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 single domain antibody 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

  • VHH 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
    Single Domain Antibody Screening
  • 4
    Single Domain Antibody Expression & Purification
  • 5
    Single Domain Antibody 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 Construction

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



Yeast Display Library Construction

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




Customized VHH Antibody Discovery Service - Phage Display Method

ServiceProgressDliverableTime
Alpaca ImmunizationAntigen emulsification;
Animal immunization;
Blood collection;
PBMC isolantion		Immunization report~ 2 month
Library Construction
Isolation of total RNA;
cDNA synthesis;
VHH fragment amplification;
Plasmid construction;
Phage library amplification
and purification		Phage library
All the original data
Project report
~ 1.5 month
Phase Display
        &
Panning
Antigen immobilization;
Phase selection by 3 rounds of
panning
Project report~ 2 weeks
Screening and
Characterization
Phase clone screening by ELISA;
VHH DNA sequencing
VHH sequences
Project report		~ 1 month



Customized VHH Antibody Discovery Service - Yeast Surface Display Method

ServiceProgressDliverableTime
Alpaca ImmunizationAntigen emulsification;
Animal immunization;
Blood collection;
PBMC isolantion		Immunization report~ 2 month
Library Construction
Isolation of total RNA;
cDNA synthesis;
VHH fragment amplification;
Plasmid construction
Phage library
All the original data
Project report
~ 2 weeks
Yeast Display Library
Construction		Yeast transformation and library
generation		Yeast display library;
Project report
~ 2 weeks
Panning by MACS and
FACS		Magnetic selection followed by
FACS for cell sorting
Project report~ 1 month
Screening and Analysis
of Selected Clones		Individual yeast clone analysis;
Sequencing
VHH sequences;
Project report		~ 1 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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