Receptors in Immunodiagnostics: Antibodies, Antibody Fragments, Single Domain Antibodies and Aptamers

Abstract

Immunodiagnostics that can detect the target analytes such as organic molecules, pathogens, synthetic chemicals, in the sample with high sensitivity and specificity, that are easy to perform, and are highly stable so that the detection can be carried out near point of care are continuously sought after. The biosensors or the receptors that capture or bind to the analyte in the sample are one of the most crucial components of a successful diagnostic assay. Such receptors have to be easy to produce, should be cheap, should have high affinity towards the analyte, stable at various reaction conditions and also be able to be immobilized on the solid surface. This chapter gives an overview about the basic structure, methods to produce and usage of such receptors in the immunodiagnostics. The receptors that have been discussed include antibodies, antibody fragments, single domain antibodies and nucleic acid aptamers.

1 Introduction

Traditionally, assays for detecting proteins, carbohydrates and other organic molecules were carried out using specific antibodies generated against them in animals. Hence, such detection assays are called as immunodiagnostic or immunodetection assays. With the recent developments in the field of diagnostics, now it has also been possible to detect the analytes using nucleic acids such as aptamers and other similar molecules. Hence, collectively such detection assays have been termed here as immunodiagnostic assays. An ideal immunodiagnostic assay should be able to detect a target analyte from a complex sample such as blood, urine; it should be able to detect the lowest possible amount of the target in a sample, the assay should be easy to carry out and the assay has to be stable at room temperatures to be able to be carried out near the point of care. All such parameters rely heavily on one of the basic components of a diagnostic assay, a detection receptor. An immunodiagnostic method is basically an affinity based specific capture of the target molecules in a sample by the detection receptor. Conventionally, antibodies and other derivatives have been employed as detection receptors in the diagnostic assays. The detection of a desired analyte from a complex sample such as saliva, urine, tissues, etc. with a high affinity and specificity is only possible using biorecognition elements such as antibodies and other fragments, aptamers. This is due to the fundamental property possessed by the antibodies of interacting in a non-covalent way with the organic molecules like proteins, carbohydrates and other small molecules. In the context of antibody recognition, the organic molecule to which the antibody binds is commonly called as an antigen and the exact small regions where the antibody binds are called as epitopes. The unique biorecognition property of antibodies in turn can be attributed to their evolutionarily gained physiological function, that is, to specifically recognize foreign objects and pathogens entering the organism’s body. Similarly, the nucleic acid aptamers that form tertiary structures can also bind to several organic molecules. This function of nucleic acids may also have been attained evolutionarily due to the physiological functions such as transcription and translation. The nature of an antigen–antibody interaction dictates the overall sensitivity and the specificity of the diagnostic assay. Hence, the desirable features of an ideal diagnostic assay such as lower limits of detection and high sensitivity can be achieved using high affinity biorecognition elements as the detection receptors. One of the greatest advantages of using antibodies in the immunodiagnostic assays is that the antibodies can be easily produced using animals as well as other recombinant technologies such as phage display, the knowledge for which is available for decades. These antibodies can be custom generated to recognize a specific epitope on a target protein or similar analyte molecules of choice. There are two key features of a typical immunodiagnostic assay: the formation of a complex between the receptor and the analyte and the detection of such complex formation. Immobilization of the detector molecules on the support matrices has transformed the way the immunodiagnostic assays are developed. The immobilization of the capture ligand helps in the generation of stable stationary antibody–antigen complexes which in turn facilitates their detection. To summarize, it is important to understand the structure, biochemical binding interactions, affinities and production process of biorecognition receptors for designing a diagnostic assay. In order to understand these issues, this chapter focuses on structural–functional aspects and methodologies to produce the detection receptors that are used in the immunodiagnostic assays such as whole mammalian antibodies (e.g. IgG), antibody derivatives (e.g. Fab and scFv fragments), single domain antibodies from camelids and sharks (e.g. V_HH and VNARs) and aptamers. The term ‘whole antibody’ has been used for an immunoglobulin molecule so as to differentiate between an antibody and the antibody fragments. Since the success of a diagnostic assay is based on the nature of its receptor.

2 Antibodies

2.1 Structure and Function of Antibodies

Antibodies or immunoglobulins (Ig) are globular proteins produced by the adaptive immune system of mammals, birds and certain other species that are used to specifically recognize and destroy a range of foreign objects such as pathogens, cancerous cells and allergens. The specific recognition of a target with high affinity and relative ease of production makes antibodies one of the widely used detecting agents in immunodiagnostics. The basic structure of an antibody consists of four polypeptides, two identical heavy chains and two identical light chains. Each heavy chain is bound to a light chain by a disulphide bond and other non-covalent interactions to form a heterodimer. Two such heterodimers are in turn bound together by disulphide bonds to form a monomeric structure of an antibody (Fig. 1a). In mammals, based on the molecular composition, the antibodies have been further classified into five main isotypes as IgA, IgD, IgE, IgG and IgM. The monomeric form of an IgG class antibody has an approximate molecular weight of 150 KDa. Each heavy chain and a light chain typically consist of an amino terminus variable region (V_H and V_L) and a C-terminal constant region (C_H and C_L) (Fig. 1a). Distinct stretches of 5–15 amino acids in the V_H and V_L regions show hypervariability in the sequence among different antibodies. These are called complementarity determining regions (CDRs) (Fig. 1a). The CDRs are involved in the interaction with the antigen or stabilizing the antigen–antibody interaction. Thus the V_H and V_L regions together form a fragment variable (F_v) that is uniquely specific for a particular antigen and hence is involved in specific antigen binding. The constant region (fragment crystallizable; F_c) confers additional cell-mediated functionality to the antibody such as opsonization and phagocytosis by other immune cells.

