Single Domain Antibodies as New Biomarker Detectors

Biomarkers are defined as indicators of biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers have been widely used for early detection, prediction of response after treatment, and for monitoring the progression of diseases. Antibodies represent promising tools for recognition of biomarkers, and are widely deployed as analytical tools in clinical settings. For immunodiagnostics, antibodies are now exploited as binders for antigens of interest across a range of platforms. More recently, the discovery of antibody surface display and combinatorial chemistry techniques has allowed the exploration of new binders from a range of animals, for instance variable domains of new antigen receptors (VNAR) from shark and variable heavy chain domains (VHH) or nanobodies from camelids. These single domain antibodies (sdAbs) have some advantages over conventional murine immunoglobulin owing to the lack of a light chain, making them the smallest natural biomarker binders thus far identified. In this review, we will discuss several biomarkers used as a means to validate diseases progress. The potential functionality of modern singe domain antigen binders derived from phylogenetically early animals as new biomarker detectors for current diagnostic and research platforms development will be described.


Introduction
Early and accurate diagnosis of disease is important for providing appropriate treatment to individuals with most human diseases. Clinical diagnosis remains a mainstay method for clinical care in many settings, such as among febrile patients in endemic areas [1]. However, the overlap of clinical symptoms of many diseases makes misdiagnosis likely and frequent, thereby impeding treatment decisions and epidemiologic information [2].
Microbiological methods represent definitive diagnostic method for various infectious diseases in laboratory settings [3]. Samples tested are predominantly blood, serum, stool and urine that can be Venoms are biological toxins secreted by certain kinds of poisonous animals, such as snakes, scorpions, spiders, hornets and wasps, which usually use them for attacking their prey by targeting the victim's lymphatic system through biting or "injection" using a special "weapon" or so-called sting [97][98][99]. Snakebite envenomation remains a life-threatening medical emergency worldwide [100]. To prevent inappropriate treatment, which can result in allergy, paralysis or even death of the victims, the establishment of rapid, reliable and specific detection of envenomation is necessary. A diagnostic test is therefore essential to improving emergency management of envenomation to facilitate the provision of appropriate antivenom therapy [101].
Techniques for detection of different snake venoms have been extensively reviewed. Antigen capturing ELISA has been claimed to be the best method for detection of snake venom [102,103]. The first commercial diagnostic kit for detection of snake venoms was introduced by the Commonwealth Serum Laboratories (CSL) in Australia in 1991 [104]. This kit was specially designed to detect the venoms of the five most dangerous snakes in Australia and Papua New Guinea, including Australian Tiger snake, Brown snake, Black snake, Death adder and Taipan [105,106]. The CSL snake venom diagnostic kit (SVDK) promises to provide a rapid, easy-to-use, low-cost and long-shelf-life test. As snake venoms are complex mixtures, a polyclonal antibody is used in each well in SVDK to distinguish particular types of snake venom [107]. The SVDK has been widely evaluated in both humans and animals in many clinical studies, and has shown high sensitivity and specificity [107][108][109]. This kit has been reported to be able to detect 2.5 ng/mL venom in less than 20 min, and has a specificity of 100%. Due to high stability of immunoreagents and low cross reactivity, these characteristics have made SVDK widely used by health workers in tropical countries [107].
Several alternative diagnostic kits have also been developed to identify the venoms of the four common snakes from Asia [110][111][112]. Unlike SVDK, whole blood can directly be used as sample in the AB-microELISA kit. The sensitivity of this assay for detection of venom has been shown to be 10 ng/mL. In addition, 600 µL of whole blood is sufficient for this kit, and results are available within 30 min [113]. Apart from whole blood, serum, urine blister fluids and bite site swabs can also be used as assay samples. However, further studies are ongoing to validate the prototype of AB-microELISA kit for field use [110].

Infectious Diseases
Despite decades of advances, infectious diseases continue to represent leading causes of morbidity and mortality throughout the world [114][115][116]. Millions of people are living under threat of a diversity of diseases caused by bacteria (e.g., Staphylococcus aureus and Salmonella typhi), viruses (Human Immunodefiency Virus and Hepatitis C virus), fungi (Candidasis, Coccidioides and Pneumocystis) and parasites (malaria and helminths). Infections lead to disability, death and social and economic disruption [117,118]. According to WHO statistics, approximately 15 million people are killed by such diseases in developing countries annually [119]. Moreover, sexually transmitted infections such as syphilis [120], and tropical parasitic infections such as schitosomiasis also cause enormous morbidity [121]. Due to the diversity of environmental conditions in varying developing countries, a need has arisen for the development of simple, accurate, and stable diagnostic tools. The deployment of highly sensitive and specific diagnostic tests is also needed to counteract the spreading of drug resistance of infectious diseases [122].

