solution is optimized to deliver the most flexible

(product number HNS1MAG-95K) is a new 6-plex kit that requires

Luminex Assay available. Use the online ordering tool to see all avail-

only 25 μL of neat human cerebrospinal fluid (CSF) per assay. The

able analytes and configurations. 21

biomarkers contained in the kit are:

Don’t see an analyte of interest on the list? Simply inquire about

®

20

• GFAP (Glial Fibrillary Acidic Protein) • UCHL1 (Ubiquitin carboxyl-terminal hydrolase isozyme L1) • DJ1 (Parkinson’s Disease Protein 7) • NSE (Neuron Specific Enolase) • alpha-synuclein • TGM-2 (Transglutaminase 2) Analytes sound familiar? While four of the analytes were contained in a previous neuroscience panel, this product has higher sensitivity due to modifications of the assay and kit components. In particular, the detectability of alpha-synuclein in CSF is much improved.

custom assay services.22 Features of the R&D Systems Luminex Assays include: • Available in polystyrene or magnetic bead types • Select up to 50 biomarkers with magnetic beads or 100 in the polystyrene format • Biomarkers available for human, mouse, and rat species • Assays require <50 μl of sample for duplicate readings • Microspheres and biotin detection antibodies are supplied as premixed cocktails for ease of use

HNS1MAG-95K is the only assay currently on the market that enables simultaneous detection of these 6 key neuroscience biomarkers in a single sample, allowing investigators to expand their research while conserving valuable CSF samples.

Reproduced with permission, courtesy of Millipore®

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

18

Curiosity

Luminex: xMAP® MultiFLEX™ BioAssays Looking for a ready-made assay to support your emerging infectious disease research?

The xMAP® MultiFLEX™ BioAssays are a menu of customizable RUO panels for infectious disease and biothreat research:23 • Febrile Agent Panel 1 (Full panel is 18-plex) • Febrile Agent Panel 2 (Full panel is 9-plex) • Febrile Agent Panel 3 (Full panel is 9-plex) • Vector-borne Panel 1 (Full panel is 10-plex) • Vector-borne Panel 2 (Full panel is 12-plex) • Mosquito-borne Panel (Full panel is 17-plex) Contains Zika virus target with multiple primers and probes for expanded coverage You pick the panel and the targets – we develop a custom kit for you. All kits ship with three internal controls for reliable results. An optional external control to verify the entire process from extraction to results is also available. Assays are compatible on both the MAGPIX® and Luminex® 200™ instruments.

Contact Luminex Technical Support for additional details and product availability.

MultiFLEX is a trademark of GenArraytion Inc. Manufactured by GenArraytion Inc., distributed exclusively by Luminex Corporation.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

19

Curiosity

Can’t get out of the lab? No travel budget left this year?

Not a problem!

Check out these digital resources: xMAP® partner webinars, virtual conferences, and how-to videos

VIRTUAL CONFERENCES Cancer Research & Oncology Virtual Conference October 5-6, 2016

Clinical Diagnostics & Research November 2-3, 2016

VIDEO RESOURCES ThermoFisher has a digital library featuring how-to and educational videos for the MAGPIX® System.

Active Motif’s archived webinar discusses multiplex profiling of epigenetic modifications, specifically measuring assay specific and off-target effects in the same sample.

JoVE (Journal of Visualized Experiments) has a number of xMAP®based method demonstrations.

LUMINEX PARTNER SPONSORED WEBINARS How to Non-Invasively Monitor Stem Cell Differentiation Using Luminex® Technology Richard Fuerstenberg, Manager of Multiplex Development, R&D Systems Joy Aho, Manager of Stem Cell Research and Development, R&D Systems

• Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method • Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting • Measurement of Low Concentration Human Serum Cytokines using a Millipore High-Sensitivity Milliplex Assay • Multiplex Detection of Bacteria in Complex Clinical and

Cross-validation of Antibodies Using DigiWest

Environmental Samples using Oligonucleotide-coupled

Anthony Couvillon, PhD, Scientific Marketing Project Manager,

Fluorescent Microspheres

Cell Signaling Technology Markus Templin, PhD, Head Assay Development, NMI Natural and Medical Sciences Institute at the University of Tuebingen

Luminex’s YouTube channel features curated playlists focused on xMAP technology, instruments, assays, troubleshooting, and tips. www.youtube.com/luminexcorporation

In Person Events World Vaccine Congress October 10-12, Barcelona, Spain IDWeek Oct 26-30, New Orleans, Louisiana

Association for Molecular Pathology (AMP) Nov 10-12, Charlotte, North Carolina xMAP Connect November 16-17, Amsterdam, Netherlands 6th Munich Biomarker Conference November 29-30, Munich, Germany

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

20

REFERENCES: 1. Cancer Moonshot. Medium (Internet) Cited 2016

13. Burnette WN. “Western blotting”: electrophoretic transfer

September 30. Available from: https://medium.com/cancer-

of proteins from sodium dodecyl sulfate-polyacrylamide

moonshot?source=logo-lo_dnt_7fb69dd3d45e-1f18ef516c94.

gels to unmodified nitrocellulose and radiographic detection

2. Blue Ribbon Panel. National Cancer Institute (Internet) Cited 2016 September 30. Available from: http://www.cancer.gov/research/ key-initiatives/moonshot-cancer-initiative/blue-ribbon-panel.

