Biosensors and Bioelectronics 50 (2013) 460–471

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Biosensors and Bioelectronics

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Site-directed antibody immobilization techniques for immunosensors

Asta Makaraviciute, Almira Ramanaviciene n

NanoTechnas-Center of Nanotechnology and Materials Science, Department of Analytical and Environmental Chemistry, Faculty of Chemistry,Vilnius University, Naugarduko Street 24, LT-03225 Vilnius, Lithuania

a r t i c l e i n f o

Article history:Received 22 April 2013Received in revised form14 June 2013Accepted 26 June 2013Available online 5 July 2013

Keywords:AntibodySite-directed immobilizationImmunosensor

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esponding author. Tel.: +370 67203653; fax: +ail address: almyra@imi.lt (A. Ramanaviciene)

a b s t r a c t

Immunosensor sensitivity, regenerability, and stability directly depend on the type of antibodies used forthe immunosensor design, quantity of immobilized molecules, remaining activity upon immobilization,and proper orientation on the sensing interface. Although sensor surfaces prepared with antibodiesimmobilized in a random manner yield satisfactory results, site-directed immobilization of the sensingmolecules significantly improves the immunosensor sensitivity, especially when planar supports areemployed. This review focuses on the three most conventional site-directed antibody immobilizationtechniques used in immunosensor design. One strategy of immobilizing antibodies on the sensor surfaceis via affinity interactions with a pre-formed layer of the Fc binding proteins, e.g., protein A, protein G, Fcregion specific antibodies or various recombinant proteins. Another immobilization strategy is based onthe use of chemically or genetically engineered antibody fragments that can be attached to the sensorsurface covered in gold or self-assembled monolayer via the sulfhydryl groups present in the hingeregion. The third most common strategy is antibody immobilization via an oxidized oligosaccharidemoiety present in the Fc region of the antibody. The principles, advantages, applications, and arisingproblems of these most often applied immobilization techniques are reviewed.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4602. Techniques of site-directed immobilization of antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

2.1. Site-directed immobilization via proteins binding the Fc region of immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4622.2. Site-directed immobilization of antibody fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4642.3. Site-directed immobilization via oxidized oligosaccharide moieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

3. Comparison of different antibody immobilization strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4664. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

1. Introduction

Despite the long-lasting interest and multiple efforts in immuno-sensor design, few examples of commercialization have been reported(Deacon et al., 1991; Ruano-Lopez et al., 2009; Johnson et al., 2011), themost widely known immunosensor being the pregnancy test for thedetection of human chorionic gonadotropin (hCG). At first glance, thismight look surprising as immunosensors seem to be a promising andattractive technique, offering high specificity due to the use of very

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specific immune molecules, non-destructive approach to sample,simple operation, uncomplicated sample preparation and, mostimportantly, high sensitivity, especially when different signal transdu-cers have recently been combined to yield low limits of detection(Ramanaviciene et al., 2012). However, in order to achieve thecharacteristics mentioned above, some important issues have to beresolved because the sensitivity, stability and longevity of an immu-nosensor for themost part depends on the amount of the immobilizedimmune molecules on the surface, their conformational stability,remaining activity after the immobilization procedure and theirorientation on the sensor surface, especially considering immunoglo-bulins, which are asymmetrical molecules with their recognition sitestaking different positions in space following different immobilization

Fig. 1. Schematic representation of an antibody molecule. Fab—fragment, antigen-binding; Fc—fragment, crystallizable; CHO—carbohydrate moiety; VL—variabledomain of the light chain; VH—variable domain of the heavy chain; CL—constantdomain of the light chain; and CH1, CH2, CH3—first, second and third constantdomains of the heavy chain.

Fig. 2. Two main approaches to antibody immobilization. A—random, and B—site-directed antibody immobilization.

A. Makaraviciute, A. Ramanaviciene / Biosensors and Bioelectronics 50 (2013) 460–471 461

procedures, and thus leading to hindered interactions with the analyte.Schematic representation of an antibody molecule is depicted in Fig. 1.

Although both antigen and antibody can be immobilized on thesensor surface (Lu et al., 1996; Rao et al., 1998; Jung et al., 2008a;Holford et al., 2012; Zeng et al., 2012), in this review we willmainly focus on site-directed antibody immobilization. It is a veryattractive technique, especially in clinical applications as it issimple, precise, allows direct analyte detection, and could be usedin cases when the immune response is minimal.