Diagnostics for Viral Disease
Since the first case of AIDS was reported in early 1980s, HIV/AIDS has caused 1.7 million HIV-related deaths in 2011 alone and estimated about 34 million people were living with HIV worldwide in 2010 [123]. AIDS, referring to acquired immunodeficiency syndrome, is a dangerous infectious disease that eventually causes death without treatment. The etiological agent of AIDS is known as human immunodeficiency virus (HIV), a retrovirus with a single-stranded RNA (ssRNA) [124]. However, many persons who are infected with HIV are not aware of the infection until late in the course of disease.
To increase access to early treatment and prevention, rapid HIV tests play an integral role in HIV prevention activities in both clinical and non-clinical settings [125,126]. ELISA assays are extensively used to screen for the appearance of specific antibodies. To perform a typical indirect assay, the serum collected from a patient is incubated to detect a response to an HIV target antigen; for example, p24, gp24 or gp120. A positive antibody response is then detected by an enzyme-labeled anti-human antibody or an enzyme-labeled antigen [127,128].
Instead of using blood or serum as a sample, HIV infection can also be diagnosed by detecting the presence of anti-HIV antibodies in a patient's saliva or urine samples [129,130]. For example, the OraSure ® assay is a specific salivary test that is designed to determine anti-HIV IgG antibodies from saliva [131]. However, HIV diagnostic tests based on urine and saliva are still not comparable to blood samples in terms of detailed information and the specific characteristics of the HIV subtype responsible for the infection [132].
With the aid of rapid tests, screening tests can be conducted in hard-to-reach patient populations. Unlike high-throughput EIA screening tests, RDTs can perform well for HIV diagnosis even when the volume of samples is low [133]. With the administration of combination anti-retroviral therapies, the transmission rate of HIV can be reduced from over 25% to less than 2% for those infected pregnant patients [134]. Recently, the US FDA has approved four types rapid tests for screening HIV-1 infection, including Murex ® Single Use Diagnostic System HIV-1 Test (Murex Diagnostics, Inc., USA), OralQuick ® Rapid HIV-1 Antibody Test (OraSure Technologies, Inc., USA), Reveal ® Rapid HIV-1 Antibody Test (MedMira Laboratories Inc., Canada), and UniGold Recombigen ® HIV (Trinity Biotech PLC, Ireland) [135].
However, false-negative results can occur in individuals who are in the acute phase of infection [136]. Diagnostic tests that directly detect the HIV p24 antigen in serum samples have been shown to be superior to antibody-based detection tests for early infection [137]. For instance, the commercial Vidas Duo assay (bioMerieux Inc., March-L'Etoile) is a fourth-generation ELISA. This assay possesses the advantage by decreasing the diagnostic window to an average of 7 days. In addition to detecting anti-HIV antibodies, this kit targets the HIV p24 antigen that is present in the blood of an HIV infected individual in the early phase [138].

