VHH Antibody Discovery VHH Antibody Discovery

VHH Antibody Discovery

Antibody Discovery
  • Self-owned Alpaca Breeding Farm
  • 100+ projects delivered/year
  • High Diversity and Large Capacity


VHH antibodies, also known as single-domain antibodies, are a promising next-generation therapeutic antibody technology for cancer immunotherapy and various other applications. These antibodies possess unique characteristics that set them apart from conventional antibody formats, such as their size, solubility, intrinsic stability, and the ability to be easily tailored into pluripotent constructs. They can recognize uncommon or concealed epitopes and effectively bind to cavities or active sites of enzyme targets. Additionally, VHH antibodies offer advantages in drug discovery due to their ease and rapidity of development, along with straightforward manufacturing processes.

VHHs are the smallest antigen-binding fragments derived from heavy-chain-only antibodies found in camelids, such as alpacas, llamas and camels. First discovered in 1993, VHH antibody fragments offer unique characteristics which are particularly advantageous in therapeutics, diagnostics, and research tools. This is due to their small size and structure which allows them to penetrate tissues and reach targets that may be challenging for conventional antibodies, in addition to their stability and ability to bind with high affinity to specific targets.1

VHH Antibody Discovery Overview

Biointron has established itself as a leading entity in the arena of single domain antibody discovery. Leveraging our state-of-the-art phage display technology, our team of scientists specializes in both the production and discovery of unique single domain antibodies.



Self-owned Alpaca Breeding Farm

  • 300+ alpacas available
  • One alpaca just for one project

100+ projects delivered/year

  • Various immunization/panning/screening methods
  • Multiple assays for validation (ELISA, SPR, FACS, Internalization, ADCC, etc.)

High Diversity and Large Capacity

  • Maximizing success with high diversity and large capacity
  • Library size at least 109

Biointron Alpaca VHH Affinity Distribution

  • 0%AF-5
  • 4%AF-6
  • 16%AF-7
  • 33%AF-8
  • 32%AF-9
  • 13%AF-10
  • 2%AF-11
  • 0%AF-12
  • 0%AF-13

Data source: 615 VHH antibodies (as part of our developed VHHs)

Calculation example:

Total number of VHH of affnity 10-9 M from different target
Total number of VHH of from different target (615 unique sequence)

From our platform, nanobody hits with affinity ranges from 10-8 to 10-11 M have been generated, with most falling within 10-8 and 10-9 M. It should be noted that the presented data represents only a portion of projects from one client and may have sampling bias. The distribution of hits with different affinity may not reflect the variance between different targets. The data is from SPR affinity measurement.

Working Flowchart

Antigen expression or provided by client
Animal immunization
PBMC isolation
mRNA Extraction and Reverse Transcription
Library generation
Library screening and Tailored biopanning ( solid phase panning & liquid phase panning)
Positive Clone Sequencing and Sequence Analysis
VHH expression
VHH Validation


VHH antibodies are used in a wide variety of applications, such as clinical therapeutics and immunodiagnostics, as well as environmental monitoring, due characteristics such as their small size and ability to undergo genetic engineering.2

Recombinant Antibody Drugs: Within antitumor therapeutics, Caplacizumab is a bivalent VHH antibody treating thrombotic thrombocytopenic purpura and thrombosis.3

Chimeric antigen receptor (CAR) T therapy: Within a CAR construct, VHH antibodies are typically used as the antigen binding domain of CAR-T due to their unique properties and manufacturing feasibility.4

VHH Antibody Discovery
Service Details

Service Step Service Description Timeline Deliverables
Phase Ⅰ: Antigen preparation /Antigen validation
  • Antigen can be prepared by BioIntron or provided by our client
2 weeks for preparation / 2-3 days for validation
  • 1mg purified protein antigen
  • If antigen provided by client, it should be at least 4mg with SDS-PAGE >95%, endotoxin level <1EU/mg
Phase 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
8-10 weeks
  • 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
Phase Ⅲ: 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 library
3-4 weeks
  • VHH capacity reach to 108 - 109
Milestone Ⅳ: Library Biopanning and Screening
  • at least 3 round panning (liquid or solid phase panning)
  • ELISA verification, identification and evaluation of positive clones screening
  • positive clone sequencing and sequencing result analysis
3-4 weeks
  • 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 weeks
  • Purified antibody (SDS-PAGE >95%, endotoxin level <1EU/mg)
Affinity Ranking (optional) Affinity measured by Biacore 8K 0.5 weeks

