Antibodies are immunoglobulins that specifically bind to antigens. They are produced by plasma cells differentiated from B cells and exist in the blood and other body fluids of vertebrates, as well as on the cell membrane surface of their B cells. Antibodies are composed of two identical heavy chains (H chains) and two identical light chains (L chains). The heavy chains are longer and have a larger relative molecular weight, while the light chains are shorter and have a smaller relative molecular weight. Antibodies have a Y-shaped symmetrical structure.

Nanobodies (Nbs) are a new type of antibody first discovered in camel blood by Belgian scientists Hamers and others. Compared with traditional antibodies, this type of antibody does not have a light chain, but only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The molecular weight is only about 15 KDa, so it is called a nanobody, which is the smallest unit known to bind to the target antigen.

Nanobodies have Unique Structural Advantages:

1. Small Relative Molecular Mass and Low Immunogenicity

The sequence homology between the framework region (FRs) of Nbs and the human VH region exceeds 80%, and their three-dimensional structures can overlap. It has been proven in mice that Nbs have low immunogenicity due to their high homology with the traditional VH region and high conformational stability.

2. Good Solubility

There are some important differences between the VHH of Nbs and the VH of traditional antibodies. Compared with VH, the four amino acid residues (V37F or V37Y, G44E, L45R, W47G) of the VHH framework region 2 (FR2) are replaced. These four amino acids are hydrophilic, making the surface of Nbs more hydrophilic and increasing its water solubility.

3. Strong Antigen Recognition Ability

Similar to traditional VH, VHH includes 4 FRs and 3 complementary determining regions (CDRs). Compared with VH, the CDR1 and CDR3 of VHH are longer, and the binding of CDR3 to the cysteine residues of CDR1 or FR2 is achieved through disulfide bonds, which makes its structure very stable. Traditional Fab fragments and typical single-chain antibodies (scFv) can only recognize surface antigens because their antigen binding sites are concave or flat. VHH has a protruding antigenic determinant cluster mainly composed of CDR3 loops, which can bind to concave antigen epitopes, so Nbs can specifically recognize some hidden antigen epitopes that cannot be recognized by traditional antibodies. Therefore, Nbs is more suitable for binding to the groove part of the antigen surface, such as the catalytic reaction site of the enzyme, than the scFv antibody binding site, thereby blocking its catalytic activity.

4. High Stability

Nbs can form different conformational patterns to protect the stability of amino acids. Under extreme environmental conditions, such as high temperature or extreme acidity and alkalinity, ordinary polymer antibodies will expose hydrophobic surfaces, form macromolecules and precipitate, and lose their original normal functions; while nano antibodies can be refolded after chemical and thermal denaturation, and by re-forming a disulfide bond between CDR1 and CDR3, the structural stability ensures the stability of its functional activity.

5. Strong Penetration

Nbs can penetrate the blood-brain barrier. Studies have shown that immunization of brain vascular endothelial cells with camel-derived Nbs found that Nbs can be released on the basolateral side of vascular endothelial cells through transcytosis. In vitro studies have shown that nano antibodies can penetrate the blood-brain barrier and can carry some substances such as M13 phage particles to the brain.

6. Easy to Produce

Nano antibodies are easy to express in large quantities in protein expression systems such as yeast expression systems and prokaryotic expression systems due to their simple structure, small relative molecular mass and lack of glycosylation. Nano antibodies have low production costs and are easy to promote and apply.

Nanobodies are widely used in basic research, disease diagnosis and treatment due to their unique advantages. Some have entered clinical phase I or II trials, including oncology, neurology and immunology. In addition, Nbs can also be used for virus detection and diagnosis, such as HIV, vaccinia virus, green monkey disease virus and dengue fever; nanobodies are also used in the detection of small molecule compounds such as environmental pollutants and biotoxins, which improves the sensitivity of detection and the simplicity of operation; in addition, nanobodies have also been studied and applied in food toxicology research and food analysis and detection. For example, studies have shown that some Nbs can bind to bacteria and inhibit the enzyme production ability of bacteria, thereby playing a certain therapeutic role.

Tek Biotech has been committed to the production of alpaca VHH antibodies for more than 9 years and has an experienced technical team with rich biological technology background, which can provide one-to-one special services. Tek Biotech has a stable mammalian CHO cell and prokaryotic cell nanobody expression platform, which can provide customers with one-stop services from antibody sequence analysis, linker design, label selection design, vector selection design to exploration and optimization of recombinant nanobody expression conditions, recombinant antibody purification, etc. In addition, Tek Biotech also has a complete quality system and has established a variety of purity analysis methods such as SDS-PAGE and HPLC. It has rich experience in nanoantibody quality evaluation systems. At the same time, it also has a variety of nanoantibody purification methods such as affinity chromatography, hydrophobic chromatography, and ion chromatography, which can be flexibly combined and can also provide large-scale production services.