Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection
Introduction
Antibodies are widely used as specific capture molecules in immunoassay devices for rapid point of use diagnostics in the fields of medicine, agriculture and the environment. A wide variety of devices have been reported with different configurations to meet the needs of different applications (Rogers, 1998). There is a continual drive for new technologies capable of sensitive detection of the target molecule combined with simplicity and rapid delivery of test results. Furthermore, to decrease costs, a generic platform device for a range of applications is desirable. Biosensors based on electrical measurement and direct read out of binding reactions combined with recombinant antibodies as the specific recognition element have the potential to meet these needs.
Advances in antibody gene cloning and expression have led to the development of recombinant antibodies, comprising the binding fragments of antibody molecules (scFv or Fab). They can be obtained by cloning the genes from hybridomas or, de novo, by selection from large synthetic, naïve or immune libraries of antibody genes (reviewed by Bradbury and Marks, 2004 and Hoogenboom, 2005). Antibody fragments with a wide range of antigen specificities and affinities of importance to medical, agricultural and environmental research have been isolated from synthetic, naïve or immune libraries (Nissim et al., 1994, Vaughan et al., 1996, Toth et al., 1999, Charlton et al., 2001, Strachan et al., 2002, Ziegler and Torrance, 2002).
The scFv fragments are small monovalent molecules, and scFv's obtained by selection from phage display libraries can be of variable affinity with the strongest binders usually being obtained from very large libraries (Bradbury and Marks, 2004). For many applications the affinity of selected scFv is sufficient particularly if they are subcloned into plasmid vectors for expression as multivalent molecules (Plückthun and Pack, 1997, Neri et al., 1995, Kramer et al., 2002). For example, modification of scFv through genetic engineering to add a C-terminal cysteine residue followed by a pentahistidine tag enabled expression of monomeric and dimeric molecules with antigen-binding activity in Western blots and ELISA and the covalently linked scFv had four-fold higher binding activity compared with the monomers (Kipriyanov et al., 1994). Biotinylation of scFv through the cysteine residue allowed detection with streptavidin conjugates, but the binding reactivity was weaker than the unmodified monomeric scFv (Kipriyanov et al., 1994). The biotin-streptavidin system was used to produce a functional scFv protein layer assembled on glass by successive applications of biotin derivatised BSA, streptavidin followed by scFv fused to a 10 amino acid streptavidin peptide tag. However, when the scFv molecule itself was biotinylated it was not functional (Piervincenzi et al., 1998). Another approach is to immobilise antibody fragments using a hexahistidine tag at the C terminus, and functional Fab fragments were shown to reversibly bind to a chelator thioalkane assembled on a gold surface (Kröger et al., 1999).
When immobilised directly onto solid supports, the binding activity of scFv molecules is often impaired and miniantibody fusion proteins have been devised that have helped to alleviate this problem for successful application in DAS ELISA. For example, scFv have been used effectively in assays when expressed as fusions to the light chain constant domain (CL) (McGregor et al., 1994) or a leucine zipper domain (Kerschbaumer et al., 1997) for coating ELISA plates, and alkaline phosphatase (Kerschbaumer et al., 1996, Griep et al., 1999) for antigen detection. These modifications result in dimerisation and lead to increased functional affinity and stability. Combinations of such fusion proteins in fully recombinant ELISAs for the detection of plant viruses gave equivalent results to ELISAs based on immune reagents (Toth et al., 1999, Uhde et al., 2000, Saldarelli et al., 2005).
The selection of scFv fragments from phage display libraries thus offers the potential to access very large numbers of molecules with different binding specificities quickly and cheaply, avoiding the need for animal immunizations. The scFv can be genetically engineered to produce tailored constructs as described above. Such fusion proteins can be coated directly onto plastic, silica or carbon surfaces by simple adsorption, which as discussed above is adequate for ELISA-based assays, but this method results in molecules adsorbed in random orientations, decreasing the proportion of bound functionally active molecules. In contrast, covalent coupling through defined C-terminal amino acid residues has the advantage of orientating the antigen-binding site away from the support surface and in principle leading to an increase in the proportion of bound functionally active molecules. This is an important consideration in designing active surfaces for nanoscale biosensor applications, and, for example, a 30 amino acid spacer molecule fused to the C terminus of scFv was employed for studies on individual antigen-antibody binding forces by atomic force microscopy (Ros et al., 1998).