There are two main approaches that can be used in antibody-based sensor surface preparation: random and site-directed anti-body immobilization. The main principles of these approaches arepresented in Fig. 2. This review is mainly centered on site-directedantibody immobilization on planar supports. Despite many pub-lications reporting the benefit of the site-directed antibody orien-tation on flat surfaces, it has been suggested that on threedimensional supports different antibody orientations result inonly minor differences of specific activity (Johnsson et al., 1995).In contrast, the significance of antibody orientation on a threedimensional support has been shown in another publication (Pateland Andrien, 2010). Thus it is extremely difficult to evaluate therole of antibody orientation on irregular surfaces. However, certainstudies investigating this problem have been included in thereview for illustrative purposes. On the other hand, planar sup-ports are of a smaller surface area, so the influence of antibodydensity and steric effects are very explicit and in this case site-directed antibody immobilization is a valuable tool for adjustingthese characteristics that should definitely be considered. Differentsupports have been reported to be used for immunosensor design,for example, noble metals, especially gold (Hafaiedh et al., 2013),glass (Tedeschi et al., 2003; Zhao et al., 2006), silicon (Yakovleva

et al., 2002; Dhanekar and Jain, 2013), silicon nitride (Caballeroet al., 2012; Kurihara et al., 2013), indium–tin oxide (Bandodkaret al., 2010). The immobilization support is usually determined bythe method used for signal transducer and can be modified fordifferent purposes.

The simplest sensor preparation technique is based on randomadsorption of antibody molecules on the sensor surface. Althoughadsorption does not require the use of multiple materials andcomplex reactions, it also results in serious disadvantages, such asdenaturation of proteins, very low stability and random proteinorientation (Buijs et al., 1996; Hlady and Buijs, 1996; Wiseman andFrank, 2011).

The most commonly used technique is, however, antibodyamine coupling to the sensor surface previously modified withdifferent coatings that allow biomolecule immobilization, such as,self assembled monolayers, dextran or various polymers (Zhouet al., 2006; Kyprianou et al., 2013). Niemeyer's group developedstrategy based on protein conjugation to oligonucleotides, forfurther hybridization and immobilization on a surface (Niemeyeret al., 1994). Such an approach has been successfully used forhybrid molecule cleavage and regeneration on a sensor (Bomberaet al., 2012).

A self assembled monolayer (SAM) is a layer formed ofn-carbon atom alkyl chains with certain functional groups, inmany cases a group enabling the molecules to attach to the sensorsurface and a carboxyl group that later can be used for theformation of a peptide bond with amino groups randomly scat-tered on the antibody molecule surface. Different variants of SAMsare available, not only modified with different functional groupsfor various immobilization strategies but also exhibiting disparateresponses to non-specific binding (Silin et al., 1997; Stigter et al.,2005). Direct antibody immobilization via SAMs has multipleadvantages, such as a well-known, tested and quite simpleimmobilization technique, stability and reusability of immunosen-sors due to covalent bonds. However, a critical drawback is therelatively small sensitivity caused by random antibody orientationand subsequent decreased availability of antibody active sites toantigen in comparison to site-directed antibody immobilizationmethods (Tsai and Pai, 2009; Kausaite-Minkstimiene et al., 2010),although in some cases very efficient random immobilizationbased immunosensors have been reported (Billah et al., 2008;Mattos et al., 2012).

In order to avoid random antibody immobilization and improveantigen binding site availability, site-directed antibody immobili-zation methods are being constantly developed, e. g., incubation ofthe antibody with its antigen prior to immobilization on a mixedself-assembled monolayer, so that the active sites would remainprotected from the active groups of the support (Yoon et al., 2011),cyclic voltammetry assisted coupling of the hydroxyl groups of theoligosaccharide moieties present on antibodies to the cyanogroups of the poly-(2-cyano-ethylpyrrole) (Um et al., 2011), theuse of UV irradiation to break the disulfide bridges upon adsorp-tion by nearby tryptophan residues, this way freeing the sulfhydrylgroups that can be directly coupled to the gold surface inan controlled manner (Della Ventura et al., 2011), calixarene deriva-tives able to orient an antibody in a site-directed manner (Oh et al.,2005), and fusion proteins, such as pentamerized bispecific anti-bodies (decabodies) (Hussack et al., 2009), ZZ-alkaline phosphatase-His fusion protein exhibiting Fc binding (Yang et al., 2013) orcutinase-single chain antibody fusion proteins that allow theoriented immobilization of cutinase to phosphonate ligands(Kwon et al., 2004) to name a few. However, the most popularand widely employed site-directed antibody immobilization tech-niques are immobilization via Fc binding proteins, via antibodyfragments and via oxidized oligosaccharide moieties. These anti-body immobilization methods, their advantages and problems,

Table 1Comparison of different site-directed and random antibody immobilization techniques.

