**4.1. VHH heavy-chain domain in camelids**

Conventional immunoglobulins comprise two major parts such as the antigen-binding fragment (Fab region) and fragment crystallisable region (Fc region), with a typical molecular weight of 150 kDa. The Fab domain is responsible for antigen binding and therefore its specificity. This domain is divided into heavy (H) and light (L) chains with the molecular weights of 25 kDa each [85]. The stability of the molecular complex of an immunoglobulin is conferred by four inter-domain disulfide bonds in the hinge regions. The heavy chain can be subdivided into one variable (VH) region and three constant (C) regions (CH1, CH2 and CH3) while the light chain contains one variable region (VL) and only one constant region (CL). Lacking direct antigen-binding functions, the main role of the Fc domain is to provide effector functions such as binding to cellular receptors on macrophages and complement activation, and determination of the half-life of an antibody [86].

In addition to conventional heterotetrameric antibodies, the sera of Camelidae were discovered to possess special IgG antibodies known as heavy-chain antibodies (HCAbs). Although HCAbs contain both a constant (Fc) and variable domain, these antibodies are slightly different from conventional IgG by devoid of the L chain polypeptide and the first constant domain (CH1) (**Figure 1**). Therefore, the isolated variable domain region of camelids HCAbs is known as VHH (variable domain of the heavy chain of HCAbs) or Nanobody® (Nb; Ablynx) [87]. VHH constitutes a binding surface to interact with the target antigen. The molecular weight of VHH is 15 kDa, 10 times lower than that of a conventional antibody. It was thereby considered the smallest possible antibody fragment and has attracted the interests of many scientists [88, 89]. Moreover, the capability of camelid antibodies to retain the reversibility and binding activity after heat denaturation has enabled new applications where transient heating may occur [90].

The major advantage of a VHH antibody is their greater solubility compared to classical VH [17]. This is due to the hydrophilic amino acid substitution present in the framework 2 region. Meanwhile, the single coding exon of less than 450 base pairs facilitates genetic engineering of VHH fragments [91]. In addition, on account of its smaller antigen binding surface area, the unique CDR3 region enables the heavy domain of camelids to penetrate into antigen cleft regions that are not easily recognized by conventional antibodies [92, 93]. From a phylogenetic prospect, since camelids are related to the primate lineage [94] it is possible to produce humanized VHH, a process that may be easier to perform than the complicated manipulation required to "humanize" murine or other more distant species to reduce an alloresponse, such as the human anti-mouse antibody (HAMA) response [95].

Furthermore, due to their high intrinsic domain stability, camelids VHH are now under investigation as probes for diagnostics [18, 96]. The diagnostic potential of camelids VHH as probes in immunodetection systems offers the possibilities of improving the diagnosis of infection [97], cancers [98] and food contaminants [99]. Although VHHs do not originate from humans, the humanizations strategies of VHHs have successfully been undertaken by designing a humanized scaffold region onto the antigen-binding loops (CDRs) of specific VHHs can be grafted [100]. In addition, non-humanized and humanized VHHs with therapeutic potential have been applied in multiple areas, including hematology [101], inflammatory diseases [102], infectious diseases [103], *in vivo* imaging [104], neurological disorders [105] and oncology [106, 107].