Fig. 1

The structure of an antibody molecule and various antibody fragments. (a) The whole antibody molecule is a dimer of heterodimers, made up of a heavy chain connected to a light chain via disulphide bonds (Black lines). Each heavy chain and a light chain have a variable portion (V_H, Red and V_L, Blue accent) and a constant region (C_H, Dark blue and C_L, gold). The V_H and V_L form the antigen binding fragment variable, F_V. The constant region of the heavy chain forms the fragment crystallizable, F_C. The variable region contains three hyper-variable regions, that are responsible for antigen binding, called as complementarity determining regions (CDRs; CDR1, CDR2 and CDR3). (b) A Fab fragment is a monovalent antigen binding domain made up of V_H − V_L and first constant region domains. A monovalent Fab fragment that also has some part of the Fc region and the functional thiol groups is called as Fab’ fragment. A bivalent form of the Fab’ fragment is called as F(ab’)2. The V_H and V_L regions linked together via a short peptide linker are called as Single Chain Fragment Variable (scFv)

A typical antibody–antigen interaction is governed by non-covalent bonds such as electrostatic forces, hydrogen bonding, hydrophobic forces and Van der Waals forces. The complex formation between antigen–antibody is reversible and can dissociate depending on the strength of the interactions as well as the reaction conditions such as pH, temperature, etc. The overall strength of the interaction shown by antibody towards the antigen or vice versa is generally termed as affinity. The affinity of an antibody to a particular antigen is commonly expressed in terms of equilibrium dissociation constant (K_D). In simple terms, K_D is the concentration of the ligand required to saturate half of the available binding receptors. So, if the antibody is said to possess high affinity towards a particular antigen, it takes lower concentration of the antibody or the antigen to saturate the available binding sites. Thus higher affinity in turn means the value of the K_D will be lower. For a sensitive diagnostic assay, it is desirable to have high affinity antibodies that are capable of detecting lower concentrations of the antigens.

2.2 Production and Use of Antibodies in Immunodiagnostics

Antibodies are produced by the plasma cells of the immune system. Upon encountering a specific antigen, the B cells differentiate into either memory cells or plasma cells which then secrete antigen specific antibodies. Generating antigen specific polyclonal antibodies by immunizing animals has been one of the standard methods for obtaining antibodies for the immunodiagnostics. After the immunization, polyclonal antibodies can be obtained from the serum and other fluids of the animal. These antibodies can be further purified if required, using methods such as affinity chromatography. Polyclonal antibodies can be considered as a mixture of different antibodies secreted by various plasma cell clones that each recognizes a different epitope of the same antigen (Fig. 2a). Thus such polyclonal antibodies targeting multiple epitopes of the same antigen can make the immunodiagnostic assay more sensitive. However there are some disadvantages of using polyclonal antibodies. There can be significant variations in the immune response generated by the animals used for immunizations. This can be due to the differences in the genetic background of the animals or could simply be due to the immunological health of the particular animal. This can result in a lot-to-lot variation of the polyclonal antibody mixture. Secondly, the polyclonal antibodies can consist of a mixture of antibodies that are specific for the antigen as well as those that are cross-reactive with other antigens. This can make the diagnostic assay less specific. The later problem can be overcome to a certain extent by affinity purification of the polyclonal antibodies against the desired antigen or by prior cross-adsorption of the antibodies against the undesirable antigen.

Fig. 2

Generation of antibodies from immunized animals. (a) Polyclonal antibodies can be obtained from the serum and other fluids of the animals immunized with the antigen. Polyclonal antibodies contain a mixture of antibodies that identify different epitopes of the same antigen. (b) Monoclonal antibodies that can recognize the same epitope of the antigen can also be generated from immunized animals such as mouse and rabbit. Fusion of antibody secreting cells with myeloma cells generates hybridoma cells that are immortal and can potentially secrete unlimited amounts of antibodies in an in vitro culture

The discovery of hybridoma technology by Kohler and Milstein in 1975 paved the way for generation of monoclonal antibodies (MAbs) specific for a particular antigen (Köhler and Milstein 1975). The hybridoma technology principally involves fusion of a primary B cell with a myeloma cell to create hybrid cells (hybridoma) that are immortal and can potentially secrete unlimited amounts of antibodies in the culture (Fig. 2b). Thousands of antibody secreting hybridoma cells can be generated by immunizing the animal with an antigen of choice and then using the B cells from the immunized animal to create hybridoma cells. Then by performing cloning or limiting dilution of single hybridoma cells a unique clonal population of cells (monoclonal) all of which secrete antibodies that recognize a single epitope can be created. In this way, MAbs specific for a desired antigen can be developed. Development of MAbs allowed for generation of uniform lots of antibodies without any variation. Also, the in vitro culture of hybridoma cells allowed for production of large quantities of antibodies without much dependence on the animals. The ability to produce custom designed MAbs for a specific antigen led to the development of specific and sensitive diagnostic assays that were not possible with the polyclonal antibodies. One of the disadvantages of using MAbs in the diagnostic assays is that if the antigen to be detected has undergone any mutations, it may no longer bind to the MAb and can thus escape the detection. To avoid such problems, a cocktail of MAbs can be used in the diagnostic assay to make it more specific and sensitive.

