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 nanobody 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 preparation of nanoantibodies has a broad application market, especially in the field of drug research and development. Due to the strong penetration of nanoantibodies, 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 nanoantibody development is carried out through alpaca immunization because animals such as alpacas are easy to breed and immunize, and the profits are high. Nanoantibody libraries can be constructed through phage display technology. Antibody libraries are divided into natural antibody libraries, immune antibody libraries, synthetic antibody libraries, and semi-synthetic antibody libraries.

There are many types of immunogens used in animal immunization. Viral immunogens are a commonly used type of immunogen, including whole virus inactivated vaccines, subunit vaccines, viral vector vaccines, and mRNA vaccines. The virus is inactivated by physical or chemical methods, its antigenicity is retained, and its pathogenicity is removed. The whole virus inactivated vaccine can stimulate the body to produce an immune response, but the virus needs to be completely inactivated, and the preparation process is relatively complicated. Subunit vaccines only use viral surface proteins as antigens, so this type of vaccine is safer, but adjuvants need to be added during use to enhance the immune effect. Adenoviruses, lentiviruses, etc. are used as vectors after genetic modification. The target antigen gene is inserted into the viral genome, and the immune response is induced through viral replication and expression. The immune effect of viral vector vaccines is efficient and long-lasting, but its preparation process is complicated and requires strict control of biosafety. The mRNA encoding viral antigens is directly introduced into cells, so that the cells express antigens and stimulate the occurrence of immune responses. mRNA vaccines can be prepared quickly, but it is difficult and requires ensuring the stability and delivery efficiency of mRNA. Virus-type immunogens have become a hot topic in the field of infectious disease prevention and control, and efficient and safe viral vaccines have also been used in a variety of disease fields.

Fig. 1 Antibody response to glycan pores in HIV-1 Env and Ebola GP

Precautions for Preparing Viral Immunogens:

When preparing whole virus inactivated vaccines, the choice of inactivation method is crucial. Cytotoxicity tests and infectivity tests can be used to verify whether the virus has been completely inactivated. When preparing subunit vaccines and viral vector vaccines, it is necessary to ensure that the antigen is correctly expressed in the host cells and is highly purified, so a suitable expression system and purification technology are important. When using vaccines, an appropriate dose of adjuvant can be added to improve the immune effect of the vaccine.

When preparing immunogens, it is necessary to ensure that the virus titer can reach a level that can provide sufficient antigen stimulation, thereby inducing an effective immune response. However, if the virus titer is too high, it may cause an overly strong immune response and even cause disease. Therefore, it is necessary to control the virus titer within a safe range while ensuring the immune effect. Accordingly, the amount of immunogen should not be too much, as long as it can induce an immune response. In addition, the use of highly pure immunogens can reduce nonspecific reactions and improve the specificity of immune responses. Antibodies prepared by immunizing animals with viral immunogens have a strong neutralizing effect and can bind to antigens on the surface of viral particles, preventing the virus from binding to host cells, thereby blocking the virus from entering cells. Antibodies can also play an antiviral role through other mechanisms, such as mediating immune cell phagocytosis and clearance of viral particles, interfering with the viral replication cycle, etc. These mechanisms work together to effectively resist viral infection.

TekBiotech has been committed to nanoantibody library construction and nanoantibody library screening services for many years and has rich experience in nanoantibody development. 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 preparation 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 single-domain antibody development 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 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 nanoantibodies that satisfy customers. We can also express and purify the screened nanoantibodies 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.