Case Study

  • Case 1:Anti-X Antigen
    After panning and screening, 53 unique sequences were obtained & expressed in HTP CHO expression system.
    Validation by ELISA against Antigen X
    49 of 53 VHH Antibodies bound to X in ELISA
    Validation by SPR against Antigen X
    • 5Not Binding
    • 9E-7
    • 21E-8
    • 16E-9
    • 2E-10
    Affinity range 5.36x10-7 M to 7.66x10-10 M.
    48 of 53 VHH Antibodies bound to X in SPR
    Validation by flow cytometry against CHO-K1/X overexpression cell
    43 of 53 VHH Antibodies Bound against CHO-K1/X Cell
    Validation by Agonist activity assay
    31 of 53 antibodies showed significant agonist activity (above 1.5 times Isotype value).
    Validation by Blocking assay
    18 of 53 antibodies showed significant blocking activity.
  • Case 2:VHH Antibody Discovery Multi-transmembrane target A

    First, we immunized alpacas with DNA-encoding Target A, and then boosted their immune response by Target A-overexpressing cell line. Alpaca serum can specifically bind to Target A-overexpressing cells but not to blank cells.


    Phage Library construction from PBMC

    Library size is ~1.1x10E10

    Insert rate >95%

    Antigen Immunization
    DNA encoding Target A 1&2
    CHO-K1-Target A 3, 4, 5
    Cell panning Input (CFU/ml) Output Output/Input
    1 2.00E+12 9.00E+07 4.50E-05
    2 1.33E+12 2.10E+08 2.10E+08
    3 2.10E+08 2.10E+08 2.10E+08
    Total clone OD450
    (293T-Target A) >0.5
    (293T-blank) >0.1
    Combined with Target A
    clone number
    Clone %
    0635-Human Target A 88 2 0 2 2.27% 1
    0635-Human Target A 88 18 12 6 6.82% 2
    0635-Human Target A 88 55 41 14 15.91% 10
    Among the purified antibodies, 4 of them can bind specifically to target A.
“As a dedicated scientist in the forefront of VHH antibody discovery, I am proud to contribute to Biointron’s commitment to advancing healthcare through cutting-edge biotechnology. Our VHH antibodies epitomize precision, innovation, and the relentless pursuit of scientific excellence.”
Yang Xiang
Yang Xiang
Antibody Discovery Team


  • What are VHH antibodies?

    A VHH antibody is the antigen binding fragment of heavy chain only antibodies. They are derived from the immune system of camelids such as alpacas. As they are highly stable and much smaller than traditional antibodies, VHH antibodies are ideal for a wide range of applications, including diagnostic tests, therapeutics, and research tools.

  • What are recombinant antibodies?

    Recombinant antibodies are monoclonal antibodies produced in vitro through synthetic genes and antibody fragments, instead of using hybridomas. They can take several different formats, such as full-length immunoglobulins (Ig), monovalent antibody fragments such as single-chain fragment variable (scFv) and fragment antigen-binding (Fab), and multimeric diabodies (dimeric scFvs) or triabodies (trimeric scFvs).5

  • How are VHHs isolated from camelids?

    There are several ways to isolate VHHs from camelids against a target of interest and to build a library for screening, such as immunized, naïve, and synthetic/semi-synthetic libraries. The most common approach is by immunizing camelids and building a library based on the repertoire of heavy-chain immunoglobulins.6


  • Atarhouch, T., Muyldermans, S., Robinson, G., Hammers, C., Songa, E. B., Bendahman, N., & Hammers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363(6428), 446-448. https://doi.org/10.1038/363446a0
  • Bever, C. S., Dong, X., Vasylieva, N., Barnych, B., Cui, Y., Xu, L., Hammock, B. D., & Gee, S. J. (2016). VHH antibodies: Emerging reagents for the analysis of environmental chemicals. Analytical and Bioanalytical Chemistry, 408(22), 5985. https://doi.org/10.1007/s00216-016-9585-x
  • Bannas, P., & Hambach, J. (2017). Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Frontiers in Immunology, 8, 309808. https://doi.org/10.3389/fimmu.2017.01603
  • Bao, C., Gao, Q., Li, L., Han, L., Zhang, B., Ding, Y., Song, Z., Zhang, R., Zhang, J., & Wu, X. (2021). The Application of Nanobody in CAR-T Therapy. Biomolecules, 11(2), 238. https://doi.org/10.3390/biom11020238
  • Muyldermans, S. (2021). A guide to: Generation and design of nanobodies. The Febs Journal, 288(7), 2084-2102. https://doi.org/10.1111/febs.15515
  • Arbabi-Ghahroudi, M. (2022). Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments. International Journal of Molecular Sciences, 23(9). https://doi.org/10.3390/ijms23095009

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