Immobilizationformat

Methodology Advantages Disadvantages

Site-directedVia Fc

bindingproteins

Affinity interactions with a preformed layer ofproteins specific to the Fc regions of Ab, e.g.,proteins A, G, A/G, L, anti-Fc, recombinantproteins

Improvement in sensitivity in comparisonto random immobilization (Song et al.,2011; Quang Huy et al., 2012)Does not require antibody modification(Deisenhofer, 1981; Kato et al., 1995; Sauer-Eriksson et al., 1995)Surface regeneration for multiple analyses ifcross-linking is used (Nakanishi et al., 1996;Pulido-Tofino et al., 2000)

Single use if non-cross linked (Pulido-Tofino et al., 2000;Kausaite-Minkstimiene et al., 2010)Cross-linking might reduce sensitivity (Song et al., 2011).Protein G is reported to be prone to non-specificinteractions (Quinn et al., 1999)Specific to certain classes of antibodies only (Goding,1978; Bjorck and Kronvall, 1984)

Viaantibodyfragments

Chemical reduction or genetic engineeringbased disruption of disulfide bridges andimmobilization via sulfhydryl groups

Improvement in sensitivity in comparisonto random immobilization (Peluso et al.,2003; Tsai and Pai, 2009; Balevicius et al.,2011)Affinity towards antigen is adjustable ofrecombinant Fab′ (Rouet et al., 2012)Surface regeneration for multiple analyses(Kausaite-Minkstimiene et al., 2010)

Steric hindrance possibility because of a very compactlayer (Nakanishi et al., 1996; Vikholm, 2005)Chemical reduction resulting in the potential loss ofbiological activity of Ab fragments, especially in the caseof mAbs (Desilva and Wilson, 1995; Lu et al., 1995;Bonroy et al., 2006)Low stability of genetically engineered fragments(Quintero-Hernandez et al., 2007)Possible Ab denaturation upon direct contact with gold(O'Brien et al., 2000), which is likely to cause non-specific binding (Vikholm-Lundin, 2005)

Via anoxidizedoligosacchar-ide moiety

Chemical or enzymatic oxidation of anoligosaccharide moiety and coupling to amineor hydrazine terminated supports

Improvement in sensitivity in comparisonto random immobilization (Pulido-Tofinoet al., 2000; Ho et al., 2010)No direct modification of amino acids(O'Shannessy and Quarles, 1985; Hoffmanand O'Shannessy, 1988)Surface regeneration for multiple analyses(Pulido-Tofino et al., 2000)

Different reactions conditions, such as temperature, pHand periodate concentration, might strongly affectoxidation and can yield inconsistent results (Wolfe andHage, 1995)Antibody structure damage during oxidation, especiallyfor certain mAbs (Hudson and Barker, 1967; Abrahamet al., 1991)

Random Covalent attachment via amine couplingPhysiosorption

In some cases shows good sensitivity (Billahet al., 2008; Mattos et al., 2012)Surface regeneration for multiple analysesafter covalent attachment (Pulido-Tofinoet al., 2000)

Lower sensitivity in comparison to site-directedimmobilization methods (Quang Huy et al., 2012)In case of physiosorption denaturation of proteins, verylow stability and random protein orientation (Buijs et al.,1995; Buijs et al., 1996)

Ab—antibody, mAb—monoclonal antibody.

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possible solutions, comparison among different techniques andnew approaches will be reviewed in this publication. Since theauthors tried to relate the advantages and disadvantages of themethods with the immobilization mechanism, a clearer and amore compact comparison of the discussed techniques is pre-sented in Table 1.

