In 1993, a natural antibody lacking a light chain (VL) was discovered in the serum of camelids. It has a simple structure and consists of only two heavy chains (VH). It still has antibody activity in the absence of Fc. Due to its lack of Fc domain, its size is very small, about 25 kDa, so it is called a nanobody (VHH). Later, this antibody was also found in animals such as alpacas and sharks. Nanobodies contain only one heavy chain variable region and two conventional CH2 and CH3 regions. Although there is no VL domain, they can still bind to antigens. They are single-domain antibodies with high stability and are also the smallest binding unit with antibody activity known so far. Both nanobodies and traditional antibodies are composed of 4 relatively conservative framework regions (FRs) and 3 complementary determining regions (CDRs), but compared with traditional antibodies, the CDR3 of nanobodies has a loop structure region, which is longer than the general variable region and has better binding performance. In addition, nano antibodies have the characteristics of weak immunogenicity. Humanized antibody treatment can further reduce the immunogenicity of recombinant antibodies, thereby reducing the impact on drug treatment effects.

Nano antibodies can be screened and obtained through phage display technology. The antibody library is divided into natural antibody library, immune antibody library, synthetic antibody library and semi-synthetic antibody library. The RNA isolated from the hybridoma is used as an amplification template for the antibody gene after reverse transcription. At the same time, a large antibody gene library containing a large number of nano antibodies is created, which can then be screened and identified through phage display technology. In addition, ribosome display technology and yeast surface display technology can also obtain high-affinity nano antibodies. Compared with full-length antibodies, nano antibodies are easier to penetrate into tumor tissues and can be used in targeted cancer treatment drugs. At the same time, nano antibodies also play an important role in detection reagents and in vitro diagnosis. It is reported that phage display technology can be used to screen nano antibodies against Aspergillus flavus, which can then be used in the field of food safety to detect the content of aflatoxin in agricultural products.

Expression and purification of nanobodies:

Nanobody expression systems include prokaryotic recombinant antibody expression systems and eukaryotic recombinant antibody expression systems. Different expression vectors, fusion tags, and host bacteria will produce nanobodies with different activities. Different VHH antibody expression strategies can be formulated according to different needs. His tags are widely used in affinity chromatography purification. VHH fusion His tags can enable antibodies to specifically bind to metal ions and then purify them. The operation is simple and suitable for most nanobodies that require efficient purification. The Fc fragment is the heavy chain constant region of IgG, which can enhance the stability of antibodies. Nanobodies fused with Fc fragments can bind to Fc receptors, trigger immune responses, and enhance their stability and half-life in the body. They are suitable for nanobodies that work for a long time in the body, such as nanobodies for immunotherapy and drug delivery. Avi-tag is a peptide tag that can specifically bind to the biotin ligase BirA to achieve biotinylation of nanoantibodies, and then the biotin-streptavidin system can be used for purification, detection, targeted delivery, etc. The combination of biotin and streptavidin has high specificity and high affinity, and is suitable for proteomics, drug screening, disease diagnosis, etc. In addition, fluorescent tags, enzyme cleavage tags, etc. have different functions and characteristics. In order to ensure the expression and correct folding of VHH antibodies, it is necessary to select a VHH antibody expression system according to one's own needs and optimize the expression conditions. The Escherichia coli recombinant antibody expression system is the first choice for prokaryotic expression. When expressing eukaryotic proteins, the corresponding expression system will be selected according to the characteristics of eukaryotic proteins and different eukaryotic protein expression requirements. After that, nanoantibody expression and purification are carried out. Commonly used antibody purification methods are divided into antibody affinity purification and antibody separation and purification, including Protein A/G purification, ion exchange chromatography, gel filtration, precipitation, and hydrophobic interaction chromatography. According to the characteristics of the antibody and different needs, select an appropriate purification method to obtain a high-purity antibody.

Fig. 1 Nanobody expression and identification diagram

Fig. 2 Nanobody purification Western Blot identification diagram

Tek Biotech has many years of research experience in the field of antibodies, has built a complete antibody customization platform, and provides various types of antibody preparation and development services. In order to ensure that customers can get better antibodies, we also provide a series of nanobody expression and purification, antibody sequencing and other services. Tek Biotech has a complete recombinant antibody expression platform, and can use different protein expression systems to express and purify nanoantibodies according to customer needs. Mammalian expression systems can correctly and effectively identify the synthesis, processing and secretion signals of eukaryotic proteins, have similar structures and functions to natural proteins, can be glycosylated, phosphorylated and acetylated, and other post-translational modifications, and the expression and purification process is simple, and it is easy to transfect recombinant plasmids. However, mammalian expression systems have the disadvantages of low yield and long preparation cycle. TekBiotech has focused on the optimization and research of mammalian expression systems for many years, and can provide customers with high-yield and high-quality nanobody expression services. We are good at using HEK293 and CHO cell lines for transient and stable expression of proteins. We can also use the E. coli prokaryotic expression system to prepare soluble proteins, and by optimizing the protein expression conditions, avoid protein inclusion body expression and ensure protein activity. TekBiotech's protein expression platform can be used for small-scale and large-scale production of nanoantibodies to meet the different needs of customers. We can also formulate professional antibody humanization strategies for customers, and humanize nanoantibodies. The optimized nanoantibodies have extremely low immunogenicity, which is almost equivalent to human antibodies, ensuring the affinity and specificity of nanoantibodies to antigens. In order to verify the effectiveness of VHH antibodies after humanization, we will characterize the antibodies through ELISA, WB, immunoprecipitation, flow cytometry, immunohistochemistry, endotoxin detection, etc. to ensure the effectiveness of humanized nanoantibodies.