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Introduction to single domain VHH property:        Immunoglobulin (Ig) is a protein that has two distinct structural components, the fragment antigen-binding (Fab) domain and the fragment crystallizable (Fc) area, which can be separated by proteolytic cleavage with papain and pepsin. The binding specificity of the entire Ig molecule, especially the two variable domains on the top—variable heavy chain (VH) and variable light chain—is solely dependent on this domain, whereas the Fc region triggers biological processes upon antigen binding, Fab is responsible for antigen recognition (VL).         As we know camelid antibodies are different from conventional antibodies as they are made up of two heavy chains only. These antibodies do not contain CH1 region, instead, they have an antigen binding domain known as the VHH region. So, VHH antibodies are also known as single domain antibodies.         Single-chain variable fragments (scFvs) and camelid heavy-chain variable domains (VHHs), often known as nanobodies, are the most widely used types of designed and recombinant antibody forms. Single-chain variable fragment (scFv), which is made up of VH and VL, was previously thought to be the smallest antibody fragment with the same antigen-binding selectivity as the entire Ig molecule. However, it has been shown that a single V-like domain can maintain the affinity of an entire antibody molecule by the discovery of the camelid VHH and shark variable novel antigen receptor (VNAR) (Asaadi et al., 2021).

Properties of VHH antibodies:Size:The size of nanobody is about 30kDa weight. Due to their smaller size and the fact that they contain only three antigen-binding loops, VHHs are more easily genetically modified to increase their natural propensity to attach to antigen. The smaller size of VHHs also contributes to their short half-life in blood, which is a result of renal filtration and degradation. This property can be advantageous because it increases tissue permeability, but it can also be disadvantageous since it causes complications in some clinical regimens that call for prolonged antibody circulation because their molecular weight is below the glomerular filtration threshold size (65 kDa).

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