2. Techniques of site-directed immobilization of antibodies

2.1. Site-directed immobilization via proteins binding the Fc regionof immunoglobulins

Proteins A and G have been firstly and most widely used inaffinity chromatography, especially for antibody purification(Aybay and Imir, 2000; Burckstummer et al., 2006; Evazalipouret al., 2012), but numerous uses of these proteins in immunosen-sor design have also been reported. By binding the Fc region of animmunoglobulin these proteins immobilize immunoglobulins onthe surface in a site-directed manner with antigen-binding regionsdirected towards the analyte (Fig. 3A) (Sauer-Eriksson et al., 1995).

Proteins A and G are biomolecules characteristic to certainspecies of pathogenic staphylococcal and streptococcal bacteria.Protein G in its native form is expressed in group C and GStreptococci and it recognizes both Fab and Fc domains of

immunoglobulins of all human IgG subclasses, rabbit, mouse,and goat IgGs, having approximately 10-fold lower affinity forthe Fab region (Bjorck and Kronvall, 1984). Protein G has threeimmunoglobulin G binding domains: I, II and III (Goward et al.,1991). Primarily, protein G binds antibodies in the Fc region, morespecifically at the interface between CH2 and CH3 domains (Katoet al., 1995; Sauer-Eriksson et al., 1995); however, some binding tothe Fab region can occur (Derrick and Wigley, 1994; Derrick et al.,1999). Native protein G also binds albumin (Akerstrom et al., 1987),but this specificity along with the ability to bind Fab has been removedfrom the widely used recombinant protein G usually expressed inEscherichia coli (Goward et al., 1990).

Protein A is expressed in Staphylococcus aureus and has five IgGbinding domains (A–E) (Bjork et al., (1972); Sjoquist et al., (1972);Moks et al., 1986). Each domain can bind amino acid residuespresent in CH2 and CH3 domains of IgG Fc region (Deisenhofer,1981) and Fab region (Graille et al., 2000). Protein A, unlike proteinG, exhibits a narrower antibody binding range within the IgG class.For example, it does not bind human IgG3. However, it demon-strates affinity towards other immunoglobulin classes—IgA, IgM,IgE, although the highest affinity is still exhibited towards IgG.Protein A is also specific to IgG2a, IgG2b and IgG3 of mice (Goding,1978; Surolia et al., 1982). While protein G shows overall higheraffinity to IgG, protein A usually is less expensive and, as a generalrule, it is more often used in immunosensing applications. In

Fig. 3. Schematic representation of site-directed antibody immobilization meth-ods. (a) Via Fc binding protiens, (b) via antibody fragments and (c) via oxidisedoligosaccharide moieties.

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addition, these proteins are only applicable to antibodies of certainorigin. It has also been suggested that protein G is more prone tonon-specific interactions (Quinn et al., 1999). Another importantconsideration when deciding between proteins A and G in immuno-sensor applications is the optimal antibody binding pH. The maximalIgG binding efficiency of protein G is at pH 4 and 5 (Akerstrom andBjorck, 1986) and protein A at pH 8 (Wright et al., 1977).

A recombinant protein A/G combining four immunoglobulinbinding domains from protein A and two from protein G has alsobeen constructed. It can bind all the immunoglobulin classes thatboth proteins bind, e.g., it binds all human IgG subclasses and IgA,IgM, IgE. In some cases it also exhibits stronger affinity (Lindmarket al., 1983; Sikkema, 1989). This could be particularly useful forbiosensing applications as a wide variety of different surfacescould be easily prepared via this reagent.

Another option of site-directed immobilization via Fc bindingproteins is the use of antibodies against the Fc region of immu-noglobulins (anti-Fc). However, few studies reporting the applica-tions of protein A/G (Gonzalez-Martinez et al., 1998; Penalva et al.,2000a; Penalva et al., 2000b) or anti-Fc (Ben-Dov et al., 1997;Quinn et al., 1999; Patel and Andrien, 2010) in immunosensinghave been published (Fig. 3).

