Nanobody Preparation Service
In camelids, both conventional "Y"-shaped antibodies and heavy-chain antibodies exist(HCAbs, Heavy-Chain Antibodies).The percentage of HCAbs and conventional IgG in camel serum differs: in camels, the proportion can reach 50–80%, whereas in South American camelid species it is only 10–25%. This difference is related to their genetic background.
Structurally, HCAbs comprise a single heavy-chain variable domain(VHH), a hinge region, and two conventional constant domains (CH2 and CH3),with a molecular weight of approximately90kDa.
At the genetic level, in some B cells, IGHV3H rearranges with D and J genes to form the VHH domain, which is ultimately co-expressed with HCAb-specific IGHG2 (or IGHG3) genes.During mRNA splicing, the CH1 region is excised, ultimately forming HCAbs.
Nanobody (Nb or VHH) is a heavy-chain single-domain antibody containing only the VHH region fragment. It is composed of the heavy-chain variable region of camelids (camels, llamas, alpacas, and their close relatives).Nanobody crystals have a diameter of 2.5 nm, a length of 4 nm, and a molecular weight of approximately 12–15 KD.
The region of the nanobody V domain with higher sequence variability is the hypervariable region (Hypervariable Regions, HV), and the three HVs of a nanobody are surrounded by relatively conserved regions (Conserved Framework, FR). From the spatial structure in Figure 2, the V domain consists of nine β-strands (A-B-C-C’-C’’-D-E-F-G), four of which form one β-sheet and the other five form another β-sheet. The two sheets are linked by loop structures between β-strands and disulfide bonds (formed by conserved Cys23 and Cys94). In this structure, the HVs are located at H1 to H3 connecting the B-C, C’-C’’, and F-G strands (labeled blue, green, and red in the figure). The amino acid cluster at the N-terminus of the domain forms a continuous VHH FR and HV region structure similar to VH, but there are clear differences between the two in the FR2 and CDR regions. In the FR2 region of conventional antibodies, there are highly conserved hydrophobic amino acids (Val47, Gly49, Leu50, Trp52), which are involved in binding to the light-chain variable region. In VHH, however, these amino acids are replaced by smaller amino acids and/or hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52). After this replacement, VHH cannot interact with VL, and helps VHH maintain antibody hydrophilicity (non-stickiness) in the absence of VL, and reduces VHH aggregation. Another difference between VH and VHH lies in the HV loops. The CDR1 and CDR3 regions of VHH have longer HV regions, and these longer regions form a more efficient antigen-binding interface, approximately 600–800 Å2, similar in size to the space provided by IgG antibodies. The extended CDR3 can extend into antigen cavities, such as the active-site cleft of enzymes and the ligand-binding cavity of receptors. However, the enlarged CDR regions also have greater flexibility, and this flexibility can also negatively affect antigen–antibody binding. In VHH, disulfide bonds formed between CDR3 and CDR1 or CDR2, or even FR2, can solve this problem. At the same time, these disulfide bonds can optimize the topology of the binding site to the greatest extent and promote the orientation of CDR3 toward the antigen.
Nanobodies are used in biopharmaceutical R&D(genetic engineering drug development, ADC drug development), clinical in vitro diagnostics(colloidal gold assay, enzyme-linked immunosorbent assay, electrochemiluminescence assay), tumor research, immunology research, etc.
Intracellular Applications of VHH
Diagnostic Applications of VHH
Therapeutic Applications of VHH
Summary of Nanobody Advantages