Diagnostics for Bacterial Disease
Typhoid fever is a serious systemic illness caused by the Salmonella enterica serotype Typhi. This disease represents the most common cause of community-acquired bacteremia in developing countries [139]. The annual global incidence of typhoid fever is over 21.5 million cases, resulting in more than 200,000 deaths [140]. The emergence of multidrug resistance S. typhi has complicated treatment [141]. Therefore, rapid and accurate diagnosis is essential to provide early antimicrobial treatment, for preventions of mortality cases, and for the control of disease transmission [142].
In the past, the Widal test was the most widely used serologic test for detection of host antibodies. This agglutination test targeted typhoid antisera such as lipopolysaccharide (O) and flagellar (H), and Vi antigens of S. typhi [143,144]. However, tests was limited by false-positive results due to cross-reaction of the antigenic determinants with non-typhoid Salmonella or other tropical diseases such as malaria and dengue [145,146]. False-negative reactions may also occur if the blood sample is collected too early from the infected individuals [147]. The Widal test is not a satisfactory test for diagnosing typhoid fever in endemic areas [148]. Recently, a new generation of rapid serologic tests has been developed, for example Linear Cromotest ® (Linear Chemicals, Barcelona, Spain). This test aims to detect host IgM and IgG antibodies which are specific to O and H antigens of S. typhi. The highest reported specificity (50%) and sensitivity (95.2%) limited the accuracy of diagnosis of typhoid fever at two sub-Saharan African sites [149].
TUBEX ® (IDL Biotech AB, Bromma, Sweden) is semiquantitative colorometric rapid test that use polystyrene particle agglutination to detect anti-O9 IgM antibodies specific for group D of S. typhi. This kit enables rapid diagnosis of typhoid fever patients, with only 3 min per test at room temperature [149]. The TUBEX ® test kit is designed for detection of antibodies in the patient's serum by inhibiting the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle [150]. Thus, an acute stage of S. typhi infections can be indicated by elevated levels of anti-O9 IgM antibodies in combination with typical clinical symptoms of typhoid fever [151,152]. However, the TUBEX ® colorimetric reaction may be subject to false-positive results due to hemolyzed samples in individuals with recent S. enteritidis infection [150]. Despite the promising test performance, the requirement for additional laboratory equipment has limited this test kit being used in resource-limited endemic regions [149].
Another rapid test for diagnosing S. typhi of typhoid fever is Typhidot ® (Malaysian Biodiagnostic Research, Bangi, Malaysia). This is a dot EIA test that detects either host IgM or IgG antibodies against the S. typhi antigen [153]. The target antigen used in the assay is neither an O nor H antigen, but a 50 kDa outer membrane protein (OMP) antigen of S. typhi. This recombinant protein is coated on a nitrocellulose strip for detection of the antigen-antibody complex by employing an anti-human antibody conjugated peroxidise and a chromogenic substrate [154]. Due to host immune response, the 50 kDa OMP is a good antigen to identify S. typhi specific antibodies in the sera of individuals with typhoid [155,156]. Since the IgG antibody can persist in the host for more than 2 years, the detection of IgG antibodies can lead to false positive results by confusing between acute or convalescent cases [157]. An upgraded version of Typhidot-M ® has been introduced. It activates antibodies to allow accessibility of OMP antigens to the specific IgM. With this approach, detection of acute typhoid infection can be obtained within 3 h [158]. Both Typhidot ® and Typhidot-M ® are simple, fast, specific, sensitive, and economical dot diagnostic assays for providing early detection of S. typhi infections. Evaluation studies on the Typhidot ® and Typhidot-M ® tests in clinical settings showed that these tests performed better than the Widal test and conventional gold standard culture methods [158]. Both Typhidot ® and TUBEX ® kit have reportedly given good performance for diagnosis of typhoid fever in small cohorts of hospitalized patients [159,160].

Diagnostics for Parasitic Diseases
Malaria remains a severe parasitic disease leading to high morbidity and mortality in tropics [161]. This protozoan parasitic disease is transmitted by female Anopheles mosquitoes. According to the World Malaria Report 2012, it was estimated that about 219 million cases of malaria, causing 660,000 deaths, occurred throughout the world in 2010. Africa is the most prevalent region, where up to 90% of all malaria deaths occur [161]. Four species of Plasmodium parasites are well-known as causative agents for human malaria, namely P. falciparum, P. malariae, P. ovale, and P. vivax. However, the simian species P. knowlesi has been recently identified as a new species that can cause malaria infection in humans [162]. This mainly occurs in Malaysian Borneo [163] and other South East Asian countries [164,165]. Of these species, P. falciparum is the most pathogenic, accounting for the majority of febrile illness and death [166]. Therefore, it is crucial to understand the important parameters in the transmission of the disease, and develop effective diagnostic strategies for its prevention and control.
Malaria rapid diagnostic tests (RDTs) using antigen capture technology were developed in the early 1990s, and have led to much-improved access to diagnostic tests for malaria. Such devices are intended to provide simple, swift, accurate and reliable diagnosis of malaria in areas where microscopic diagnosis is not applicable [167,168]. Other advantages of RDTs are that they do not require complex methodologies, intensive training, and electricity supply, thus representing promising diagnostic tools in remote areas [169,170].
A variety of antigens have been investigated as candidate targets for malaria RDTs. Plasmodium falciparum histidine rich protein 2 (PfHRP2), Plasmodium lactate dehydrogenase (pLDH), and parasite fructose 1,6-biphosphate aldolase (Aldolase) are predominantly used as biomarkers in malaria RDTs. PfHRP2 is water soluble protein that is specific to P. falciparum, and is produced by parasite 2 h after invasion of red blood cell [171,172]. The molecular weight of the secreted Pf HRP2 varies from 60 to 105 kDa [173]. pLDH is a soluble glycoprotein enzyme produced by the asexual and sexual stages of parasites [174]. Different isomers of pLDH can be identified in all human malaria species [175]. Aldolase is an enzyme of the parasite glycolytic pathway that is also synthesized by all human malaria species [176]. Although three types of tissue-specific aldolase isoenzymes can be found in all higher vertebrates, P. falciparum and P. vivax possess only one aldolase isoenzyme, which is also similar to that possessed by Giardia lamblia and Trypanosoma brucei [177].
Nowadays, malaria RDTs have been developed into a range of test formats, including dipstick, strip, card, pad, well, or cassette devices [175]. PfHRP2-detecting tests were the first type of RDT to become available specifically for P. falciparum detection (ParaSight-F ® and ICT ® ), where mAb against PfHRP2 were used as signal and capture antibodies [178,179]. It was followed soon after by pLDH, and Aldolase detection tests, such as OptiMAL ® which are able to detect all four human Plasmodium species (pan-malaria) [180][181][182]. Polyclonal antibodies have been used as capture antibody in the qualitative and quantitative immunoassay test for targeting pLDH [183]. Meanwhile, monoclonal antibodies against parasite Aldolase that are pan-specific have been used in a combined "P. f/pan" immunochromatographic test to detect non-P. falciparum spp., along with PfHRP2 [184]. Recently, the increased demand for RDTs has resulted in more than 200 malaria RDT products from 60 manufacturers currently being available in the global market [185].

Currently Available Antibody Binders for Detection of Biomarkers
Antibodies can be classified into three different categories: polyclonal antibodies, monoclonal antibodies, and recombinant antibodies [186]. Polyclonal antibodies (Polyclonal Abs) are heterogeneous antibody mixtures that are derived from multiple plasma cell lines. Owing to their complexity, polyclonal antibodies have excellent properties for recognizing complex antigens carrying numerous epitopes [187]. A monoclonal antibody (mAb) is a homogeneous antibody generated from a single B lymphocyte clone. Antibodies produced in mAb format have extremely high specificity against a single epitope on antigens [188]. Recombinant antibodies or antibody fragments (rAbs) are antibodies generated using molecular techniques in laboratory. They are aimed at improving the sensitivity, selectivity, stability and immobilization properties in diagnostic applications, for example, in biosensors [189].
In making the decision to use or generate polyclonal, monoclonal or recombinant antibodies, several factors should be considered, including commercial availability, animals to raise, types of applications, time length and cost [186]. A comparison of parameters for producing different source of antibodies is shown in Table 1. "+" represents the least, "++" represents the moderate, and "+++" represents the most (Adapted from [186] with permission from publishing journal).

Monoclonal Antibodies
The first description of mAb production was by Nobel Prize winner Kohler and Milstein in 1975 [190]. The fusion technique between splenic B cells and myeloma cells, termed the hydridoma technique has revolutionized immunology. The production of mAbs is not influenced by the animal sources used, thus, giving mAbs a better homogeneity and consistency in scaled-up production [191]. mAb technology has been widely applied in biomedical research and the pharmaceutical industry.
Unlike polyclonal Abs, the monospecificity of mAb enables targeting a single epitope. This permits a range of applications, including targeting members of a protein family and evaluating changes in molecular conformation and protein-protein interactions. However, the functionality and sensitivity of mAbs can be reduced by small changes in the structure of antigen determining regions, or even by minor changes in pH or salt concentration. One advantage is that mAbs can be produced at greater concentration and much higher purity than polyclonal Abs [191]. Disadvantages of mAb can be overcome using combinations of multiple mAbs specific to desired antigens. However, this pooling method can be difficult, costly, and time consuming [191]. Nowadays, the mass production of mAbs through the ascites method has been largely replaced by in vitro technology such as bioreactors due to the constraint of needing use of mice as host animals [192][193][194][195][196].

Limitations of Conventional Monoclonal Antibodies
As bivalent antibodies, IgG represents the most abundant immunoglobulin proteins (approximately 85%) found in all mammalian serum ( Figure 1) [197]. Due to their ability to confer high affinity and retention times, monospecific IgGs are the preferred reagents in biomedical research, as well as in therapeutic and diagnostic applications. However, several practical drawbacks are apparent for diagnostic reagents based on conventional IgG antibodies. The complex architecture and large molecular size (~150 kDa) may result in weak bindings, when small sizes or small amounts of protein antigens are not easily recognized by the concave surfaces of CDR loops [198,199]. diagnosis of typhoid fever in small cohorts of hospitalized patients [159,160].