A modern more commonly used method of generation of antibodies is the phage display method (McCafferty et al. 1990; Winter et al. 1994). It is a method that combines recombinant DNA technology with the bacteriophage-bacterial expression system to discover novel, target specific ligands. Recombinant technology allows for generation of limitless diversity of the antibody ligands at the genetic level, whereas bacteriophage system allows for phenotypic expression/display of all such ligands (Fig. 3). Millions of filamentous bacteriophage that each display an antibody fragment on their outer coat protein are used to identify the antigen specific antibodies. In the phage display screening methodology, an antibody encoding phagemid library is prepared by fusing the antibody V genes to the gene expressing one of the outer coat proteins (gIII or gVIII) of the bacteriophage. In this way, the antibody is displayed on the coat protein of the bacteriophage which in a way links the antigen binding phenotype of the antibody to its genotype. Depending upon the source of the antibody genes, the phage display library can be immune, naïve or a synthetic library. In the immune library, the sequences of the antibody ligands originate from the immune cells of the immunized animal or even from the human volunteers immunized or exposed to the antigen. In the synthetic phage display library, the antibody sequences are designed in silico which may or may not exist physiologically. A naïve library can have the antibody sequences derived from primary B cells that have not encountered the antigen. An antibody library that is a mixture of two or more of these types can also exist. However despite the origin of the genes, owing to the preparation of a bulk antibody library the heavy chain–light chain pairing of the parental antibody is most often lost. But this could also give rise to novel or better heavy chain–light chain ligands. The phage library displaying variety of ligands is then panned repetitively (3–8 cycles) against the desired antigen (Fig. 3). A panning is an assay similar to ELISA where the phages are allowed to bind to the antigen via the displayed specific antibody, while the non-specific phages that do not bind to the antigen get washed away (Fig. 3). After several rounds of such panning, specific antibody fragments get enriched which can then be sequenced to get the nucleotide sequence of the V region. After obtaining the sequences, the antibodies can be suitably expressed from prokaryotic or mammalian system. One of the advantages of using phage display method for generating antibodies is that it can be faster than the other traditional methods. Phage display method may also allow for discovering higher affinity ligands. This can simply be done by sequentially panning the phage display library with decreasing concentration of the antigen. Alternatively, the binding domains of the antibody ligands can be mutated at the genetic level during the generation of phagemid library. Although phage display technique offers several advantages over the other antibody generation methods, there are few disadvantages as well. Phage display method generally allows for generation of smaller antibody fragments such as Fab and scFv, which are not as stable as whole IgG. These smaller antibody fragments show low avidity of antigen binding as compared to the whole antibody. This may result in missing out on some of the best binders due to less avidity. Thus, ultimately the binders arising from phage display library may have to be expressed as a full length antibody or fusion proteins in vitro in mammalian cells, which can decrease their yield and increase the time.

Fig. 3

Generation of antibodies from phage display library. The process of antibody generation using phage display library begins with a generation of a V domain library in phagemid. Introduction of such phagemids into a suitable prokaryotic host followed by infection of helper phages generates a bacteriophage library displaying antibody fragments on their surface. Such phages are then panned against a target antigen, wherein the unbound phages are washed away and the bound phages are amplified and rescreened. After finishing the panning rounds, the variable region antibody sequences can be sequenced from the selected phages. The selected antibodies can then be expressed as fragments or full length antibodies using suitable prokaryotic or mammalian expression system

The whole antibodies have been principal components of traditional diagnostic immunoassays which are based on principles of ELISA. In these assays, antibodies are immobilized on the solid support to capture the analytes in the reactions. Then a secondary antibody conjugated to an enzyme provides a colorimetric readout of the assay. There has also been progress with other detection methods where the secondary antibodies are conjugated to a radioisotope or a fluorophore to make the diagnostic assay more sensitive. The modern diagnostic assays are based on the lateral flow of the fluids that simultaneously allow for the capture and detection of the analyte in the sample. A well-known example of such kind of assay is a pregnancy detection kit which involves detection of human chorionic gonadotropin (hCG) hormone using specific immobilized antibodies. Another significant improvement in terms of the label free detection is the use of Surface Plasmon Resonance (SPR) technology. In this method, the antibodies are immobilized on the gold surface. When the antigen binds to the antibody, the change in the mass of the complex affects the light emission behaviour of the gold surface. To be able to be used as a detecting sensor in an immunoassay, the whole antibody needs to be immobilized on a solid support. In this immobilization process, the antibody activity is often lost due to the denaturation of the molecule or due to the improper orientation of the antigen binding site on the antibody after immobilization. One of the commonly used methods for immobilization of antibodies is by using protein G (from Streptococcus sp.) or protein A (from Staphylococcus aureus) (Shen et al. 2017). The protein G/A binds to the Fc portion of the antibody. So by pre-treating the solid surface with protein G/A, antibodies can be immobilized on the solid matrix of the diagnostic assay. Protein L (from Peptostreptococcus magnus) that binds to the light chain can also be used for immobilization. Another method for immobilization has been to make use of high affinity biotin–streptavidin interaction. The antibodies to be immobilized can be biotinylated and by pre-treating the solid surface with streptavidin the antibodies can be immobilized.

