How To Automate SMC^® Immunoassays on the AAW™ Automated Assay Workstation

This protocol details how to automate SMC^® ultrasensitive immunoassays with the AAW™ automated assay workstation. Automation reduces hands-on time while ensuring assay robustness and reproducibility. See details and data below.

Intro to Automating SMC^® Assays

The goal of automating lab-based assays is to increase efficiency and reproducibility while reducing manual hands-on steps. The AAW™ automated assay workstation is a modular robotic liquid handler powered by Opentrons^®, that is designed for high-throughput and technical workflows such as high sensitivity immunoassays.

SMC^® high sensitivity immunoassays use a quantitative fluorescent sandwich technique to measure target proteins in a variety of sample types. Due to the highly sensitive nature of SMC^® assays, precision and accuracy in pipetting is important to achieve optimal results. The work below describes the reproducible performance of SMC^® assays by automating them with the AAW™ workstation to achieve key performance metrics, while reducing manual intervention steps.

Automation and SMC^® Materials Required

Reagents

Choose your SMC^® assay. Examples of SMC^® assays verified on the AAW™ Automated Assay Workstation include:

Sample Preparation Labware

Hardware and Instruments

Labware

• 96-Well V-Bottom Plate (Prod. No. AXYP96450VCS)
• SMC^® 384-well Commercial Plate (refer to kit protocol)
• NEST 12-Well 15 mL Reservoir (Opentrons^® Prod. No. 99900076)
• NEST 2 mL 96-Well Plate (Opentrons^® Prod. No. 99900103)
• Sphere Magnetic Plate (Prod. No. 90-0003-02, provided with BioTek^® 405™TSUVS Plate Washer )
• Opentrons^® 50 μL Tip Rack, Filtered (Prod. No. 99100104)
• Opentrons^® 1,000 μL Tip Rack, Filtered (Prod. No. 99100106)
• Aluminum Adhesive Plate Seals (Fisher Sci Prod. No. 276014)
• 5 mL Luer-Lok™ Syringe (Fisher Sci Prod. No. 14-829-45)
• 0.2 μm Syringe Filter (Prod. No. SLGPR33RS)

Preparing the AAW™ Workstation for the SMC^® Assay

The AAW™ workstation was used to automate SMC^® immunoassays. Assay protocols were adapted into Python code, imported into the Opentrons^® app and onto the workstation. The instrument performed all liquid handling steps, including serial curve dilutions, as well as on-deck shaking, and magnetic bead pull down. Plate washing is performed off-deck using the BioTek^® 405™TSUVS Plate Washer for SMC^® and MILLIPLEX^® Technology (Prod. No. 95-0004-06). To prepare the instrument for SMC^® assay performance:

1. Download the SMC^® kit protocol file from the Millipore^® Protocol Library and load in the Opentrons^® app on your computer. The protocol can then be sent to the AAW™ robot via the “Send to Opentrons FLEX” or “Start Set-up” functions.
2. Load required modules and pipettes as listed in the app or AAW™ touchscreen. Load the Opentrons FLEX™ Heater-Shaker into deck slot D3. Ensure the module is plugged in, powered on, and visible in app or touch screen modules.
3. Once the protocol is sent to the robot, the assay can be initiated by selecting the protocol and "Start Set-up" button. A list of parameters will be shown whereupon specific assay selections can be made as listed below:
   1. Multi-Channel Mount: Left (Default) or Right
   2. Detection Antibody: SMC^® Assay Kit of interest (16 options)
   3. Run Type: Can Run Standard Curve + Samples (Default) or Standard curve only
   4. Dry Run: On or Off (Default) for use as practice runs with shortened incubation and wash steps
   5. Seal Plate for Capture Step: On or Off (Default) for option to manually seal plate before capture step. Otherwise, assay proceeds immediately to capture step after plate set-up.
   6. Dilute Samples: On or Off (Default is Off for Neat samples)
   7. Dilution Ratio: Input 2 (Default) if Samples + Dilute Samples chosen
      Note: If other sample dilution ratios are required (1:3-1:1,000), please run the SMC^® Sample Dilution Protocol prior to running the SMC^® protocol
   8. Number of Samples: Input 1-20
4. It is recommended to run a dry run of the protocol to familiarize oneself with the assay steps on the AAW™ workstation. A dry run can be run dry with no liquid or with water/buffers. Dry runs have shortened mixing, incubation steps, and a return tips to racks function for further practice. 
5. Prior to starting a run, a Labware position check must be performed at least once by following the step-by-step instructions on the touchscreen or in the Opentrons^® app. Once labware offsets are confirmed, the AAW™ workstation stores these values for future runs and will not have to be repeated unless desired. Labware position check can be run with empty labware during a dry run or with reagent-ready labware.

