In 1993, a natural antibody lacking light chains (VL) was found in camel serum. It has a simple structure and consists of only two heavy chains (VH). It is very small, about 15 kDa, so it is called a nanobody (VHH) or a single-domain antibody. 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 highly stable single-domain antibodies and the smallest binding unit with antibody activity known so far. Nanobodies and traditional antibodies are both 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, nanobodies 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.

Fig. 1 Traditional monoclonal antibodies, heavy chain antibodies, nanoantibody structure diagram

Nanoantibody library is constructed by 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 nanoantibodies is created, which can then be screened and identified by phage display technology. In addition, ribosome display technology and yeast surface display technology can also obtain high-affinity nanoantibodies. Compared with full-length antibodies, nanoantibodies are easier to penetrate into tumor tissues and can be used in targeted cancer therapeutic drugs. At the same time, nanoantibodies also play an important role in detection reagents and in vitro diagnosis. It is reported that nanoantibodies against Aspergillus flavus can be screened by phage display technology, and then applied to the field of food safety to detect the content of aflatoxin in agricultural products.

Advantages of Single-domain Antibodies:

Nano antibodies only contain CH2, CH3 and variable regions, and are long and flat. From a structural perspective, single-domain antibodies have many advantages over other antibodies. The molecular weight of single-domain antibodies is very small, usually around 12-15kDa, which is only one-tenth of traditional IgG antibodies. Therefore, single-domain antibodies can better penetrate tissues and cell gaps and reach areas that are difficult for traditional antibodies to reach, such as deep in tumor tissues or inside cells. The structure of single-domain antibodies is relatively stable and can still remain active at extreme pH values and temperatures. Single-domain antibodies have a longer CDR3 region and special disulfide bonds. Based on these characteristics, single-domain antibodies can form a tight and stable antigen-antibody complex. Therefore, although single-domain antibodies have only one antigen binding domain, they can specifically bind to target molecules. Single-domain antibodies have good solubility, which helps their distribution and metabolism in the body. Because the structure of single-domain antibodies is simple, it is easy to modify and optimize them through genetic engineering technology. For example, the function of single-domain antibodies can be enhanced or their characteristics can be changed by adding tags or fusing other proteins.

Preparation of Single Domain Antibody Library:

First, animal immunization is performed. Appropriate camelid animals, such as camels and alpacas, should be selected for immunization. The corresponding antigens are used to stimulate the animals to produce immune responses against specific antigens, thereby producing antigen-specific B cells. After a certain period of time, peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood of the immunized animals. Total RNA in PBMCs is extracted, and the integrity and purity of RNA should be paid attention to during the extraction process. The extracted RNA is used as a template and reverse transcribed into cDNA. Then, the VHH gene is amplified by PCR using cDNA as a template. Generally, two rounds of PCR amplification are performed to improve the specificity and yield of the antibody. The amplified VHH gene fragment is cloned into a phage display vector to construct a phage display library. The constructed phage display library is transformed into host cells, such as Escherichia coli, yeast or mammalian cells, so that the VHH gene is expressed in the host cells and fused with the phage surface protein. The host cells are stimulated to express the fusion protein through appropriate induction, and the VHH is displayed on the phage surface. Using the phage display platform, through multiple rounds of screening and enrichment, VHH positive clones with high affinity to specific target proteins are screened from the library. Finally, the screened VHH positive clones are sequenced and analyzed to obtain their gene sequence information.

TekBiotech has been committed to nano antibody library construction and nano antibody library screening services for many years. Based on our mature antibody discovery service platform, hundreds of alpaca nano antibody library construction services are successfully delivered every year. Tek Biotech has established a complete and mature nano antibody display technology service platform. Based on phage display technology, we can provide major experimental links including antigen design, alpaca immunization, alpaca nano antibody library construction and screening, and active function verification, and provide customers with high specificity and high affinity alpaca nano antibody library construction services. And we will conduct a comprehensive analysis of the nano sequence information and verify it with a variety of experiments, such as EC50 determination, affinity analysis, flow blocking verification, etc. We have a variety of phage antibody library construction platforms including M13, T4, T7 and λ phage, which can meet the different needs of customers and provide personalized nano antibody library construction services. TekBiotech is good at constructing different types of phage display libraries, such as immune libraries, natural libraries, semi-synthetic libraries, synthetic libraries, etc. The nano antibody library we constructed has a large capacity and can produce high-affinity nano antibodies. We can provide customers with a variety of phagemid vectors including pMECS, pComb3X and pCANTAB 5E. We have strains such as TG1 Escherichia coli, XL1-Blue and ER2738, which can be used for phage infection after expansion and cultivation. The antibody library we constructed has a large capacity of up to 10^9, with a high insertion rate of the target fragment, which is conducive to screening out nano antibodies that satisfy customers. We can also express and purify the screened nano antibodies according to customer needs. In addition to prokaryotic expression systems, we also have a variety of eukaryotic expression systems for antibody proteins, such as mammalian cells, yeast cells, plant and insect cell expression systems, etc., which can produce high-quality nano antibodies for customers.