Antibodies have been used in the detection of various infectious diseases such as anthrax, leptospirosis; parasitic helminths; protozoans as well as novel emerging diseases. Antibodies have also been part of diagnostic assays in food and drug industry, environmental monitoring as well as in in vivo imaging. Recent developments such as antibody screening using droplet microfluidics technology can potentially allow for high-throughput antibody discovery in shorter time (Shembekar et al. 2016, 2018). This methodology neither requires immunization of the animal nor generation of a phage display library. The concept revolves around compartmentalization of a whole antibody binding assay in the aqueous droplets (~100 μm in diameter) that are surrounded by oil. Since the whole assay is compartmentalized, single B cells can be screened for antibody discovery on high-throughput scales. Because the antibody screening can be directly performed on single antibody secreting cells, primary cells can be employed in the screening process and the generation of hybridoma cells or the phages is not required. There has also been an attempt to use droplet microfluidics to preserve the heavy chain–light chain linkage by encapsulating single B cells in droplets (Rajan et al. 2018). Such microfluidics based technologies are being improved over the last few years and could provide a significant impetus to the field of antibody discovery. Each antibody discovery method has its own advantages and disadvantages. Thus a combination of traditional antibody generation methods along with the modern methods can offer a great novelty and diversity in generating the reagents for an immunodiagnostic assay.

3 Fab Fragments

3.1 Structure and Function of Fab Fragments

The whole antibody can be cleaved or synthesized into smaller antibody fragments that can still bind the cognate antigen. The smaller antibody fragments can allow for ease in production and their engineering. One of the most useful antibody fragments is called Fab (Fragment antigen binding) fragment. A Fab fragment is a monovalent antigen binding unit that consists of V_H and V_L domains, along with first constant region. Domain from heavy chain C_H1 and the constant region from the light chain C_L bound together with disulphide bonds (Fig. 1b). The molecular weight of a Fab fragment is approximately 50 kDa. A 55 kDa Fab monovalent fragment that also has some part of the Fc region and functional thiol groups is called as Fab’ fragment. The functional sulphydryl group on the Fab’ fragment can be used for alkylation or conjugation to other proteins and can also help in immobilization of the fragment on the solid supports used in the diagnostic assay. A bivalent form of the Fab fragment that also contains some part of the Fc region is commonly called as F(ab’)2 fragment and is approximately 110 kDa.

3.2 Production and Use of Fab Fragments in Immunodiagnostics

The Fab fragments can be generated mainly via two ways: using enzymatic cleavage of the whole parent antibody or using recombinant DNA technology. A less commonly used method combines these two methods, where fragments are primarily generated using recombinant synthesis and then they are further broken down using proteolytic cleavage. In order to proteolytically produce Fab fragments from the whole antibody, a non-specific, thiol endopeptidase called Papain is used (Brezski and Jordan 2010; Vlasak and Ionescu 2014). Papain cleaves the peptide bond before the hinge region thus giving two Fab and the Fc region (Fig. 4a). If the intact Fc fragments are required, then this is the method of choice. Papain can also be used to generate F(ab’)2. However, the production of F(ab’)2 fragments is not very consistent due to the unavoidable over digestion of the parent molecule. Pepsin is another protease that cleaves the antibody molecule after the hinge region yielding an intact F(ab’)2 fragment and degraded Fc region (Fig. 4b) (Vlasak and Ionescu 2014). The F(ab’)2 fragment can then be mildly reduced to obtain monovalent Fab’ fragments. The pure Fab fragments from both these methods can then be obtained by gel filtration or chromatographic methods. Another enzyme called Ficin is a preferred protease to generate F(ab’)2 and Fab fragments due to its higher consistency of degradation of antibodies over the papain. In presence of varying concentrations of cysteines, the ficin can effectively either produce F(ab’)2 fragment or 2 Fab fragments (Fig. 4c) (Mariant et al. 1991). The problem with the proteolytic cleavage method for generating antibody fragments is that some enzymes work only for particular antibody class or species and thus cleavage protocols have to be optimized for each and every antibody class. Another problem with the enzymatic or proteolytic cleavage method is that it can lead to the destruction of the antigen binding domains to some extent. In order to immobilize the Fab-related fragments on the solid surface, fusion with another protein is a preferred choice. Because it has been shown that the direct immobilization of the Fab-related fragments denatures their structure (Crivianu-Gaita and Thompson 2016). Several proteins such as albumin and protein G have been used to immobilize the fragments on the solid surfaces. Similar to the whole antibody molecule, these fragments can also be immobilized using biotin–streptavidin interactions. Due to their smaller size, they can be immobilized at higher densities as compared to the parental antibody molecule. Due to the presence of functional thiol groups, Fab and Fab’ fragments could be immobilized on the maleimide or iodoacetyl activated surfaces (Welch et al. 2017). Out of all the Fab-related fragments, F(ab’)2 fragments could potentially result in higher avidity antigen interaction, as compared to the Fab and Fab’ fragments. This is simply because of the bivalent structure of the F(ab’)2 fragment as compared to the monovalent Fab and Fab’ fragments. The Fab fragments have been mainly used in immunoassays as compared to the Fab’ fragments which have been used in the optical, electrochemical type of biosensors. Fab’ fragments have also been used in the detection of insulin and TNF-α, which were fluorescence and luminescence based assay (Lee et al. 2005; Erikaku et al. 1991). In a competition based assay, the target analyte, phenytoin was already coated on the surface. Anti-phenytoin Fab’ fragments were then allowed to bind to the target. When the sample flowed over the surface, presence of phenytoin displaced the Fab’ fragment which resulted in the signal detection (Schiel et al. 2011).

