Diagnosis by biomarker detection has become a new trend in a wide range of diagnostics. Antibodies are the primary tool used as recognition reagents to detect biomarkers, and are widely used as analytical tools in clinical settings. Therefore, monoclonal antibodies are widely used as a main source of antibodies in rapid diagnostic tests for detecting various infectious diseases such as malaria, dengue, HIV and so on.

In order to achieve greater binding efficacy, molecular scientists have continually explored new binders with smaller size and better durability.

More recently, natural single domain antibodies (sdAbs) were discovered in a few ancient vertebrates. They are known as VHH antibody from camelids and VNAR from sharks. These sdAbs are composed of heavy chains only, thereby making them the smallest antibodies thus far.  

The unusual antibodies derived from these groups of animal have been reported to provide promising specificity and sensitivity for their target antigens. The advantages of natural sdAbs including better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs offer the posibility of advances in finding new binders for use in research, diagnostic and clinical.

Schematic representation of the comparison conventional antibody IgG with natural single domain antibodies derived from camelids and sharks.

VHH Heavy Chain Domain in Camelids

A unique subclass of immunoglobulin containing only a heavy chain domain and lacking a light chain was found in the circulatory system of camelids. This antibody has been named as “heavy chain only” antibodies (HCAbs). The isolated variable domain region of camelids HCAbs is known as VHH (variable domain of HCAbs) or Nanobody® (Nb; Ablynx). The molecular weight of VHH is 15 kDa, ten times lower than conventional antibody.

Due to their high intrinsic domain stability, camelid VHH is now under investigation as a probe for diagnostics. The diagnostic potential of camelid VHH as a probe in immunodetection systems offers possibilities for improving the diagnosis of infection, cancers, and caffeine contaminants in the food and beverage industries .

Applications of VHH Antibodies or Nanobodies®

To monitor infections, VHH antibodies naturally derived from camelids (nanobodies) may enable superior detection of species-specific antigens to classical monoclonal antibodies in immunodiagnostic tests.

Sleeping sickness and Chagas disease, which often occurs in rural Africa, a both are severe parasitic diseases caused by protozoa of the genus Trypanosoma. The antigenic variation strategy adopted by this parasite represents a major barrier to the immune system to eliminate it. Therefore, it is difficult for specific mAbs to detect genus-specific antigens. Novel nanobody clones, that showed specificity to T. evansi at a species level and genus-specific reactivity against various Trypanosoma species, were isolated by adopting an in vitro selection method. Due to their small sizes, those VHH antibodies were shown to be capable of penetrating into the conserved epitopes of antigens that are inaccessible to classical mAbs.

Cysticercosis, that is widespread in many low-income countries, is a serious tissue infection caused by larval cysts of the pork tapeworm. Monoclonal antibodies that are currently deployed in sandwich ELISAs are poorly specific at a species level to identify Taenia solium, the major species threatening human health. To recognize a specific marker on T. solium, was developed an in vitro selection of VHH antibodies from immunized dromedaries. After in vitro selection, the VHH antibodies 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 cysticerosis.

Apart from being used as diagnostic reagents for infectious diseases, VHH antibodies have been identified as alternative binders to analyze the compositions of substances in the food and beverage industries. Due to their excellent thermal stability, nanobodies showed superior performance at measuring caffeine concentration in hot and cold beverages.

VHH antibodies have recently been applied in ELISA methods to detect a wide range of small molecules, including explosive materials (trinitroluene or TNT), agents of bioterrorism (Botulinum A neurotoxin), toxins (ricin, cholera, staphylococcal enterotoxin B), scorpion toxin, and viruses (HIV, rotavirus, Vaccinia, and Marburg). Owing to the combination of several favorable properties, VHH antibodies have also been applied in some sophisticated devices to diagnose diseases. The development of biosensors coupled with nanobodies (nanoconjugates system) has enabled significant improvement in the performance of a device at identifying harmful bacteria (Staphylococcus aureus) at down to a nanometer scale within 10 min.

Nevertheless, mAbs remain common binding agents for identifying and tracing tumor-associated proteins for noninvasive in vivo imaging. However, due to their limitations, particularly their large size (150 kDa) and their Fc regions, mAbs penetrate poorly into solid tumors. The emergence of nanobodies offers the possibility of resolving such problems, and thereby promises the development of probes for diagnosing tumor markers such as EGF receptors. This will enable cancer staging predictions in the blood circulation such as prostate-specific antigen.

Source https://www.ncbi.nlm.nih.gov/pubmed/29039819