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 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 nano antibodies has a broad application market, 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 development is carried out through alpaca immunization because animals such as alpacas are easy to breed and immunize, and the profits are high.

The immunogens used in alpaca immunization mainly include natural antigens, recombinant antigens, synthetic antigens, and small molecule antigens. The purification of natural antigens is difficult and the cost is also high. Although recombinant antigens are different from natural antigens in conformation, recombinant antigens can be mass-produced. Synthetic antigens can be prepared by in vitro synthesis methods, mainly small molecule proteins or polypeptide antigens, whose structures are controllable, but may be complicated to design. Small molecule antigens are mostly small molecule compounds such as oligosaccharides and nucleotides. Since they are not immunogenic themselves, they can only be used as immunogens after being connected to macromolecular carriers.

Fig. 1 Structure of immunoglobulin G and sites available for modification

Preparation of Synthetic Antigens:

When preparing protein immunogens, the differences between the target protein and the host system will lead to differences in the expression level and stability of the protein, so the preparation of high-expression and high-purity proteins is relatively difficult. Since some proteins require specific post-translational modifications to maintain their antigenicity, the selection of a suitable protein expression system is crucial. In order to maintain the natural conformation of the protein and stimulate the body to produce a corresponding immune response, it is necessary to add auxiliary factors and special conditions. When preparing small molecule immunogens, small molecules need to be coupled with carrier proteins to improve immunogenicity, but the coupling efficiency may be low, the process is relatively complicated, and the stability of the coupled immunogens during transportation is difficult to maintain. When preparing polypeptide immunogens, if the polypeptide to be prepared is a long-chain polypeptide, the preparation process is relatively complicated, especially for polypeptides with complex sequences, which are difficult to synthesize and purify, and not all polypeptide sequences have sufficient immunogenicity. In order to have the correct epitope display, it is necessary to design a special carrier so that the polypeptide can display the antigen epitope in an appropriate conformation.

For a specific expression system, optimizing the codons of the target gene and selecting or designing a strong promoter can increase the expression of the protein. The use of adjuvants can enhance the body's immune system's response to the immunogen during the immunization process, and can also gradually enhance the immune system's response through multiple immunizations.

When conducting subsequent polypeptide and small molecule screening, it is necessary to ensure that specific antibodies against the target polypeptide or small molecule can be identified and separated during the screening process to avoid interference from nonspecific antibodies. During the screening process, the stability of polypeptides and small molecules must also be maintained to avoid degradation or denaturation. When screening, it is best to choose a highly sensitive screening method so that low concentrations of antibodies or antigens can also be detected.

Key Points of Immunogen Preparation:

When preparing protein immunogens, different protein expression systems should be selected according to the characteristics of the target protein, such as Escherichia coli, yeast, insect cells or mammalian cells. The stability and solubility of the protein can be improved by adjusting the temperature of induction expression and optimizing the induction time. The use of appropriate salts, pH buffers and stabilizers, such as glycerol, during the purification process can maintain the natural conformation of the protein. The introduction of specific functional groups on small molecules by chemical synthesis can improve the immunogenicity of small molecule immunogens. Commonly used carrier proteins in small molecule design include KLH and BSA. These large molecular proteins can carry multiple small molecule epitopes and enhance the immunogenicity of small molecules. Biocompatible cross-linkers can be used to achieve stable coupling of polypeptides and carrier proteins to ensure the stability and activity of polypeptide immunogens in vivo.

Select an appropriate polypeptide sequence length for polypeptide sequence design, generally between 15-20 bases, to ensure that it contains enough antigenic determinants. Adjuvants can be used to enhance the immune system's response to immunogens during the immunization process.

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 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 nanoantibodies with high affinity. 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 target fragments, 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 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 nanoantibodies for customers.