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MOLECULAR DIAGNOSTICS

Earlier disease detection and prediction are the preeminent goals for next-generation diagnostic technologies. For diagnostic systems based on detecting protein biomarkers, the likelihood of making earlier disease detection a reality depends on the following synergistic activities: discovering clinically relevant protein biomarkers that can differentiate between disease states, and developing and validating diagnostic systems with the sensitivity and precision to detect the relevant biomarkers across all possible disease states.

Effectively integrating these two components is essential for success. However, diagnostic technologies currently lag compared with the many advances in biomarker discovery. There is a large unmet need for next-generation diagnostic systems that provide the required sensitivity and precision for quantifying biomarkers being delivered by the discovery process. Diagnostic systems need to quantify samples over a large dynamic range so that candidate biomarkers can be fully validated across disease states of interest, even in healthy, normal patients. This article discusses the adaptation of a life sciences technology to address the unmet diagnostic needs and contribute to the future of the IVD market.

Higher Sensitivity, Lower Sample Volume

Figure 1. (click to enlarge) Comparison of a traditional ELISA with the Erenna immunoassay system.

Singulex Inc. (Alameda, CA) has developed the Erenna immunoassay system, which is currently available as a life sciences, research-use only platform. This technology integrates a microparticle-based sandwich immunoassay format (which increases surface capture compared with plate-based assays) with single-molecule counting instrumentation, a detection method that keeps background signal low (see Figure 1).¹ In this assay system, the immunoassay complex formed on the microparticle surface results in the release of fluorescent-labeled detection antibodies, which are passed via capillary flow through a 2-µm interrogation space where single molecules are counted by confocal microscopy.

A large dynamic range of 4+ logs is obtained by combining single-molecule counting (low range) with photon counting (mid-range) and total light measurements (high range). The discreet counting of individual antigen-antibody binding events contributes to the sensitivity and precision of the Erenna immunoassays.

Through collaborations with academia and pharmaceutical companies, Singulex has shown applications of this technology for detecting clinical biomarkers to address unmet needs in biomarker validation and utility in several disease areas.^1-7 With this technology, protein assays have been developed that are 10–1000 times more sensitive and use 20-100 times less sample than other assays.¹ The Erenna system can also achieve such levels of sensitivity using less than 5 µl of sample.^2-3 This significant reduction in sample volume allows more-efficient use of precious samples that are held in biorepositories or are difficult to procure.

Figure 2. (click to enlarge) Erenna cTnI healthy levels compared to two leading Dx cTnI assays.

The dynamic range of the Erenna system offers several advantages compared with other systems that require separate kits to measure high and low analyte ranges. With this system, one kit can measure the full dynamic range, which is important for making clinically relevant comparisons between healthy and diseased states. For example, studies involving cardiac troponin I (cTnI) have demonstrated that the normal healthy range of 1-12 pg/ml is far below the cTnI levels in patients experiencing acute myocardial infarction (see Figure 2).⁴ Such low measurements are highly specific, and not the result of background or nonspecific interactions with such assay constituents as serum or antibodies.⁵ The cTnI assay can also be used to stratify with the risk of transient myocardial ischemia in patients under stress testing.^6,7

Table I. (click to enlarge) Adapting the Erenna immunoassay system to meet diagnostic needs.

Currently, the Singulex systems run more than 1 million tests per year, and the company is in the process of establishing a CLIA-certified reference laboratory to expand such capabilities. Singulex expects to continue adapting its life science assays to advanced IVDs by building on its technologies to meet the unique needs of each diagnostic setting (see Table I).

The cTnI assay will be the first assay to be transferred from a life sciences platform to a diagnostics platform, and will build on the strengths of the Erenna system. The diagnostics platform will feature improved automation and reduced turnaround times to less than 30 minutes for a high-sensitive assay. Other features will include standardization, full laboratory integration, and a laboratory information system (LIS) interface. The system will be designed to function in a central laboratory, and will potentially include stat and point-of-care capabilities in the future. Most importantly, during this transition, the sensitivity of each assay will be maintained, enabling advanced detection across a full spectrum of disease states including normal individuals.

