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Coupled with the SMCxPRO™ or Erenna® Systems with SMC™ Technology, the Microlab® STARlet allows researchers to monitor and detect fluctuations in very low levels of established disease biomarkers. The automated workflow of the Microlab® STARlet saves time that can be spent on higher-value tasks, such as results analysis.

Verification of the Hamilton Microlab® STARlet for Use With the SMCxPRO™ and Erenna® Immunoassay Systems Powered by Single Molecule Counting (SMC™) Technology

Introduction

Cytokines are signaling molecules that mediate and regulate the immune system. Interleukins, which regulate immune and inflammatory responses, and interferons, which are responsible for adaptive immunity, are two broad cytokine categories that are particularly challenging to measure using traditional ELISAs or ligand binding assays because they are present in low levels within the human body.

An alternative to quantifying these low-level cytokines is the patented Single Molecule Counting (SMC™) Technology from MilliporeSigma. The SMC™ Technology method is similar to a traditional ELISA and offers reduced background signal and increased detection signal in comparison.

  • In process, target analytes in solution are captured onto an antibody-bound plate or microbead.
  • Fluorescently labeled detection antibodies are then added to the immune complex to translate each biomarker into a signal.
  • During a modified elution step, the bound antibodies are released from the immune complex where the fluorescent signal from single, tagged molecules are detected by either the SMCxPRO™ or Erenna® instruments. The total signal detected is calculated as a direct indication of biomarker levels, with a limit of detection down to < 1 fM*.

When manually performed, the SMC™ immunoassays are labor-intensive. This prevents researchers from focusing on high-value activities such as result interpretation, thus reducing overall laboratory efficiency. Manual methods also introduce risk of operator error and elements of variability from one user to the next and among subsequent assay runs.

Here, we demonstrate use of the Erenna® Immunoassay System with SMC™ Immunoassays integrated onto the Hamilton Microlab® STARlet liquid handling workstation in order to create a hands-free, automated, assay-ready workflow (see Workflow at end). The STARlet is equipped with up to four independent air displacement pipetting channels for high precision and reliability without reagent crossover.

Compressed O-Ring Expansion (CO-RE®) Technology creates an air-tight seal between the disposable tips and pipetting channel mandrels without using mechanical force, to maximize sample care and integrity, and also ensure accurate, reproducible liquid level dispensing. Barcode reading provides full sample tracking and eliminates the risk of sample mishandling or manual documentation errors.

Two ELx405™ HT Microplate Washers (BioTek Instruments, Winooski, VT) and four heated shakers were integrated into the deck of the STARlet for added workflow efficiency and convenience (see Deck Layout).


Finally, in order to facilitate user-friendly operation, minimize operator intervention, and reduce input errors, the STARlet software was pre-programmed with the SMC™ workflow steps to create a standardized solution.

Using plasma samples and controls, we demonstrate that the assay-ready automated workstation delivers results on par with those achieved through manual methods, while maximizing assay reproducibility, reducing active labor time, and eliminating risks of error and variability from manual intervention.

Benefits-Based Highlights

  • Robust, sensitive, and reproducible method to quantify biomarker concentration without sources of error and variability associated with manual processing.
  • Refocus manual labor on high-value activities such as result interpretation.

The STARlet automates and simplifies your workflow when coupled with the SMCxPRO™, an affordable, fast and compact benchtop instrument, or the Erenna® Systems with SMC™ Technology, both of which facilitate high sensitivity protein measurement. Additionally, both instruments feature small sample volume requirements, allowing for efficient operations.

Materials and Methods

Automated and manual workflows were compared using the SMC™ Interleukin 6 (IL-6) Immunoassay Kit (P/N 30-0572-01-TED), SMC™ Tumor Necrosis Factor (TNF-α) Immunoassay Kit (P/N 30-0571-01-TED), and SMC™ Interleukin 1-β (IL-1β) Immunoassay Kit (P/N 30-0573-01-TED) from Singulex. In each assay, the manufacturer’s protocol was followed.

Standard, Sample, and Control Preparation

Standard protein curves were created manually as follows. IL-6 standard protein was thawed and diluted to 100 pg/mL in standard diluent to make the top standard, followed by ten 2-fold serial dilutions, down to 0.1 pg/mL. TNF-α standard protein was thawed and diluted to 200 pg/mL in standard diluent to make the top standard, followed by ten 2-fold serial dilutions, down to 0.31 pg/mL.

IL-1β standard protein was diluted to 50 pg/mL in standard diluent to make the top standard, followed by ten 2-fold serial dilutions, down to 0.05 pg/mL. Each standard curve also included a zero blank. Human K2 EDTA plasma samples from five healthy individuals (BioreclamationIVT P/N HMPLEDTA2, Westbury, NY) and 3 plasma controls (MilliporeSigma, Hayward, CA) were tested as described in Table 1. Three vials of each sample and plasma control were thawed, lightly mixed, and filtered through a 96-well, 1.2 μm Durapore® membrane filter plate (Millipore, #MSBVN1210) according to each kit protocol.

SMC™ Immunoassay Workflow

For each automated assay type, a 4 Row, Pyramid Bottom 292 mL High Profile Reagent Reservoir (E&K Scientific, P/N EK-2216) was loaded onto the STARlet with assay-specific reagents. Using 300 μL conductive non-filtered CO-RE tips (P/N 235950), a total of 100 μL of microparticles per well were added to four 96-well v-bottom polypropylene microplates (E&K Scientific, P/N EK2470, Santa Clara, CA), followed by 100 μL of each respective 12-point standard protein curve in triplicate.

