How to Choose an Assay Development Platform

The Luminex Team knows assay development. Check out some tips from our experts below.

If you need to design a new assay, there are several factors you’ll want to consider before selecting the platform you’ll develop it on. Based on our experience, these are the most important:

Analyte of interest: Is your assay meant to detect DNA, proteins, or something else? While xMAP® Technology is flexible and allows you to detect a range of analytes, other platforms may be specific to certain sample types. Be sure to do your research before selecting the platform you’ll use for an assay.

Number of analytes: If you need a multiplex assay to detect several analytes at once, that may affect your assay optimization workflow. When sample volume is particularly low—such as with precious samples—a multiplex platform can help reduce the amount of sample required for the assay.

Sensitivity: If your target of interest requires high sensitivity, then this will have to be factored in as you design the assay. If possible, identify the relevant concentration range of your target in the matrix you are testing. This can be done by a quick literature search. Then, evaluate available technologies to best meet your specific assay needs.

Ease of use: This is a really important consideration for selecting an assay platform. How easy is it to use the instrument and to analyze the data it generates? Some platforms offer universal software to do the analysis, which might make life easier if your lab already has other instruments that also use that software. Be sure to match the platform to the skill set available.

Flexibility: Some platforms—like those offered by Luminex—offer an open-architecture design that can be configured to perform a wide variety of protein or nucleic acid assays quickly, cost-effectively, and accurately. Having the ability to build your own assays, or have an assay developer of your choosing build an assay, is key to the flexibility of your custom projects.

Reagent availability: It would be frustrating to develop your assay only to discover that you can’t run it because reagents are unavailable. Unfortunately, this is not uncommon. For some systems, reagents seem to be permanently on backorder. Check to make sure that all components of your assay are easy to acquire before you make your platform selection.

Software: If your assay will be under FDA review, then even the software you use has to be compliant. Consider a platform that offers 21 CFR Part 11-compliant software, and an instrument that is FDA-cleared.

Vendor support: You’ll want to ensure the platform you choose comes with strong technical support for both the instrument and the chemistry used in the assay. To determine this, look through online scientific forums such as those on ResearchGate, or contact authors who have published results from the platform you’re considering and ask about their experience with the vendor.

Community support: In addition to vendor support, it’s helpful to have robust literature to guide you in assay development. When you’re considering a platform, take a moment to find out whether there are sufficient publications that show validated assays. These will make a huge difference as you develop your own assay on that platform.

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3D Organoid Cultures Offer Clues to Heterogeneous Therapy Response in Cancer Patients

Luminex-powered DigiWest® assays show significant intratumoral differences in response

A publication in PLoS Genetics sheds much-needed light on the heterogeneity of drug response within tumors. The scientists used organoid cultures with several analytical tools, including DigiWest protein expression assays, which are based on xMAP® Technology from Luminex.

“Heterogeneous pathway activation and drug response modeled in colorectal-tumor-derived 3D cultures” comes from lead author Dirk Schumacher, senior author Christian Regenbrecht, and collaborators at several institutions in Germany and the U.S. The team aimed to characterize how organoid cultures — 3D cell cultures derived from patient tumor samples — could be used to understand heterogeneity of therapeutic response within an individual patient. Because organoids represent multiple cell populations from a tumor, they were expected to offer a more accurate reflection of biological response to treatment than simpler assessments, such as those based on a single genetic variant.

“The heterogeneity of tumors remains a severe obstacle in routinely translating preclinical data to patient treatments,” the scientists note in the paper. “While the organoid cultures largely recapitulated the genomic profiles of donor tumors, the overall treatment responses and inhibitory effects on the intracellular signaling system were quite variable.” Indeed, sampling multiple regions from the same organoid revealed as much as a 30-fold difference in drug response metrics.

Study Methods

For the study, the team looked at organoids derived from colorectal cancer (CRC) samples and focused on characterizing the KRAS/MAPK-signaling pathway as well as treatment effects from compounds targeting the PIK3CA and RAS/MAPK pathways as well as receptor tyrosine kinases. “MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition,” the team reports. “In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel ‘sibling’ 3D cultures established from a single KRAS-mutant CRC.”

Scientists generated expression data for dozens of proteins using DigiWest, a multiplex approach developed at the University of Tübingen to replace the laborious western blot. The analysis was performed with the FLEXMAP 3D® instrument.

“Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients,” the team concludes.

DigiWest is a trademark of NMI registered in Germany and the EU.

