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. VHH antibody 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.

According to different molecular weights and properties, immunogens are divided into proteins, polysaccharides, nucleic acids (DNA and RNA), and cell lines. Protein immunogens include enzymes, proteins, bacterial toxins and other substances that are antigenic and can cause immune responses in the body. Nucleic acid immunogens include DNA and RNA. DNA immunogens express antigenic proteins by introducing target genes into host cells, and RNA immunogens express antigenic proteins in cells through mRNA. Cell-based immunogens contain a variety of antigenic epitopes and can stimulate the body to produce complex immune responses. Virus-like particles (VLPs) are a commonly used cell-based immunogen.

Fig. 1 Structure-based immunogen design

Preparation of Immunogens:

When preparing DNA immunogens, it is necessary to select a suitable vector system to ensure that the target DNA is correctly cloned into the vector and that the DNA immunogen is effectively delivered to the host cells. The host cells must also be able to correctly express and process the generated antigens. The transfection efficiency and expression level can be improved by optimizing the design and construction of the vector. Immunogen delivery methods such as electroporation and liposome-mediated transfection also need to be correctly selected to improve the delivery efficiency. Since RNA is easily degraded, stabilizers should be added to ensure the integrity of RNA when preparing RNA immunogens. When storing RNA immunogens, conditions such as temperature and pH should also be strictly controlled. The success rate and efficiency of RNA packaging can be improved by optimizing the packaging system. When cell line type immunogens are cultured, cell contamination should be prevented to ensure normal cell growth. When extracting immunogens from cells, appropriate and gentle extraction methods should be selected to maintain the natural structure and activity of the protein.

The immunogenicity of the prepared immunogen can be enhanced by introducing specific epitopes on the antigen through genetic engineering. According to the expression properties of the antigen, a suitable carrier system is selected to improve the expression level and stability of the antigen. The culture conditions of the expression system, such as temperature, pH value, culture medium components, etc., can optimize the expression and folding of the antigen after appropriate adjustment.

Key Points of Immunogen Preparation:

When preparing DNA immunogens, plasmid DNA should first be constructed according to the antigen type and its function. The coding gene of the target antigen is cloned into an appropriate plasmid vector, and promoters, enhancers, etc. are added to ensure that the target antigen is efficiently expressed in the host cell. The vector can be modified, such as adding fluorescent tags, for subsequent tracking and detection. Plasmid DNA is amplified by bacterial culture, and plasmid DNA is extracted by alkaline lysis or plasmid extraction kit. Purification methods such as gel filtration and ultracentrifugation are used to remove impurities and obtain high-purity plasmid DNA. After the immunogen is inoculated into the animal, the host's immune response is monitored regularly, and antibodies are collected and purified from the blood of the immunized animal for subsequent use. When preparing RNA immunogens, cap structures, poly (A) tails, etc. can be added to RNA to improve its stability and immunogenicity.

TekBiotech has been committed to VHH antibody 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 nano-antibody preparation 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. In addition, 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 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 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. 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.