Antibodies are immunoglobulins produced by B lymphocytes and can participate in humoral immunity. They are composed of variable domains, light chain variable regions (VL) and heavy chain variable regions (VH). Antibodies recognize target antigens through their variable fragments. In the 1980s, BIrd et al. designed single-chain antibodies (scFv) as a genetic fusion of VL and VH, which linked VL and VH through short flexible peptides. scFv is the smallest binding unit with antibody activity. Because it lacks the Fc domain, its size is very small, about 25 kDa. Compared with full-length antibodies, it is easier to penetrate tissue barriers and penetrate tumor tissues. It can be used in targeted cancer therapeutic drugs and also plays an important role in detection reagents. In addition, based on the characteristics of scFv without Fc fragments, its immunogenicity is low and the risk of causing immune response in the human body is relatively small. scFv can also be efficiently expressed in a variety of expression systems, including prokaryotic expression systems and eukaryotic expression systems, such as Escherichia coli, yeast, insect cells, mammalian cells, etc.

Fig. 1 Comparison of the structure of traditional antibodies and single-chain antibodies

Applications of Single-Chain Antibodies:

scFv can be linked to chemical toxins to form immunotoxins, which can then target and kill tumor cells. In addition, scFv can also be linked to T cells to construct chimeric antigen receptor T cells (CAR-T) for cancer treatment. scFv can neutralize specific antigens on the surface of viruses, thereby inhibiting the replication and spread of viruses. scFv can be fused with markers such as fluorescent proteins for early diagnosis and localization of diseases. Phage display technology can display scFv on the surface of phages, and drug screening becomes more convenient and faster by constructing a single-chain antibody library.

Preparation of scFv Antibody Library:

First, peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood of immunized animals, and total RNA in PBMCs is extracted. During the extraction process, attention should be paid to the integrity and purity of RNA. The extracted RNA is used as a template and reversely transcribed into cDNA. Then, cDNA is used as a template to amplify the VH and VL genes through PCR. Generally, two rounds of PCR amplification are performed to improve the specificity and yield of the antibody. Use a specific Linker sequence to connect the VH and VL genes to form a complete scFv gene, insert the scFv gene into the phagemid vector, and construct an expression vector. The vector can contain promoters, terminators, replication origins, etc. to ensure the correct expression of the scFv gene in the host cell. Transform the constructed expression vector into host cells, such as Escherichia coli, yeast or mammalian cells. The expressed scFv antibody is based on the phage antibody library construction technology to construct a single-chain antibody library. The solid-phase antigen can be incubated with the antibody library, and after several rounds of washing, elution, and amplification, single-chain antibodies with specificity and affinity are screened. Select positive clones from the screened single-chain antibody library and sequence them to verify whether the sequencing results are consistent with the expected scFv gene sequence.

Animals That Can Produce scFv Antibodies:

Animals of different species have differences in immune systems and antibody characteristics, and these differences will affect the efficiency and specificity of single-chain antibody production. Commonly used animals for single-chain antibody production are rabbits, sheep, and rats. Among them, rabbit IgG has high sensitivity and specificity in immune response, and the structure of rabbit IgG is relatively simple and stable. Therefore, rabbits are ideal for preparing high-affinity and high-specificity single-chain antibodies. Rabbit single-chain antibodies are widely used in the diagnosis and treatment of diseases that require high sensitivity and high specificity. Rats are one of the commonly used experimental animals. Its IgG has multiple subtypes, and different subtypes also have certain differences in structure and function. The immune system of rats can produce diverse antibodies. Therefore, rats are widely used in the production of single-chain antibodies with specific functions and characteristics. In experimental studies simulating human disease states, rat single-chain antibodies are the most common. Sheep IgG also has multiple subtypes, and the immune system of sheep can target multiple antigens. Sheep single-chain antibodies have potential application value in livestock disease prevention and control.

Table 1. Comparison of animals that can produce single-chain antibodies

TekBiotech has a mature single-chain antibody library construction system, which can prepare scFv antibody libraries for spleen cells and human B lymphocytes of immune animals. And we will conduct a comprehensive analysis of the scFv antibody 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-chain antibody library construction services. TekBiotech specializes in constructing different types of scFv phage display libraries, such as immune libraries, natural libraries, semi-synthetic libraries, synthetic libraries, etc. The scFv antibody library we construct has a large capacity and can produce high-affinity scFv 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 construct has a large capacity of up to 10^9, with a high insertion rate of target fragments, which is conducive to screening out scFv antibodies that satisfy customers. We can also express and purify the screened scFv 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, which can produce high-quality scFv antibodies for customers.