**5.1. Applications of camelids VHH domains or nanobodies®**

To monitor infections, single domain antibodies naturally derived from camelids (nanobodies) may enable superior species-specific antigen detection than classical monoclonal antibodies in immunodiagnostic tests. Trypanosome infection causes African sleeping sickness and Chagas disease. Both are severe parasitic diseases caused by protozoa of the genus *Trypanosoma*. Sleeping sickness disease is mainly found in rural Africa. The antigenic variation strategy adopted by this parasite represents a major barrier to the immune system to eliminate it. Consequently, it is difficult for specific mAbs to detect genus-specific antigens [127]. By adoption of an *in vitro* selection method, novel nanobody clones were isolated that showed specificity to *T. evansi* at species level, and genus-specific reactivity against various *Trypanosoma* species [128].

Cysticercosis is a serious tissue infection caused by larval cysts of the pork tapeworm, which is prevalent in many low-income countries [129]. Monoclonal antibodies that are currently deployed in sandwich ELISAs are mainly genus-specific against *Taenia* sp., but poorly specific at a species level to identify *Taenia solium*, the major *Taenia* species threatening human health [130, 131]. To circumvent such limitations, an *in vitro* selection of nanobodies from immunized dromedaries was developed to recognize a specific marker on *T. solium*. After *in vitro* selection, the nanobodies showed no cross-reactivity against other livestock *Taenia* species, while having a very specific response to a specific 14 kDa glycoprotein (Ts14) in *T. solium*. Therefore, nanobodies showed potential as an alternative to genus-species mAb for developing unambiguous ELISA tests for human cysticercosis [97].

Apart from diagnostic reagents for infectious diseases, nanobodies have been identified as alternative binders to analyze the compositions of substances in food and beverages industries. Due to their excellent thermal stability, nanobodies showed superior behavior to classical mouse mAbs in ELISA to measure caffeine concentration in hot and cold beverages [132].

Camelid sdAbs have recently been applied in ELISA methods to detect a wide range of small molecules, including explosive materials (trinitroluene or TNT) [133], agents of bioterrorism (Botulinum A neurotoxin) [90], toxins (ricin, cholera and staphylococcal enterotoxin B) [134], scorpion toxin [135] and viruses (HIV, rotavirus, Vaccinia and Marburg) [136–138]. Owing to the combination of several favorable properties, camelid nanobodies have also been employed as molecules to diagnose diseases. In small molecule development, the advanced features of highly stable and unique conformational structure of nanobodies have permitted overcoming many problems faced by traditional whole antibodies and scFv fragments such as cross-reactivity and nanoparticle agglutination. The development of biosensors coupled with nanobodies (nanoconjugates system) has enabled significant improvement in the performance of a device to identify harmful bacteria (*Staphylococcus aureus*) to a nanometer scale within 10 min [139].

Nevertheless, mAbs remain the common binding agents to identify and trace tumor-associated proteins for noninvasive *in vivo* imaging. However, limitations, particularly large size (150 kDa) and Fc regions, result in mAbs poorly penetrating into solid tumors [140]. The emergence of nanobodies offers the possibility of resolving such problems, and thereby enables nanobodies to diagnose tumor markers such as EGF receptor [141]. This will enable cancer staging predictions in blood circulation such as prostate-specific antigen [142]. More applications using camelids VHH targeting antigens from various diseases are summarized in **Table 2**.