For each assay kit, 100 μL of sample or plasma control filtrate was added to each of the four microplates. The microplates were then incubated on the STARlet deck for two hours at 25oC with shaking to allow binding of the target biomarker. The assay plates were then transferred to the microplate washer, where the microbeads were magnetically retained, and unbound material was removed in a single wash step.

After washing, 20 μL Alexa Fluor 647-labeled detection reagent was added to the wells, using 50 μL conductive non-filtered CO-RE tips (P/N 235947), and the microplates were incubated for one hour in order to bind the microbead-captured analyte. After incubation, the assay plates were again transferred to the microplate washer, where the microbeads were magnetically retained and washed four times in order to remove any unbound detection reagent.

The microparticles were then automatically transferred from the 96-well assay microplates to new microplates to avoid eluting non-specific plate bound detection reagent. Detection reagent specifically bound to the target analyte was then eluted and transferred to a 384-well polypropylene microplate (ThermoFisher Scientific P/N 264573, Waltham, MA). The 384-well microplate was manually transferred to the Erenna® Instrument for detection. Alternatively the plate could also be read on the SMCxPRO™ Instrument.

The entire workflow was also performed using manual methods and one microplate per assay.


Three signal outputs were obtained from the Erenna® Instrument: Detected Events (DEs; low end signal), Event Photons (EPs; low end and mid-range signal), and Total Photons (TPs; high end signal). Using the SgxLink™ algorithm, unknown concentrations were interpolated from the standard curve.

1. SMC™ Human IL-6 High Sensitivity Immunoassay Kit: Immunoassay kit for the quantitative determination of Interleukin 6 (IL-6) in human EDTA plasma. Singulex: Alameda, CA. Dec 14, 2017. Kit P/N: 30-0572-01-TED.

2. SMC™ Human TNF-α Immunoassay kit for the quantitative determination of Tumor Necrosis Factor (TNFα) in human EDTA plasma. Singulex: Alameda, CA. May 15, 2017. Kit P/N: 30-0571-01-TED.

3. SMC™ IL-1β High Sensitivity Immunoassay Kit: Immunoassay kit for the quantitative determination of Interleukin 1β (IL-1β) in human EDTA plasma. Singulex: Alameda, CA. Dec 14, 2017. Kit P/N: 30-0573-01-TED.

Results and Discussion

Using data obtained from the single, manually processed microplate, and the four replicate microplates that were automatically processed on the STARlet liquid handling system, an interpolated average for each protein standard concentration was calculated and plotted as a standard curve.

Interpolated averages from each sample and control that were manually and automatically processed were then plotted against the standard curve. When comparing manual and automated results, analyte concentration, SD, and CV values produced robotically are all on par with those produced manually. All samples tested in the IL-6 and TNF-α assays resulted in interpolated values well above the sensitivity of the assays.

IL-6 and TNF-α protein concentrations are generally more abundant in human samples, and assay results are well above the lower limit of quantitation (LLOQ) of the SMC™ assays. But IL-β protein concentrations in human samples are known to be very low, resulting in IL-β assay results near the LLOQ of the assay (0.2 pg/mL) with some below. However, samples that are quantifiable did show a good correlation between the Hamilton and manual method assay methods.

IL-6 Results

The IL-6 slope approaches 1, indicating a strong correlation of interpolated IL-6 values between manual and automated runs of both plasma and control samples. Each of the four plates produced by automated means had similar interpolated IL-6 values for both plasma samples and controls. LLOQ = 0.08 pg/mL; see Table 2 and Figure 1 for data.

TNF-α Results

The TNF-α slope approaches 1, indicating a strong correlation of interpolated TNF-α values between manual and automated runs of both plasma and control samples. Each of the four plates produced by automated means had similar interpolated TNF-α values for both plasma samples and controls. LLOQ = 0.2 pg/mL, LOD 0.05 pg/mL; see Table 3 and Figure 2 for data.

IL- β Results

The IL-1β slope approaches 1, indicating a strong correlation of interpolated IL-1β values between manual and automated runs of both plasma and control samples. Samples in which the signal was below the LLOQ are listed as "not quantifiable." LLOQ = 0.2 pg/mL, LOD 0.1 pg/mL; see Table 4 and Figure 3 for data.

Conclusion

The SMCxPRO™ platform and Erenna® Systems with SMC™ Technology allow researchers to monitor and detect changes in extremely low levels of established disease biomarkers linked to disease progression, such as interleukins and interferons. When the assay technology is integrated as an automated workflow using the Microlab® STARlet, results are comparable to those obtained using manual methods, and within acceptable limits as established by the manufacturer.

Automated Diagnostic Tool

The automated workflow eliminates the risk of errors and variability due to manual manipulations, operating virtually as an automated diagnostic tool that allows researchers to refocus their efforts on high-value activities.

Simplify and automate your life science with the Hamilton Microlab® STARlet SMCxPRO™ platform or Erenna® Systems with SMC™ Technology.

ordering-information-for-the-smcxpro-and-hamilton-starlet-integration

Authors: James A. Araujo, B.S.1, Sarah J. Hamren, B.S.1, E. Bradley Meyer, B.S.2, Kevin W.P. Miller, Ph.D.2 1MilliporeSigma, Burlington, MA, USA, 2Hamilton Company, Reno, NV, USA