Automating the ultrasensitive SMC® immunoassay workflow with the Hamilton Microlab® STARlet liquid handling workstation provides a robust, sensitive, and reproducible method to quantify biomarker concentration without sources of error and variability associated with manual processing. Find out more about how it refocuses manual labor on high-value activities such as result interpretation below.
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.
Single Molecule Counting (SMC®) technology provides the path to quantifying these low-level cytokines. The SMC® technology method is similar to a traditional ELISA and offers a reduced background signal and an increased detection signal in comparison. In this process, target analytes in solution are captured onto an antibody-bound plate or magnetic 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 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 (determined from calibrator set: 0–300 fM of 150 kD antibody labeled with fluorophore).
When manually performed, the SMC® immunoassays can be labor-intensive. This prevents researchers from focusing on high-value activities such as result interpretation, thus reducing overall laboratory efficiency. Manual methods also introduce the risk of operator error and elements of variability from one user to the next, and among subsequent assay runs. Automating immunoassay workflows helps reduce these risks/variabilities and allows researchers to shift their focus to high-value activities.
SMC® immunoassays can be integrated onto the Hamilton Microlab® STARlet liquid handling workstation to create a hands-free, automated, assay-ready workflow. This simplified automated workflow is as follows:
The STARlet workstation 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 workstation for added workflow efficiency and convenience.
Finally, 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, the assay-ready automated workstation was shown to deliver 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.
Automated and manual workflows were compared using the SMC® Interleukin 6 (IL-6) Immunoassay Kit, SMC® Tumor Necrosis Factor (TNF-α) Immunoassay Kit, and SMC® Interleukin 1-β (IL-1β) Immunoassay Kit. In each assay, the protocol was followed.
Standard protein curves were created manually as follows:
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 were tested. 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 according to each kit protocol.
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 workstation with assay-specific reagents. Using 300 µL conductive non-filtered CO-RE® tips (Hamilton, 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 25°C 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 (Hamilton, P/N 235947), and the microplates were incubated for one hour 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 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 (discontinued) 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.
For the data analysis, three signal outputs were obtained from the Erenna® instrument (now discontinued): 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. As the automation is for the immunoassay workflow, it can be utilized equally successfully with the second generation SMCxPRO® instrument.
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, quantifiable samples did show a good correlation between the Hamilton automation and manual method assay methods.
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.
The LLOQ was 0.08 pg/mL. See Table 1 for data.
Table 1. Comparison of manual versus automated processing of the IL-6 assay using plasma samples.
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.
The LLOQ was 0.2 pg/mL and the LOD was 0.05 pg/mL. See Table 2 for data.
Table 2. Comparison of manual versus automated processing of the TNF-α assay using plasma samples.
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."
The LLOQ was 0.2 pg/mL and the LOD 0.1 was pg/mL. See Table 3 for data.
* < LLOQ — Interpolated sample value is below the 0.20 pg/mL limit of quantification.
Table 3. Comparison of manual versus automated processing of the IL-1β assay using plasma samples.
The SMCxPRO® and Erenna® systems with SMC® technology allow researchers to detect and monitor changes in extremely low levels of established disease biomarkers such as interleukins and interferons. When the assay technology is integrated as an automated workflow using the Microlab® STARlet workstation, results are comparable to those obtained using manual methods, and within acceptable limits, as established by the manufacturer. The automated workflow eliminates the risk of errors and variability due to manual manipulations, and also allows researchers to refocus their efforts on high-value activities.
This was part of a collaboration with Hamilton Company in Reno, NV, USA. Data was obtained from E. Bradley Meyer, B.S. and Kevin W.P. Miller, Ph.D. in 2019.