Author Boren Lin, PhD, Kinnari Watson, PhD

Application Overview Enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) is a common analytical tool for detecting target molecules (such as antigens) in liquid samples, often for immunodiagnostics or research. The assay typically uses a 96-well plate format, and the workflow typically includes reagent transfer and mixing, as well as some incubation and wash steps at constant temperature. These steps require a high degree of consistency in sample processing. In order to achieve automation, we used the OT-2 automated pipetting workstation with a thermal shaker to develop and test a fully automated ELISA protocol. This protocol uses Takara Bio's sandwich EIA kit to detect human fibronectin (FN), Tecan's A competitive ELISA kit detects cortisol in saliva, and another competitive ELISA kit from Cell Sciences is used to quantify neutralizing SARS-CoV-2 antibodies in serum samples.

The key finding is that the OT-2 pipetting workstation equipped with an 8-channel pipette and a thermal shaker has the ability to automatically complete ELISA with high precision. In higher throughput settings, the protocol can process up to 96 samples with minimal operation time.

Application Description Enzyme immunoassays (EIAs) or enzyme-linked immunosorbent assays (ELISAs) are techniques for the detection and quantification of target molecules (e.g., antigens) in liquid samples through highly specific antibody-antigen interactions. This assay is usually performed on a 96-well or 384-well plate, where we immobilize the antigen, either by directly attaching the antigen to a solid surface or by using a capture antibody pre-coated on the solid surface. Through this process, it separates the antigen from other non-targeting molecules in the sample.

Sandwich ELISA is the most commonly used format of this assay. The general steps are as follows: 1. If the supplier does not provide a well plate pre-coated with the antibody, we need to fix the capture antibody (that is, the antibody that specifically binds to the target) on the well plate through passive adsorption (for example, 8-well or 12-well strips assembled on a frame compatible with 96-well microplate readers). 2. Add the sample to be analyzed to the plate coated with antibodies to capture the target antigen. 3. Expose the immobilized target antigen to a detection antibody conjugated to an enzyme (such as horseradish peroxidase or HRP). 4. Add enzyme substrate to generate a signal that can be measured and quantified.

The human fibronectin (FN) EIA kit (Takara Bio, San Jose, California, USA) is a kit for the in vitro quantitative determination of soluble human FN in serum, urine, cell culture supernatants, and other biological fluids. This kit provides sufficient reagents and materials for the design and validation of EIA protocols on the OT-2 platform.

FN is widely distributed on the cell surface, extracellular matrix and plasma, and is involved in cell matrix adhesion, cell migration, regulation of cell morphology and other cell functions. Takara's FN EIA kit uses two mouse monoclonal antibodies against human FN to form an antibody-antigen-antibody sandwich complex: the capture antibody is pre-coated in an 8-piece tube, and the detection antibody is combined with peroxidase. By exposing this complex to a peroxidase substrate, a photometric signal is generated whose absorbance is proportional to the amount of target (i.e., human FN) in the sample.

Competition ELISA (Enzyme-linked Immunosorbent Assay) measures the concentration of the antigen by detecting signal interference. Briefly, the target antigen competes with a reference (antigen) pre-coated on a well plate to bind to the enzyme-conjugated antibody. The higher the concentration of the target antigen in the sample, the weaker the reference antigen's ability to bind to the antibody, resulting in a weaker signal. In some competitive ELISA kits, such as Tecan's Cortisol Saliva ELISA tested in this study, the enzyme is not carried by the antibody, but by the reference substance, which competes with the target antigen for the antigen-antibody interaction. Another ELISA reagent we tested on OT-2 is the SARS-CoV-2 Alternative Virus Neutralization Test Kit from Cell Sciences (Newburyport, MA, USA), which is used to measure SARS-CoV-2 post-infection or circulating neutralizing antibodies in response to vaccination. Neutralizing antibodies inhibit the SARS-CoV- 2 enters the cell. The kit provides a pre-coated viral spike protein RBD plate in the form of a competitive ELISA, introducing HRP-conjugated ACE2 to compete with neutralizing antibodies to capture the RBD.

Although there are many ways to design ELISA, they all follow a similar workflow. Routine ELISA steps including reagent pipetting, incubation, and repeated washing can be easily accomplished on the OT-2. Continuous washes are performed between each reagent step to remove unbound molecules. To prevent remaining solution from being carried over to the next reagent step, it is important to remove all excess liquid. Opentrons' 8-channel pipettes enable efficient and precise pipetting to meet the needs of reagent pipetting and washing in ELISA. In addition, Opentrons temperature control modules or thermal shakers can be added to enhance automation and provide the thermostatic function required for sample incubation in experiments (e.g. set to 37°C to promote antigen-antibody interaction).

Materials and Methods Figures 1-3 show schematics of the three ELISAs tested on the Opentrons OT-2, including an overview of the experimental procedures and deck layout.

Liquid transfer was performed with an 8-channel pipette. The ELISA plates used a sealing membrane with a slit seal (BioChromato, Fujisawa, Japan), which allowed pipette tips to pass through the sealing membrane while preventing evaporation during incubation. The ELISA plate was then mounted on a thermal shaker and heat and stirring were provided as specified in the instructions (see Table 1). Immediately after completing the experiment, the ELISA plate needs to be manually moved to the microplate reader and the absorbance value measured at a wavelength of 450 nm.

To test the FN EIA kit, FN in human plasma was first dissolved in deionized water to prepare a 1 mg/mL stock solution, which was then further diluted to 500 ng/mL and 2-fold serial dilutions were performed. For the cortisol ELISA test, use standards and controls of known cortisol concentration provided in the kit. Serum samples that have previously tested positive or negative for SARS-CoV-2 antibodies will be used in virus neutralization ELISA experiments. The running time of each experiment at different sample sizes is predictable (see Table 1).

Experimental results processing FN EIAs on OT-2. Sample processing quality can be assessed by assessing the correlation between the final readout and the concentration of the target molecule and the consistency of this correlation between different experiments. The goodness of fit of the linear regression can be determined by plotting the mean absorbance (n=3) versus FN concentration and calculating the standard deviation.

The results demonstrate the accuracy and precision (R-squared >0.99, CV<10%) (Figure 4A), and repeatability (Figure 4B) of OT-2 in experiments performed with the kit. A competitive ELISA on OT-2 was tested using serial dilutions of cortisol. Results show consistency in liquid handling (n=3, CV<10%), with excellent negative linear correlation between test readings and log-transformed sample concentration on a semi-log plot (R-squared >0.99 ) (Figure 5A), and were reproducible between experiments (Figure 5C).

To detect the inhibition of ACE2-RBD binding in a competition ELISA, serum samples were processed three times on OT-2, the absorbance was measured on a microplate reader, and the coefficient of variation was obtained. The results reconfirmed the accuracy of OT-2 liquid handling (CV<10%) (Figure 6). According to the manufacturer's instructions, the experiment was valid because the OD450 values ??of each control group were within the standard range (positive control <0.3, negative control >0.9). The inhibition rate is calculated as follows:

This experiment successfully detected antibodies capable of neutralizing the ACE2-RBD interaction in samples previously diagnosed as positive for SARS-CoV-2 antibodies (Table 1).

Experimental Summary By using OT-2 for competitive ELISA and neutralizing antibody detection, we concluded that: 1. Dilution processing and absorbance measurements performed on OT-2 showed highly consistent results, confirming OT-2 liquid processing accuracy and repeatability. 2. According to the requirements of the manufacturer's instructions, the OD450 values ??of the control group in the experiment were all within the valid range, which verified the validity of the experiment. 3. Using experimentally measured inhibition rates, neutralizing antibodies present in samples previously diagnosed as positive for SARS-CoV-2 antibodies were successfully detected. 4. The human serum sample in Table 2 was confirmed to be positive, further confirming the results of its previous ELISA test. In summary, this experiment demonstrates superior accuracy and reliability in detecting neutralizing antibodies, and the sample processing capabilities of OT-2 are comparable to traditional manual methods.