3. McNamara DT, Kasehagen LJ, Grimberg BT, et. al. Diagnosing infection levels of four human malaria parasite species

with antibody and radioiodinated protein A. Anal Biochem.

1981;112(2):195-203.

14. Treindl F, Ruprecht B, Beiter Y, et. al. A bead-based western for high-throughput cellular signal transduction analyses. Nat Commun. 2016;10:1038.

by a polymerase chain reaction/ligase detection reaction

15. Detection of soluble isoforms of immuno-oncology checkpoint

fluorescent microsphere-based assay. Am J Trop Med Hyg

markers. Affymetrix (Internet) Cited 2016 September 30.

2006;74(3):413-21.

Available from: http://www.ebioscience.com/media/pdf/

4. Ménard D, Barnadas C, Bouchier C, et. al. Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people. Pro Natl Acad Sci USA 2010;107(13):5967-71.

5. Smith SE, Neier SC, Reed BK, et. al. Multiplex matrix network analysis of protein complexes in the human TCR signalosome. Sci Signal 2016;9(439):rs7.

6. Hebert PD, Cywinska A, Ball SL. Biological identifications through DNA barcodes. Pro R Soc B 2003;270(1512):313-21.

7. Widespread Seafood Fraud Found in New York City. Oceana (Internet) Cited 2016 September 30. Available from: http://oceana.org/reports/ widespread-seafood-fraud-found-new-york-city.

8. Lu R, Neff NF, Quake SR, Weissman IL. Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat Biotechnol 2011;29(10):928-33.

9. Yu C, Mannan AM, Yvone GM, et. al. High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nature Biotechnol.

2016;34,419-23.

10. The men behind Western Blotting. ASBMB (Internet) Cited 2016 September 30. Available from: http://www.asbmb.org/ asbmbtoday/asbmbtoday_article.aspx?id=16084.

11. Renart J, Reiser J, Stark GR. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.

Proc Natl Acad Sci USA 1979;76(7):3116-20.

12. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA.

LMNX06918-FLYER-PPX_Coagulation_0716b_WEB.PDF. 16. ProcartaPlex Human Immuno-Oncology Checkpoint Panel (14 plex). Affymetrix (Internet) Cited 2016 September 30.

Available from: http://www.ebioscience.com/human-immunooncology-checkpoint-markers-panel-14-plex-procartaplexmultiplex-kit.htm.

17. ProcartaPlex Simplex Bead Set Reagent Kits. Affymetrix (Internet) Cited 2016 September 30. Available from: http://www. ebioscience.com/product-line/procartaplex/simplex.htm.

18. Custom Services: Immunoassay Development. Affymetrix (Internet) Cited 2016 September 30. Available from: http:// www.ebioscience.com/custom-services/custom-immunoassaydevelopment.htm.

19. QuantiGene Plex Assay. Affymetrix. (Internet) Cited 2016 September 30. Available from: http://www.ebioscience.com/ application/gene-expression/quantigene-plex-assay.htm.

20. Luminex Assay Customization Tool. R&D Systems (Internet) Cited 2016 September 30. Available from: https://www. rndsystems.com/luminex/analytes?wwwcatno=LXSAHM. 21. Luminex Custom Services. R&D Systems (Internet) Cited 2016 September 30. Available from: https://www.rndsystems.com/ services/luminex-assays-custom-development-services.

22. HNS1MAG-95K | MILLIPLEX MAP Human Neuroscience Magnetic Bead Panel 1 - Neuroscience Multiplex Assay.

EMD Millipore (Internet) Cited 2016 September 30.

Available from: http://www.emdmillipore.com/US/en/ product/,MM_NF-HNS1MAG-95K. 23. xMAP MultiFLEX BioAssays. Luminex (Internet) Cited 2016 September 30. Available from: https://www.luminexcorp.com/ research/applied-markets/xmap-multiflex-bioassays/.

1979;76(9):4350-4.

xMAP Insights | Q3 2016 | Luminex supports life science research with its Research Use Only (RUO) product portfolio.

Not for use in diagnostic procedures.

21

Š2016 Luminex Corporation. All rights reserved. Luminex, xMAP, MAGPIX, and FLEXMAP 3D are trademarks of Luminex Corporation, registered in the U.S. and other Countries.

Luminex 200 is a trademark of Luminex Corporation. MultiFLEX is a trademark of GenArraytion Inc. DigiWest is a Trademark of NMI Registered in Germany.

www.luminexcorp.com