How To Automate ELISAs on the AAW™ Automated Assay Workstation

This automated ELISA protocol explains how to automate our Conferma^® ELISAs and other ELISAs on the AAW™ workstation. See why walkaway ELISA automation helps enhance the reproducibility of ELISA assay performance.

Introduction to Automating ELISAs

Lab automation, such as ELISA assay automation, can increase efficiency and consistency while reducing human errors that often occur during hands-on steps. The AAW™ automated assay workstation is a modular robotic liquid handler and assay automation system powered by Opentrons^®, specifically designed for high-throughput and technical workflows, including immunoassays.

Our ELISA kits primarily utilize a sandwich ELISA technique to accurately measure target antigen levels in various sample types, including serum and plasma. Given the assay's sensitivity, precise and accurate pipetting is crucial for obtaining reliable results.

Below outlines the enhanced reproducibility and performance of the ELISA workflow by automating the assay protocol with the AAW™ workstation, which streamlines key performance metrics while reducing manual hands-on time and human errors. This protocol was verified on the following soluble human biomarkers in human serum: TNF-α, IL-6, IL-8, MCP-1, Leptin, Insulin, Amylin, Ghrelin (Active/Total), and S100B.

Automation and ELISA Materials Required

View related ELISA kits, labware, and hardware for running this automated ELISA protocol. 

Labware

• Opentrons^® Tough 22 mL 12 Well Reservoir (Opentrons^® Prod. No. 999-00260)
• 290 mL One-Well Reservoir (Opentrons^® Prod. No. 999-00207)
• NEST 2 mL 96-Well Deep Well Plate, V-Bottom Reservoir (Opentrons^® Prod. No. 999-00103)
  • For TNF-α, IL-6, MCP-1, Leptin, Amylin, Ghrelin, and S100B ELISA kits
• 96-Well V-Bottom Plate (Prod. No. AXYP96450VCS)
  • For Insulin and IL-8 ELISA kits
• Opentrons^® 50 μL Tip Rack, Filtered (Prod. No. 99100104)
  • For Ghrelin and Insulin ELISA kits
• Opentrons^® 200 μL Tip Rack, Filtered (Prod. No. 99100105)
• Opentrons^® 1,000 μL Tip Rack, Filtered (Prod. No. 99100106)

In addition, this protocol utilizes Opentrons^® Universal Flat Heater-Shaker Adapter Type B. This adapter is included with the Heater-Shaker module as part of purchasing the Assay-Ready AAW™ Workstation.

In addition to the above, it is optional to use BioTek^® 405^™ TSUVS Plate Washer for plate washing if the user chooses to wash off-deck. It is also optional to use Opentrons Flex™ Waste Chute for tip disposal. 

Software

Our Belysa^® Immunoassay Curve Fitting Software is a perfect complement to our ELISAs and provides a user-friendly tool to help easily evaluate assay data and compare standard curves. This software was used in the data generated below.

Protocol Uploading and AAW™ Workstation Setup

The AAW™ workstation can be used to automate manual ELISA protocols. New users should refer to the AAW™ user manual for detailed instructions on loading protocols to your AAW™ workstation, however we’ve provided guidance here.

To prepare the instrument for an automated ELISA experiment:

