Wild-type CoV-2-NP was obtained from RayBiotec. Avi-His-Tag-Alpha, Beta, and Gamma CoV-2-NP were obtained from BPS Bioscience. Delta CoV-2-NP and Omicron CoV-2-NP were obtained from Sino Biological and Acro Biosystems, respectively. Female Hartley guinea pigs were purchased from Japan SLC, Inc. DyLight 488 and 550 microscale antibody labeling kits, DyLight 650-labeled anti-guineapig IgG and DAPI were obtained from ThermoFisher Scientific. Horseradish peroxidase-labeled anti-guinea pig antibody was obtained from Abcam. Heat-inactivated SARS-CoV-2 particles were acquired from Zeptmetrix. The plasmid encoding myc-tagged CoV-2-NP (pCMV-CoV-2-NP) was obtained from Sino Biological.
Immunization And Isolation Of Antigen-specific Plasma Cells
Anesthetized guinea pigs were immunized three times intramuscularly at the tail base with 100 µl of a 50:50 water-in-oil TiterMax Gold adjuvant emulsion containing 50 µg of wild-type CoV-2-NP. One week after the final immunization, the iliac lymph nodes were surgically removed and used for the isolation of antigen-specific plasma cells as previously described [17]. Briefly, lymph node cells were fixed with ice-cold phosphate-buffered saline (PBS) containing 2% paraformaldehyde and incubated for 10 minutes on ice, suspended in PBS containing 0.1% Triton X-100 (PBST) and stained with DyLight 488-labeled CoV-2-NP, DyLight 550-labeled CoV-NP, DyLight 650-labeled anti-guineapig IgG and DAPI. CoV-2-NP-specific plasma cells, defined as COV-2-NPHigh, COV-NPLow and IgGHigh, were single-sorted using a JSAN Cell Sorter (Bay Bioscience). Flow cytometry data were analyzed using FlowJo Software version 10.7.1 (BD Biosciences).
Generation Of Monoclonal Antibodies
Amplification of immunoglobulin heavy and light chain variable genes by rapid amplification of 5’ cDNA ends PCR was performed as previously described [18]. High-throughput production of recombinant antibodies was performed by TS-jPCR as previously described [18]. Briefly, cognate pairs of linear immunoglobulin heavy and light chain genes were cotransfected into HEK293 cells, and the cell culture supernatant was used for enzyme-linked immunosorbent assay (ELISA) and cell ELISA four days after transfection. For large-scale antibody production, immunoglobulin heavy and light chain genes were inserted into pET-IgG and pET-IgK vectors by target-selective homologous recombination cloning as described previously [19]. Transient transfection of the plasmids into CHO-S cells was performed with the CHOgro High Yield Expression System (Takara Bio). Recombinant mAbs were purified from the CHO cell culture medium by using MabCaptureC™ Protein A chromatography resin (ThermoFisher Scientific). The purified antibodies were labeled with either alkaline phosphatase or biotin by using Alkaline Phosphatase Labeling Kit-SH or Biotin Labeling Kit -NH2 according to the manufacturer’s instructions, respectively (Dojindo).
ELISA
Each well of 96-well plates (Corning) immobilized with 15 ng of COV-2-NP or COV-NP were blocked with PBS containing 1% bovine serum albumin (BSA). Crude or purified antibodies were added to the plates, incubated at 25°C for 120 min to allow the antibody to react with the antigen, and then washed three times with PBST. Horseradish peroxidase-labeled anti-guinea pig antibody was added to the plates and incubated at 25°C for 60 min. The plates were washed three times with PBST and developed with TMB (SURMODICS). The reaction was stopped with 1 N sulfuric acid, and the absorbance values at 450 nm and 620 nm were measured. PBS was used as a blank control.
Cell Elisa
A plasmid encoding the N-terminal domain or C-terminal domain of CoV-2-NP was constructed by PCR using pCMV-CoV-2-NP as a template. Each plasmid was transfected into HEK293 cells using FuGENE 6 Transfection Reagent (Promega) and cultured for two days. After fixation with PBS containing 4% paraformaldehyde and permeabilization with PBST, cells were stained with anti-CoV-2-NP mAb and anti-Myc mAb, and signals were developed with goat anti-guinea pig IgG DyLight 647 and goat anti-mouse IgG DyLight 488. Images were captured with an Operetta High Content Imaging System (PerkinElmer) or a BZ-X700 All-in-one fluorescence microscope (KEYENCE).
Peptide Microarray Analysis Of The Linear Epitopes
Epitope mapping of mAbs against CoV-2-NP was performed by PEPperMAP peptide microarray F-PEP-CEM-3 (Filgen). The CoV-2-NP sequence was converted into 7, 10 or 13 amino acid peptides with peptide-peptide overlaps of 6, 9 or 12 amino acids. Microarrays containing 672 different CoV-2-NP peptides were incubated with the purified mAb (10 µg/ml) for 16 h at 4°C with orbital shaking at 140 rpm, and the signal was developed by goat anti-guinea pig IgG (H + L) DyLight 680. Images were captured with an Innopsys InnoScan 710-IR microarray scanner.
Surface Plasmon Resonance (Spr) Analysis
All SPR experiments were performed with a Biacor T100 (Cytiva). Biotinylated CoV-2-NP was immobilized on streptavidin SA sensor chips (Cytiva). Briefly, two flowcells were prepared, one of which served as a negative control, while biotinylated CoV-2-NP was injected into the other to obtain an immobilization level of 150 response units (RU) at a flow rate of 30 µl/min. The interaction assays involved injections of 5 different dilutions of mAb (0, 0.3, 1, 3, 9 nM), followed by a 3 min washing step with HBS-EP + buffer at a flow rate of 30 µl/min to induce the dissociation of the complexes formed. At the end of each cycle, the sensor chip surface was regenerated by injection of 0.1 M citric acid, pH 3. The sensorgrams corresponding to the CoV-2-NP signal were subtracted from the negative control. The single cycle kinetic curves were fitted to a bivalent analyte model to estimate the association (kon), dissociation (koff) rate and equilibrium dissociation constants (KD).
Sandwich Assay And Pocube Setting
Biotin-labeled anti-SARS-CoV-2 capture mAb (50 ng) and alkaline phosphatase-labeled anti-SARS-CoV-2 detection mAb (3.2 ng) were mixed with different dilutions of recombinant CoV-2-NP in 65 µL of sample buffer (Tris-HCl buffer containing 0.1% BSA, pH8.5) to form an immune complex. The immune complex was captured on a filter on which an anti-biotin goat polyclonal antibody was immobilized. The filter was then washed with 160 µL of a washing solution (MOPS buffer containing 0.05% Tween 20,pH7.2), and a luminescence signal was produced by adding a chemiluminescent substrate for alkaline phosphatase (APS-5, Lumigen) to the filter. The signal was measured using a photomultiplier tube settled in POCube. Sample buffer was used as a blank control.
Statistical analysis
The limit of detection (LOD) was determined as the mean value of the negative controls (n = 10) plus 3.3 times the standard deviation. The limit of quantitation (LOQ) was determined as the mean value of the negative controls (n = 10) plus ten times the standard deviation.