1.Immunogenicity of Nanobody

Immunogenicity is related to molecular weight and chemical structure, and the smaller the molecular weight, the smaller the immunogenicity. Because the molecular weight of nanobody is only 1/10 of that of conventional antibodies and only one domain, the probability of stimulating the body to form specific antibodies or causing humoral and cellular immune responses is greatly reduced. At the same time, the nanobody has no Fc segment, avoiding the complement reaction caused by Fc, and has low immunogenicity and good biocompatibility with human. Therefore, nanobody (Nbs) are beneficial in reducing their immunogenicity compared to conventional antibodies. However, nanobody are foreign substances to the human body, which may still trigger immune response. Moreover, studies have shown that long-term repeated use of nanobody as drugs will increase immunogenicity and affect treatment, so it is very important to analyze the immunogenicity of nanobody.

Figure 1: Structures of conventional antibodies (left), heavy chain antibodies (middle), and nanoantibodies (right)

In the process of antibody preparation, the larger the immunogenic molecule, the more the number of determinant. The more types of activated B cells there are, the more types of antibodies there are. Therefore, the selection and design of immunogens is the relatively core link of antibody research and development, which determines whether specific antibodies can be produced in the immune preparation process. The number and number of antibody types and the strength and weakness of the specificity of the obtained antibodies also affect the intensity of the later purification and verification process.

The selection of immunogens includes polypeptide peptide segment, prokaryotic expression of whole protein, eukaryotic expression of whole protein, cell, etc. Combined with the characteristics of the immunogen molecule, the application of antibodies can provide the type of epitope expected to be recognized, determining which type of immunogen to use and the sequence of the immunogen.

2.The Type of Immunogen

The immunogen types of nanobody include protein, small molecule, polypeptide, virus, DNA, RNA and cell line type immunogen. Combined with the characteristics of the immunogen molecule, the application of antibodies can provide the type of epitope expected to be recognized, determining which type of immunogen to use and the sequence of the immunogen. For example, the experimental process of IHC involves a denaturation process of its own antigen, or that of the target in natural cells. Therefore, when designing, it tends to be designed as a linear epitope, such as the use of peptides as an immunogenic way to carry out the development of immunohistochemical antibodies. Prokaryotic/eukaryotic recombinant proteins have good antigenicity in most production. In experiments involving conformational binding, unknown non-specific binding may occur, which increases the difficulty of subsequent screening. As immunogen, the cells are suitable for the development and production of flow antibody.

3.Cell Line Type Immunogen

When you do not know what antigens you need, such as when you want to prepare antibodies against tumor-specific antigens, you often need to use tissues, whole cells, or cell components as antigens for animal immunization.

If tissue is used as an antigen, the fresh tissue needs to be removed, washed with saline, and then separated into individual cells with a strainer and pestle. If the whole cell is used as the immunogen, the serum in the medium needs to be washed away, and it is recommended to directly use the whole cell as the immunogen rather than breaking it, because the whole cell has a stronger granular and exogenous. Tissue-derived cells and cultured cells are used as immunogens for animal immunization through peritoneal immunization, and subcutaneous immunization is not recommended. For cell components as antigens, the emphasis is on the separation of cell components. Cell membrane and cytoplasm can be separated by hypotonic method (only a low concentration buffer is required, and the addition of MgCl2 can stabilize the nucleus so that it does not break), or Ca2+ can be homogenized, and then centrifuged, and the cell membrane will form a precipitate below. The separation of intracellular organelles depends mainly on density gradient centrifugation. Some organelles have relatively close density differences, so finer density gradient centrifugation may be required.

Sometimes special methods can be used to help. The sedimentation coefficient of mitochondria and peroxidase bodies is very close, and it is not easy to separate them by density gradient centrifugation, so Triton WR1399 can be injected into mice. This detergent cannot be metabolized by mice and accumulates in the mitochondria of the liver, so the mitochondrial density becomes smaller at this time, and it is easy to separate from the peroxisome.

4.Difficulties and Key Points in the Process of Cell Line Type Immunogen Production

This is a method that uses a cell line as an immunogen, usually by producing antibodies from a cell line that expresses a target protein. Cell line type immunogens are useful for studying the function and localization of proteins within cells. However, it is more complex in terms of building and maintaining stable cell lines.

Using cell lines as immunogens requires more processes, including culture and cell extraction. Cell culture: The maintenance and culture of cell lines requires special attention to ensure the health of cells and high expression of immunogens. Extraction method: Extraction of immunogens from cells requires the use of appropriate methods to preserve the natural structure and activity of the protein. Different cell lines have different characteristics, and choosing the right cell line is crucial.

The customized nanoantibody service provided by TekBiotech covers the entire process from immunogen preparation to nanoantibody development. First, TekBiotech ensures that it can stimulate a strong immune response in the target animals through efficient immunogen design and preparation. Subsequently, advanced technical means, such as phage display technology, are used for efficient screening and cloning to obtain nanoantibodies with strong specificity and high affinity. Throughout the service process, TekBiotech, relying on its mature technology platform and rich experience, can quickly respond to customer needs and provide customized solutions to help customers efficiently obtain ideal nanoantibodies. Whether in drug development, diagnostic reagent development or therapeutic research, TekBiotech's customized nanoantibody service can provide strong support for customers.