Reexamining Threshold and Cutoff Values

For many protein biomarkers of diseases, current diagnostic guidelines based on the quantification of molecular biomarkers are lacking due to three distinct factors. First, assay sensitivity is often what determines the lower limit of the threshold and the diagnostic cutoff value.^1,4,8 Second, the threshold system only reveals the tip of the iceberg regarding disease states. As in cTnI, a potential gold mine of information resides beneath the 99th percentile threshold value for a clinically relevant biomarker.^6,7 This valuable information, which can be quantified by single-molecule counting, could be used to diagnose, stratify, determine risk, or aid in disease prevention.

Third, development of a disease state is not an on-off proposition. Rather, a continuum of disease development makes disease onset and diagnosis difficult to determine. In other words, when does a patient cross the line to a full-fledged disease state? The fuzzy line marking disease onset is a common cause of diagnostic failures. Even when diagnostic cutoff values are rigorously defined, as in the case of cTnI, potentially valuable diagnostic information resides below the threshold, which could affect diagnosis. Development criteria for diagnostic sensitivity of immunoassay systems should be redefined in terms of quantifying biomarkers in easily collected and routine samples, such as serum from healthy individuals.

Measuring Healthy Cytokine Levels

The life sciences effort toward developing diagnostics feeds the early-stage assay development pipeline, which can later be transitioned into the clinical setting. Singulex is currently focused on developing assays for cytokines, a complex family of secreted signaling molecules that trigger various responses from the immune system in a combinatorial manner. The level of secretion of specific cytokines can have implications for a variety of disease pathologies, including inflammation, cancer, diabetes, and autoimmune disorders.

However, baseline concentrations from the plasma of healthy human subjects for many cytokines have yet to be defined by prior assay technologies. Establishing such baseline levels of cytokines will provide an important comparison before validating levels across disease states associated with inflammation. This new information will benefit future development of diagnostics that use inflammation as an indicator for such diseases as rheumatoid arthritis, autoinflammatory disease, and asthma.

Some studies have also shown a role for cytokines in staging and/or diagnosing cancer.⁹ For example, based on the Luminex platform, the Bio-Plex assay by Bio-Rad Laboratories Inc. (Hercules, CA) has been used to implicate elevated IL-6, IL-17, and G-CSF as indicative of breast cancer–positive patients.¹⁰ The benefit of this technology is the ability to multiplex, so a large number of cytokines were surveyed. However, this outcome came at the expense of sensitivity, and as a result, this study did not include levels from healthy subjects free of any symptoms. Better, more-sensitive immunoassay technologies could improve the clinical relevance and diagnostic value of these biomarkers in the future.

The most sensitive traditional enzyme-linked immunosorbent assays (ELISA) for cytokines are the Quantikine Immunoassay Kits by R&D Systems (Minneapolis), which offer high sensitivity with lower limits of detection (LoD) capable of detecting some cytokines. However, these assays have a limited dynamic range and require more than one assay kit to quantify a cytokine across a broad dynamic range that encompasses both healthy and disease states.

Other immunoassay technologies are similarly limited by the sensitivity of the detection system being used. Without the ability to detect normal cytokine ranges, only deviations above the LoD of the assay can be detected. As a consequence, small but clinically significant increases above normal levels go undetected. This measurement gap is a crucial factor limiting the clinical utility of cyto­kines and the future of cytokine-based diagnostic assays.

Although elevated levels of cyto­kines have been reported in many disease states including cancer, few studies have looked into normal serum levels of cytokine expression in healthy subjects.^9,10 One study used the Erenna system to examine such normal levels of cytokines, and it demonstrated the feasibility of quantifying the plasma concentration of cytokines in healthy patients.⁸ The Erenna microparticle-based immunoassay quantified a specific panel of cytokine concentrations in normal human plasma samples. The human plasma was obtained from normal blood bank donors with no apparent health issues. The samples (100 µl) were assayed for a panel of cytokines with the Erenna system, which utilized paramagnetic microparticles as the solid-phase format in combination with single-molecule counting. The assay panel consisted of IL-1-beta, IL-6, IL-8, and IL-17 capture and detection antibodies in a sandwich assay format.