SMC^® High Sensitivity Kit Set-Up Procedure

 The SMC^® immunoassays were performed according to the product instructions manually or by using the adapted protocol on the AAW™ workstation (see below). For this application study, SMC^® assays were repeated 3 times on the AAW™ workstation. All steps of the assay were performed on the instrument deck, except for washing steps performed on the BioTek^® plate washer. All assays were read on the SMCxPRO^® instrument and data was acquired via xPRO^® software. The product list of required labware and materials can be found above.

Reagent Preparation

Note: Reagents were prepared according to each SMC^® kit’s individual manual.

1. Warm all reagents to room temperature (RT) prior to use.
2. Store the Detection Antibody away from light until ready to use after the Post-Capture Wash. Touchscreen instructions will prompt when to dilute Detection Antibody.
3. Prepare 1X Wash Buffer (from 10X Wash Buffer).
4. Mix Antibody Coated Beads on a rotisserie spin rotator, or manually by repeated pipetting.
5. Load reagents into the Standard+Sample and Reagent Reservoir Plates as shown in example Figure 1 or the AAW^TM workstation touchscreen.
   1. Exact volumes can be found in the Opentrons^® app or AAW^™ touchscreen upon protocol start-up.
   2. Sample diluent is only required if running diluted samples at 1:2 dilution in this protocol, otherwise, please run the SMC^® Sample Dilution protocol prior to this assay.  Sample Dilution reagent locations are compatible with this Automated SMC^® Protocol.

Note: If running a Standard only protocol, volumes can be found below in the Tips and Tricks section.

Sample Preparation

Prepare samples by one of the following methods: 1) Centrifuge samples at >13,000 x g for 10 minutes immediately prior to use or 2) If using a filter plate with prefilter: Stack the filter plate on top of a 96-well v-bottom receptacle plate. Place 400 µL of sample into a filter plate well and spin for ≥ 10 minutes at 1,100 x g. Samples can be loaded into the reagent plate as shown in Figure 1 if used neat or 1:2. If other sample dilutions are required, please use the SMC^® Sample Dilution Protocol or manually dilute samples prior to running the SMC^® assay. Sample Dilution Protocol can be found on the Millipore^® Protocol Library page.

Robot Deck Configuration and Assay Protocol

Once all reagents and samples have been prepared and added to the appropriate wells, the deck layout on the AAW™ workstation can be set up. Deck layout is shown in Figure 2.

1. Load 2 boxes of 50 µL tips in deck spots A1 and B1.
2. Load a box of 1,000 µL tips in deck spot A2.
3. Load the NEST 12-trough reservoir in B2.
4. Load the magnetic plate in C2.
5. Load the SMCxPRO^® 384-well read plate (without the skirted lid) in D1.
6. Load the NEST 2 mL 96-well reagent plate in D2.
7. Load the 96-well v-bottom assay plate onto the Heater-Shaker (D3).

1. Once all labware and reagents are in their proper location, the robot door can be closed. If the Start Set-up function has already been activated and parameters selected, the assay can begin by selecting the blue run Arrow. Assay workflow is outlined in Figure 3.