Diagnostics for Parasitic Diseases
Malaria remains a severe parasitic disease leading to high morbidity and mortality in tropics ( Figure 1) [161]. This protozoan parasitic disease is transmitted by female Anopheles mosquitoes. According to the World Malaria Report 2012, it was estimated that about 219 million cases of malaria, causing 660,000 deaths, occurred throughout the world in 2010. Africa is the most prevalent region, where up to 90% of all malaria deaths occur [161]. Four species of Plasmodium parasites are wellknown as causative agents for human malaria, namely P. falciparum, P. malariae, P. ovale, and P. vivax. However, the simian species P. knowlesi has been recently identified as a new species that can cause malaria infection in humans [162]. This mainly occurs in Malaysian Borneo [163] and other South East Asian countries [164,165]. Of these species, P. falciparum is the most pathogenic, accounting for the majority of febrile illness and death [166]. Therefore, it is crucial to understand the important parameters in the transmission of the disease, and develop effective diagnostic strategies for its prevention and control. Malaria rapid diagnostic tests (RDTs) using antigen capture technology were developed in the early 1990s, and have led to much-improved access to diagnostic tests for malaria. Such devices are intended to provide simple, swift, accurate and reliable diagnosis of malaria in areas where microscopic diagnosis is not applicable [167,168]. Other advantages of RDTs are that they do not require complex methodologies, intensive training, and electricity supply, thus representing promising diagnostic tools in remote areas [169,170]. More importantly, there is a concern in the application of RDTs in tropical countries regarding their shelf life, because some of these antibodies are susceptible to degradation by excessive temperatures (>40 • C), or by storage for extended periods under conditions of high humidity [200]. To overcome humidity, most RDT devices are now protected in a hermetically sealed plastic packet, or a desiccant is used to ensure that the test strips remain dry [201]. However, it is still a challenge to protect RDTs from high ambient temperatures, resulting in reduced performance of RDTs, especially in tropical countries, where temperatures regularly rise to 45 • C. Hence, for most RDT devices a storage temperature between 4 to 30 • C is recommended. However, this condition is difficult to meet, especially in endemic areas where refrigerated storage systems may not be available [202,203].
To address these problems, initial attempts to generate single domain antibody fragments by separating expression of individual VH or VL units was introduced Ward and co-workers [204]. However, this approach reportedly resulted in solubility problems in aqueous solvents, higher cost, a more time-consuming process, and the requirement of sophisticated protein engineering approaches [205]. Moreover, its failure to recognize selected mAbs on conserved epitopes of specific antigens due to unbound reactivities mediated by the Fc region hinder its utility for diagnostic applications [206,207].
With the emergence of DNA engineering, surface display has been widely used to discover new antibody fragments for the purposes of diagnostic and therapeutic application. As a consequence, a range of different types of new antibodies has been investigated, aiming to overcome the limitations presented in the conventional antibodies.

Phage Display Technology for New Biomarker Binder Discovery
Screening phage display libraries is a powerful tool for identifying specific binders from libraries that contain a large diversity of targets [33,208]. Library construction is achieved by splicing a repertoire of genes (genotype) that encodes the peptide into a gene that encodes a capsid structural protein (phenotype). The "displayed" peptides are included in the capsid layer on the phage surface. Ideally, these proteins should not interfere with the phage structure [209].
Recombinant DNA technology has enabled phage library construction whereby billions of variant peptides and proteins are able to be presented on the surface of the phage [210,211]. From this diversity library, binders specific to proteins of interest with high affinity can be selected by biopanning. This technique facilitates understanding of protein-ligand interactions [212], antigen-antibody interactions [213,214], and permits improvement of the affinity of proteins to their binding partner [215,216]. For instance, phage display antibody libraries with diversities as high as 10 10 can be established using display technology [217,218]. Recently, surface display technology has expanded to include ribosome display [219], yeast surface display [220], and mammalian cell display [221]. Such technologies have enabled the exploration of new antibodies that may not otherwise have been discovered, from humans and animals including shark, camel, llama, and lamprey [222][223][224][225].
Antibody phage display libraries have been used extensively for isolation of high-affinity specific binders against unique antigens from different targets [226][227][228][229][230]. Three types of antibody libraries are typically constructed: naïve, synthetic, and immunized libraries [231]. A naïve antibody library refers to the repertoire of antibody genes derived from non-immunized donors. Synthetic antibody libraries are constructed using synthesized V-gene fragments with randomized CDRs, whereas immunized libraries are based on a host immunized with the target antigen of the disease [232]. The principle of the phage display is represented in Figure 2, indicating the workflows of library construction, biopanning, and clone screening prior to protein expression and purification for functional assays. library refers to the repertoire of antibody genes derived from non-immunized donors. Synthetic antibody libraries are constructed using synthesized V-gene fragments with randomized CDRs, whereas immunized libraries are based on a host immunized with the target antigen of the disease [232]. The principle of the phage display is represented in Figure 2, indicating the workflows of library construction, biopanning, and clone screening prior to protein expression and purification for functional assays.

