Single Domain Antibody

Single Domain Antibody

Alpaca heavy chain antibody (HcAb) is a naturally occurring special antibody composed of only two heavy chains, including only one heavy chain variable domain (VHH in Antibody) and two conventional CH2 and CH3 domains . Alpaca heavy chain antibodies specifically bind to antigens through a variable domain (VHH) on the heavy chain, which can exist stably in vitro alone, named nanobody or VHH. The nanobody crystal has a diameter of 2.5nm and a length of about 4nm. The molecular weight of the nanobody is only 1/10 (about 15KD) of the conventional antibody, but it has complete antigen recognition ability.



Advantages of Single Domain Antibody

  • 1
    High stability
    Small batch to batch variation
    Suitable for industrial production
  • 2
    Higher Tissue Penetration
  • 3
    Suitable for modification
  • 4
    High Specificity
  • 5
    Low immunogenicity Easy humanization

Applications of Single Domain Antibody

  • Bispecific and Multispecific Antibody Drugs

  • Nanobody ADC Drugs

  • Immune Cell Therapy (CAR-T)

  • Molecular Imaging

  • Precise mRNA delivery

  • Affinity Chromatography

  • Target transferrin receptor (TfR) or insulin receptor (IR)
    to assist drugs to cross the blood-brain barrier (BBB)

Alpaca Immunization

  • 1
    Alpaca lmmunization
  • 2
    Library Construction
  • 3
    Nanobody Screening
  • 4
    Nanobody expression & purification
  • 5
    nanobody characterization
  • 6
    Humanization& affinity maturation

Alpaca Base

Immunization Service Process

  • Preparation of antigen emulsions:

    An equal volume of Freund's adjuvantwas added to emulsify the antigen.

  • Immunization and blood collection:

    lmmunization site: the left and right sides near the lymph nodes in the neck.


    Blood collection site: jugular vein of alpaca.

  • lsolation of PBMCs:

    Collect 50 mL of anticoagulated blood to separate peripheral blood mononuclear cells (PBMCs) and store them in RNAisoPlus.

Library Construction

AlpalifeBio has rich experiences in the construction of VHH libraries,
and adopts optimized primers to ensure diverse high-quality phage display libraries.
  • 1
    Alpaca lmmunization
  • 2
    Library Construction
  • 3
    Nanobody Screening
  • 4
    Nanobody expression & purification
  • 5
    nanobody characterization
  • 6
    Humanization& affinity maturation

Phage Display Library Generation

Construct diverse VHH DNA sequences in phagemids, transform into TG1 competent cells, culture and induce VHH display on phage surface.



Yeast Library Construction

Insert diverse VHH DNA sequences into the display plasmid vector containing the protein scaffold-Aga2 gene, and transform into the competent yeast to prepare the yeast display library.


        


Technical Advantages

Extracted high quality RNA lays a solid foundation for library construction.
Optimized primers can ensure VHH library diversity.




Coustomized Nanobody Discovery Service

Service Progress Dliverable Time
Alpaca immunization Antigen emulsification
Animal immunization,
blood colletion
PBMC isolantion and preservation. 		Immunization report ~2 month
Library construction
 Isolation of total RNA
cDNA synthesis
PCR
Vector construction
Phage library amplification
and purification 		Phage library
All the original data
Project report
 ~1.5 month
Library screening Antigen presrntation &
phage selection by
panning

DNA sequencing
 Nanobody DNA
sequences

Plasmids
Project report
At least 20 sequences
guaranteed
 ~1.5 month

VHHs Screening

  • 1
    Alpaca lmmunization
  • 2
    Library Construction
  • 3
    Nanobody Screening
  • 4
    Nanobody expression & purification
  • 5
    nanobody characterization
  • 6
    Humanization& affinity maturation

VHH Phage Library Screening

① Screening process: Use immunotube panning or magnetic bead panning; detect the phage titer before and after each round of panning, and calculate the degree of enrichment.

② Single clones were randomly picked after multiple rounds of bio-panning of the library, verified by ELISA, and positive clones were sequenced. Or enriched phage library DNA can be used as template for PCR which will be used for NGS analysis.


        



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