2. For the detection step, while the plate is washing, prepare the detection antibody according to the kit instructions and transfer the diluted detection antibody solution into Well 5 in the NEST 12-trough reservoir.
3. Continue to follow the steps as guided by the AAW^™ workstation touchscreen for the detection and elution/transfer steps. Pauses are built in for the plate to be manually sealed with an aluminum seal prior to the on-deck detection and elution incubation steps. Follow the timings indicated on the touchscreen for when to return after incubation steps.
4. Once the protocol is complete, add the plate holder (skirted lid) to the bottom of the plate and firmly seal the read plate with aluminum adhesive plate seal using a plate roller. Centrifuge plate for 1 minute at RT, approximately 1,100 x g.
5. Remove the plate holder from the sealed reading plate and load it onto the SMCxPRO^® Immunoassay System. Start read.

Automated SMC^® Assay vs Manual Assay Results

The automated SMC^® protocol on the AAW™ workstation was verified on six different SMC^® kits including IL-6, cTnI, IL-17A, IL-22, IL-23, and IL-17F. Assays were run in triplicate on the workstation and compared to manual performance. For each run on the AAW™ instrument, each IL-6 assay demonstrated a lower limit of quantification (LLOQ) that was below the published and manual LLOQ of 0.077 pg/mL and resulted in an average of 2 outliers removed (Figure 4). cTnI achieved LLOQ results where two of the three runs matched the published LLOQ of 0.694 pg/mL (Figure 4). While one run was slightly above this threshold, it was equal to that of the manual assay LLOQ (1.39 pg/mL). For the IL-17A assay, all automated performances were lower than the published LLOQ of 0.069 pg/mL (Figure 4), with an average of just over 1 outlier removed per assay. All automated assays resulted in a lower number of outliers removed than manual assays. Overall, these results indicate that the automated assays performed reliably and consistently across all tested parameters and the results are summarized in Table 1.

Similarly, assays IL-22, IL-23, and IL-17F each reached their published LLOQs or better for each automated assay run (Figure 5). The average number of outliers for each assay was 0, ~1, and ~3, respectively, higher than manual assays with 2, 3, and 6 outliers removed/run. These results are also summarized in Table 1. These assays confirm that AAW^™ automation resulted in improved reproducibility and consistency while still maintaining high assay performance and robustness.  

Improved assay reproducibility is further highlighted in the comparison of IL-6 LLOQ achieved by manual (n=5) and automated (n=4) assays (Figure 6). The standard deviation using AAW™ workstation is statistically reduced compared to manually performed assays (p<0.05). Finally, healthy serum and plasma samples were compared between manual and automated assay performance using the IL-6 SMC^® kit (Figure 7). Sample correlation results confirm the AAW^™ workstation performs as well as manual with an R-squared value of 0.9915.

Finally, comparison across different workstation performances of the same SMC^® assay demonstrated the robustness of the automated protocol. Performance on AAW™ workstations 1 and 2 resulted in overlayed standard curves with the same LLOQ (Figure 8A). In addition, correlation between the standard curves on the two instruments was high with an R² value of 0.9966 (Figure 8B).

Summary

The AAW™ workstation is a robotic liquid handler with a user-friendly interface capable of performing high-throughput and technical workflows allowing for more hands-off time. An automated protocol was developed to perform the key liquid handling and incubation steps of the SMC^® assay on the AAW™ workstation. All assay steps were performed on-deck except for wash steps. The AAW™ workstation was able to achieve manual LLOQ or better across three runs, with similar or better %CV to manual handling by an experienced user.

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Tips & Tricks

• It is recommended to run a dry run of the protocol to familiarize oneself with the assay steps on the AAW™ workstation. A dry run can be run dry with no liquid or with water/buffers. Dry runs have shortened mixing and incubation steps, as well as returns tips to racks for further practice.
• If tips are not centered in well during dispense steps, recheck Labware Position Check to ensure proper location.
• If running a standard only plate, you may remove unnecessary tips from the tip box rows (D-H) for bead dispense (1,000 μL tip rack, column 12), detection dispense (1,000 μL tip rack, column 11), and elution/transfer steps (50 μL tip racks columns 1-12) to minimize reagent waste.
  • Minimum volumes required for standard only run include: 6 mL of 1x beads, 2.5 mL of 1x detection, 2 mL of Elution Buffer B, and 2 mL of Buffer D.
• An aluminum plate seal is recommended on the AAW™ automated assay detection and elution incubation steps. Keep the tabs on and fold over the edges of the plate.