Fig. 4

Generation of Fab fragments from whole antibody by proteolytic cleavage. (a) A Fab fragment is generally cleaved off proteolytically from a whole antibody using a Papain enzyme. This process can also yield intact Fc fragment. (b) An enzyme called Pepsin is used to cleave F(ab’)2 fragments from a whole antibody. Since the enzyme cleaves the whole antibody after the hinge region. (c) An enzyme called Ficin can cleave the whole antibody resulting into either F(ab’)2 or Fab fragments, based on the concentration of cysteine residues

4 Single Chain Variable Fragment (scFv)

4.1 Structure and Function of scFv

An antibody fragment smaller than the Fab fragment that contains only antigen binding V domains is commonly called as single chain variable fragment (scFv). The V domains of an antibody possess relatively weak interactions. Also due to the sequence variability in the Fv region, the fragment is unstable and its expression results in the dimerization of V_L domains. Hence, the V domains have been stabilized using different strategies such as disulphide linkers, peptide linkers and multimeric formations (Nelson 2010; Essen and Skerra 1993). A typical scFv contains a V_H and V_L domain connected from N to C-terminus of one another through a peptide linker. Thus the scFv retains the antigen binding function of an antibody but lacks the constant region. The most common linkers to produce monomeric scFvs are peptide linkers that are 15–30 amino acids long. The peptide linkers are commonly made up of Glycine and Serine residues such as (Gly4-Ser)₃ due to the flexibility that they impart to the antigen binding domains. However, charged residues such as glutamic acid and lysine have also been used by some groups to enhance the solubility of the scFv (Ahmad et al. 2012). The orientation of the V_H and V_L domains also plays a significant role in the functionality of the scFv. The comparative studies about the scFv orientations using the same linkers have shown that the V_L—linker—V_H orientation provides higher antigen binding capacities than the V_H—linker—V_L. This is because the N-terminus of the V_L domain and the C-terminus of the V_H domain are involved in antigen binding (Desplancq et al. 1994). The linkers 0–10 amino acids tend to give rise to multimeric forms of scFvs. The multimeric forms can provide greater avidity for the antigen binding. These non-covalently linked dimers (diabodies) or trimers (tribodies) are difficult to generate in controlled conditions. Since the different multimeric formats are all in equilibrium. However, certain strategies such as PEG based fusion of two scFv fragments and inclusion of cysteine residues in the linker to allow disulphide bond formation could help in controlling the formation of multimeric forms.

4.2 Production and Use of scFv Fragments in Immunodiagnostics

Recombinant technology using phage display method is the most commonly used method for production of scFv fragments. A library of linked V_H and V_L regions is expressed in a bacterial system. As described above, with the help of a filamentous bacteriophage, this library of scFv fragments is then displayed on the phage particles. The phages are panned against the desired antigen to enrich the specific scFv fragments. The selected scFv fragments can then be expressed in soluble forms in suitable bacterial hosts. Using another approach, scFv fragments have been produced from hybridoma cells, splenocytes of immunized mice as well as from human peripheral B cells. In these cases, the V region of the antibody was cloned and expressed directly in a suitable prokaryotic host. To date, scFv fragments have been expressed using bacterial, mammalian, yeast, plant and insect cell systems. The bacterial system remains a preferred choice for scFv expression, since the engineering of the V region and the protein level modifications in the expressed scFv such as His- and other tags, fusion with another protein are easy to perform. There are different factors to be considered for the expression of the scFv fragments in the bacterial system. Expression of these fragments in the cytoplasm of the cells results in the production of the scFv protein as an insoluble aggregates in the inclusion bodies. The properly folded scFv fragments then have to be renatured from these inclusion bodies. Alternatively, the fragments can be directed using a signal peptide to be produced in the periplasmic space of the cells that lies between inner and outer membranes of gram-negative bacteria. This method of expression allows correctly folded scFv fragments to be produced in the oxidizing environment (Ahmad et al. 2012).

One of the major advantages of expressing scFv fragments in bacterial system is that they can be conveniently expressed as a fusion protein along with other target proteins. This can be useful for immobilization of these scFv fragments for an immunodiagnostic assay. For example, scFv fragments expressed with streptavidin peptide were immobilized on the biotin surfaces and scFv fragments fused with His-tags were immobilized on the solid surfaces using zinc- iminodiacetic acid (IDA) method or Ni-NTA coated gold surfaces (Crivianu-Gaita and Thompson 2016). Such methods non-covalently immobilize the scFv fragments on the solid surfaces. Similarly, using various other strategies scFv fragments have been covalently immobilized on the solid surfaces. Such strategies include modification of the C-terminal thiols and addition of reactive N-terminal Serine to the scFv fragments (Ros et al. 1998). Most of the strategies involving covalent immobilization of the scFv fragment on the solid surface give rise to a correct orientation of the scFv, with the antigen binding domain accessible to the antigen. One exception to this rule is with the covalent immobilization using free amines on the scFv for coupling on the solid surfaces, which may result in higher density of the immobilized fragments but does not ensure correct orientation (Howell et al. 1998).

