Assay Development: DIY or Use a Custom Service?

Abstract

You’ve poured countless hours into your research project and need to create an assay to move your research forward. Some people save time by farming out the assay design to experts at custom service providers. Others try the do-it-yourself (DIY) route to save some money. Although this decision is particular to each lab and project, there are some resources available to scientists to assist them through their assay-development challenges.

DIY

A wealth of tools awaits DIY-minded researchers. PerkinElmer offers them three platforms to develop their own assays: the LANCE® and  DELFIA® assay platforms, which use time-resolved fluorescence (TRF) to detect biomolecular interactions, and the AlphaLISA® assay platform for homogeneous, bead-based proximity assays. In addition, PerkinElmer’s AlphaPlex™ technology is available for multiplexing in DIY assay development; the company also offers custom services (see below).

For researchers developing their own assays, ForteBio (a division of Pall Life Sciences) offers the Octet platform for label-free analysis. Pharma companies developing antibody-based, biotherapeutic drugs use the Octet system for determining binding kinetics and affinity of therapeutic antibodies for target proteins. “Recent developments have led to treatments with lower side effects and less toxicity, so there is a big push for it, but the antibodies require a lot more characterization compared to small-molecule drugs,” says Dominic Andrada, senior product manager at ForteBio.

Interest in using bispecific antibodies therapeutically has driven researchers back to the assay-design drawing board. “Bispecifics are more complex and can target the tumor cell while activating the immune system against the cancer, resulting in better efficacy and different types of treatments,” says Andrada. But bispecifics are so different from monoclonal antibodies (mAbs) that researchers need to create different types of assays for them.

An advantage of the Octet system is speed in both characterization and quantitation. Andrada believes this can relieve bottlenecks in measuring multiple antibody characteristics. “Changes at the Fc region, the region which determines isotype and effector function, affect many antibody properties, but the time to results for measuring these changes at the Fc can be long,” says Andrada. Besides characterization or binding assays, Octet users have developed their own two-minute quantitation assays (for a 96-well plate), which save time. Other quantitation assays—such as with ELISA or HPLC—take significantly longer.

Custom services

Although the term “custom services” evokes images of dollar signs, they’re not necessarily more expensive than DIY solutions. “Our custom assay-development services can be cost-effective because, while a customer could spend six months developing an assay, our team could do it in as little as a few weeks,” says Bethanne Mullen, product manager in Immunoassay and Biomarker Custom Assay Development Services at MilliporeSigma (life sciences business of Merck KGaA).

MilliporeSigma offers immunoassay and biomarker development services such as ELISAs; multiplex panels using Luminex xMAP® technology; assays for its automated, high-throughput Gyrolab® workstation; and ultrasensitive, quantitative assays for the Singulex® platform. Recent requests include multiplex panels for biomarker screening and the adaptation of existing assays to new platforms for better performance or automation. The Cell Design Studio™ (of recently acquired Sigma-Aldrich) also offers custom-engineered cell lines for molecular and cell-based assays.

MilliporeSigma’s service does the work while letting customers make important decisions about assay development, such as performance criteria. “Increasing the size of the panel, or the plex size, may introduce cross-reactivity or some loss in sensitivity,” says Mullen. Multiplex assays can require a delicate balance between different areas of performance that only the researcher can decide.

Custom assay-development services also are offered by Maryland-based Precision for Medicine. These include immunophenotyping, immunological assays like ELISpot (enzyme-linked immunospot), ADCC (antibody-dependent cell-mediated cytotoxicity) assays, multiplex ELISAs and regulatory T cell-suppression assays. The company works with customers to develop the right technology for each assay. “Too many people want to use their favorite technology, regardless of the problem,” says Precision’s vice president of research, Deborah Phippard. Instead, Precision considers many factors, including assay goals, future clinical trial use and fresh vs. frozen samples.

For example, sometimes it’s better to use frozen rather than fresh samples. “I always advise that an assay that works on frozen cells will almost certainly give you the most reproducible data,” says Phippard, because frozen samples allow batch processing for less variability. Sometimes freezing samples results in the loss of biomarkers of interest, “but we compare fresh to frozen cells upfront, so that we really understand the performance of an assay,” she adds.

Often customers want help adapting an existing research assay for a clinical setting. “This means standard operating procedures need to be written, reagents used will have to pass quality assurance, the assay standardized and validated with full documentation, and performed by qualified technicians under [regulatory] standards,” says Phippard.

PerkinElmer offers the OnPoint custom assay-development service for a range of assays, including protein-protein interaction, enzymatic, epigenetic, pharmacokinetic, immunogenicity and biomarker assays. OnPoint is used “by researchers new to our technologies who may be trying to develop a complex assay, and by researchers experienced with our technology who are looking to maximize their own internal resources by outsourcing custom assay development,” says Lindsay Nelson, senior application scientist at PerkinElmer.

Nelson notes the importance of quality material sources for custom assay development and subsequent screening. “When developing custom assays, we test raw material from multiple sources and from multiple preparations to ensure that the quality of the developed assay is consistent across different batches,” she says.

Try the combo

Free online tools for assay development are easily accessible and becoming more intuitive for DIY-ers. As a backup, tool providers are still available to provide expert support, if needed. For example, Thermo Fisher Scientific’s Ion AmpliSeqTM Designer is a free online tool for designing next-generation sequencing primers to assay genotypes, genes or chromosome regions, RNA expression, gene fusion and copy number variation. AmpliSeq returns the recommended set of multiplex primers after analyzing the targets and conditions specified by the researcher.

Although AmpliSeq can be a DIY resource, some researchers seek help from Thermo Fisher Scientific’s custom services group. “They may be going into high-volume production and want to tighten up their specs,” says Andy Felton, vice president of product management at Thermo Fisher Scientific. “They might develop an assay with the online tool, but then hand it over to the custom services group to optimize the performance before going into production.”

AmpliSeq has features that enable researchers to make assay design trade-offs so they can obtain the best results. For instance, to get maximum sequencing coverage across target regions, other regions may go off-target—but the researcher makes the choice. “The online tool will tell you very clearly, for example, you can cover 85% of everything you want without any off-target reads, but if you want 90% coverage, you might have to deal with 10[% to] 20% of the reads going off-target,” says Felton. “We’ll do those calculations and give you a chance to make those trade-offs for yourself.”

Another online tool, Agena Bioscience’s Assay Design Suite, designs genotyping and mutation-detection assays for the company’s MassARRAY® System. Researchers enter information for single nucleotide polymorphisms (SNPs) of interest, and the tool returns recommended primers that can be ordered from oligonucleotide vendors. “You can specify the plex level and optimize in different ways to see which assay designs are best for you,” says Christina Littler, associate manager in clinical market development at Agena Bioscience.

Although the online interface is user-friendly, some researchers prefer to use Agena Bioscience’s custom assay-development services. Agena Bioscience pays attention to the requests it receives to learn more about researchers’ needs. “We saw that customers were running somatic mutation variance panels over and over again, so we made the OncoFOCUS™ Panel,” says Littler, who expects the Assay Design Suite’s flexibility to broaden the company’s custom services in the future.

Flexibility in designing assays enables researchers to incorporate new technologies. Phippard believes that future assay development will include more detailed analyses of cellular and molecular interactions, such as live-cell studies of fluorescently tagged proteins, drug discovery using induced pluripotent stem cells and the diagnostic and therapeutic uses of exomes. A look at today’s custom assay-development tools reveals many powerful options from which to choose, even now.