Protocol for monitoring of neutralizing antibodies to various receptor-binding domains using the tANCHOR system

Abstract

Neutralizing antibody analysis against SARS-CoV-2 variants requires assays that rapidly adapt to protein mutations. Here, we present a protocol for monitoring neutralizing antibodies to various receptor-binding domains using the tANCHOR system. We describe steps for displaying variant receptor-binding domains on HeLa cells and producing tagged soluble angiotensin-converting enzyme 2 (ACE2). We then detail the procedures for establishing a cell-based ELISA to measure serum neutralization efficiency, using ACE2 competition as a readout. For complete details on the use and execution of this protocol, please refer to Ivanusic et al.¹.

Keywords: Antibody; Cell Membrane; Cell-based Assays; High-Throughput Screening; Immunology; Microbiology; Protein expression and purification.

Graphical abstract

Figure 1

Overview of the workflow for cloning RBD coding DNA fragments into the tANCHOR vector system The RBD coding fragment is inserted between the restriction sites EcoRI and EcoRV. The ligation mix is plated on agar containing 100 μg/mL ampicillin. Colonies are propagated in TB medium with 100 μg/mL ampicillin, and plasmid DNA is isolated for sequence confirmation.

Figure 2

In-frame cloning of the RBD into the tANCHOR vector system The RBD or protein of interest is inserted between the restriction sites EcoRI and EcoRV. The coding region has to be in frame with the 4× glycine linker.

Figure 4

Appearance of transfected cells after puromycin selection Images show typical cell clusters observed one week post-selection (top row). Untransfected control cells (bottom row) are completely eliminated by puromycin and washed out during cell culture medium changes.

Figure 5

Quantification of secreted ACE2_1-740-V5-His protein The protein was isolated from the supernatant of a stably transfected HEK293T cell line and separated by SDS-PAGE (4%–15% Mini-PROTEAN TGX gel, Bio-Rad) and stained with Coomassie Brilliant Blue R-250. For quantification, signal intensity is compared to a BSA standard. If the stained ACE2_1-740-V5-His band from 500 μL supernatant aligns with the 4 μg BSA band, the sample volume should be adjusted accordingly. Protein molecular weights were estimated using the PageRuler Prestained Protein Ladder (10–180 kDa).

Figure 3

Workflow for the production of supernatant containing secreted ACE2_1-740-V5-His protein The stable HEK293T cell line secretes soluble ACE2_1-740-V5-His protein, which is used as one of the components to evaluate antibody competition in the binding of ACE2 to the RBD.

Figure 7

Subcellular localization of tANCHORed RBD in transfected HeLa cells (A) Epifluorescence image showing tANCHORed RBD fused to mCherry after fixation in a 96-well plate (Zeiss Axio Observer). (B) Confocal laser scanning microscopy (CLSM) image showing membrane-localized RBD expression (Zeiss LSM780). Cell nuclei are stained with Hoechst 33342. Scale bars: (A) 100 μm; (B) 10 μm.

Figure 6

Key steps in a cell-based assay for evaluating the neutralizing activity of human serum against SARS-CoV-2 The workflow illustrates the protocol used when cells are transfected on Day 2 following seeding.

Figure 8

Representative results obtained from collected human serum samples Neutralization activity is assessed using HeLa cells expressing surface-displayed tANCHORed RBDs. Left panel: Neutralization measured against Wuhan-RBD. Right panel: Lower neutralization activity observed against Omicron-RBD. Groups are compared for significance by an unpaired two-tailed Student's t-test, ∗∗∗∗: p < 0.0001.

Figure 9

Improving cell layer stability during washing steps The cell layer is more stable when cells are attached to the well wall. Position the washing pipe as close as possible to the wall of the 96-well plate.