Resources Blog Antibody Basics: Part 2​ - Antibody formats: VHHs​

Antibody Basics: Part 2​ - Antibody formats: VHHs​

Biointron 2024-02-02 Read time: 10 mins

Welcome to Antibody Basics by Biointron, Part 2. Here we’ll give an introduction on the smallest antibody format, VHH. We've mentioned full length antibodies in our previous video, with IgG, IgM, IgD, IgE, and IgA. However, several other engineered formats have been developed:

  • Monovalent antibody fragments such as single-chain fragment variable (scFv) and fragment antigen-binding (Fab), as well as Fragment with two Fab regions, lacking the fragment crystallizable (Fc) region, and scFv-Fc (single-chain variable fragment - Fc)

  • Multimeric formats such as diabodies (dimeric scFvs) or triabodies (trimeric scFvs).

  • Multi-specific antibodies (bispecific, trispecific)

  • Antibody-drug conjugates (ADCs)

  • Single-domain antibodies (sdAbs), aka VHHs

A VHH antibody is the antigen-binding fragment of heavy chain-only antibodies

  • Derived from heavy-chain only IgG antibodies found in the Camelidae family

  • Beta-sandwich fold supports presentation of 3 CDRs to bind antigens

  • Convex-shaped paratope formed allows grant to small clefts on the antigen


History of VHHs

  • Late 20th Century: Discovery of camelid antibodies and initial recognition of their potential.

  • 1990s: First VHH sequences published in 1996, and cloning and expression took place.

  • Early 2000s: VHH antibodies gain popularity in research, diagnostics, and therapeutics due to their small size and unique binding properties.

  • Mid-2000s: Investment in VHH antibodies for therapeutic use increases, leading to preclinical and clinical trials.

  • 2010s to Present: Diversification: Ongoing advancements in VHH antibody engineering and applications contribute to their adoption in various fields.


VHHs are ideal for a wide range of applications, including diagnostic tests, therapeutics, and research tools.

Screenshot 2024-02-01 at 3.36.13 PM.png

VHH antibody immune libraries are produced by phage display:

  1. Antigen Expression

  2. Animal Immunization

  3. PBMC Isolation

  4. mRNA Extraction and Reverse Transcription

  5. Library Generation

  6. Library Screening and Biopanning

  7. Positive Clone Sequencing and Sequence Analysis

  8. VHH Expression


There are currently only four approved VHH-based antibody therapeutics.

  1. Caplacizumab: FDA in 2019. Bivalent, anti-VWF factor, humanized antibody to treat acquired thrombotic thrombocytopenic purpura.

  2. Envafolimab: Approved in 2022 in China. Anti-PD-L1 antibody to treat various solid tumours and chronic hepatitis B; soft tissue sarcomas and biliary tract cancer.

  3. Ozoralizumab: Approved in Japan in 2022. Trivalent, bispecific, humanized anti-TNFα antibody to treat rheumatoid arthritis. Binds to two subunits of TNFα to potently neutralize its action.

  4. Ciltacabtagene autoleucel (cilta-cel): Approved by FDA in 2022. VHH-based, BCMA-directed genetically modified autologous CAR T-cell therapy to treat relapsed/refractory multiple myeloma.

Several VHH-based drugs are in various stages of clinical trials.


VHH antibodies are particularly useful as diagnostics.

  • Lateral flow immunoassays: Rapid diagnostic test that specifically detects the presence of an antigen of interest within a mixture. VHH antibodies are highly stable, possess a large paratope repertoire, prevent cross-reactions with host, and can be adsorbed onto gold nanoparticles.

  • Enzyme-linked immunoassay (ELISA): Competitive ELISA: a labeled antigen competes with target antigen for binding to immobilized antibodies. Sandwich ELISA: two VHH antibodies sandwich the target antigen, with one immobilized on a solid support and the other labeled with an enzyme.

  • Biosensors: A device with a bioreceptor specific to a target antigen affixed to a semiconductor, and binding causes an electric potential change. VHH antibodies can withstand high temperatures and pH changes allows for storage in less-than-ideal environments.

  • In vivo diagnostic imaging: Medical/nuclear optical imaging techniques, PET, and SPECT use fluorescently or radioactively labeled VHH antibodies. The small size of VHH allows for rapid tissue penetration, ease of modification/conjugation, and rapid renal clearance.

VHH antibodies can thus be applied to disease diagnosis, pharmaceutical quality control, food and environmental analysis, and disease imaging and progression monitoring.

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