Table II. (click to enlarge) Erenna cTnI healthy levels compared to two leading Dx cTnI assays.

In all four assays, the concentrations of the cytokines were quantifiable in all of the plasma specimens. The sensitivity (LoD in pg/ml), dynamic range (pg/ml), linearity, precision (%CV), and recovery of the cytokines were determined (see Table II). This study also found the healthy ranges (pg/ml) for the cytokines in normal plasma (see Table III). The following normal distributions were observed for the cytokines assayed in the panel: IL-1-beta (n=16), IL-6 (n=32), IL-8 (n=32), and IL-17 (n=9).

Table III. (click to enlarge) Cytokine distribution in healthy human subjects.

The healthy ranges for the cyto­kines as determined by the Erenna system can be compared with the lowest LoD of the system itself and three other systems. The Erenna system was consistently below the lowest value of the healthy range, while the other assays could detect from within the healthy range of all individuals studied. In these cytokines studied to date, the Erenna system has the sensitivity capable of quantifying cytokines in serum samples to levels below those quantified in healthy subjects. These studies are preliminary, and more studies are needed to validate fully these assays and broaden the number of cytokines included in the panel.

Diagnostic Applications

Table IV. (click to enlarge) Sensitivity versus healthy range of select EIAS assays.

The Erenna system has provided the sensitivity and precision to quantify accurately baseline levels of biomarkers in serum from healthy subjects, which were previously unquantifiable with other immunoassay systems.^1-8 The Erenna system’s LoD for a sample set of assays with respect to the healthy range (pg/ml) is shown in Table IV. The LoD of each assay falls below the minimum quantified value of the healthy biological range for each assay. While many of Singulex’s current assays require 20-100-µl samples, this sensitivity allows the development of small-volume assays that retain the ability to detect biomarker concentrations within the normal range. Such capabilities are useful in studies in which only small amounts of sample are available from disease repositories, small-animal studies, and investigations of responses to environmental contaminants.

This article has demonstrated that a useful strategy is to quantify early in development a preliminary healthy reference range for each analyte, and to use this healthy range to gauge success. The study above used this strategy to quantify a preliminary healthy reference range of the cytokines IL-1-beta, IL-6, IL-8, and IL-17, which were previously not quantifiable with other assay systems.⁸ Since this study was preliminary, the results need to be validated in a larger, matched serum panel.

In this manner, Singulex is looking into further expanding the clinical diagnostic utility of the Erenna system by validating more cytokine immunoassays as relevant inflammatory, autoimmune, and oncology biomarkers. In particular, an interesting future application of this technology is to develop a validated multipanel cytokine assay that can quantify a full panel of cytokines for the first time in normal healthy subjects, which has been unattainable with traditional ELISA or multiplex bead immunoassays.¹⁰ The resulting panel of cytokine immunoassays could quantify a wide range of inflammatory biomarker data, spanning the full spectrum of healthy to diseased states.

Conclusion

The next step is to validate further and improve the Erenna technology, in addition to obtaining CLIA certification and FDA approval. By further advancing clinical and industry access to this detection technology, the Erenna system will have a large impact on clinical diagnostics and pharmaceutical development.

Looking forward, other discoveries in the life sciences could be used to translate efficiently assays into robust diagnostics. The goal of protein diagnostics should be to develop immunoassays that can span the full dynamic range of clinically relevant biomarkers from healthy to diseased states. This goal requires the development and validation of immunoassay systems such as the Erenna system with the sensitivity to detect the lowest concentrations of these proteins in healthy individuals and with the dynamic range to quantify elevated levels in diseased states. Since assay sensitivity is often the limiting factor, developers of diagnostic immunoassay systems should set a new diagnostic standard for detection that targets an LoD that is below the lower limit of the range found in healthy subjects. By taking this development strategy, the Erenna immunoassay system has been positioned to usher in the next generation of diagnostic systems.

Sara J. Agee, PhD, is a research scientist at Singulex Inc. (Alameda, CA). She can be reached at s.agee@singulexcom.	Quynh Anh Lu, PhD, is the assay development and service laboratory manager at Singulex Inc. (Alameda, CA). She can be reached at a.lu@singulex.com