Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy

Xin Zeng; Lingfang Li; Jing Lin; Xinlei Li; Bin Liu; Yang Kong; Shunze Zeng; Jianhua Du; Huahong Xiao; Tao Zhang; Shelin Zhang; Jianghai Liu

After the first round of the standard biopanning, a competitive biopanning protocol that included steps of competitive binding, magnetic separation, elution and amplification ( Fig.1) , was applied to isolate the epitope-specific antibodies. Briefly, 100μl of ACE2-hFc protein (5μg/ml) was coated on the 96-well Maxisorp plates. The wells were washed and blocked with 1% PVA, and then the mixture of antibody library (10 10 pfu per well) and free RBD-His protein (100ng per well) was added. After a 2-hour competitive binding, the supernatant was transferred into a 1.5ml microfuge tube containing the pre-washed Ni-NTA magnetic beads (GenScript) and incubated on a shaker at room temperature for 1 hour. Beads were collected using the magnetic separation rack and washed by the PT buffer for 8 times. Bound phages were eluted with 100mM HCl (100μl per tube) after 5-min incubation. Beads were collected using the magnetic separation rack, and the supernatant was transfer into a tube for neutralization. Half the neutralized phage solution was mixed with 1ml of actively growing NEB alpha F' cells and amplified as the standard biopanning protocol. 10μl of the bacterial culture before infection with helper phages was taken, diluted, and grown on the LB plates containing 50μg/ml carbenicillin at 37°C overnight. The single clones were picked up next day for the phage ELISA assay. Fig.1 Schematic presentation of a competitive biopanning strategy. A specific binder of target protein was added during the binding step for the selection of blocking antibodies. In this work, the immobilized ACE2-hFc captured RBD-His and the antibodies binding RBD at different epitopes, forming a complex like a "sandwich". However, when an antibody recognized the same or similar epitopes within RBD as the ACE2 did, it could block RBD-ACE2 interaction. The antibodies would bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads.