Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein

Abstract

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM K D , as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 10 3 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.

Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies

“…Two recent studies reported the isolation of single-domain antibodies (sdAbs) that recognize the SARS-CoV-2 N protein (Figures 1A, B) (18,19). One study used an in vitro selection method to isolate one sdAb (here termed sdAb-N3; Supplementary Table 1) that recognizes the N protein with high affinity (EC 50~5 0 nM as measured by luciferase-based ELISA assay) (19).…”

“…One study used an in vitro selection method to isolate one sdAb (here termed sdAb-N3; Supplementary Table 1) that recognizes the N protein with high affinity (EC 50~5 0 nM as measured by luciferase-based ELISA assay) (19). The second study isolated three sdAbs (here termed sdAb-B6, sdAb-C2, and sdAb-E2; Supplementary Table 1) from an immunized llama, which each recognize the N protein with extremely high affinity (K d of 0.8-1.6 nM as measured by surface plasmon resonance) (18). The four sdAbs show no obvious similarity in their three variable complementaritydetermining regions (CDRs; Figures 1B, C), suggesting that they likely each recognize distinct epitopes on the N protein.…”

“…Two recent studies reported the isolation of a total of four single-domain antibodies (sdAbs) that target SARS-CoV-2 N with high affinity (18,19). Here, we map the epitopes of these sdAbs and show that each sdAb recognizes a specific domain of SARS-CoV-2 N. We report high-resolution crystal structures of two sdAbs bound to the N RBD, and of one sdAb bound to the N CTD.…”

“…Vectors encoding sdAbs B6, C2, and E2 in pET22b were generously provided by Ellen Goldman (18). These vectors encode each sdAb with a pelB leader sequence at the N-terminus for periplasmic expression, and a His 6 -tag at the C-terminus for purification (Supplementary Table 1).…”

“…(A) Domain schematic of SARS-CoV-2 N, with the RNA-binding domain in magenta, the dimerization domain in blue, and the three intrinsically disordered domains (N-IDR, serine/arginine rich (SR)-IDR, and C-IDR) in white. (B) Sequences of four sdAbs targeting the SARS-CoV-2 N protein(18,19) showing the variable sequences within the Complementarity-Determining Regions (CDRs). (C) Example sdAb structure (sdAb-B6 shown) with complementarity-determining regions (CDR) 1, 2, and 3 colored blue, green, and orange, respectively.…”