As a diagnostic receptor, scFv fragments offer several advantages such as they are easier to produce in bulk, convenient to engineer, different immobilization strategies are well known and can be immobilized at higher densities on the solid surfaces as compared to the whole antibodies which means a sensitive diagnostic assay. scFv fragments have reportedly been successfully used as a diagnostic biosensors in the diagnosis of various infectious diseases and pathogens such as cholera, influenza virus, Bacillus anthracis, Hepatitis B virus and Entamoeba histolytica. scFvs from Chicken origin have also been demonstrated as useful biosensors for detecting Mycobacterium bovis (Ahmad et al. 2012).

5 Single Domain Antibodies

5.1 V_HH Antibodies: Structure, Function and Use in Immunodiagnostics

Certain mammals such as camels, llamas and dromedaries produce antibodies with only heavy chains and lacking the light chains. These antibodies are called as single domain antibodies or heavy chain only antibodies (HCAbs). Typically these antibodies contain two heavy chains joined via disulphide bonds. Such HCAbs usually have one variable domain (V_H) and two constant region domains (C_H) (Hamers-Casterman et al. 1993) (Fig. 5). The isolated antigen binding V_H of such HCAbs is commonly known as V_HH (variable domain of the heavy chain of HCAbs) or Nanobody^® (Ablynx). The finger-like projection of N-terminal V_H domain is responsible for antigen binding. The molecular weight of a V_HH antibody is approximately 15 kDa, making it one of the smallest antibody fragments. One of the major advantages of V_HH antibody is that the smaller antigen binding area allows for binding to epitopes that are not accessible by other antibody formats such as catalytic sites of enzymes. From a therapeutic point of view, these antibodies are useful as they can penetrate into tissues. Due to the presence of hydrophilic amino acids in the framework of V_HH antibodies, they show higher solubility as compared to the classical V_H fragment (Muyldermans 2001). The V_HH antibodies have been mainly produced either from naïve or immunized phage display libraries. After immunizing the camelid with a desirable purified antigen or crude protein extracts or denatured antigens, V_HH fragments can be easily cloned into a phage display library. The antigen specific V_HH fragments can then be obtained by panning this phage display library against the target antigen. Due to their monomeric structure, small size and solubility they can be easily expressed using prokaryotic systems. Owing to the small size of about 450 base pair of their single coding exon, their genetic engineering is also easier (Muyldermans 2001). V_HH antibodies have also been generated using other display systems such as bacterial display system, ribosome as well as yeast surface display. V_HH antibodies are suitable biosensors due to their easier production and robust structure that can withstand harsh environmental conditions. They can be immobilized at higher densities as compared to the conventional antibodies, making the diagnostic assay more sensitive. Conversely, due to the smaller size of V_HH antibodies, they are likely to be inaccessible to the antigen after immobilization on the solid surface. Also, their intrinsic hydrophilicity can be problematic for their immobilization. Hence, hydrophobic C-terminal peptide tags such as his or myc or fusion to Fc portion has been used for their adsorption on the solid surfaces, with the Fc region providing the best results (Saerens et al. 2005). V_HHs have been used in diagnosis of infectious pathogens, parasites, cancer antigens as well as toxins. V_HH antibodies were isolated using an immune library against Trypanosome parasites which cause African sleeping sickness and Chagas disease. Some of the novel nanobody clones that were obtained were specific for the immunized trypanosomal antigen, whereas some clones showed genus specific broad reactivity. The broad reactivity of the clones was due to the penetration of the V_HH antibodies to access the conserved residues (Saerens et al. 2008). In certain cases, V_HHs have shown more specificity as compared to conventional antibodies. For example, V_HHs have successfully been used to specifically distinguish between Brucella and Yersinia species in livestock (Abbady et al. 2012). Biotinylated V_HHs have also been employed as capture ligands on streptavidin beads to identify CEA biomarkers in cancer patient’s sera (Even-Desrumeaux et al. 2010). A V_HH based agglutination reagent has also been developed for HIV diagnosis. A V_HH that is specific for red blood cell antigen is fused to the HIV p24 antigen. The presence of anti-p24 antibodies in patient sera then mediates red blood cell agglutination reaction (Habib et al. 2013). Such kind of diagnostic assays can be developed for various other pathogens. One of the advantages with V_HH antibodies is their thermostability. This has also been demonstrated by using anti-caffeine V_HHs which showed binding to caffeine even at 70 °C in the hot beverages (Ladenson et al. 2006).

Fig. 5

Single domain antibodies from camelid and shark species. The human IgG antibody is a dimer of heterodimer made up from a heavy and a light chain. The antibodies found in camelid and shark animal species are made up of two heavy chains only. The camelid antibody consists of one variable and two constant regions, whereas shark antibody consists of one variable and five constant region domains in each heavy chain domain. The variable regions of these antibodies called as V_HH and VNAR are one of the smallest antibody fragments with a molecular size of about 15–12 kDa, respectively

