The immune system of camelids produces two types of antibodies in response to pathogens : one is traditional IgG (composed of two heavy chains and two light chains), and the other is a single domain antibody (HCAb) composed of only heavy chains. This type of antibody is completely independent of the light chain, and its antigen binding function is completely completed by the variable heavy-chain domain (VHH). This VHH domain is the source of nanobodies. When camelids are immunized with antigens, B cells expressing specific VHH are enriched in their peripheral blood lymphocytes. The mRNA of these cells was extracted and the VHH gene fragment was amplified by reverse transcription PCR. Subsequently, the VHH gene was inserted into the phage coat protein gene by phage display library technology, so that the VHH fragment was displayed on the phage surface. By using antigen immobilization screening (such as ELISA) or flow cytometry sorting, VHH sequences that specifically bind to antigens can be efficiently screened.

Compared with the variable region (VH + VL double domain) of traditional antibodies, VHH can achieve antigen binding only by relying on a single domain. Its intermolecular complementary determining regions (CDRs) are long, especially CDR3 often forms a convex finger structure, which can penetrate into the hydrophobic pocket or complex epitopes on the surface of the antigen. In addition, the framework regions of some VHHs contain hydrophilic amino acid mutations, which reduces non-specific adsorption and improves solubility. 

Naturally screened VHHs need to be further optimized for affinity or stability by site-directed mutagenesis or in vitro evolution (such as Error-prone PCR). The final optimized gene was produced by E.coli, yeast and other expression systems. Because nanobodies are disulfide-independent, resistant to high temperature and extreme pH, their production and purification costs are significantly lower than traditional antibodies.

The production process of nanobodies mainly includes several key steps such as immunization, library construction, screening and expression. 

The first step in the production of nanobodies is to activate the immune system of camelids through an immune response. Usually, antigens (which can be proteins, peptides or other molecules) are injected into animals. The animal 's immune system recognizes the antigen and produces an antibody response against the antigen. Camelidae animals are particularly suitable for the production of nanobodies because they can produce small antibodies with strong specificity and high affinity.

Animals after immunization will produce a large number of antibodies. Next, researchers can collect antibody genes that bind to antigens from the blood of animals or directly from their B cells. Since the nanobody is composed of a heavy chain variable region (VHH), the extraction of heavy chain genes is crucial at this stage. 

By isolating and cloning the heavy chain genes of B cells, researchers can construct a diverse antibody gene library. This library contains a large number of heavy chain antibody genes that may bind to antigens, and these genes will be transferred into the expression system for screening and enrichment.

After screening the ideal nanobody, the researchers transferred its gene into E.coli, yeast or mammalian cells for expression. The target nanobodies were extracted and purified by culturing the expression system and using affinity chromatography. This process can effectively produce a large number of nanobodies with high purity.

Purified nanobodies are widely used in medicine, diagnosis, treatment and other fields. Due to its small molecular characteristics, nanobodies can penetrate tissues, have better biocompatibility, low immunogenicity, and strong stability. Therefore, nanobodies have shown significant advantages in targeted therapy, disease diagnosis, and vaccine development. 

Alpha Lifetech has made significant strides in the field of biotechnology by providing advanced Nanobody Production Service. These assays are highly specialized and designed to cater to the needs of research professionals and scientists working in pharmaceuticals and biomedicine.