Resources

3D Organoid Cultures Offer Clues to Heterogeneous Therapy Response in Cancer Patients

MAGPIX® Systems See New Life Through Equipment Donation Program

MilliporeSigma and Seeding Labs send gently used instruments to labs in need

Thanks to a nonprofit organization called Seeding Labs and our Licensed Technologies Partner MilliporeSigma, previously-owned xMAP® Technology systems are helping scientists in laboratories in developing countries around the world answer important new research questions.

Seeding Labs is a Boston-based nonprofit dedicated to empowering every scientist to transform the world. They have a number of programs, including Instrumental Access, their flagship program which makes affordable, high quality lab equipment available to carefully vetted university and research institutes in developing countries.

Mike Vaughn, western regional sales manager at MilliporeSigma, has been working closely with Seeding Labs as part of his company’s partnership with the nonprofit. “We partner with Seeding Labs to advance global science. One way we are doing that is through donating used lab equipment,” he says. Seeding Labs fills up entire shipping containers of donations with much-needed equipment and consumables that are then shipped to the Instrumental Access awardees. “People say it would take 10 years to get as much equipment as they get in a single day from this donation,” he says.

Teaming up with a biotech giant like MilliporeSigma is invaluable for Seeding Labs. Vaughn and his colleagues are able to secure high-quality used instrumentation through demo programs and trade-in offers. They then repair and repack those instruments, and donate them to labs chosen by the nonprofit. We’re honored that our MAGPIX® Systems have gotten new life through this program; Vaughn has already facilitated the donation of several of them, which found a second home at university labs in Africa, and has more in the donation pipeline. In addition to helping scientists build research infrastructure in underfunded labs, this program also helps researchers who want to upgrade their equipment without sending older systems to a landfill.

MAGPIX® Systems are a good fit for the Seeding Labs program, according to Vaughn, because they’re simple instruments that require minimal maintenance and can be easily installed by users. Because Luminex and MilliporeSigma offer so many video tutorials for working with these systems, like on Seeding Labs’ TeleScience platform, researchers anywhere can easily get them up and running, troubleshoot basic problems, and learn to build their own multiplex assays. “It helps them get great results the first time out,” Vaughn says.

Resources

Learn More About Seeding Labs Here

METHODS Journal: A New Multiplexing Protocol Performs Protein and Gene Level Measurements on a Single Luminex Platform

Researchers demonstrate a high-throughput method for measuring mRNA and proteins using the Luminex xMAP® Platform

You shouldn’t have to choose between analyzing gene expression or screening for proteins. In a Methods publication, researchers demonstrate how to measure mRNA expression and analyze proteins with a single sample set using Luminex xMAP® Technology.

The article comes from lead author Damon Cook, senior author Thao Sebata, and collaborators at Thermo Fisher Scientific. While it is well-known that multi-dimensional data sets are widely valuable, the authors note that there are obstacles generating and utilizing them using conventional technology. “Bottlenecks to traditional immunoassays and gene expression assays include large sample consumption, time-consuming experimental procedures, and complex data analysis,” the team reports. A better approach—they add—would “ideally be able to work with different types of starting material, and make maximum use of often precious, difficult to obtain, and limited clinical tissue samples.”

The scientists turned to xMAP Technology for multiplex analysis of proteins and mRNA expression. “Using xMAP technology to measure secreted protein and mRNA expression levels on a Luminex instrument … overcomes previous limitations, as these assays are sensitive, specific to a target, and quality control-tested for the end-user,” they write.

For this protocol, the team paired the QuantiGene™ Human 80-plex Panel for measuring gene expression with the ProcartaPlex™ Human Immune Monitoring 65-plex Panel for analysis of corresponding proteins. The assays require a low sample volume and enable high-throughput analysis of targets. Both assays were prepared from a single sample and then run on the Luminex xMAP multiplexing platform.

“Often, measurement of mRNA expression or protein levels alone may not tell a complete story, as mRNA expression levels may not translate to protein,” the scientists note. “Our study demonstrated [that] a single sample can provide both gene expression and protein profiles.”

Resources

Read the Full Article Here.

Webinar: Fast, Economical Malaria Testing with xMAP® Technology

Multiplex assay allows for detailed investigations of transmission and drug resistance

Multiplexing technology from Luminex has been critical for the development of assays designed to study human SNPs involved in parasite transmission as well as the spread of drug resistance markers in Plasmodium falciparum. That was a key message in this presentation from Lynn Grignard, a research fellow at the London School of Hygiene and Tropical Medicine.