The first use of Fc binding proteins for immunosensing applica-tions was reported in 1987 by Muramatsu et al. (1987). Theydesigned a quartz crystal microbalance (QCM) based piezoelectricbiosensor for human IgG detection by a protein A layer immobi-lized via 3-aminopropyltriethoxysilane. It was demonstrated thatthe system could be used for determining concentrations of IgG ofdifferent subclasses. This technique was later improved by Prusak-Sochaczewski and Luong, who proposed an idea of using protein Anot as a capture ligand but as an intermediary protein for antibodyimmobilization (Prusak-Sochaczewski and Luong, 1990). Theseauthors demonstrated applied protein A in a piezoelectric

immunosensor design for human serum albumin detection. Sincethen proteins A and G have been used in immunosensor designbased on different forms of signal transducers, e. g., surfaceplasmon resonance (SPR) (Quinn et al., 1999; Lee et al., 2003;Soh et al., 2003; Oh et al., 2004b; Jung et al., 2008b; Bergstrom andMandenius, 2011), ellipsometry (Wang and Jin, 2003; Bae et al.,2005), microfluidics chemiluminescence (Yakovleva et al., 2003),fiber optic biosensors with fluorescence detection (Anderson et al.,1997), electrochemical (Chen et al., 2010; Mendes et al., 2012),piezoelectric (Attili and Suleiman, 1996; Kanno et al., 2000;Carrigan et al., 2005), lateral flow immunoassay (Ryu et al., 2011)and immunosensors based on combinations of different techni-ques, e.g., SPR and electrochemical (Tang et al., 2006).

Most often, proteins A or G are immobilized on the sensorsurface via preformed layers of dextran (Bergstrom andMandenius, 2011), self-assembled monolayers (Jyoung et al.,2006), different polymers (Yakovleva et al., 2003) or hydrogels(Carrigan et al., 2005), and Langmuir–Blodgett films (Owaku et al.,1995; Oh et al., 2004a). However, other techniques of protein A orG immobilization have been reported, including thiolation of theamino groups of the protein and immobilizing proteins directly onthe gold coated sensor surface (Oh et al., 2004b; Fowler et al.,2007; Lee et al., 2007), modifying protein G with tags, for example,biotin (Jung et al., 2006) or DNA oligonucleotides complementaryto oligonucleotides present on the sample surface (Jung et al.,2007). Another popular approach is the design of recombinantproteins exhibiting Fc binding protein properties, e.g., glutathioneS-transferase with binding domains from protein G (Ha et al.,2006), protein B5C1 consisting of five B-domains from protein A(Kanno et al., 2000), or small recombinant peptides (Yang et al.,2005; Jung et al., 2008b).

It is worth mentioning that orientation plays an important rolenot only in antibody immobilization on the sensor surface but alsoin the Fc binding protein immobilization step. However, thesensitivity of the system depends more on the orientation of theantibodies than of the Fc binding proteins. There might be severalexplanations to this. First, it is important to consider antibodysurface density. Antibody immobilization via amine couplingresults in a layer of asymmetrical molecules positioned in differentorientations. It has been shown that surface density of theantibodies immobilized on solid supports depends on antibodyorientation, for example, up to 2.75 times bigger surface densitycan be achieved when antibodies are in “end-on” orientation incomparison to “side-on” orientation (Buijs et al., 1995). Thus, byconventional immobilization methods not only less antibodies arelikely to attach but also some of the immobilized antibodies areoriented in such a manner that they become completely unavail-able to antigens. In addition, amine coupling might disrupt someantigen binding sites as amine groups from the binding sites mightparticipate in the coupling reaction. Antibodies are also likely tocause steric hindrance impeding antigen binding. In contrast,when a randomly directed layer of Fc binding proteins is used,the main disadvantage is that some of the Fc binding sites areunavailable to antibodies. Subsequently not all antibody bindingsites get occupied but all the antibodies that bind are oriented in asite-directed manner with their antigen recognition sites unda-maged. In addition, a randomly oriented Fc binding protein layerstill enables reaching better sensor capacities in comparison torandom antibody orientation despite the fact that the maximalantibody binding efficiency is not reached (Song et al., 2011).A lower surface density of antibodies on the sensing surface islikely the decrease of the steric hindrance, which in turn results inthe increase of the response to antigen in comparison to randomlyoriented antibodies (Spitznagel and Clark, 1993).

