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.”

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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.”

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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.”

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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.

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Webinar: Improving Rheumatoid Arthritis Diagnostics with Multiplex Assays

METHODS Special Issue: Multiplexing to Quantify Cytokines and Chemokines in Mouse Brain Study

Assay requires minimal sample volume for 33-analyte interrogation

Scientists from the National Institute of Neurological Disorders and Stroke and Bio-Rad Laboratories published a helpful protocol for a method to analyze more than 30 cytokines and chemokines in mouse brain tissue. The technique can be adapted to other sample types as well.

The paper, titled “Method to quantify cytokines and chemokines in mouse brain tissue using Bio-Plex® multiplex immunoassays,” was published earlier this year in Methods. The authors cite the need to conduct in-depth investigations of cytokines and chemokines—even when sample volumes are low. To address this need, they turned to multiplex immunoassays built using Luminex xMAP® Technology.

“These assays allow researchers to analyze an entire network of cytokines/chemokines in a single sample, which conserves tissue, streamlines workflows, and accelerates research,” the scientists note. “In addition to saving time and money, multiplex immunoassays help advance vaccine development, drug discovery, basic research, and clinical trials by providing a quantitative snapshot of immune mediators expressed in samples of interest.”

For this project, the team focused on Plasmodium berghei, a parasite that causes cerebral malaria in mice. Two groups of mice—those with and without CD8+ T cells—were infected with the pathogen, and a third group was used as a control. Subsequently, they analyzed the expression of 33 chemokines and cytokines using the Bio-Plex® multiplex immunoassay to characterize the inflammatory profile, as well as the role of CD8+ T cells.

“We observed elevated chemokine and cytokine levels in the brains of infected mice during development of cerebral malaria,” the scientists report. “Twenty different analytes were upregulated, with the majority being chemokines involved in the recruitment [of] immune cells.” They also found that CD8+ T cells are important for promoting inflammation at the onset of cerebral malaria.

The paper also includes a discussion about the utility and logistics of multiplex immunoassays. The approach was successful in overcoming the sample volume obstacle: scientists needed just 50 µl of tissue sample from each mouse to evaluate all 33 chemokines and cytokines in a single assay. Authors also encourage anyone planning to try this protocol to ensure detailed planning and proper timing of all steps. “For optimal results, it is important to plan and allow sufficient time to perform instrument validation/calibration, design plate layouts, and perform mixing/dispensing steps with precision,” they write.

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METHODS Special Issue: New Multiplex Assay for Vaccine Immunogenicity Testing

Recently developed test lowers cost and reduces sample volumes needed for evaluating combination vaccines

Scientists at the Serum Institute of India have developed a new multiplex immunoassay to perform the mouse immunogenicity testing recommended in many countries for quality control in the production of combination vaccines. They chose xMAP® Technology to design the serological assay, which features higher throughput, faster analysis, and lower sample volume compared to serial ELISA testing.

How the Serological Assay Works

The team reported this work in a recent publication in Methods. The pentaplex assay they created “simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T), and diphtheria (D) toxoid in the Tdap vaccine-immunized animals,” the authors write.

While combination vaccines are important for a host of reasons, preclinical evaluation of them can be challenging. Even later in the development phase, these multivalent vaccines can be difficult to monitor. For that reason, the World Health Organization and European Pharmacopeia both encourage non-lethal immunogenicity testing in mice “for monitoring lot-to-lot consistency during manufacturing,” the scientists note. In conventional testing, “the assay involves immunization of mice with serial dilutions of the vaccine, [where the] serum IgG antibody response against each pertussis vaccine component is then measured by [ELISA].”

Multiplexing to Reduce Cost and Time

This multiplexing project was launched to eliminate the need for ELISAs, which are costly, tedious to run, and require a significant amount of serum. The team selected xMAP Technology due to its prior success in this area: “Several studies have previously described the accuracy and high-throughput advantage of this platform for evaluating vaccine-elicited binding antibodies,” the authors report.

