1.Methods for Nanobody Preparation

Nanobodies, also known as single-domain antibodies (sdAbs), are developed differently from traditional monoclonal antibodies produced via hybridoma technology. Typically, nanobodies are generated by immunizing alpacas, constructing a phage display library, and screening for target-specific nanobodies through phage display technology. After expression and purification, the nanobodies undergo binding validation to confirm their interaction with the target antigen.

Single-domain antibodies are commonly obtained by immunizing alpacas. Following immunization, the alpaca’s immune system undergoes natural antibody maturation. B lymphocytes are then isolated, and RNA is extracted. Through reverse transcription, cDNA is synthesized, which serves as the template for PCR amplification to obtain diverse nanobody gene fragments. These fragments are subsequently cloned into a phagemid vector to construct a phage library. Phage display screening is then employed to isolate suitable antibodies, followed by nanobody validation. The entire process primarily includes alpaca immunization, phage library construction, antibody screening, expression and purification, and functional validation.

Figure 1: Schematic diagram of nanobody generation

2.Nanobody Production Workflow

2.1 Alpaca Immunization Process

Antigen preparation;Alpaca immunization;Blood collection; Serum separation;Lymphocyte isolation

2.2 Phage Library Construction Process:

RNA extraction;cDNA reverse transcription;Antibody fragment amplification;PCR amplification;Cloning into phagemid vector;TG1 transformation;Phage library amplification and purification

Figure 2: Electrophoresis Analysis of RNA Extraction

2.3 Antibody Screening and Identification Process：

Coating immunotubes;Blocking;Antigen-phage incubation;Washing;Elution;ELISA validation

2.4 Antibody Expression and Functional Verification

Expression: Establish mammalian expression systems for antibody production. KMD Bioscience has developed a comprehensive mammalian expression platform, utilizing cell lines including but not limited to FreeStyle™ 293-F and ExpiCHO-S™, combined with our proprietary high-expression vectors (featuring full-length CMV promoters and optimized secretion signal peptide sequences) for recombinant antibody expression and production.

Figure 3: Expression Results of Fc-VHH Nanobody

Purification: The nanobodies were purified using a nickel column.

Affinity measurement: The purified nanobodies were subjected to Biacore affinity analysis.