However, site-directed Fc binding protein immobilizationsignificantly improves the antibody–antigen binding ratio (Jung

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et al., 2009; Tajima et al., 2011) and a number of Fc binding proteinorientation techniques have been proposed, such as exposingprotein A to tyrosinase that converts tyrosine residues present ina distance from the Fc binding sites to o-quinones that are reactivetowards primary amines (Ahmed et al., 2006), tagging theN-terminus of protein G with cysteines for site-directed immobi-lization on gold surface (Lee et al., 2007), coupling oligonucleotideto protein G N-terminus, which is complementary to oligonucleo-tide immobilized on the sensor surface (Jung et al., 2007), orvarious Fc binding fusion proteins, e. g., with elastin, that can beimmobilized via temperature triggered hydrophobic interactions(Gao et al., 2005), His tag for immobilization on the histidinebinding matrixes (Johnson et al., 2003; Roth et al., 2012). Inter-estingly, controlling the lateral spacing of the Fc binding moleculescan further enhance sensitivity of the system. A study by Kim et al.(2012) has revealed that upon antibody immobilization via a site-directed by a N-terminal cysteine protein G layer, antigen bindingefficiency was increased 2.2 times in comparison to antibodiesimmobilized via a randomly oriented protein G layer. After theadditional lateral spacing of protein G molecules by 9-acid den-dron molecules, antigen binding efficiency was increased 1.5 times.As a result, synergistic effect of orientation and lateral spacing ofprotein G enabled the 10-fold improvement in the detectioncapability of recombinant mouse interleukin-2 in comparison tosensor surfaces based on protein G immobilized randomly vialysine residues. All in all, an optimal antibody surface density thatis closely related to antibody orientation is an important char-acteristic largely affecting the sensitivity. The surface density hasto be high enough for good binding capacity of the sensor but atthe same time not too high in order to avoid the decrease insensitivity caused by steric effects.

Despite the advantages of orienting the affinity ligand towardsthe analyte solution, Fc binding proteins have one importantdrawback limiting their use. Since both Fc binding protein-antibody and antibody-antigen interactions are affinity based,after the regeneration of the surface (e.g. dissociation of formedimmune complex), Fc binding protein-antibody complex is alsodisassembled. This is a critical disadvantage when developing amultiple use immunosensor. One way of solving this might be theuse of various bifunctional cross-linking reagents, most commonlydimethyl pimelimidate (DMP) (Catimel et al., 1997; Bergstrom andMandenius, 2011). The first use of DMP for antibody–protein Acoupling was reported for affinity chromatography column pre-paration in 1982 (Schneider et al., 1982). DMP has imidoestergroups on both ends and cross-links via amino groups afterantibody is immobilized on the layer of the Fc binding protein.However, before considering cross-linking one must have in mindthat cross-linkers bind randomly and that they might affectantigen recognition sites subsequently having a negative effecton immunosensor sensitivity (Song et al., 2011).

2.2. Site-directed immobilization of antibody fragments

Another attractive method of site-directed antibody immobili-zation has been developed by employing native antibody disulfidebridges. These bridges are located in the antibody hinge regionbetween the heavy chains and maintaining the structure of theantibody molecule. In some cases disulfide bridges of the hingeregion of intact antibodies can be reduced and half-antibodymolecules be self-assembled on the gold surface via free sulfhydrylgroups (Fig. 3B) (Karyakin et al., 2000; Ho et al., 2010; Baleviciuset al., 2011; Baleviciute et al., 2013; Baniukevic et al., 2013).However, this immobilization method is more commonly appliedto antibody fragments and not to intact molecules (Saerens et al.,2008). Most often it are Fab′ fragments that are used, i.e.,fragments consisting of one heavy and one light chain retaining

specific antigen binding ability. Fab′ fragments can be prepared inseveral ways. The simplest and most widely used way is to digestintact antibodies with pepsin, bromelain or ficin (Waller et al.,1968; Mariant et al., 1991) yielding F(ab′)2 fragments and severalFc peptides. The F(ab′)2 fragments are purified using affinitychromatography and after the purification are reduced to Fab′fragments by incubating with thiol-containing chemical reduc-tants, most commonly with dithiothreitol (DTT), 2-mercaptoethanolamine (2-MEA) or cysteine (Cebra et al., 1961;Mandy and Nisonoff, 1963; Nisonoff and Dixon, 1964). The maindifference among these reductants is that DTT is disulfide cleavagereagent that enables the formation of intramolecular ring struc-ture during the reduction reaction. This structure results in theshift of the reduction reaction equilibrium towards the products;so subsequently lower concentrations of reductants are needed forcomplete cleavage of disulfide bonds (Cleland, 1964). A thiol-freereagent often used in preparation of antibody fragments is tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Trialkylphosphinesare known reduce organic disulfides, and, unlike many othertrialkylophosphines, TCEP is water-soluble, odorless and stable inair and water (Burns et al., 1991). One of its advantages is a widerpH interval of action in comparison to DTT (Cherkaoui et al., 2010).It is important to note that the reduction reaction yields a mixtureof antibody fragments and that the relative abundance of eachspecies depends on the reduction conditions and the type ofantibody (Virella and Parkhouse, 1973)