After development, the multiplex assay was evaluated using serum samples taken from animals with varying dilutions of immunization. “The assay was able to predict the dose-dependent trend in IgG responses against all five antigens, which will be important to define the potency and quality of [each] vaccine against a reference vaccine batch of proven clinical efficacy,” the scientists note. Validation was performed according to international regulatory guidelines.

“The assay described here meets all the requirements of specificity, selectivity, reproducibility, and accuracy, and is thus a viable alternative to conventional ELISA for detection of DTaP3 antibodies in mouse serum samples,” the authors conclude.

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METHODS Special Issue: New Multiplex Assay for Vaccine Immunogenicity Testing

Custom Assays: ‘If You Dream It, We Can Multiplex It’

This video presentation details assay development services available from MilliporeSigma

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With new biomarkers being published every day, fewer and fewer scientists can get exactly what they need from an off-the-shelf immunoassay kit. That’s why MilliporeSigma offers extensive custom xMAP® Technology assay design and development services, which are showcased in this 10-minute video presentation from Mike Godeny, Head of the Milliplex Reagent Business.

Recognizing that researchers have unique needs and widely varied interests in analytes, Godeny described two approaches MilliporeSigma has for delivering custom assays. The simpler method involves reconfiguring existing analytes from as many as three panels into a single, new multiplex assay.

The more intensive service requires the development of new analytes, multiplexing and verification of performance, and careful validation. For this service, Godeny and his team consult with customers about desired markers, sample type, and species before launching into assay development and design.

Importance of Customer Consultation

In the presentation, he offered examples of multiplex assays developed for Bayer to replace gold-standard ELISA tests, walking through the various steps of the process and the extensive validation protocols followed to ensure that the new assay met a customer’s needs, generated data that was comparable to the previous tests, and performed as well in the customer’s lab as it did in the hands of MilliporeSigma scientists.

Development Team Leads to Success

Godeny also shared some interesting statistics from the custom assay development team, which has developed everything from single-plex to 48-plex xMAP assays. They’ve developed more than 50 new analytes and had a success rate better than 98 percent.

If you’re curious about custom assay services, we encourage you to take a few minutes to watch Godeny’s informative presentation.

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Custom Assay Development Case Study: Custom-Made Milliplex Map Assays Aid Unique Toxicity Tests

METHODS Special Issue: Multiplexing Enables More Robust Surveillance for Avian Influenza

New assay developed for routine poultry monitoring

In the journal Methods, scientists from Wageningen Bioveterinary Research report the development of a multiplex assay for avian influenza. The assay is built on Luminex’s xMAP® Technology, and allows for serotyping all 16 hemagglutinin (HA) and 9 neuraminidase (NA) protein subtypes simultaneously.

Testing for avian influenza (AI) is becoming increasingly important. In the Netherlands, the 2003 bird flu outbreak was so devastating to the poultry industry that the government responded by launching a surveillance program to monitor certain related viruses in these birds. “In this program, all poultry flocks are screened for the presence of antibodies against AI, at least once a year, depending on the type of poultry…” the scientists note.

Multiplexing allows for fast and reliable scale-up

Given the scope of testing required, conventional ELISA testing was unable to adequately scale-up to meet the needs of serotyping. ELISAs are notoriously labor-intensive, and have to be performed separately for every possible protein subtype. As a result, they require so much sample volume that it’s often impossible to fully test for all subtypes.

These complications led the team to explore multiplex technology as a way to create their own test. “This novel assay allows the detection of antibodies against the 16 HA and 9?NA subtypes simultaneously, in one single test,” the scientists report. “The assay is high-throughput and efficient in cost and time compared to the traditional assays for serotyping.” It also requires considerably less sample volume.

Performance evaluation of the multiplex assay compared to conventional ELISA tests revealed that the multiplex assay has higher sensitivity thanks to a lower detection limit. “We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera,” the scientists write. Comparison testing was done using sera samples from chickens, turkeys, guinea fowl, pheasants, swans, and ducks.

The authors concluded that “the assay will allow complete serotyping of different poultry species sera samples, which will improve the monitoring of [avian influenza] subtypes circulating in poultry in the Netherlands significantly.”

