In 1993, a natural antibody lacking light chains (VL) was found in the serum of camelids. 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 nano antibody (VHH) or a single domain antibody. Later, this antibody was also found in animals such as alpacas and sharks. Based on the unique structure and broad application prospects, the research on nano antibodies has become a hot topic, especially in the field of drug research and development. Due to the strong penetration of nano antibodies, it has become an ideal choice for the treatment of tumor diseases, which helps to improve the efficacy of drugs and reduce side effects. Although there are antibodies with similar structures in sharks, most single domain antibody production is carried out through alpaca immunization because animals such as alpacas are easy to breed and immunize, and the profits are high.

Fig. 1 Traditional monoclonal antibody, heavy chain antibody, nano antibody structure diagram

Advantages of Alpaca Immunity:

The antibodies produced by the alpaca's immune system against specific antigens have high specificity and affinity, which helps in the treatment and diagnosis of diseases. Camelids such as alpacas can produce nanoantibodies with only heavy chains, which have the advantages of high temperature resistance, strong stability, and strong tissue penetration ability. Since the structure of nanoantibodies is very simple, it is relatively easy to modify them, such as antibody humanization. By efficiently expressing nanoantibodies in yeast systems, large-scale single-domain antibodies can be obtained at a low cost. Nanoantibodies produced after alpaca immunization have strong penetration ability, so they can reach deep tissues that general antibodies cannot reach, which is helpful for the treatment of tumor diseases. In addition, nanoantibodies have high stability and water solubility and can adapt to extreme temperatures and pH values.

Construction of Nanoantibody Library:

Nanoantibody library construction can be carried out through phage display technology. Antibody libraries are divided into natural antibody libraries, immune antibody libraries, synthetic antibody libraries, and semi-synthetic antibody libraries. The RNA isolated from the hybridoma is reverse transcribed and used as an amplification template for antibody genes. In this process, a large antibody gene library containing a large number of nanoantibodies is created, and then it can be screened and identified through phage display technology. In addition, ribosome display technology and yeast surface display technology can also obtain high-affinity nanoantibodies.

In order to obtain high-affinity nanoantibodies, animals must be immunized first. Appropriate alpacas and other animals must be selected for immunization, and corresponding antigens must be used to stimulate animals to produce immune responses against specific antigens, thereby producing specific B cells. After a certain period of time, peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood of immunized animals. Total RNA from PBMCs is extracted, and attention must be paid to the integrity and purity of RNA during the extraction process. The extracted RNA is used as a template and reversely 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 cell and fused with the phage surface protein. The host cell is 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, 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.

Immunization Technology for Preparing Single-domain Antibodies:

When immunizing animals, adjuvants can be used to enhance the immunogenicity of antigens. Commonly used adjuvants are Freund's adjuvant and water-soluble adjuvant. Freund's adjuvant forms an oil-in-water emulsion, which slowly releases antigens in the body, prolongs the antigen stimulation time, and is conducive to shortening the cycle of antibody production. It can non-specifically enhance the animal's immune response to antigens, thereby increasing the affinity and yield of antibodies. However, after being injected, Freund's adjuvant is prone to cause inflammatory reactions at the injection site, and may even cause tissue necrosis and ulcers, and Freund's adjuvant is not suitable for the preparation of human vaccines and antibody drugs. Compared with Freund's adjuvant, water-soluble adjuvant has lower toxicity and better biocompatibility, and is suitable for the preparation of human vaccines and antibody drugs. In addition, the preparation of water-soluble adjuvant does not require a complex emulsification process, and is relatively simple to prepare. The local reaction caused by water-soluble adjuvant after injection is also relatively mild. However, compared with Freund's adjuvant, water-soluble adjuvant does not form an oil-in-water emulsion, and the antigen is released faster in the body, which may lead to a shorter immune stimulation time. Therefore, it is necessary to increase the antigen dose or extend the immune cycle to induce the body's immune response.

Commonly used immunogens include protein immunogens, small molecule immunogens, peptide immunogens, viral immunogens, DNA/RNA immunogens, and cell line type immunogens. Among them, the protein purity of protein immunogens should be high, and it is best to simulate natural antigen epitopes. Small molecule immunogens need to be coupled with larger carrier proteins to enhance their immunogenicity. Viral immunogens need to ensure that the virus has been inactivated and cannot cause disease.

TekBiotech has been committed to nanoantibody library construction and nanoantibody library screening services for many years, and has rich experience in single-domain antibody production. Based on our mature antibody discovery service platform, hundreds of alpaca nanoantibody library construction services are successfully delivered every year. TekBiotech has established a complete and mature nanoantibody display technology service platform. Based on phage display technology, we can provide major experimental links including antigen design, alpaca immunization, alpaca nanoantibody library construction and screening, and active function verification, and provide customers with high-specificity and high-affinity alpaca nanoantibody library construction services. And we will conduct a comprehensive analysis of the nanosequence 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 nanoantibody 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 nanoantibody library we constructed has a large capacity and can produce high-affinity nanoantibodies. 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. The antibody library we have constructed has a large capacity of up to 10^9, with a high insertion rate of target fragments, 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 various eukaryotic expression systems for antibody proteins, such as mammalian cells, yeast cells, plants and insect cell expression systems, etc., which can produce high-quality nano antibodies for customers.