Monoclonal Antibodies against Nucleocapsid Protein of SARS-CoV-2 Variants for Detection of COVID-19

Abstract

Mitigation strategies of the coronavirus disease 2019 (COVID-19) pandemic have been greatly hindered by the continuous emergence of SARS-CoV-2 variants. New sensitive, rapid diagnostic tests for the wide-spectrum detection of viral variants are needed. We generated a panel of 41 monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (NP) by using mice hybridoma techniques. Of these mAbs, nine exhibited high binding activities and were applied in latex-based lateral flow immunoassays (LFIAs). The LFIAs utilizing NP-mAb-7 and -40 had the best sensitivity and lowest limit of detection: 8 pg for purified NP and 625 TCID₅₀/mL for the authentic virus (hCoV-19/Taiwan/4/2020). The specificity tests showed that the NP-mAb-40/7 LFIA strips did not cross-react with five human coronavirus strains or 20 other common respiratory pathogens. Importantly, we found that 10 NP mutants, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2) variants, could be detected by NP-mAb-40/7 LFIA strips. A clinical study (n = 60) of the NP-mAb-40/7 LFIA strips demonstrated a specificity of 100% and sensitivity of 90% in infected individuals with cycle threshold (Ct) values < 29.5. These anti-NP mAbs have strong potential for use in the clinical detection of SARS-CoV-2 infection, whether the virus is wild-type or a variant of concern.

Keywords: COVID-19; LFIA; SARS-CoV-2; antibody; nucleocapsid protein; rapid test.

Figure 1

Generation of mAbs against SARS-CoV-2 NP. (A) Binding activity of 41 anti-NP mAbs was performed by ELISAs that were coated in recombinant NP-His protein (2 μg/mL). Each anti-NP mAb was serially diluted from 500 to 20 ng/mL. (B) Thirty-four anti-NP mAbs were used as the primary antibody for NP-His immunoblots. Anti-6 × His mAb was used as a positive control. (C) Comparative ELISAs were performed to evaluate the binding activities of mAbs against NP-His. Recombinant NP-His (0.5 μg/mL) was coated on the ELISA plates. Normal mouse IgG (NMIgG) was used as a negative control. (D) The binding activity of nine mAbs against recombinant full-length NP, the RNA-binding domain (RBD) of NP, and dimerization domain (DD) of NP by ELISA. (E) NP-expressing Ver-E6 cells were probed with 1 μg/mL anti-NP mAbs and then stained with FITC goat anti-mouse IgG. NP expression was confirmed by staining with anti-6 × His Ab and rhodamine fluorescence detection. Abbreviation: NTD, N-terminal domain; SR rich, Serine/Arginine-rich region; CTD, C-terminal domain.

Figure 2

Detection limit of two LFIAs using different capture mAbs. (A) Schematic diagram of the lateral flow rapid test configuration. (B) Binding activities of anti-NP mAb-7, -40, and -53 to NPs from seven types of human coronaviruses (HCoV). Each recombinant NP-His was coated at the same concentration on the ELISA plates. Anti-NP mAbs were added at 100 ng/mL to the probe antigens. (C) SARS-CoV-2-infected Vero E6 cells were collected in a lysis buffer, and the NP concentration was determined by sandwich ELISA. The samples were diluted to given concentrations and analyzed by two antigen rapid tests with different capture mAbs. The rating chart showed an intensity range from 0 to 5 grading of the positive and negative results on the test line. A color intensity ≥ 0.5 was judged as a positive result. A color intensity < 0.5 was judged as a negative result. (D) Authentic SARS-CoV-2 virus concentrations ranging from 0 to 5000 TCID₅₀/mL were used to detect the sensitivities of two antigen rapid tests. The numbers in red indicate the limits of detection (LODs).

Figure 3

Specificity of NP mAb-40/7 LFIA strips. (A) Detection of viral NP of human coronavirus strains was performed by loading 10 ng of the indicated recombinant protein onto NP mAb-40/7 LFIA strips, followed by a 10-min reaction time. (B) The indicated recombinant SARS-CoV-2 NP variants were loaded onto NP mAb-40/7 LFIA strips at 1 ng. Mutations of D3L, R203K, G204R, and S235F have been identified in lineage B.1.1.7 (lane 2). A mutation of T205I was reported in lineage B.1.351 (lane 3); P80R was identified in lineage P.1 (lane 4); and D63G, R203M, D377Y, and R385K were found in lineage B.1.617.2 (lane 5). NC indicates the negative control. (C) NP mAb-40/7 LFIA strips were used to detect clinically identified alpha variant lineage B.1.1.7 and epsilon variant B.1.2 by loading the indicated viral titer (PFU/mL). NC indicates the negative control. (D) Several respiratory associated viruses were not detected by NP mAb-40/7 LFIA strips. SARS-CoV-2 served as a positive control (lane 1). The detecting concentration of the indicated viruses was 10⁵ copies/reaction of SARS-CoV-2, adenovirus type 5, rhinoviruses A40, respiratory syncytial virus type B1, parainfluenza virus type 3, and influenza A virus H1N1. Influenza B virus utilized 10⁶ copies/reaction for detection.

Figure 4

Clinical verification of the antigen rapid tests. (A) NP mAb-40/7 LFIA strips were used to probe five frozen samples with Ct values between 14.6 and 23.4 for the viral E gene. (B) NP mAb-40/7 LFIA strips were used to probe ten freshly collected positive patient samples with Ct values between 16 and 29.5 for the viral E gene. Days* means the days after onset to sampling. (C) Recognition sites of anti-NP mAbs on SARS-CoV-2 NP. SARS-CoV-2 NP contains 419 amino acid residues (a.a.). Each domain length is shown underneath the illustration. Amino acid mutations in SARS-CoV-2 NP are labeled for the different variants of concern (VoCs). The binding domains of the anti-NP mAb we studied are listed in the low panel.