Nanobody Preparation

Nanobody Service Introduction

• Nanobodies are superior to traditional antibodies in many ways. Based on the unique structure of VHH single-domain antibodies derived from camelid heavy chain antibodies, nanobodies combine the advantages of traditional antibodies and small molecule drugs. They almost perfectly overcome the shortcomings of long development cycles, low stability, and the strict storage conditions of traditional antibodies. Nanobodies are gradually emerging as a new force in the development of therapeutic biopharmaceuticals and clinical diagnostic reagents.
  Compared to conventional antibodies, nanobodies have the following advantages:
  Small molecular weight, capability of penetrating the blood-brain barrier; High expression in prokaryotic or eukaryotic systems; strong specificity and high affinity; Strong specificity and high affinity; Weak immunogenicity in humans, etc.

M13 Phage Display Technology

• Phage display technology is a method of expressing exogenous peptides or proteins by inserting their encoding genes into the appropriate location of the phage coat protein structural gene using genetic engineering techniques. The exogenous peptides or proteins can form fusion proteins on the coat protein of the phage during correct expression in the reading frame, and are presented on the surface of the phage along with progeny phages upon the reassembly of the phage.
  Then, using the target molecule, an appropriate panning method is used to wash away the non-specifically bound phage. The bound phages are then eluted with acid-base or competing molecules, and the neutralized phages are amplified by infecting Escherichia coli. After multiple rounds of enrichment, the proportion of phages that can specifically recognize the target molecule is gradually increased, eventually yielding peptides or proteins that recognize the target molecule.

Construction of Phage Library and Selection of Nanobodies

• Medicilon provides unimmunized, full-age (24-36 months old) camels for antigen immunization. According to the standard immunization methods, each item is immunized with 4-6 injections. During the immunization period, ELISA titer determination services are provided to ensure that the anti-serum meets the qualified ELISA titer before blood collection and PBMC separation.
  Medicilon uses the pIII protein of M13 phage for surface display of VHH antibodies (nanobodies). Our scientists constructed a phagemid vector with double tags of HA and 6*His tag, and prepared a competent TG1 host strain with a transfection efficiency of 10⁸. After 2-3 rounds of transfection and amplification, it can be used for high-quality VHH antibody display libraries with an effective library capacity greater than 10⁸. After several rounds of nanobody library panning, at least 20 phage strains with high affinity can be obtained.

Comparison of Camel and Alpaca VHH Antibody Libraries

• Comparison Item	Camel VHH Antibody Library	Alpaca VHH Antibody Library
  Antigen tolerance	High, multiple antigens can be mixed with high-dose immunization	Higher, multiple antigens can be mixed with medium immunization dose
  HCAb serum globulin ratio	60%~80%	20%~30%
  Neutralizing antibody titer	High	High
  Blood collection volume	200ml blood can be collected at one time	80-100ml blood can be collected at one time
  Effective storage capacity	High, immune library 108-109	High, immune library 107-108
  Animal cost	Lower	High
  Antigen requirement	4-6mg, purity>85%	4-5mg, purity>85%

Schematic diagram of nanobody preparation service process

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  Image source: MA Lin-lin, et al. Construction and screening of phage display library for TIM-3 nanobody. Acta Pharmaceutica Sinica2018, 53 (3): 388-395.

Camelidae nanobody library preparation project process

• Step	Content	Cycle	Delivery
  Step 1: Antigen preparation	The customer provides the antigen or Medicilon prepares the antigen	3-4 weeks	If the customer provides the antigen, the quality standard: 1. >5mg; 2. SDS-PAGE >95%
  Step 2: Animal immunization	Immune unvaccinated camels 5 ml unimmunized serum ELISA test serum titer	10-12 weeks	If the animal dies due to antigen toxicity, a certain fee will be charged According to the ELISA results, client decides how to build the library
  Step 3: Antibody cDNA preparation	Peripheral blood isolation of PBMC Extract total RNA and reverse transcribe into cDNA	3-4 weeks	Provide PBMC stored in Trizol Provide cDNA prepared by high-fidelity RT-PCR
  Step 4: Phage library construction	Construction of phagemids Phage library construction		VHH fragment insertion rate ≥75% Effective library capacity≥108CFU/mL
  Step 5: Phage library panning	Phage display nanobody, panning, at least two rounds of panning ELISA verification, identification, screening and sequencing of positive clones (200 clones were sequenced) Sequencing of positive clones and analysis of sequencing results	4-6 weeks	At least 20 sequences are guaranteed

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