December 1, 2014 (Vol. 34, No. 21)
Advanced Platform Designed to Provide 1,000-Fold Improved Quantification
Biomarker concentrations are a key determinant measurement in many aspects of the drug discovery and development process. Specific biomarker measurements are key components for discovery, translational research, and safety assessment to name a few. For preclinical and clinical samples, the immunoassay remains the method of choice for these measurements due to the ease of setup, sample handling, and assay transfer. However, limited sensitivity of an immunoassay format can hamper the ultimate utility of a biomarker. This can have serious implications for programs where an expected biomarker change cannot be observed, simply because the assay to measure it is not sensitive enough.
Singulex’s Research Use Only (RUO) Erenna® Immunoassay System overcomes these sensitivity limitations and allows researchers to take a second look at biomarkers that may not have been easy to measure by traditional immunoassay formats. The Erenna system uses a proprietary Single Molecule Counting (SMC) technology to quantify low-abundance biomarkers; in many cases providing quantification improvements several orders of magnitude over traditional technologies.
These quantification improvements can be achieved without the need to screen new antibodies. Improving the quantification limits of a biomarker assay by 100- to 1,000- fold or more can have profound implications to the way the biomarker is used. The quantification improvement can allow for measurement of endogenous or baseline levels of the analyte and more precisely track small changes in the analyte concentration. This could lead to a better understanding of a disease progression, and even allow researchers to identify relationships between disease and biomarkers that would have been impossible with less sensitive formats.
Quantification improvements for immunoassays in general will also enable better biotherapeutic monitoring strategies, such as microdosing for safety and efficacy assessment, and longer-term monitoring of drug clearance.
Singulex’s SMC technology combines the straightforward elements of an ELISA with an advanced detection capability. It utilizes a basic sandwich immunoassay format with two antibodies specific to the analyte—a capture antibody coated on a plate or bead, and a detection antibody conjugated to a fluorescent tag. A stepwise immunoassay protocol is used to capture the analyte in a sandwich format. SMC technology differs from a classic immunoassay protocol after the final wash step; prior to detection, a unique elution buffer is added to the reaction, which breaks apart the complex.The eluate is transferred to a second plate, and then aspirated through a narrow capillary on the Erenna system (Figure). A confocal laser is used to specifically excite the detection antibody as it passes through a discrete region of the capillary. The narrow read area allows for the digital quantification of individual molecules passing through the beam.
This protocol and detection method allows for low levels of analyte to be quantified. Analog detection is utilized when high levels of analyte are present, further extending the dynamic range of the assay. In contrast to traditional ELISAs and other advanced immunoassay platforms, the sample is concentrated into a smaller volume with SMC technology, and transferred out of the assay plate. These steps reduce the background and improve the signal-to-noise ratio as compared to other techniques.
Since the bound detection antibody is eluted from the plate in order to quantify the sample, the Erenna system provides researchers the ability to choose the assay format best suited for their specific analyte and project. Depending on quantification needs, a researcher can opt for a plate-based or bead-based capture surface. While other advanced immunoassay platforms require the operator to choose their format prior to committing to use the instrument, the flexibility of the Erenna system allows the researcher to balance costs, time, and other operational constraints against their previous format or platform, while still enabling a more quantitative assay than their previous method.
Converting ELISA to SMC Technology
As a proof of concept to demonstrate the ease of transition to the SMC technology on the Erenna system, we tested an existing ELISA that we built using commercially available antibodies against mouse IL-17A. This analyte was chosen because the endogenous levels are not detectable by traditional ELISA techniques, and sample volume is limited. Thus, a quantification improvement on the Erenna system was expected to provide data where otherwise unattainable, and allow for reduced sample volume requirements, retaining precious sample to be used for other biomarker studies.
As seen in the table, the initial ELISA provided a lower limit of quantification (LLOQ) of 62.5 pg/mL. In testing the same antibody pair in an Erenna plate-based assay format, and without further optimization, we improved the quantification limit by more than 30-fold, to 2 pg/mL (it was necessary to label the detection antibody with a fluorescent tag, specific to the Erenna system). This quantification improvement was enough to now provide a precise and accurate assay result for samples that had been previously undetectable using the ELISA.
Further improvement in sensitivity was achieved by switching the assay to an Erenna bead-based assay format. This lowered the quantification limit to 0.05 pg/mL—a full 1,250 times better than the initial ELISA. The sensitivity improvements with SMC technology formats also enable the reduction of sample volume. For mouse IL-17A, a quantifiable measurement can be obtained with as little as 1.6 µL of sample, whereas the ELISA provided no data with as much as 100 µL of sample.
In addition to the improved quantification limits, the running costs can be reduced by using SMC technology over a traditional ELISA format. The SMC technology labeling chemistry is straightforward, and directly labeling the detection antibody does not reduce its performance.
Direct labeling reduces the number of steps in the assay and the requirement for multiple reagents to affect the signal output. The reagent costs for ELISA detection are significant, and eliminating those reagents reduces the cost of running plates. The reduced sample volume requirements also have an impact on overall assay running costs, especially with preclinical samples as the amount of serum or plasma that can be extracted from a mouse is limiting.
Microparticle beads are a more costly reagent than a simple microplate, however in this particular example the assay does not require that format as the plate-based assay was able to achieve a high degree of improvement over the ELISA, providing quantifiable data for all samples measured.
In summary we have demonstrated that a more sensitive and quantitative immuno-
assay is achievable by making minimal changes to an assay. To make a 30-fold improvement in the quantification of the assay, the ELISA detection chemistry was changed to a fluorescent label and read on the Erenna system. To make a 1,250-fold improvement in assay quantification, we changed the chemistry to a fluorescent label and captured on a bead instead of a plate, and read on the Erenna system. The choice between these formats will depend ultimately on the sensitivity improvement required for the specific analyte or study, balanced against cost, ease of operation, and sample volume availability.
As demonstrated here, minimal chemistry changes allowed assay transfer to an improved detection technology, providing a second look at a biomarker or sample matrix that has traditionally been considered difficult to measure. The Erenna system is therefore a potential tool to quantify the previously undetectable analyte.
For Research Use Only. Not for use in diagnostic procedures.