1. To load any program onto the AAW™ workstation, users should first download the Opentrons^® application on a computer and establish access to the AAW™ robot through Wi‑Fi, ethernet, or USB cable.
2. Download your ELISA kit protocol from the Millipore^® Protocol Library and upload it to the Opentrons^® App on your computer. The protocol can then be transferred to the AAW™ workstation by clicking the “Send to Opentrons FLEX” or “Start Set-Up” buttons.
3. Load required modules and pipettes as listed in the app or AAW™ touchscreen. Load the Opentrons Flex™ Heater-Shaker into deck slot D1. Ensure the module is plugged in, powered on, and visible in app or touchscreen modules.
4. Once the robot receives the protocol from the computer, select “Start Set-Up” button to initiate the experiment. Users will be presented with a list of parameters to customize their specific experimental settings:
   1. Dry Run: Off (Default) or On for practice runs with shortened incubation time without reagent and tip consumptions.
   2. Number of Samples: Enter the number of samples to be run in the experiment, (e.g. 0-38 for TNF-α ELISA kit). Maximum number of samples supported differ between kits. Follow the protocol instructions to add appropriate number of samples.
   3. P1000 1-ch Position: On the right or On the left (Default is On the right).
   4. Wash Step: Provides three options for plate washing:
      1. Wash on Deck (Default): AAW™ workstation will perform wash on deck, allowing for continuous walkaway automation.
      2. Plate Washer: AAW™ workstation will pause, users will wash the plate with the plate washer of their choice.
      3. Manual Wash: AAW™ workstation will pause, users will wash the plate manually.
   5. Substrate Incubation (Minutes): Input substrate incubation time (e.g. 5-25 minutes for TNF-α kit, default is 16 minutes).
   6. Tip Disposal: Choose tip disposal method between trash bin and waste chute.
   7. Matrix Effect: Yes (Default) if samples included in this run are with significant serum matrix component.
      • (This runtime parameter only applies to Human Insulin ELISA kit).
   8. It is recommended to conduct a dry run of the protocol to become familiar with the assay steps on the AAW™ workstation. A dry run can be performed without any liquid or with water/buffers. These dry runs include shortened mixing and incubation steps, as well as a function to return tips to racks for additional practice.
   9. Before starting a run, it is required to perform a Labware position check at least once. Follow the step-by-step instructions on the touchscreen to perform the Labware position check. Once the labware offsets are confirmed, the AAW™ workstation saves these values for future runs. Offsets do not need to be adjusted repeatedly for the same protocol unless desired.

   The AAW™ instrument performs all liquid handling steps including serial curve dilutions, on-deck shaking, as well as on-deck plate washing. There is one manual intervention step in the assay to remove any excess wash buffer and to add the light sensitive substrate solution to the deck.

   Off-deck washing with the BioTek^® 405™TSUVS Plate Washer or manual washing are offered as options to users.

ELISA Assay Setup Procedure

Find details on sample prep and reagent prep for these ELISA protocols.

Sample Preparation

• For serum samples, allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1,000xg. Remove serum.
• For plasma samples, centrifuge the blood for 10 minutes at 1,000xg within 30 minutes of blood collection. Remove plasma.
• Store unused samples at ≤ -20 °C. Avoid multiple ≥ 2 freeze/thaw cycles. When using frozen samples, it is recommended to thaw the samples completely, mix well by vortexing, and centrifuge prior to use in the assay to remove particulates.
• Refer to the protocol manual for more detailed, kit-specific instructions.

Reagent Preparation

Note: Reagents were prepared according to each ELISA kit’s individual manual.

1. Warm all the reagents to room temperature prior to use.
2. Reconstitute all standards and quality controls according to the instructions in the protocol booklet or certificate of analysis.
3. Reconstitute serum matrix according to the instructions in the protocol booklet.
4. Prepare 1X Wash Buffer (from 10X Wash Buffer).
5. Keep the Substrate away from light until ready to use after the Post-Enzyme Wash. The workstation will pause and instructions on touch screen will prompt when to add Substrate.
6. Load reagents into the Standard+Sample and Reagent Reservoir Plates as shown in example Figure 1 or the AAW™ workstation touchscreen.

• Exact volumes can be found in the workstation touchscreen in Map View once the protocol is loaded.
• When running under a certain number of samples (e.g. 14 samples for TNF-α kit), only one wash buffer and waste reservoir is needed.
• Do not load Substrate at the beginning of the experiment. Due to the light sensitive nature of Substrate solution, it is optimal to keep Substrate from light until right before it is being used to ensure the best result. The robot will pause and instruct users to add Substrate right after the Post-Enzyme Wash.

Deck Configuration and Assay Protocol

Once all reagents and samples have been prepared and added to the appropriate wells, follow the deck layout displayed in the Opentrons^® App to for placement of labware, as shown in Figure 2.

1. Load 3 boxes of 200 µL tips in deck spots A1, A2, and B2.
   i. For kits that use 50 µL tips, load two boxes of 200 µL tips in deck spots A1 and A2, and one box of 50 µL tips in deck spot B2.
2. Load a box of 1,000 µL tips in deck spot A3.
3. Load the Opentrons^® 12-well reagent reservoir in C1.
4. Load the NEST 2 mL 96-well sample plate or 96-Well V-Bottom Plate in B1.
5. Load the 96-well ELISA assay plate provided by the kit onto the Heater-Shaker (D1) with Universal Flat Type B adapter.
6. Load the 290 mL One-Well Reservoir filled with appropriate volume of wash buffer in deck spots C2 and B3.
7. Load the 290 mL One-Well Reservoir (Wastes) in deck spots D2 and C3.

