Elsevier

Biosensors and Bioelectronics

Volume 85, 15 November 2016, Pages 32-45
Biosensors and Bioelectronics

Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements

https://doi.org/10.1016/j.bios.2016.04.091Get rights and content

Highlights

  • scFv fragments, Fab' fragments, and aptamers are common biorecognition elements.

  • scFv fragments are the most customizable due to recombinant synthesis methods.

  • Fab' fragments are the cheapest and quickest to develop for biosensor use.

  • Aptamers have the highest affinity for their target analytes and are most stable.

Abstract

The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab’ fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

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)

  • K.M. Arndt et al.

    J. Mol. Biol.

    (2001)
  • M.J. Berry et al.

    J. Chromatogr.

    (1993)
  • U. Brinkmann et al.

    J. Mol. Biol.

    (1997)
  • B. Catimel et al.

    J. Chromatogr. A

    (1997)
  • E.J. Cho et al.

    Anal. Chim. Acta

    (2006)
  • V. Crivianu-Gaita et al.

    Biosens. Bioelectron.

    (2016)
  • V. Crivianu-Gaita et al.

    Biochem. Biophys. Rep.

    (2015)
  • V. Crivianu-Gaita et al.

    Biosens. Bioelectron.

    (2015)
  • M. Czerwinski et al.

    New Biotechnol.

    (2009)
  • J. De Jonge et al.

    Mol. Immunol.

    (1995)
  • P.P. Dillon et al.

    J. Immunol. Methods

    (2003)
  • T. Erikaku et al.

    Biochem. Biophys. Res. Commun.

    (1991)
  • L.-O. Essen et al.

    J. Chromatogr. A

    (1993)
  • Y.S. Grewal et al.

    Biosens. Bioelectron.

    (2014)
  • R.J. Hosse et al.

    Anal. Biochem.

    (2009)
  • R. Iwata et al.

    Colloids Surf. B

    (2008)
  • J. Kang et al.

    FEBS Lett.

    (2007)
  • Y. Kikuchi et al.

    J. Biosci. Bioeng.

    (2005)
  • T. Konig et al.

    J. Immunol. Methods

    (1998)
  • Y. Kumada

    Biochim. Biophys. Acta

    (2014)
  • Y. Kumada et al.

    J. Immunol. Methods

    (2014)
  • Y. Kumada et al.

    J. Biosci. Bioeng.

    (2011)
  • C. Larsson et al.

    Anal. Biochem.

    (2005)
  • L.J. Lawrence et al.

    FEBS Lett.

    (1998)
  • M. Lee et al.

    Anal. Biochem.

    (2000)
  • W. Lee et al.

    Colloids Surf. B

    (2005)
  • J. Maly et al.

    Mater. Sci. Eng. C

    (2002)
  • S. Medina-Casanellas et al.

    Anal. Chim. Acta

    (2013)
  • M.A.D. Neves et al.

    Biosens. Bioelectron.

    (2015)
  • M. Nisnevitch et al.

    J. Biochem. Biophys. Methods

    (2001)
  • R.J. Owens et al.

    J. Immunol. Methods

    (1994)
  • L. Petersson et al.

    Biochim. Biophys. Acta

    (2014)
  • K. Petrov et al.

    J. Mol. Biol.

    (2004)
  • J. Pezzini et al.

    J. Chromatogr. B

    (2009)
  • R.T. Piervincenzi et al.

    Biosens. Bioelectron.

    (1998)
  • V.S. Prisyazhnoy et al.

    J. Chromatogr. -Biomed.

    (1988)
  • M.H. Ryan et al.

    Mol. Immunol.

    (2008)
  • J.B. Adlersberg

    Ric. Clin. Lab.

    (1976)
  • S. Balamurugan et al.

    Anal. Bioanal. Chem.

    (2008)
  • S.M. Ballantyne et al.

    Analyst

    (2004)
  • A.G. Benny et al.

    N. Z. J. Med. Lab. Technol.

    (1987)
  • R.J. Brezki et al.

    mAbs

    (2010)
  • F.V. Bright et al.

    Anal. Chem.

    (1990)
  • P. Burgstaller et al.

    Angew. Chem.

    (1994)
  • J.A. Burns et al.

    J. Org. Chem.

    (1991)
  • J.D. Carter et al.

    J. Nucleic Acids

    (2011)
  • H. Chen et al.

    Biochemistry

    (1996)
  • T.-T. Chen et al.

    J. Am. Chem. Soc.

    (2015)
  • M. Choe et al.

    Cancer Res.

    (1994)
  • T.C. Chu et al.

    Cancer Res.

    (2006)
  • Cited by (0)

    View full text