Natural Single Domain Antibodies
The evolution of immunoglobulins from invertebrates began~550 million years ago [233]. With the emergence of antibody surface display technology, interest has increased in new binders from less commonly used animals, including V HH from camelids, and V NAR from sharks. The unusual antibodies derived from these groups of animals have been reported to provide promising specificity and sensitivity for target antigens [36-38]. The availability of new binders derived from lower vertebrates is now discussed.

V HH Heavy Chain Domain in Camelids
As in all mammals, members of the camelid family produce immunoglobulin G which comprises two heavy chains and two light chains fused with disulfide bonds. However, unlike the V H and V L domains in conventional antibodies, a unique subclass of immunoglobulin containing only a heavy chain domain and lacking a light chain was found in the circulatory system of camelids. Owing to its peculiar structure, this antibody has been configured as "heavy chain only" antibodies (HCAbs) [234]. The capacity of camelid HCAbs to retain the reversibility and binding activity after heat denaturation has enabled new applications where transient heating may occur [235][236][237].
HCAbs are slightly different from IgG, in that they also include both a constant (Fc) and variable domain. The isolated variable domain region of camelids HCAbs is known as V HH (variable domain of the heavy chain of HCAbs) or Nanobody ® (Nb; Ablynx) [238]. Similar to the products of protein engineering the V H domain from other mammals, the N-terminal of V HH is naturally utilized as a binding surface to interact with the target antigen [234]. The molecular weight of V HH is 15 kDa, ten times lower than that of an intact conventional antibody (Figure 1). It was thereby considered the smallest possible antibody fragment, and has attracted the interest of many scientists [239][240][241].
The major advantage of the V HH antibody is its greater solubility compared to classical V H [205]. 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 V HH fragments [240,242]. 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 [243,244]. From a phylogenetic prospect, it is conceivably possible to produce humanized V HH [245], a process that may be "easier" than the complicated manipulation required to "humanize" murine or other more distant species to reduce an alloresponse, such as the human antimouse antibody (HAMA) response [246]. Furthermore, due to their high intrinsic domain stability, camelid V HH is now under investigation as a probe for diagnostics [247,248]. The diagnostic potential of camelid V HH as a probe in immunodetection systems offers possibilities for improving the diagnosis of infection [249], cancers [250], and caffeine contaminants in the food and beverage industries [251,252].

