Nanobody (VHH) Library Generation

Nanobody (VHH) Library Generation

Nanobody (sdAb or VHH) is a promising next-generation therapeutic antibody technology for cancer immunotherapy and other applications. Nanobodies are distinguished from other conventional antibody formats by their unique properties of size, solubility, intrinsic stability, easy tailoring into pluripotent constructs, recognition of uncommon or hidden epitopes, binding into cavities or active sites of enzyme targets, ease and speed of drug discovery, and ease of manufacture.

BioIntron is a well-recognized leader in the field of single domain antibody discovery. Based on our advanced phage display technology, our scientists are specialized in the production and discovery of specific single domain antibodies.

Our Advantages

Self-owned Alpaca Breeding Farm
Self-owned Alpaca Breeding Farm

(more than 100 Alpacas available)

Guaranteed 20+ Unique Binders
Guaranteed 20+ Unique Binders

(combined solid phase and liquid phase panning methods)

High Diversity And Large Capacity
High Diversity And Large Capacity

(multiple libraries generated after 2nd, 3rd, 4th immunization)

Nanobody (VHH) Library Generation Working Flowchart


Nanobody (VHH) Library Generation Working Flowchart

Nanobody (VHH) Library Generation Service Detail

Service StepService DescriptionTimelinePriceDeliverable
Phage I: Antigen preparation /Antigen validationAntigen can be prepared by BioIntron or provided by our client.2 weeks for preparation and 2-3 days for validation

Inquiry

1mg purified protein antigen.

If antigen provided by client, it should be at least 4mg with SDS-PAGE >95%, endotoxin level<1EU/mg

Phage II: Alpaca immunization and PBMC isolation /preservation

5ml pre-immune bleed 

the ELISA titer after 2nd ,3rd and 4th immunization will be monitored and the report will send to client 

the 50ml PMBC after 2nd ,3rd and 4th immunization will be extracted and stored in Trizol .

8-11 weeksInquiry

Alpaca's death caused by antigen, there will be setup fee about $5,000.

Decision gate: based on the ELISA titer on the 2nd ,3rd and 4th immunization. Our client can decide to use which cell for library generation

Phage 3: VHH Phage Library Generation

RNA extraction and cDNA preparation

VHH amplification

Electro competent E. coli

Library Diversity and capacity measurement

clone NO counting and sequencing to measure the capacity of the librar

3-5weeksInquiry

VHH capacity reach to 109

Milestone 4: Library Biopanning and Screening

at least 3 round panning (both liquid phase panning and solid phase panning)

ELISA verification, identification and evaluation of positive clones (at 200 clones for sequencing) screening 

positive clone sequencing and sequencing result analysis

10-12weeksInquiry

Guarantee to provide at least 20 unique sequence.

VHH-Fc Antibody Production (optional)   

Gene synthesis, subcloning, plasmid preparation 

transient expression and purification

QC analysis

2 weeksInquiryPurified antibody (SDS-PAGE >95%, endotoxin level <1EU/mg)
Affinity Ranking (optional)Affinity measured by Biacore T2001-2 weeksInquiry
Note: If the ELISA titer after 4th immunization is <1:5,000, we will suggest to terminate the project and charge the fee for phase I and phase II.

Case Study: Unique VHHs screening from alpaca immunization

Project description: A biotech client provide two Antigens, H (human origin) and M (mouse origin) in order to obtain VHH recognized both M and H.

VHH library screening result:


68 Unique sequences


68 Unique sequences



33 Unique CDR3


33 Unique CDR3


Alpaca A (3rd immunization): 293 double positive clone, 38 unique sequence and 9 unique CDR 3.

Alpaca B (3rd&4th immunization): 200 double positive clones, 40 unique sequence and 27 unique CDR 3.

Totally, obtain 68 unique sequences and 33 unique CDR3 from Alpaca A and Alpaca B.

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