The development of a highly sensitive and quantitative SARS-CoV-2 rapid antigen test applying newly developed monoclonal antibodies to an automated chemiluminescent flow-through membrane immunoassay device

Abstract

Background: Rapid and accurate diagnosis of individuals with SARS-CoV-2 infection is an effective way to prevent and control the spread of COVID-19. Although the detection of SARS-CoV-2 viral RNA by RT-qPCR is the gold standard for COVID-19 testing, the use of antigen-detecting rapid diagnostic tests (Ag-RDTs) is emerging as a complementary surveillance tool as Omicron case numbers skyrocket worldwide. However, the results from Ag-RDTs are less accurate in individuals with low viral loads.

Results: To develop a highly sensitive and accurate Ag-RDT, 90 monoclonal antibodies were raised from guinea pigs immunized with SARS CoV-2 nucleocapsid protein (CoV-2-NP). By applying a capture antibody recognizing the structural epitope of the N-terminal domain of CoV-2-NP and a detection antibody recognizing the C-terminal tail of CoV-2-NP to an automated chemiluminescence flow-through membrane immunoassay device, we developed a novel Ag-RDT, CoV-2-POCube. The CoV-2-POCube exclusively recognizes CoV-2-NP variants but not the nucleocapsid proteins of other human coronaviruses. The CoV-2-POCube achieved a limit of detection sensitivity of 0.20 ~ 0.66 pg/mL of CoV-2-NPs, demonstrating more than 100 times greater sensitivity than commercially available SARS-CoV-2 Ag-RDTs.

Conclusions: CoV-2-POCube has high analytical sensitivity and can detect SARS-CoV-2 variants in 15 min without observing the high-dose hook effect, thus meeting the need for early SARS-CoV-2 diagnosis with lower viral load. CoV-2-POCube is a promising alternative to currently available diagnostic devices for faster clinical decision making in individuals with suspected COVID-19 in resource-limited settings.

Keywords: COVID-19; Lateral flow immunochromatography; Monoclonal antibody; Omicron; Point-of-care test; Rapid antigen test; SARS-CoV-2.

Fig. 1

Single-cell-based development of anti-CoV-2-NP mAbs. a FACS gating strategy for the isolation of Wuhan CoV-2-NP-specific plasma cells. Plots (I)–(IV) represent the sequential gating strategy. (I) FSC vs. SSC with gate R1 represents lymphocytes. (II) Single cells were selected via DAPI staining (R2). (III) Cells labeled with CoV-NP were excluded from the R3 gate. (IV) The CoV-2-NP^High CoV-NP^Low and anti-guinea pig IgG^High fraction was defined as Wuhan CoV-2-NP-specific plasma cells (R4 gate). Percentages show the content of each cellular subpopulation among the total number of lymph node cells. b Representative agarose gel electrophoresis of the cognate pairs of immunoglobulin heavy chain variable (VH) and light chain kappa variable (VL) genes amplified from the single-cell-sorted R4-gated cells in (A). Line M: 1 kb DNA ladder (New England Biolab). Arrowheads indicate the size of the amplified DNA fragments. c The antigen specificity of the mAbs produced from the R4-gated cells. Cognate pairs of immunoglobulin heavy and light chain genes were cotransfected into HEK293 cells, and the CoV-2-NP binding activities of the expressed mAbs (5 ng) were analyzed by ELISA. The red line indicates the cutoff line for the screening

Fig. 2

The selection of appropriate detection and capture antibodies using a sandwich assay. a Reactivity and specificity of the candidate mAbs. Forty candidate mAbs were tested with either Wuhan CoV-2-NP or CoV-NP to evaluate cross-reactivity by ELISA. Data are expressed as the mean of two measurements. b Pairwise analysis of mAbs against Wuhan CoV-2-NP. Sandwich assay was used for pairwise coupling of thirteen capture mAbs against sixteen detection mAbs. The numbers inside the grid are the luminescence activities. The heatmap represents the performance of antibody pair. Red, > 80,000; orange, 80,000–60000; green, 59,999–40,000; blue, 39,999–20,000; gray, not determined. Data are expressed as the mean of two measurements. The highest luminescence was considered as the optimal mAb set

Fig. 3

Epitope mapping of mAbs. a Antibody epitope analysis of Wuhan CoV-2-NP by cell ELISA. HEK293 cells expressing either the full-length (Full), N-terminal domain (NTD) or C-terminal domain (CTD) of myc-tagged Wuhan CoV-2-NP were stained with the indicated mAbs. Representative florescence images are shown. b Epitope mapping of #1 using solid-phase peptide arrays. Solid-phase supports containing synthetic peptides spanning the C-terminus of Wuhan CoV-2-NP were screened for reactivity with #1. Major epitopes were defined as having greater than ten times the reactivity of guinea pig IgG. c Schematic diagram of the CoV-2-NP domain. Each mAb epitope determined by peptide array and/or immunostaining is shown with a red bar. d Epitope residues of #1 on CoV-2-NPs of SARC-CoV-2 variants and NPs of other human coronaviruses. Epitope residues that are conserved are shown in black, and those that are not conserved are shown in red. e Cross-reactivity of mAbs with CoV-2-NPs of SARS-CoV-2 variants. The indicated mAb was reacted with a series of CoV-2-NPs immobilized on plates, and the binding was analyzed by ELISA. The results are expressed as the mean ± SD of four replicates relative to Wuhan CoV-2-NP. f Cross reactivity of #1 and #31 with NPs of human coronaviruses. The indicated mAb was reacted with a series of NPs immobilized on plates, and the binding was analyzed by ELISA. The results are expressed as the mean of two replicates

Fig. 4

Antibody affinity determination by SPR

Fig. 5

Determination of the technical sensitivity of CoV-2-POCube. a A schematic of CoV-2-POCube. Immunocomplex formation in the test sample followed by the sample loaded into a reaction vessel (I). Immunocomplex trapping on an anti-biotin antibody-coated membrane followed by washing to remove contaminants (II). Luminescence signal measurement (III). Reaction time requires 10 min for (I) and 5 min for (II) ~ (III), totaling 15 min. b The calibration curve of CoV-2-POCube for CoV-2-NP detection. Serially diluted recombinant CoV-2-NP of Delta or Omicron variant were subjected to CoV-2-POCube analysis. c COV-2-POCube signal intensity of serially diluted heat-inactivated Wuhan SARS-CoV-2 particles. Each dilution was analyzed for CoV-2-NP using a COV-2-POCube. d Correlation between COV-2-POCube signal and CoV-2-NP concentrations. COV-2-POCube signal intensity of serially dilute Delta CoV-2-NPs are shown. All data are presented as the mean ± SD of three replicates