V NAR Heavy Chain Domain in Sharks
A class of naturally occurring single variable domain antibodies was discovered in the serum of elasmobranch cartilaginous fish during the early 1990s [253][254][255]. These natural functional repertoires were termed immunoglobulin new antigen receptors (IgNARs). IgNARs are an unconventional and unique class of proteins found in sharks, including nurse sharks (Ginglymostoma cirratum) [256], wobbegong sharks (Orectolobus maculatus) [257], smooth dogfish (Mustelus canis) [258], banded hound sharks (Triakis scyllium) [259], and horn sharks (Heterodontus francisci) [260]. Investigations has revealed that IgNARs function as antibody and immune response mediators in sharks. However, unlike camelid V HH domains, the IgNAR V region is more similar to light chain and T-cell receptor variable regions than to other VH regions [254,261].
Several desirable biological properties of IgNAR V-domains have been identified, and their potential as alternative antigen binders explored [257,258,262]. The natural habitat of sharks has resulted in them evolving an extraordinarily stable immune system such that the functionality of antibodies can be retained in a harsh environment [263]. Electron microscopic studies have indicated that the intact IgNAR exists as a disulfide-bonded homodimer that consists of a polyprotein with one variable domain (V NAR ) and five constant domains (C NAR ) (Figure 1) [264].
Similar to the camelid V HH , the V NAR has only a heavy chain domain. However, the cross-species conservation of amino acid sequences with human VH is extremely low in V NAR (~25%), whereas it is more than 80% homologous to classical V H scaffolds in camelid V HH [255,265]. It is hypothesized that IgNARs lack many residues that exist in conventional antibodies; these are replaced by other hydrophilic residues. The greatly truncated CDR2 region, herein defined as an HV2 region, has created a signature hallmark for shark V NAR . Due to this unusual structure, the single variable heavy domain proteins of shark IgNARs are currently the smallest antibody fragments observed in the animal kingdom, having a size of only 12 kDa. Yet, in combination with the peculiar feature of a long CDR3 region, these V NAR domains thought to more readily penetrate cleft regions of antigens, thereby increasing the opportunity to target small target epitopes that may not be accessible to conventional IgG [266].
In terms of heat-stability, V NAR also possesses refolding properties as found in camelid V HH . The ability to retain fully functional binding-specific activity after exposure to temperatures of up to 95 • C may make V NAR ideally suited to protein array and diagnostic applications where transient heating may occur as part of the protein immobilization process [258,267]. It is partly due to the presence of cysteine residues in these single domain antibodies, making an extraordinary structure conformation [268].
V NAR domains are more easily produced as recombinant proteins than conventional antibodies. Additionally, due to hydrophilic residues present within V NAR surfaces, high yields of expressed proteins associated with high solubility are achievable, and are thus easy to express in prokaryotic systems [257]. Therefore, the potential utility of V NAR as an alternative binder for clinical applications is now being investigated in a variety of research areas for diagnostic and therapeutic purposes.

Use of Different Binders for Specific Applications
To date, mammals remain the main source of intact antibodies for targeting diseases. However, with the aid of DNA technology, a number of new antibodies have been engineered as smaller single domain fragments to improve of immunoassays, immunosensors, and imaging probes in various applications. As described recently, the discovery of natural single heavy domain antibodies from camelids V HH , shark V NAR , and lamprey VLRs offer some advantages over conventional antibody fragments. This range of natural antibodies is expected to open applications such as enzyme inhibitors and intrabodies, and as detection units in biosensors or immunodiagnostics. In the following section, the deployment of different binders in specific diagnostic applications will be reviewed.

Applications of Camelids V HH Domain or Nanobodies ®
To monitor infections, single domain antibodies naturally derived from camelids (nanobodies) may enable superior detection of species-specific antigens to 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 reported 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 [269]. By adopting an in vitro selection method, novel nanobody clones that showed specificity to T. evansi at a species level and genus-specific reactivity against various Trypanosoma species were isolated. Due to their small sizes, nanobodies were shown to be capable of penetrating into the conserved epitopes of antigens that are inaccessible to classical mAbs [270].
Cysticercosis is a serious tissue infection caused by larval cysts of the pork tapeworm that is prevalent in many low-income countries [271]. 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 [272,273]. 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 cysticerosis [249]. Apart from being used as diagnostic reagents for infectious diseases, nanobodies 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 to classical mouse mAbs in ELISA at measuring caffeine concentration in hot and cold beverages [252].
Camelid sdAbs have recently been applied in ELISA methods to detect a wide range of small molecules, including explosive materials (trinitroluene or TNT) [274], agents of bioterrorism (Botulinum A neurotoxin) [235], toxins (ricin, cholera, staphylococcal enterotoxin B) [275], scorpion toxin [276], and viruses (HIV, rotavirus, Vaccinia, and Marburg) [277][278][279]. Owing to the combination of several favorable properties, camelid nanobodies have also been employed in some sophisticated devices to diagnose diseases. In miniature device development, the advanced features of highly stable and unique conformational structures 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 at identifying harmful bacteria (Staphylococcus aureus) at down to a nanometer scale within 10 min [280].
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 [281]. The emergence of native nanobodies offers the possibility of resolving such problems, and thereby promises the development of probes for diagnosing tumor markers such as EGF receptors [282]. This will enable cancer staging predictions in the blood circulation such as prostate-specific antigen [283]. In view of therapeutic potential, Argen-X (www.argenx.com) has recently developed SIMPLE Antibody TM (chimeric humanized IgGs) that derived from llama VH and VL domains for the treatment of severe autoimmune diseases and cancer. More applications using camelids V HH targeting specified antigens from various diseases is summarized in Table 2.