5.2 Variable New Antigen Receptors (VNARs): Structure, Function and Use in Immunodiagnostics

Similar to the camelid animals, certain species of shark fishes also produce antibodies with single variable domains which are called as immunoglobulin new antigen receptors (IgNARs). These naturally occurring IgNARs are found in many different shark species such as nurse sharks, wobbegong sharks, smooth dogfish, banded hound sharks and some other cartilaginous fishes. IgNARs have been shown to function as immune response mediators similar to antibodies in these fishes. The intact IgNAR is made up of a disulphide linked homodimer of two chains each containing a variable new antigen receptor (VNAR) that is responsible for antigen recognition and five constant domain new antigen receptor (CNAR) (Kovaleva et al. 2014) (Fig. 5). The commercial research and development of VNARs has been under propriety control of some of the companies. One of the companies that has proprietary of producing VNARs by immunizing shark fishes, Elasmogen Ltd., has a trademark name of soloMERs™ for the VNARs. Another company that is involved in the research and development of shark VNARs is Ossianix Inc. The VNARs consist of two CDRs: CDR1 and CDR3, whereas CDR2 is almost absent. Usually CDR3s of VNARs are longer (15–17 amino acids) and structurally more diverse than the human IgGs. Interestingly, there are two other hyper-variable regions (HV2 and HV4) that are also known to participate in either antigen binding or in stabilizing the antigen-VNAR complexes, apart from CDRs (Barelle and Porter 2015). The VNARs are thought to be similar to the V_L of mammalian light chain or T cell receptor V regions. However, in terms of sequence homology to human V_H, VNARs are very distant with only 25% cross conserved amino acid residues (Roux et al. 1998). The VNARs are the smallest antibody fragments with a size of about 12 kDa. Due to such a small size they can efficiently recognize cryptic epitopes which cannot be accessed by other antibody fragments. VNARs contain many hydrophilic residues as compared to the conventional antibodies thus making them highly soluble and hence easy to express in bacterial system. VNARs are very stable molecules which can withstand temperatures of up to 100 °C and also extreme conditions of pH such as 1.5, without much loss in activity (Steven et al. 2017). This is partly because of the presence of significantly higher numbers of cysteine residues in the VNAR structure. This stability also makes them highly suitable as diagnostic agents, where the diagnostic assay needs to be carried out near the point of care or the reagents need to be transported to distant locations. Similar to other antibody fragments, VNARs can also be commonly produced using prokaryotic system such as phage display system or soluble proteins. VNARs can be produced from naïve phage display library or using an immunized library. There are facilities available which can immunize shark fishes with the desired antigen. Thereafter the VNAR sequences can be amplified using PCR and cloned to produce immune phage display library. By panning such phage display library of VNARs, antigen specific single domain antibodies can be obtained. Some of this technology is under proprietary and patent control of some commercial companies. The shark VNAR domains have been reportedly used to detect markers from viral diseases and viruses such as Ebola virus haemorrhagic fever (EVHF), Sudan virus (SUDV), Tai Forest virus (TAFV) and Zaire Ebola virus with high sensitivity (Leow et al. 2017). VNARs have also been used to detect toxins such as staphylococcal enterotoxin B, ricin, botulinum toxin A and cholera toxin (Liu et al. 2007a, b). A VNAR isolated against AMA-1 protein of malarial parasite showed high binding affinity through its CDR3. The binding specificity of this VNAR was comparable with commercially available polyclonal and monoclonal antibodies as well as other smaller antibody fragments. In addition, the purified recombinant VNAR was also observed to show binding to the antigen at 45 °C as well as retained the refolding properties up to a temperature of 85 °C. Moreover, the VNAR also showed high stability under the proteolytic environment of murine stomach tissues (Griffiths et al. 2013; Henderson et al. 2007). Owing to the superior heat stability, negligible non-specificity and ability to detect cryptic epitopes, VNARs are being recognized as potent biosensors for diagnostic applications.

6 Aptamers: Structure, Function and Use in Immunodiagnostics

It is well known that nucleic acids can form tertiary structures that can bind specifically or non-specifically to proteins, nucleic acids or other organic molecules. Single stranded DNA or RNA chains which are selected by in vitro evolution to show binding to protein or other antigens are termed as aptamers. The aptamers show comparable affinities to the target antigen, similar to antibodies, in the range of nano- to pico-molar (Jayasena 1999). Aptamers are of the size of 1–2 nm, which allows for their immobilization at very high density on the solid surfaces resulting into superior sensitivity. The aptamers are developed using a selection process called as systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold 1990) (Fig. 6). In this methodology, a large random library of single stranded nucleic acids (RNA or DNA) is screened against a target of interest. Those nucleic acids that bind to the antigen are then separated from the target using various methods such as affinity chromatography, size exclusion chromatography or electrophoresis. The selected nucleic acid molecules are then amplified and repeatedly screened against the target (up to 15 cycles) (Fig. 6). The generated aptamers using the SELEX process are generally 70–80 nucleotides long, but they can also be shortened by 20–30 nucleotides by removing regions which do not participate in the functional binding. In the end, once the desired aptamer sequence is obtained, the nucleotides can be synthesized chemically within a short period of time. The SELEX process has also been improved over the last few years that allows for selection of aptamers with very high specificity and affinity. For example, it is possible to increase the affinity of the aptamers by using only the specific short regions or domains of the target analyte in the subsequent screening rounds allowing for high affinity binding (Keefe et al. 2010). Some counter-SELEX processes also allow for selection of highly specific aptamers by removal of cross-reactive aptamers that can bind other structural analogs (Jayasena 1999). Aptamers are susceptible to nuclease degradation during the immobilization or the subsequent steps of the test assay. Hence, various strategies have been developed to prevent nuclease degradation as well as to increase the stability of the aptamers such as addition of amine or fluorine or PEG groups (Pieken et al. 1991). Aptamers have been generated against variety of bioorganic targets such as metal ions, amino acids, antibodies, antibiotics and others (Crivianu-Gaita and Thompson 2016). Aptamers have also been shown to differentiate between presence or absence of reactive groups such as hydroxyl and methyl (Crivianu-Gaita and Thompson 2016). Aptamers have been shown to be very sensitive in the detection of analytes, with capability of detection of as little as 250 pM of IgE antibody and 73 pM of thrombin molecules (Wang et al. 2015; Maehashi et al. 2007).

