SARS-CoV-2 Specific Human Neutralizing Monoclonal Antibody Development Service

Phase I: ELISA screening of human polyclonal sera. The human positive respone to the antigen is critical to isolate high specificity and high affinity human monoclonal antibodies.Phase II: PBMC isolation, detection and isolation of antigen specific memory B cells by flow cytometry-based single B cell sorting, culturing single B cells in individual wells to secret antibodies, high throughput ELISA screening to isolate antigen-positive B cells. Supernatant (culture media) from ELISA-positive clones is sent to clients for evaluation of specific applications.Phase III: Cloning and sequencing the antibody variable regions (VH and VL) from antigen specific B cellsselected by clients, and transient production of recombinant human or monkey monoclonal antibodies in HEK 293 cells. Purified human or monkey monoclonal antibodies are sent to clients for confirmation and test of specific applications.You provide: human polyclonal sera and PBMC, and the antigen.Deliverable* About 80 ul supernatant from ELISA positive cells for evaluation;* About 100 ug purified human monoclonal antibodies for evaluation;* Amplified DNA fragments;* Remaining antigens;

* Study report including ELISA results and antibody VH and VL sequences.

Background

The virus causing Coronavirus Disease 2019 (COVID-19) was named 2019 novel coronavirus (2019-nCoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are four major structural proteins in SARS-CoV-2, spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.

The SARS-CoV-2 spike (S) glycoprotein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of virus entry inhibitors, neutralizing antibodies, and vaccines. There are two subunits, S1 and S2, in the SARS-CoV-2 spike (S) glycoprotein. The furin cleavage site at the boundary between the S1/S2 subunits is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. The S1 subunit, the N-terminal 14-685 amino acids of S protein, contains N-terminal domain (NTD), receptor binding domain (RBD), and receptor binding motif (RBM). The C-terminal S2 subunit contains fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), transmembrane domain (TM), and cytoplasmic domain (CP).

Although the S protein, its S1 subunit, and RBD are the most popular targets of antibodies for diagnostics test and proteins and antibodies for therapeutic drugs. The other virus proteins are potential targets for development of neutralizing monoclonal antibodies. The PBMC of recovered patients or people immunized with vaccines are great sources to screen human neutralizing monoclonal antibodies using the single B cell method.