Abstract Reliable serological tests for the detection of SARS-CoV-2 neutralizing antibodies are useful tools for public health and clinical purposes. Enzyme-linked immunosorbent assays (ELISAs) for the detection of neutralizing antibodies have been issued an Emergency Use Authorization (EUA) from the FDA. In addition to traditional ELISA antibodies (such as RBD), antigen-coupled magnetic bead-based ELISA is immobilized on the surface of a 96-well plate and has emerged as an alternative method with several advantages. We have developed a bead-based ELISA for the detection of SARS-CoV-2 neutralizing antibodies. This method requires separate instruments to perform major steps such as liquid transfer, washing, and shaking. Simplifying the experimental magnetic module and heater shaker on the OT-2 platform, a protocol was designed and tested to process serum samples collected from COVID-19 patients. RBD-coupled magnetic beads obtained from ACROBio Systems were also tested and compared using the same protocol.

Key Findings OT-2 equipped with a magnetic module and a thermal shock module demonstrated the ability to conduct bead ELISA for SARS-CoV-2 neutralizing antibodies with high accuracy. In higher throughput settings, the protocol can process up to 96 samples with minimal hands-on time.

Introduction For example, upon recognition of viral components, a cell-mediated immune response triggers the secretion of neutralizing antibodies against viral antigens. The nucleocapsid (N) protein and the spike (S) protein are present in the treatment of severe acute respiratory syndrome coronavirus. Virus 2 (SARS-CoV-2). Antibody testing can be used to detect the presence of these neutralizing antibodies in serum due to viral infection, providing information on addressing population or past SARS-CoV-2 infections to better understand the epidemiology of SARS-CoV-2. These tests are a common tool for determining vaccine efficacy in clinical trials and following mass vaccinations. Antibody testing typically uses an enzyme immunosorbent assay (ELISA), a technique that detects and quantifies target molecules (antibodies) through highly specific antibody-antigen interactions. For a typical sandwich ELISA for antibody detection, the first step is to attach the SARS-CoV-2 antigen to a solid surface in 96-well or 12-well strips, which can be assembled on a frame compatible with a 96-well microplate reader. These samples are then added to the wells, and anti-SARS-CoV-2 antibodies are captured and immobilized by binding to the antigen. The detection antibody, usually one that recognizes human IgG and carries horseradish, uses peroxidase (HRP), and in the presence of the enzyme's substrate, produces a colorimetric signal that can be measured and quantified. Absorbance is directly proportional to the level of anti-SARS-COV-2 neutralizing antibodies in the sample. Alternatively, magnetic beads can also serve as a solid surface in ELISA. Advantages include an increased available surface area and uniform distribution of beads throughout sample processing, allowing for more rapid and sensitive detection of low analyte concentrations. We developed a bead-based ELISA for the detection of neutralizing antibodies to the SARS-CoV-2 virus. The assay utilizes the receptor binding domain (RBD) S protein of SARS-CoV-2 as the capture antigen and Thermo Fisher herDynabeads (Waltham, MA, USA) as the solid surface. Despite the variety of assay designs, ELISA generally follows a similar workflow. General steps include reagent transfer, incubation, and repeated washing. Bead-based ELISA procedures require separate instrumentation to perform these steps, which is possible on the Opentrons OT-2 platform with automated magnetic modules and heater shakers. We developed a simplified assay protocol to avoid the need for multiple instruments and reduce hands-on time. The protocol was also validated using ACROBio Systems' RBD coupled magnetic beads (Newark, DE, USA) to confirm the quality of OT-2 sample processing.

Materials and Methods Schematic diagram of the ELISA tested on the Vision Center OT-2, an outline of the steps to complete the analysis, and a diagram of the deck layout (Figure 1) RBD-coupled beads were prepared by binding 60 μg of SARS-CoV-S-2S protein His-tagged RBD ( Acro Biosystems, Newark, DE, USA) to 5 mg dinabid (Thermo Fisher, Waltham, MA, USA), superparamagnetic spherical polymer particles for magnetically separated biomaterials , then stored in 2.5 mL of blocking buffer. The same protocol was also tested by using commercially available RBD precoated beads from US Biosystems (36 μg RBD per mg beads). Human anti-SARS-COV-2S1 antibody as standard antibody and mouse monoclonal anti-human antibody with HRP-conjugated IgG Fc as detection antibody were purchased from Cell Sciences (Newburyport, MA, USA) and Abcam (Cambridge) , UK), and custom assay buffers were manufactured by Tenova (Hollister, CA, USA). Liquid transfer was performed with an 8-channel pipette. The ELISA plate is placed on the magnetic module and manually moved to the heater module if stirring is required. Serum samples previously tested positive or negative for SARS-CoV-2 antibodies were obtained for this study. Immediately after the experiment is completed, the absorbance of each sample is measured at a wavelength of 450 nm using a colorimetric plate meter. The runtimes were estimated for each detection method at different sample sizes (Table 1).

Results To generate a standard curve for the test, assays were performed with 50 μL of RBD-coupled bead suspension on OT-2 transferred to each well of a 96-well 2 mL deep well plate preloaded with serial 2-fold dilutions of Anti-SARS-CoV-2 S1 antibody, see Materials and Methods for detection method. Absorbance was measured, and dose-response curves were performed. The results demonstrate that OT-2 performs this assay with accuracy (CV<10%) (Figure 2A) and there is an excellent linear correlation between assay readings and antibody concentration between 100 ng/mL and 12.5 ng/mL. /mL (r squared >0.99) (Figure 2B).

RBD precoated beads from Acer Biosystems were then reconstituted (1 mg beads per mL of blocking buffer) and tested on OT-2 using the same protocol. Serial 2-fold dilutions of human anti-SARS-COV-2S1 antibodies were prepared and exposed to 20 μL of bread suspension. These data were used to develop dose-response curves for further analysis. Consistency of sample processing on OT-2 (CV<10%) (Figure 3A) and linearity of assay readings with antibody concentration were demonstrated (100 ng/mL to 6.25 ng/mL, r-squared >0.99) (Figure 3B).

In order to perform a bead-based ELISA to determine the levels of SARS-CoV-2 S1 neutralizing antibodies, this protocol is designed for preparation of RBD-conjugated beads in house or US Biosystems' RBD conjugated bread is used prior to processing by conventional Serum samples tested by FDA-certified ELISA (positive: *). The results again show the accuracy of OT-2 liquid handling (CV < 10%) and antibody concentration obtained from the standard curve shown in Figure 4.

Antibody neutralization test for OT-2 antibodies

Bead-based ELISA on OT-2

Conclusion Bead-based ELISA can be successfully performed on the OT-2 platform equipped with a magnetic module and a heater-shaker module. The results demonstrate excellent sample processing quality and the solution is suitable for processing up to 96 samples with a minimum of practical hands-on time.