1. Once all labware and reagents are placed in the correct location, close the robot door. If the Start Set-Up function has been activated and the parameters have been selected, the assay can be initiated by clicking the blue Run Arrow. The assay workflow is illustrated in Figure 3.
2. Immediately following the post-enzyme wash, the robot will pause and unlatch the Heater-Shaker. Carefully remove the assay plate, invert it, and tap to decant any excess wash buffer before returning it to the Heater-Shaker.
   1. To ensure optimal signal development in the subsequent steps, it is essential to remove any residual wash buffer. Skip this step if using plate washer or manual washing.
   2. Next, add the appropriate volume of Substrate Solution to the designated well in the Opentrons^® 12-well reservoir. Finally, select "Resume" to proceed with the protocol.
      1. For further optimization, an additional pause-tap step is added to the Conferma^® Human IL-8 ELISA protocol during the beginning of the protocol, following standard dilution and pre-wet steps. Carefully remove the assay plate, invert it, and tap to decant any excess wash buffer before returning it to the Heater-Shaker.
3. Once the protocol is complete, remove the assay plate from the Heater-Shaker and load it onto the plate reader. Start read.
   1. For the Human Amylin ELISA, there will be an initial 22-minute incubation. Once this incubation period is complete, the Heater-Shaker will unlatch. Read the fluorescence on a plate reader to monitor signal development. If the desired signal has not been achieved, incubate the plate off deck without shaking. Continue reading the fluorescence every 2-3 minutes to track signal progression. Once the desired signal-to-noise ratio or fluorescence level is reached, return the assay plate to the Heater-Shaker and press "resume" to proceed to the final step of the protocol.

Table 1 describes the different walkaway automation times for highlighted ELISA automated protocols in the Millipore^® Protocol Library.

Results and Verification: Automated ELISA vs Manual ELISA

ELISA assays were performed according to the product instructions manually and by using the adapted protocol on the AAW™ workstation. All assay steps were performed on the instrument deck. All assays were read on the BioTek^® Synergy HTX multi-mode plate reader. Data was analyzed via the Belysa^® Immunoassay Curve Fitting Software.

The automated ELISA protocol was verified using all ELISA kits listed above. The data presented in this section were acquired from five Conferma^® TNF-α ELISAs performed on the AAW™ workstation, two of which were run with samples. Data generated from other ELISA kits are included below.

For four of the five runs on the AAW™ workstation, each assay achieved the published lower limit of quantitation (LLOQ) of 1.65 pg/mL, and one run achieved 4.94 pg/mL, which is equal to manual assay (Figure 4).

The evaluation of parallelism and relative potency across different assay runs demonstrates the consistency and reliability of the AAW™ instrument (Table 2 and Figure 5).

The parallelism values for the automated assays (AAW™ Run 2 to AAW™ Run 5) ranged from 1.007 to 1.030, indicating a stable and consistent response across multiple runs. To compare the parallelism values to manual runs, these ranged from 0.999 to 1.014. This close alignment with the manual runs reinforces the reliability of the AAW™ instrument in producing similar results as manual runs.

The relative potency values for the automated assays varied from 0.880 to 1.197, reflecting the instrument's ability to maintain reliable performance that is also comparable to manual assays (relative potency values ranging from 0.954 to 1.229).

Overall, the AAW™ robot demonstrates consistent performance between different runs and compared to manual assays, underscoring its repeatability and reliability in assay execution.

Robust assay reproducibility is further observed when comparing the coefficient of variation (%CV) between AAW™ automated runs and manual assays. Automated runs demonstrated similar variation in standards and significantly less variation in quality controls compared to manual assays (Figure 6).

Finally, human serum and plasma samples were tested on the AAW™ automated runs. The automated assay was performed concurrently with a manual run using the same kit and samples on the same day. Results demonstrated a strong correlation between the two parallel runs, evidenced by high R² values of 0.9825 for samples and 0.9998 for quality controls. These findings further verify the capability of the AAW™ workstation to deliver results comparable to those obtained from manual assays (Figure 7).