Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements
Introduction
Through the development of biosensor technology, biosensors have received significant attention as tools in analytical and diagnostic applications. Biosensors are analytical devices composed of three parts: a biosensing (or biorecognition) element, a transducer, and a signal processing unit (Skoog et al., 2007). In general, a biosensing element is chosen to specifically interact and sequester the target analyte from solution. These elements are bound to the transducer surfaces, which allow for the conversion from a chemical to electrical signal. Since biosensors are developed to provide rapid and reliable analyses of target analytes, one crucial step in the optimization of a biosensor is the choice of the biosensing element. Four of the most prominent biorecognition elements are whole monoclonal antibodies (mAb), fragment antigen-binding (Fab') units, scFv fragments, and aptamers (Fig. 1).
Immunoglobulins (Ig), or antibodies, are large proteins produced by the immune system that have extremely high affinities and specificities for their target analytes (Crivianu-Gaita and Thompson, 2015b). Of the many classes of immunoglobulins (i.e. IgE, IgM, IgG, etc.), the immunoglobulin G (IgG, ~150 kDa) is the most prominently used class in the field of biosensing. The structure of an IgG antibody consists of two heavy protein chains and two light protein chains. The two antibody halves (each half containing one heavy and one light chain) are held together via disulfide bonds in the hinge region (Fig. 1) (Adlersberg, 1976). The number of disulfide bonds in the hinge region varies depending on antibody species and antibody class (Crivianu-Gaita et al., 2015a). The paratope of the antibody – the region that recognizes and binds to the target analyte (or antigen) – involves the top of the VL and VH domains.
The antibody contains two Fab fragments, each one consisting of the VL, VH, CL, and CH1 domains. These two fragments are held together by the key hinge disulfide bridges (Adlersberg, 1976). Fab fragments may be obtained in one of two possible ways: via recombinant synthesis (Choe et al., 1994) or proteolytic cleavage of the parent antibody (Ryan et al., 2008). Fragments including disulfide bridge thiols (Fig. 1) are called Fab' fragments whereas those lacking the thiol functional group are termed Fab fragments. The thiol functional group of the Fab' fragments allows for easy immobilization onto biosensor surfaces (Crivianu-Gaita and Thompson, 2015b).
Even smaller than the Fab fragment is the antibody Fv fragment (Fig. 1), consisting of only the VH and VL domains. These fragments can only be obtained reliably via recombinant synthesis (Ward, 1992) and are held together by relatively weak non-covalent interactions (Owens and Young, 1994). As a result, several modified types of Fv fragments have been developed including, but not limited to, single-chain Fv (scFv) (Tsumoto et al., 1998), disulfide-stabilized Fv (dsFv) (Reiter et al., 1994), diabodies (divalent dimers) (Lawrence et al., 1998), and permutated Fv (pFv) fragments (Brinkmann et al., 1997). This review will focus on the scFv fragments as they are the most prominent of the Fv-derived antibody fragments used as biosensing elements.
Compared to Fab' and scFv fragments, aptamers are not derived from antibodies. Aptamers are single stranded ribonucleic acid (RNA) or 2′-deoxyribonucleic acid (DNA) chains that have affinities and specificities for their target analytes on orders of magnitude comparable to or better than antibodies (Jayasena, 1999). The size of aptamers (~1–2 nm), however, is much smaller than that of whole antibodies (~10–15 nm) allowing them to be immobilized in higher densities on surfaces, resulting in higher sensitivities and lower limits of detection (LOD) in biosensors (So et al., 2005). The same phenomenon is observed with Fab' fragments (Crivianu-Gaita and Thompson, 2015b) and scFv fragments (Kumada, 2014a). For this reason, whole antibodies will not be discussed in this review as Fab' fragments and scFv fragments are considered to be superior biosensing elements.
This review consists of the analysis of scFv fragments, Fab' fragments, and aptamers as biosensing elements. Each of these three major sections are subdivided into three smaller subsections discussing the synthesis of the particular biosensing element, a sample of immobilization techniques, and various biosensor examples. The final section of this review compares and contrasts the three biosensing elements, illustrating advantages and disadvantages for each.
Section snippets
Synthesis and engineering of scFv fragments
As stated previously, Fv fragments are inherently unstable since they are held together by only non-covalent interactions (Owens and Young, 1994). Expression and elution of Fv fragments can result in the dimerization of the VL domains, leading to Bence Jones proteins and VL dimers (Essen and Skerra, 1993, Stevens et al., 1991). The variable stability of Fv fragments is due to the difference in the sequences of the third hypervariable loops (CDR3) between antibodies, affecting the stability of
Synthesis and engineering of Fab' fragments
As stated earlier, Fab' fragments contain VL, VH, CL, and CH1 domains as well as C-terminal thiols – remnants from the antibody hinge disulfide bridges. The number of C-terminal thiols varies between different antibody species (Crivianu-Gaita et al., 2015a). These thiols are extremely useful for the oriented immobilization of Fab' fragments onto biosensor surfaces (Crivianu-Gaita and Thompson, 2015b). The primary method for the production of Fab' fragments is via enzymatic/chemical modification
Synthesis and engineering of nucleic acid aptamers
Aptamers are single stranded RNA or DNA chains created to mimic the selectivity and specificity of antibodies (Famulok et al., 2007, Jayasena, 1999). The affinities (KD) of aptamers are on the order of nanomolar and picomolar – comparable, if not better than monoclonal antibodies (Jayasena, 1999). These nucleic acid chains can be developed for an extremely wide range of molecules and can achieve binding affinities greater than those exhibited by whole monoclonal antibodies (Jayasena, 1999). The
A comparison of scFv fragments, Fab' fragments, and aptamers and their roles in the biosensor world
The previous sections have provided a well-rounded overview of the three biosensing elements. Using this information the biorecognition elements can be compared in order to determine their roles in the biosensor world. Table 1 illustrates advantages and disadvantages to using all three of the biosensing elements. The optimization of the biosensing elements and their immobilization onto transducer surfaces are arguably the most important steps in the development of a biosensor. Biosensing
Conclusions and future perspectives
This review illustrates a thorough comparison between antibody scFv fragments, antibody Fab' fragments, and aptamers. Antibody scFv fragments, composed of the antibody VL and VH domains as well as a linking peptide, are the smallest of the three biosensing elements. These recombinantly-derived fragments can be modified to include various immobilization groups (i.e. linking peptides, reactive functional groups). For this reason, scFv fragments are the most customizable compared to Fab' fragments
Acknowledgements
The authors would like to thank the Natural Sciences and Engineering Research Council (Grant no. RGPIN 46) for the support of their work. An acknowledgement to Professor Alex Romaschin of St. Michael's Hospital, Toronto is also necessary for his helpful discussions.
References (139)
- et al.
J. Mol. Biol.
(2001) - et al.
J. Chromatogr.
(1993) - et al.
J. Mol. Biol.
(1997) - et al.
J. Chromatogr. A
(1997) - et al.
Anal. Chim. Acta
(2006) - et al.
Biosens. Bioelectron.
(2016) - et al.
Biochem. Biophys. Rep.
(2015) - et al.
Biosens. Bioelectron.
(2015) - et al.
New Biotechnol.
(2009) - et al.
Mol. Immunol.
(1995)