Malaria, which led to 212 million cases worldwide in 2015, is caused by a parasite with a remarkably complex life cycle. Important steps include development in the mosquito and transmission to a human host when the mosquito feeds, followed by a liver cycle and blood stage within the human. While malaria is preventable and curable, Grignard said, it remains a life-threatening disease, particularly in Africa.

Developing Assays

In her presentation, Grignard focused on two areas she studies: how human SNPs are involved in transmission or protection against malaria, and how drug resistance is spread to the parasite. These investigations led to the development of two assays based on xMAP® Technology. “You can’t take a lot of blood,” she said, “so you need to multiplex if you want to test an ever-growing number of markers.”

The first assay targets human SNPs in genes such as G6PD, HbS, and HbC. Grignard designed the assay to incorporate both genomic PCR amplification and allele-specific primer extension, and she worked with Luminex to identify the best bead sets for her targets. Genotyping results for all markers are available in six or seven hours. She reported that the biggest rewards in assay quality came from optimizing the genomic PCR process and adding BSA during the bead hybridization protocol.

Furthering Research

Grignard is already moving to the next step, which is combining the genetic data with enzyme data to correlate a specific genotype with its effect on enzyme activity. Already, she has seen cases where the genotype doesn’t adequately explain a change in enzyme activity, but that just helps her improve the multiplex assay. “You can then go back to the drawing board, and Luminex allows you to do that because you can add more SNPs to your assay,” she said.

The second assay, which is still in the final stages of optimization, tests human blood and parasite DNA to evaluate markers of drug resistance. While she plans to use the assay to track parasites transmitted from humans to mosquitoes, it could also be used to monitor drug resistance in a population, she noted.

Ultimately, Grignard concluded, xMAP Technology offers a compelling solution for fast, economical multiplex assays that require little DNA input. Results can be generated in a single working day, and the technology is robust enough to be used with blood spots stored on filter paper, making it amenable to field use in Africa.

Resources

Multiplex Malaria MagPlex-TAG Assay to Detect Human Host and Parasite Genetic Markers

Webinar: Multiplex Serological Assay Developed to Identify Avian Influenza Subtypes

Rapid detection could help prevent transmission and reduce flock losses

In addition to being a public health threat, avian influenza is also costly and potentially devastating to people whose livelihoods depend on raising poultry. To enable faster detection of bird flu and identification of its subtypes, scientists at Wageningen Bioveterinary Research in The Netherlands developed a multiplex assay using xMAP® Technology.

In this 20-minute video presentation, veterinarian Evelien Germeraad reports on the development and validation of that assay. Wageningen Bioveterinary Research, part of Wageningen University & Research, is a national reference lab for infectious animal diseases, receiving samples for testing from all over the country. The lab tests cattle, poultry, goats, and other types of animals for illnesses ranging from rabies and tularemia to psittacosis and foot-and-mouth disease. Scientists there run as many as 300,000 assays per year, of which 20,000 are deployed for avian influenza, the main focus of Germeraad’s talk.

Poultry Industry in The Netherlands

In The Netherlands, the poultry industry is valued at about €1.5 billion. The country is home to 100 million chickens, and bird flu is not uncommon. Recent outbreaks occurred in 2003, 2014, and 2016. In the 2014 outbreak, which affected just a handful of farms, nearly 250,000 chickens had to be culled to contain the pathogen. This enormous scale of loss is why it’s so important to diagnose avian influenza quickly, Germeraad says.

One of the challenges of testing for avian influenza is the broad range of subtypes. There are 16 types of hemagglutinin in birds, plus nine types of neuraminidase, making for a large number of H+N protein subtypes of the virus. Some strains have low pathogenicity, while others are so severe that mortality can rise to 100% in just a few days, Germeraad says. Conventional serological assays — such as ELISA, hemagglutination inhibition testing, and agar gel immunodiffusion — are time-consuming, require high volumes of sample, and do not all produce subtype-specific results.

For a faster test that would identify all subtypes, Germeraad and her team turned to xMAP Technology and the MAGPIX® system. They designed a single assay with 54 beads to cover all subtypes from a small serum sample. Results come back showing median fluorescent intensity for each protein. In a validation study using 87 chicken samples, 92% of results were concordant with conventional assays. Since then, the assay has been used on hundreds of bird samples, including swans, ducks, and turkeys in addition to chickens. Going forward, Germeraad hopes to optimize the assay further to establish more accurate cut-offs and improve sensitivity.

