Compared with traditional monoclonal antibodies, the Production of nanobody has great advantages. It can be summarized as immunizing alpacas to obtain antibody genes and phage display to obtain target antibody sequences. It is mainly divided into four steps: alpaca immunization, phage library construction, antibody library screening, antibody expression and functional verification.

In order to build an immune pool, young, healthy llamas, dromedaries, alpacas, etc. must first be immunized. Typically, animals are injected 4-8 times with a mixture of target antigens and adjuvants over a 2-month time span. Generally, each injection is about 50-200μg immunogen; The exact amount obviously depends on the molecular weight of the antigen, but more on its immunogenicity and/or toxicity.

Immunogen type: The use of soluble, correctly folded recombinant proteins is preferred, and there is also direct DNA immunity. Up to 10 proteins are usually mixed to immunize animals, but more complex mixtures have also been used (e.g., protein extracts from viruses, bacteria, parasites, whole mouse spleen cells, or cancer cells).

To increase the likelihood of a specific epitope successfully acquiring a nanobodies, immunizing more than one animal is recommended. They are inbred animals, and each one produces a unique immune response, obtaining a larger panel of nano antibodies from which to select the best-performing nanobodies.

Fig. 1 Brief procedure of nanobody production

Some studies have shown a higher proportion of HCAb antibodies than classical antibodies in camels or dromedaries compared to llamas and alpacas. After immunization, 50-100 ml of anticoagulant blood (usually from the jugular vein, although lymph node biopsy is also a good raw material) is taken, lymphocytes are prepared and mRNA is extracted. The mRNA is converted to cDNA for amplification of the VHH gene (two-step nested PCR).

Some molecules, such as RNA or DNA, are not immunogenic and cannot stimulate the production of HCAb, or some compounds are too toxic, infectious, or harmful to animals or the environment, in which case a natural library of nano antibodies or a synthetic library of nano antibodies can be envisioned (Erwin De Genst, 2006).

In order to be able to screen high-affinity nano antibodies, the size of the natural antibody library is generally 109-1010, of which about 80% should encode nano antibodies. To build such a large and diverse library of natural nano antibodies, blood needs to be collected from multiple animals (at least 10 animals to avoid bias in HCAbs due to allergies or previous infections). There are an estimated 106 lymphocytes per mL of blood, and given that only a small fraction of them are B cells, about 50% of which may express HCAb, 10L of blood is needed to build a nanoantibody library of 1010 different VHH clones.

Combining in vitro antibody maturation techniques, such as DNA recombination, error-prone PCR, and random primers, can increase the diversity of complementary determining regions (CDRs), as well as the specificity and affinity of the nanobodies. Phage display and ribosome display are two main strategies for screening nanomes from natural antibody libraries (Rossotti MA,2023).

KMD Bioscience has established a complete and mature phage antibody display technology platform. In addition, KMD Bioscience has rich experience in antibody engineering construction, and can provide three-dimensional antibody upstream and downstream services, including antibody humanization service, human scFv antibody library construction service, human Fab antibody library construction service, human antibody phage library Production service, phosphorylated antibody customization service, antibody affinity maturation service, etc.(Learn more about antibody discovery services)

This article serves as a reference material for enthusiasts in scientific research. It does not substitute for professional knowledge or hands-on experimental procedures which require more detailed and professional information. In case of any content infringement, kindly reach out to the author for immediate deletion of the contentious material.


De Genst E, Saerens D, Muyldermans S, Conrath K. Antibody repertoire development in camelids. Dev Comp Immunol. 2006;30(1-2):187-98.

Rossotti MA, Trempe F, van Faassen H, Hussack G, Arbabi-Ghahroudi M. Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries. Methods Mol Biol. 2023;2702:107-147.

One Reply to “Overview: How Nanobody is Produced”

  1. Based on the phage display technology platform, KMD Bioscience can provide major experimental links including antigen design, alpaca immunity, library construction and screening, and activity function verification, and provide highly specific and high-affinity alpaca VHH antibodies to scientists around the world.

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