Typically, covalent coupling is achieved through the use of heterobifunctional linker molecules disposing two functional groups with complementary reactivities at either end of the molecule. Amongst these, activated esters of N-maleoyl-ω-amino acids have proved particularly popular and versatile, enabling the conjugation of a sulfhydryl-containing species such as a peptide containing cysteine residues via reaction of the latter with the maleimide function and formation of ester or amide bonds with a second molecule containing hydroxyl or amino groups. Such a linker was used to covalently couple a cysteine-tagged scFv to liposomes via a maleimide linker for use in immunotherapy (Xu et al., 2002).
In this paper the above concepts have been introduced to practice by describing the selection of anti-viral scFv from a synthetic library of antibody genes, production of a generic expression vector to create a scFv fusion protein containing a CL domain with a C-terminal cysteine residue, the synthesis of a maleimide linker molecule for functionalisation of gold surfaces and the covalent, oriented attachment of functional scFv. The surface was tested by repeated cycles of binding and regeneration and the results show that scFv fusion proteins can be engineered to produce robust, functional antigen-sensing surfaces.
Section snippets
ScFv library screening
The anti-Cowpea mosaic virus (CPMV) scFv were selected from Human Single Fold scFv libraries I and J (‘Tomlinson libraries’) (De Wildt et al., 2000). Both libraries are based on a single human framework for VH and VL with side chain diversity incorporated at positions in the antigen-binding site. The complementarity determining region (CDR) 3 of the heavy chain was designed to be as short as possible. The libraries are in the phagemid/scFv format and were pre-screened for binding to Protein A
Selection of scFv against CPMV from the synthetic libraries
Antibody selection was done separately for libraries I and J. Three rounds of selection gave approximately 100-fold enrichment as judged by input versus output phage titres. Approximately 100 cultures were grown from single colonies from each library and culture supernatant containing phage particles was used to detect CPMV in ELISA. Six positive clones were selected and were further tested as soluble scFv in ELISA to compare performance (Table 1). ScFv E7, F10 and F4 (obtained from library J)
Discussion
This report describes the selection of scFv molecules against a plant virus from a synthetic scFv phage display library, the construction of a generic vector for the production of a scFv fusion to a CL domain with a C-terminal cysteine residue (scFvCLcys) and a procedure for the oriented covalent attachment of functional scFv to a plain gold surface. Purified preparations of scFvCLcys were stored at 4 °C for 6 months (the longest period tested) without apparent loss of function, demonstrating
Acknowledgements
Financial support was received from Amcet Ltd. and the Scottish Executive Environment and Rural Affairs Department. We are very grateful to both Ian Tomlinson and George Lomonossoff for generously providing the phage libraries and the CPMV preparation and antiserum, respectively. We thank Karen Jackson for technical assistance.
References (37)
- et al.
Recombinant single chain antibodies in bioelectrochemical sensors
Talanta
(2001) - et al.
Antibodies from phage antibody libraries
J. Immunol. Methods
(2004) - et al.
The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep
Biosens. Bioelectron.
(2001) - et al.
Impedimetric evaluation for diagnosis of Chagas’ disease: antibody-antigen interactions on metallic electrodes
Biosens. Bioelectron.
(2003) - et al.
Atrazine analysis using an amperometric immunosensor based on single-chain antibody fragments and regeneration-free multi-calibrant measurement
Analyt. Chim. Acta
(2003) - et al.
pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins
Protein Exp. Purif.
(1999) - et al.
Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries
J. Virol. Methods
(1999) - et al.
Engineering receptors and antibodies for biosensors
Biosens. Bioelectron.
(2002) - et al.
pDAP2: A vector for construction of alkaline phosphatase fusion-proteins
Immunotechnology
(1996) - et al.
Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay
Anal. Biochem.
(1997)
Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies
Mol. Immunol.
A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F-ab fragments and leads to improved recombinant immunoglobulin stability
Biosens. Bioelectron.
Immobilization of histidine-tagged proteins on gold surfaces using chelator thioalkanes
Biosens. Bioelectron.
Nanoscaled interdigitated titanium electrodes for impedimetric biosensing
Sens. Actuators B
Spontaneous assembly of bivalent single-chain antibody fragments in Escherichia coli
Mol. Immunol.
Genetic engineering of a single-chain antibody fragment for surface immobilisation in an optical biosensor
Biosens. Bioelectron.
New protein engineering approaches to multivalent and bispecific antibody fragments
Immunotechnology
Isolation of recombinant antibodies (scFvs) to grapevine virus B
J. Vir. Methods
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