Resources

Development of A Multiplex Serological Assay for Avian Influenza [VIEW NOW]

METHODS Special Issue: Multiplex Assays Enable Simultaneous Measurement of RNA and Proteins

A more comprehensive view sheds light on the biological response to muscle injury

Scientists at the University of North Texas have established a novel method for measuring a broad range of RNA and protein biomarkers. In their study, they employed bead-based multiplex detection to assess the effects of skeletal muscle injury over time, but the same concept could be useful for many clinical applications.

The team—led by Melody Gary—published their results in an April 2019 issue of Methods.

Multiplex methods can do more with less

For this project, they used a multiplex assay based on xMAP® Technology to assess the injury response. “A current weakness of research…is the failure to include a large enough pool of biomarkers from a variety of types (i.e., lncRNA, mRNA, and protein),” the scientists note. “Our laboratory proposes to address known limitations by employing multiplex assay design to reduce sample volume, increase throughput, and reduce experimental cost compared to running singleplex assays.”

In this study, subjects ran a half-marathon and blood samples were drawn 4 and 24 hours after the event. Thanks to multiplex technology, the team was able to quantify 90 different targets, including “mRNA, lncRNA, cytokines, soluble cytokine receptors, and myokines.” One of the goals of this work was to distinguish biomarkers representative of the initial response, a delayed response, and a prolonged response—that is, biomarkers seen at both the 4-hour and the 24-hour marks.

The project provided results on injury responders, including many mRNAs, lncRNAs, cytokines, and myokines that had elevated levels in the short-term or longer-term following injury, or were consistently elevated at both readings. “These findings demonstrate the interplay between RNA and protein biomarkers in response to skeletal muscle injury,” the researchers write. “The roles of lncRNA in skeletal muscle-related injury are still emerging, but based on our observations, it is reasonable to speculate that they have effects beyond the regulation of NF-kB signaling.”

“This novel, experimental application of bead-based multiplexing is applicable to a variety of clinical models that involve muscle injury and/or wasting,” the authors conclude. “The end-goal of this approach would be to test new nutritional and pharmaceutical targets for treating muscle injury and associated inflammation.”

Resources

METHODS Special Issue: Multiplex Assays Enable Simultaneous Measurement of RNA and Proteins

METHODS Special Issue: Multiplexing Protocol for Evaluating Biological Interactions with Nanoparticles

A new multiplexing protocol offers guidelines for measuring immunomodulatory effects of nanoparticles used in clinical applications

Nanoparticles have the potential to deliver more targeted therapies to the exact location in the body where they’re most needed. To do this, it is essential to understand how these foreign particles interact with a patient’s body. A recent publication in the Methods journal is a big step forward, offering a protocol for evaluating the body’s immune response to nanotechnology.

Scientists from Texas A&M University and Bio-Rad Laboratories designed parameters for evaluating the immunomodulatory effects of nano- and microparticles using multiplex biomarker analysis. To detect biomarkers of interest, the researchers utilized xMAP® Technology from Luminex.

Considerations for monitoring the immunomodulatory effects of nanoparticles

According to the paper, the authors embarked on this project because “understanding the interactions of [particulate] materials with biological systems is crucial for the design of clinically-viable biomaterials,” they write. “The type and composition of nanomaterials plays a pivotal role in modulating the immune system, which may compromise therapeutic outcomes and can be life-threatening, [particularly] when the immunomodulation is unintentional.”

Multiplex techniques that allow for the analysis of as many as 500 biomarkers from a single sample would help address the need to characterize the effects of these particles, but certain challenges remain. For instance, the researchers note that “the adsorption of biomarkers on surfaces or within internal structures of nano- or microparticles has been explored to a lesser extent, although it can lead to biased conclusions and data misinterpretation.”

An empirical way to multiplex for large-scale biomarker analysis

With this protocol, the scientists aim to help other research teams looking to use multiplex technology to measure immunomodulatory effects of microparticles or nanoparticles by standardizing the techniques for both experiment preparation and analysis. The technique described is geared towards running 24 samples in triplicate on a 96-well plate, with appropriate standards and controls. The paper also includes precautions to help avoid artifacts, and to help with accurate data interpretation.

“The experimental details and precautions described [in the paper] allow for precise evaluation of biomarker expression in response to particulate materials, pharmaceuticals, and medical devices, and should be taken into careful consideration in pre-clinical and clinical studies, and other processes that involve the use of multiplexing techniques.”

Resources:

METHODS Special Issue: Multiplexing Protocol for Evaluating Biological Interactions with Nanoparticles

METHODS Special Issue: Multiplexing for Personalized Oncology

Assays quantify RNA expression even in challenging samples

Multiplex molecular assays have several important roles in the realm of personalized cancer medicine. A team of researchers at the University of Malta recently published a paper, titled “Bead-based RNA multiplex panels for biomarker detection in oncology samples,” in the Methods journal that offers an overview of those roles, showing that multiplex assays based on xMAP® Technology can be used to quantify RNA expression, even from formalin-fixed, paraffin-embedded (FFPE) tissues.