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METHODS Special Issue: Multiplexing Enables More Robust Surveillance for Avian Influenza

METHODS Special Issue: New Assays to Characterize Vaccination and Infection History

Single-plex targets developed will become components for future multiplex assays

Scientists in the Waterboer lab at the German Cancer Institute have developed the building blocks for a multiplex assay that will make it possible to quickly interrogate a person’s history of infection and vaccination. In a paper from Methods, the team reports developing individual assays for antibodies to several pathogens, noting that these assays could easily be combined into a multiplex assay in the future. These types of assays “are useful and versatile instruments” that can be utilized for “clinical diagnosis, personal risk evaluation, and seroepidemiological studies,” the scientists write.

While the team makes it clear that the ultimate goal is to develop multiplex serology assays that cover antibodies for many pathogens to economize time, cost, and sample volume, they first had to expand the number of available single-pathogen targets using a bead-based system. To accomplish this, they used xMAP® Technology from Luminex.

“We developed and validated bead-based pathogen-specific Monoplex Serology assays … to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus, and parvovirus B19,” they report, noting that each assay features a single antigen to improve the efficiency of future multiplexing. These assays “can be efficiently incorporated into larger multiplex serology panels,” the scientists add.

Validation was performed using commercially available reference assays. Specificity, sensitivity, and other metrics were found to be acceptable for routine clinical use. This work provides a critical foundation for future multiplex development, and will streamline the characterization of vaccination and infection history for any individual.

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METHODS Special Issue: New Assays to Characterize Vaccination and Infection History

Webinar: Protein Conformational Analysis Goes High-Throughput with xMAP Technology

Technique provides critical data for development of monoclonal antibodies and biosimilars

At a 2018 xMAP® Connect user group meeting, Xing Wang, President of Array Bridge, spoke about how his team is using xMAP Technology to streamline the development of new biologics and biosimilars for pharma/biotech scientists. The 30-minute presentation focuses on protein conformational analysis (PCA), a multiplex approach that replaces previous testing performed with ELISAs.

The need for better tools to develop biologics — Wang’s talk centered on monoclonal antibodies — is clear from the rapidly growing market, he said, noting that by 2020 it is expected that more than 70 monoclonal antibody therapies will be on the market. Pharmaceutical and biotech companies are investing heavily in the development of these therapeutics, and existing products are eventually expected to be mirrored with new biosimilars.

How PCA Helps

A major challenge in that development, however, lies in understanding the three-dimensional conformation of a protein — including how that influences its biological function and its reaction to stressors. PCA offers a straightforward way to do this by using antibodies to recognize epitopes on the surface of the mAb, Wang said. It’s also highly sensitive and high-throughput, two major advantages over many other techniques.

PCA has already been used in many biosimilar applications to the FDA and EU, including the first biosimilar mAb approved in the world with the highest standards of regulation. It has been used by the majority of biosimilar developers in the world with PCA data included in several successful approvals from US, EU, and other countries, and the technology has a number of potential applications, Wang said.

Development of PCA

The presentation also looked at how PCA was developed as Wang’s team shifted from sandwich-based ELISA tests to xMAP Technology for increased throughput. He highlighted results from real samples comparing both techniques to show that the data profiles are very similar, and also cited a publication from Bristol-Myers Squibb scientists using PCA for 48-fold higher throughput than ELISAs. The multiplex assay requires little sample and minimal hands-on time and is also very fast with high precision and broad dynamic range, Wang added.

Finally, Wang reviewed data from analyses of immunogenicity and higher order structure correlation. These studies assess drug response and spot issues associated with immunogenicity, including human whole blood assay for cytokine release and complement activation, which explain why some people respond differently to the same treatment. The antibody arrays developed by Wang’s team generated a unique signature for each monoclonal antibody, producing sensitive results and detecting changes that may be missed by other bioassays and bioanalytical technologies.

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Webinar: Protein Conformational Analysis Goes High-Throughput with xMAP Technology