A novel high-throughput single B-cell cloning platform for isolation and characterization of high-affinity and potent SARS-CoV-2 neutralizing antibodies

Abstract

Monoclonal antibodies (mAbs) that are specific to SARS-CoV-2 can be useful in diagnosing, preventing, and treating the coronavirus (COVID-19) illness. Strategies for the high-throughput and rapid isolation of these potent neutralizing antibodies are critical toward the development of therapeutically targeting COVID-19 as well as other infectious diseases. In the present study, a single B-cell cloning method was used to screen the Wuhan-Hu-1 strain of SARS-CoV-2 receptor-binding domain (RBD) specific, high affinity, and neutralizing mAbs from patients' blood samples. An RBD-specific antibody, SAR03, was discovered that showed high binding (ELISA and SPR) and neutralizing activity (competitive ELISA and pseudovirus-based reporter assay) against the Wuhan-Hu-1 strain of SARS-CoV-2. Mechanistic studies on human cells revealed that SAR03 competes with the ACE-2 receptor for binding with the RBD domain (S1 subunit) present in the spike protein of SARS-CoV-2. This study highlights the potential of the single B cell cloning method for the rapid and efficient screening of high-affinity and effective neutralizing antibodies for SARS-CoV-2 and other emerging infectious diseases.

Keywords: ELISA; RBD domain; SARS-CoV-2; SPR; Single B-cell cloning; mAbs.

Fig. 1

Screening of neutralizing antibodies against SARS-CoV-2 in patient's plasma through ELISA. (A) Determination of antibody affinity through ELISA. The patient's plasma was incubated in ELISA plates coated with recombinant RBD protein followed by detection with anti-Human-HRP antibody. (B) Schematic of competitive ELISA for checking neutralizing activity. C, Screening for neutralizing activity of antibodies present in patient's plasma. Results are represented as Mean ± SD (n = 3). ***P < 0.001 on comparison of patient's plasma with healthy donor plasma.

Fig. 2

Isolation of RBD-specific memory B cells using flow cytometry. Live IgG+ (FITC conjugated) and RBD+ (PE-conjugated) cells were selected using single B-cell sorting and sorted as single cells in a 96-well plate.

Fig. 3

Method of cloning antibody heavy and light chain sequences. (A) Schematic of RT-PCR and PCR amplification of the variable region of heavy and light chain region of the antibody. (B) RT-PCR and 2-step PCR were carried out to amplify the variable region of heavy and light chain sequences along with their natural leader sequence. Amplified sequences were cloned into a pAb20-hCHIgG1 plasmid with hygromycin (for Gamma heavy chain) and pAb20-hCK plasmid with blasticidin (for kappa light chain).

Fig. 4

Binding analysis and neutralization activity of 12 antibody clones. (A) Cell culture supernatant from the cells transfected with heavy and light chain expressing plasmid was added to ELISA plates coated with recombinant RBD at 1/10 dilution. Anti-Human-HRP secondary antibody was used for ELISA detection. (B) Neutralization activity of cell culture supernatant was determined through competitive ELISA. Results are represented as Mean ± SD (n = 3). ***P < 0.001, **P < 0.01 on comparison cell culture supernatant with media control.

Fig. 5

Determination of neutralization activity of top clones in pseudovirus reporter assay and affinity through SPR. (A) ACE-2 overexpressing cells were treated with SARS-CoV-2 pseudovirus in presence of different concentrations of purified antibodies. Luciferase activity was used as a measure of neutralizing activity. (B) SAR03 affinity against RBD was determined through SPR displayed on sensorgram plotted binding response (Response Unit; Ru) vs time (s).