The research team chose to focus on bead-based assays due to certain advantages over other RNA analysis methods. “Despite providing a wealth of data, which may be scaled up to the whole transcriptome for discovery purposes, microarrays and RNA-Seq may be too laborious and expensive for routine diagnostic use,” the authors write, noting that these techniques often struggle with the degraded RNA in challenging samples, such as FFPE blocks. The ideal solution would be robust, cost-effective, and high-throughput. “Multiplex microsphere-based assays fulfill these criteria and can be utilized by clinical laboratories to stratify [tumors] based on biomarker panels derived from research studies,” they add.

Better sensitivity with multiplexing

Looking specifically at the QuantiGene™ Plex Assay from Thermo Fisher which allows customization to quantify expression of as many as 80 genes, the scientists report that large validation studies have established the accuracy and precision of a multiplex approach. Compared to RT-PCR, the multiplex assay had better sensitivity, linearity, precision, and relative accuracy. It was also more sensitive for genes with low expression compared to a number of array-based methods.

The authors note that multiplex assays work well with stained and unstained tissue sections, so they can even be used after traditional histological analysis has been conducted. Further, the assays have been successful for liquid biopsy analyses, detecting circulating tumor cells that can provide valuable insight for patient monitoring. Thanks to the large number of genes that can be assayed at once, this method can help characterize the heterogeneity of a tumor as well.

“High sensitivity and specificity, together with multiplexing ability, make bead-based RNA assays highly useful for biomarker detection in oncology samples,” the scientists write. “We suggest that this method is a preferable option for multiplexing expression analysis of up to 80 genes, especially when using highly degraded specimens or low sample concentrations.”

Resources

METHODS Special Issue: Multiplexing for Personalized Oncology

Webinar: Improving Rheumatoid Arthritis Diagnostics with Multiplex Assays

How a European team avoided performing 16,000 ELISA tests

New efforts to improve the diagnosis of rheumatoid arthritis, as well as to guide treatment selection, are making strong progress thanks to xMAP® Technology from Luminex, according to a recent presentation from Erik Lönnblom of the Karolinska Institute. Multiplex immunoassays have allowed his team and collaborators to generate 1.5 million data points so far — a feat that would otherwise have required nearly 16,000 ELISA plates to achieve.

Lönnblom works in a lab focused on characterizing epitopes to type II collagen, which is a candidate autoantigen for rheumatoid arthritis. The protein, which is quite difficult to work with, is highly conserved; epitopes are virtually identical across mice, rats, humans, and monkeys. One of the most promising areas of study is a post-translational modification known as citrullination. Anti-citrulline protein antibodies are present in more than two-thirds of patients with rheumatoid arthritis, and they can be detected years before the onset of clinical symptoms of the disease.

Making Strides with Rheumatoid Arthritis

There are several diagnostic challenges relevant to rheumatoid arthritis. Lönnblom says the most pressing areas include earlier and more accurate disease diagnosis; prediction of response to treatment; and prediction of disease onset in healthy individuals. As part of a multi-institution consortium, Lönnblom and his colleagues have already made inroads.

That would not have happened so quickly without xMAP Technology for multiplex assays. Originally, Lönnblom says, the plan was to use ELISAs for everything. But as the scientists calculated the number of assays they’d need to run to study the 400 proteins or peptides relevant to rheumatoid arthritis, it became clear the approach was not feasible. Sample requirements alone were a deal-breaker: while each Luminex assay needs just 1 microliter of material, Lönnblom says, ELISA tests for the same biomarkers would consume half a milliliter or more, an amount that no clinical sample repository could part with.

So far, the team has run the multiplex assay on several thousand clinical samples. Already, important insights are emerging. One promising new biomarker appears to predict some established cases of rheumatoid arthritis more reliably than previously known biomarkers. Another seems to predict poor response to a specific therapy, essential information for the grueling trial-and-error method of therapies used for patients today.

Continuing Research

Looking ahead, Lönnblom and his team plan to perform technical validation of the most promising peptides, as well as clinical validation of their current findings. They also aim to build more sophisticated diagnostic models for improved treatment of patients with rheumatoid arthritis.

Resources

Webinar: Improving Rheumatoid Arthritis Diagnostics with Multiplex Assays