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.

Resources

Read The Full Article Here.

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.

Resources

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

YouTube player

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.

Resources

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.”

Resources

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.

Resources

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.

Related Content

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

METHODS Special Issue: A Universal Protocol for Attaching Any Peptide to Dyed Magnetic Beads

A new technique eliminates the use of monoclonal antibodies and is robust enough for field use

Monoclonal antibodies may soon become a thing of the past. While their high selectivity and affinity for their targets once made them the “gold standard” for detection applications, the time it takes to generate one and the variability in sensitivity has made the need for an alternative more prominent in recent years.

This is the challenge scientists at the US Army Research Laboratory tackled and reported in a recent Methods publication.

In the publication, authors Matthew Coppock and Dimitra Stratis-Cullum describe efforts to attach peptides to magnetic beads for potential use in the field. Their studies were performed using the Luminex® 200™ System since “Luminex instruments are of high interest because they can be found in some mobile lab settings and are more sensitive compared to other similar devices.”

As the scientists note, peptide-based reagents have become an appealing alternative to monoclonal antibodies for biological detection. “Peptides are advantageous due to their high thermal stabilities, which could help eliminate cold chain transport, improve long-term storage, and allow at-point detection in austere environments. Peptides also allow on-demand scalability,” they write. Using peptides in a multiplex system could permit high-throughput interrogation of samples, even in remote locations or under suboptimal lab conditions.

In the publication, Coppock and Stratis-Cullum describe a method for “the attachment of any peptide to dyed magnetic microspheres, regardless of peptide length, size, or sequence.” The technique makes use of single-arm or multi-arm PEG linkers. The authors add that “the method exploits ‘click’ chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching.” They demonstrated the method using a Streptavidin binding peptide and biotin, among other examples.

Further development of this method could expand its use to other types of reagents and microspheres, the authors note.

Resources

METHODS Special Issue: A Universal Protocol for Attaching Any Peptide to Dyed Magnetic Beads

Now Available: Custom Assay Services from Luminex

From full development to providing guidance, we can help you get to a functional assay—faster.

Now Available: Custom Assay Services from Luminex

The Luminex team is pleased to announce the launch of our custom development service, LuminexPLORE Lab, for assays based on xMAP® Technology. For more than 20 years, we have collaborated with customers and partners around the world in designing and developing high-quality, high-performing assays. Now, we are able to offer even more support and guidance for your research needs. This service will be run by Jackie Surls, our Development and Applications Scientist.

Many xMAP Technology users rely on published literature to design and develop new assays, and with good reason—there are vast resources available from peer-reviewed journals demonstrating great advances with xMAP-based assays. But not everyone has the time or bandwidth to wade through dozens of publications to find just the right method for their specific project.

We know that our customers are more than qualified to build their own assays. We have the most capable user base out there! But for those who simply don’t have time to design a new assay, or to fine-tune one that is already in development, our custom service group has you covered.

Our team has more than two decades of experience designing and developing xMAP-based assays, as well as advanced immunoassays and genomic assays. We have expertise in infectious disease, immunology, cell signaling, toxicity, allergy testing, metabolism, serology, and much more. Already, we’ve worked on several service projects involving full assay development, as well as custom coupling. We have expertise in a broad range of services:

  • Assay development
  • Assay optimization of existing homebrew xMAP assay
  • Add-on assay validation options
  • Assay conversion
  • Antibody pair screening
  • Custom coupling
  • Custom conjugation
  • Sample testing

Whether you are a new assay developer or trying to accelerate your project, our custom services program can help your team reach its goals—faster.

Please note: This service is for Research Use Only.

Related Content:

Now Available: Custom Assay Services from Luminex

METHODS Special Issue: Modifying the Luminex Workflow for Hazardous Pathogen Research

New protocol allows scientists to create assays in containment labs and perform analysis elsewhere

Scientists in the UK have developed a new protocol that utilizes Luminex multiplex assays even in specialized laboratories where space for instruments is limited. A recent hazardous pathogen study has broad implications for any research team, especially those facing space limitations.

In a paper recently published in METHODS, Stuart Dowall, Victoria Graham, and Roger Hewson from Public Health England (PHE), along with Tom Fletcher from the Liverpool School of Tropical Medicine, describe the protocol they established to perform multiplex assays in specialized Containment Level 4 (CL4) facilities, which are designed for the most dangerous pathogens.

These laboratories differ from Biosafety Level 4 (BSL-4) facilities because “work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits,” which rely on HEPA-filtered air in a positively pressurized microenvironment to protect researchers from the pathogens in their laboratory space, the authors report. “This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialized equipment.” As a result, research labs require innovative solutions to perform multiplex assays.

Multiplex assays are paving the way for improved pathogen research

Because multiplex immunoassays make a real difference in scientists’ ability to investigate the mechanisms of disease induction, the UK team came up with a different approach to perform these critical assays. Their idea was simple: what if they could prepare the Luminex assay within the containment lab’s safety cabinets, but then actually run it somewhere else?

The paper reports the validation of this method, which succeeded thanks to a fixation step. “We have developed an approach in which the Luminex assay is conducted within the CL4 laboratory, and a formalin-fixation stage is introduced to allow for analysis to be undertaken outside of containment,” the scientists write. After fixation, assay plates are loaded into a bag, sealed, and transported to another laboratory for analysis. In this study, an analysis was performed within 24 hours of fixation on a Luminex MAGPIX® System.

“Our data demonstrate that Luminex is an applicable tool for use at CL4 and that assays can be run reliably to generate reproducible, standardized data across different plates and individual experiments,” the scientists conclude. “This approach will be utilized at PHE … to determine the contribution of immunological markers in disease pathogenicity of viral hemorrhagic fever.” The research team highlighted that following this protocol, Luminex multiplex assays can be performed safely and effectively on CL4 pathogens.

Related Content

METHODS Special Issue: Modifying the Luminex Workflow for Hazardous Pathogen Research