1. Alpaca Immunization


Immunization Using Trimeric Spike Protein as Antigen

 

Immunization Protocol Lasting 46 Days

 

Dilution Ratio Post-Immunization Serum Pre-Immunization Serum
 500 1.684 1.694 1.716 0.105 0.142 0.08
1500 1.508 1.538 1.581 0.072 0.085 0.083
4500 1.186 1.178 1.231 0.107 0.093 0.056
13500 0.75 0.741 0.747 0.095 0.087 0.064
40500 0.322 0.312 0.353 0.088 0.057 0.065
121500 0.13 0.133 0.138 0.074 0.074 0.051
364500 0.071 0.075 0.085 0.067 0.063 0.056
1093500 0.071 0.08 0.075 0.063 0.071 0.088

 

 

  1. Construction of Phage Display Library

Library Diversity: 5.2 × 10^8 Colony-Forming Units (cfu)

 

VHH Insertion Rate: 100%

 

  1. Screening and Identification of Positive Monoclonal Nanobody-expressing Microbes through Phage Surface Display

 

  • A total of approximately 400 monoclonal microbes were selected and identified.
  • The positivity rate determined through ELISA was approximately 50%.
  Sanger Sequencing

 

Approximately 150 monoclonal nanobodies were obtained through Sanger sequencing.

 

 

 

  1. Recombinant Expression, Purification, and Testing of Positive Monoclonal Nanobodies
 
 
 
 
 

产量约1-10 mg纳米抗体/L细菌

 

4 4

 

  1. Identification of Antibody Binding Epitopes

Epitope

Number of Epitopes

NTD

23株

RBD

42株

NTD & RBD

15株

S2 subunit

17株

未知

17株

合计

114株

 

  1. Neutralizing Antibody Detection

A total of 8 neutralizing antibodies against the novel coronavirus were obtained.

6 2

Wild-type

6 3

South African Variant