Applications of Shark V NAR Domain
Evidence that IgNAR is part of the shark adaptive immune response was demonstrated in work where increasing levels of hen egg lysozyme (HEL) specific IgNAR developed in shark sera after 4-5 months immunization [224]. The peculiar structure of the shark IgNAR variable domain renders it amenable to the creation of synthetic peptide mimetics to target specific epitopes that are inaccessible to conventional antibodies [264]. Therefore, V NAR may be suitable as new molecular reagents for research and diagnostic applications, and for immunotherapeutic applications.
Apical membrane antigen-1 (AMA1) is a highly polymorphic 83 kDa merozoite surface protein that is essential for erythrocyte invasion by malaria parasites [312]. A V NAR isolated from a wobbegong shark showed high binding affinity to P. falciparum AMA1 through its unique CDR3 region after undergoing affinity maturation [313]. The binding specificity of a monovalent V NAR clone to P. falciparum AMA1 was comparable with commercially available binding reagents, derived from conventional polyclonal sera, monoclonal antibodies, small fragments (Fab, scFv) and peptides [314].
Foley and co-workers demonstrated that the heat stability of purified recombinant V NAR was superior to that of conventional mAbs by targeting immobilized P. falciparum AMA1 in various format at 45 • C, and the refolding property of V NAR was retained when the temperature increased to 80 • C. The excellent stability property at extreme pH and resistance to proteolytic cleavage was further evidenced by incubating V NAR with homogenized murine stomach tissues under in vivo conditions [267]. Based on these properties, it was proposed that V NAR domains have potential for development as alternate binders for malaria diagnostics platforms.
Human periodontal disease is an advanced gingivitis caused by the bacterial pathogen Porhyromonas gingivalis [315]. Late treatment often leads to dental loss due to the accumulation of lysine gingipain (KgP). KgP is a high molecular weight polyprotease produced by P. gingivalis [316]. This bacterial toxin is responsible for destruction of dental tissue of host by suppressing the secretion of specific lytic enzymes from immune system [317]. Nuttall and co-workers (2002) identified two distinct clones specific to KgP from a wobbegong shark V NAR phage display library. The high stability and binding affinity towards P. gingivalis KgP indicated the potential for V NAR as a valuable source of single domain binding reagents [318].
In recent studies, shark V NAR domains have been reported to detect markers from viral diseases at a greater sensitivity. Ebolavirus hemorrhagic fever (EVHF) is a highly lethal disease caused by Bundibugyo virus (BDBV), Sudan virus (SUDV), Tai Forest virus (TAFV), and Zaire Ebolavirus (ZEBOV) [319][320][321]. Shark V NAR and murine scFv phage display libraries have been generated against specified markers on Zaire Ebolavirus. The results indicated that the sensitivity and thermal stability of shark V NAR against viral nucleoprotein (NP) was superior to murine mAb and scFv in this class [262].
As in the case with camelids nanobodies, highly diversified shark V NAR libraries have also been used to detect different kind of toxins, including staphylococcal enterotoxin B (SEB), ricin, botulinum toxin A (BoNT/A) complex toxoid [322], and cholera toxin (CT) [258]. In addition to identifying markers from non-infectious diseases, the intrabody of V NAR has been reported to recognize immunosilent targets in humans, for example the 70 kDa translocase of outer membrane (Tom70) [323]. Owing to the findings of negligible cross-reactivity with other unspecified antigens, and superior heat stability, shark V NAR domains may be potent source of thermal sdAbs over conventional antibodies in diagnostic and biotherapeutic applications. The applications of recombinant shark V NAR against specified antigens from various diseases is summarized in Table 3.

Conclusions
Diagnosis by biomarker detection has become a new trend in a wide range of diagnostics, as it could be beneficial for personalized therapy. Conventional antibodies such as IgG and IgM derived from mammals are commonly used as antigen binders in immunoassays for identification of particular disease. With the emergence of genetic engineering, the production of monoclonal antibodies is undoubtedly overcoming many shortcomings presented in polyclonal sera. In order to achieve greater binding efficacy, molecular scientists have continually explored new binders with smaller size and better durability. Natural small-molecule single domain antibodies (sdAbs), functional but not structurally related to the conventional antibodies, have recently been discovered in some ancient animals. They are known as V HH or Nanobodies ® from camelids, V NAR from sharks, and, recently, variable-like lymphocytes (VLRs) from lamprey fish. Unlike mammal antibodies, these sdAbs are only composed of heavy protein chains, thereby making them the smallest antibodies thus far. Although most remain at the stage of proof of concept, 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.