Fig. 6

Systematic evolution of ligands by exponential enrichment (SELEX) process. Single stranded RNA or DNA molecules called as aptamers that show binding to various organic molecules are generated using a process called as systematic evolution of ligands by exponential enrichment (SELEX). A random library of single stranded nucleic acid molecules is screened against the target antigen. The unbound aptamers are washed away, whereas the bound ones are eluted out. The selected aptamers are then amplified using PCR and then rescreened against the antigen. The screening cycle can be repeated for 8–15 rounds. The selected aptamers can then be chemically synthesized

7 Comparative Analysis of Different Biosensors and Future Perspectives

The ease of production of the biosensors and their suitability in immobilization on the solid surfaces are some of the most important criteria in the selection of a biosensor for a diagnostic assay. In order to select the best biosensor for a diagnostic assay, various parameters such as specificity and affinity towards the analyte, stability and the ease of production are needed to be considered. Even though the individual biorecognition receptors can have better affinities or specificities on a case to case basis, all these parameters can be compared in a generalized way to select the best biosensors (Fig. 7).

Fig. 7

Generalized comparative analysis of different receptors. The whole antibody (Ig), Fab fragments (Fab, Fab’, F(ab’)2), scFv, V_HH and VNARs and aptamers have been compared considering the parameters such as ease of production and cost effectiveness (Fab >> aptamers), affinity (Ig >> scFv & others), specificity (scFv >> Ig), stability (V_HH and VNARs >> scFv) and ease of immobilization (scFv >> Ig)

In terms of ease of production and cost effectiveness, out of all the biosensors that have been discussed in this chapter, Fab fragments are overall easiest and cheapest to produce. Since these fragments are obtained by proteolytic or chemical cleavage, once the parent antibody is generated, the downstream cleavage methods can be applied in few days. The drawbacks with this methodology are the loss of functional activity of the fragments and the need to have large amounts of the parent antibody for this process. At the second number in terms of ease of production are the biosensors which are produced using phage display library, such as scFv and single domain antibodies that can be considered easiest to produce. Even though the generation of the phage display library is a laborious process and requires skilled manpower, once the library is generated, it can be used to generate antibody fragments against various different antigens. Also, in the downstream production, if the antibody fragments are expressed using prokaryotic system, then the overall manufacturing process becomes relatively easy. The whole antibodies that are produced either from animals or using phage display library are relatively difficult to produce. The animal immunizations and the subsequent antibody screening processes are laborious as well as costly. The whole antibody expression using the mammalian system makes the overall process even more laborious, even if the antibodies are screened using phage display library. The aptamers are costliest to produce due to the time consuming SELEX process. To identify aptamer sequences, nucleotide sequence library has to be prepared and the library needs to be screened repeatedly against the target, followed by PCR and purification of amplified products after each rescreening. However, once the desired aptamer sequence is obtained, its chemical synthesis can take few days.

In terms of affinity of the biosensing elements, aptamers and whole antibodies can be considered as the top choices. The whole antibodies isolated from immunized animals with repeated immunizations can attain affinities in the pico- to nano-molar range. Monoclonal antibodies isolated from rabbits have been consistently shown to have affinities in the pico-molar range (Feng et al. 2011). Due to the multiple rounds of selection, the aptamers can attain as much affinity as the antibodies have, which could be in the pico- to nano-molar range. Fab and scFv fragments isolated using phage display library generally tend to have lower affinities. Single domain antibodies generally have also been shown to have lesser affinity as compared to the whole antibodies. The affinities of all such antibody derivatives can be increased by adopting suitable screening procedures such as using decreasing concentrations of the antigens with the subsequent rounds of panning or dimerization of the molecules. In terms of specificity of the biosensors, the antibody fragments that are isolated from phage display library could possibly have higher specificity than the ones isolated from the animals. The Fab fragments cleaved from the polyclonal antibodies raised in animals could show non-specific binding to the closely related antigens. The aptamers also tend to have higher specificities due to a careful selection process. With regard to the stability of various antibody formats, the single domain antibodies show highest stability that can withstand high temperatures in the range of 80–90 °C as well as extreme pH. The aptamers are also considered to be highly stable biosensors that can retain binding to the target even after denaturation. The whole antibodies are on the second rank in terms of stability with the ability to withstand moderate room temperatures that can be suitable for the point of care diagnostic assay. The Fab and scFv fragments are the least stable biosensors which can be stabilized with multimerization or by fusion with Fc fragments or other proteins. With regard to the ease of immobilization, scFv, aptamers and Fab fragments are relatively easier to immobilize as compared to the whole antibodies. Since there is a scope for addition of various functional groups to these fragments which confers suitability in their immobilization. Also due to their smaller size, it is possible to immobilize them at higher densities as compared to the whole antibodies, making the diagnostic assay more sensitive.

To conclude, the immunodiagnostics are being consistently improved over the last few years. The choice of biosensors largely determines the success of a diagnostic assay. Based on the target to be detected, type of diagnostic assay and method of detection, the biosensors have to be carefully selected. Newer methods of antibody screening procedures such as microfluidics which allow for rapid high-throughput screening can provide novel biosensors without the need for immortalization or library preparation.