Another way of preparing Fab′ fragments is by employing geneengineering. It allows the production of recombinant Fab′ frag-ments having different characteristics, e.g., terminal cysteineresidues can be included. This modification enables site-directedself-assembled immobilization of Fab′ fragments on gold or SAMcovered surfaces via strategically positioned sulfhydryl groups(Torrance et al., 2006). Additional advantage of recombinant Fab′fragments is adjustable affinity towards antigen (Rouet et al.,2012). Another option of antibody-based recombinant moleculesare scFv (single chain fragment-variable) fragments (Bird et al.,1988; Huston et al., 1988). These small heterodimers consist ofantibody heavy and light chain variable domains connected by apeptide linker and are the smallest fragments capable of specificrecognition (Padlan, 1994). The advantages of the small size ofscFvs are the possibility to develop a sensing surface of highsurface density and lesser non-specific protein–protein bindinginteractions.

Fab′ fragments with sulfhydryl groups can be self-assembleddirectly on the gold covered sensor surface by forming a gold–thiolate bond (O'Brien et al., 2000; Brogan et al., 2003). Thoughthis sensor surface preparation technique is quick and non-complicated, it might also present one with some disadvantages,such as structural changes of proteins upon the contact with gold.This might cause antibody unfolding and unfolded antibodymolecules not only lose their antigen binding capacity but canalso serve as a platform for non-specific protein adsorption viaelectrostatic, hydrophobic, and hydrophilic interactions. Moreover,if the proteins are not adsorbed in a uniform manner and there issome uncovered gold surface left, the bare sensor surface spotscan be yet another reason for non-specific interactions (Vikholm-Lundin, 2005). A solution to this problem is the attachment ofantibody fragments via various cross-linking layers, e.g. SAMs(Brogan and Schoenfisch, 2005; Bonroy et al., 2006) and low- ornon-fouling surfaces mainly based on polar molecules (Zhao et al.,2012). Furthermore, mixing Fab′ fragments with protein repellentpolymers and the advantages of the system has been reported(Piehler et al., 2000). Antibody immobilization platforms thatallow the minimization of non-specific interactions are of specialinterest, particularly if real sample measurements are to beconsidered. Another support of antibody fragment immobilization

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might be gold nanoparticles because of their large surface area,which enables high antibody fragment surface density with smallsteric hindrance, their convenient shape, which comes in use forpreservation of the bioactivity of immunoglobulins (Ho et al.,2010). In spite of the fact that antibody fragment immobilizationis usually achieved via sulfhydryl groups, other immobilizationstrategies have also been reported, e.g., recombinant antibodyfragment immobilization via His tags (Shen et al., 2005; Lo et al.,2009) or biotinylation (Cho et al., 2009; Romanazzo et al., 2010).

Immobilization of Fab′ fragments was first proposed by Jimboand Saito (1988). Lu et al. showed the increased antigen bindingactivity of Fab′ fragments in comparison to randomly oriented Fab′fragments by 2.7 times (Lu et al., 1995). The group of Vilkholmattached Fab′ fragments to phospholipid layers for QCM immuno-sensor design (Vikholm et al., 1998) and later they developed thisimmobilization method by self-assembling Fab′ fragments on goldsurface along with protein repellent polymers (Vikholm, 2005) forapplication in optical signal transducers, namely SPR. The use ofFab′ fragments for other types of signal transducers, such aselectrochemical (Nassef et al., 2009) and fluorescence (Herronet al., 1993) have been reported while scFvs have also been usedcoupled with SPR (Backmann et al., 2005), piezoelectric (Shenet al., 2005), electrochemical (Benhar et al., 2001) and colorimetric(Liu et al., 2009) signal transducers.

Although it has been demonstrated that site-directed Fab′fragments show higher sensitivity (Bonroy et al., 2006), maindisadvantages of chemically prepared Fab′ fragments are related tothe ambiguous mechanism of chemical reduction resulting in thepotential loss of chemical activity of the Fab′ (Lu et al., 1995).DeSilva and Wilson reported that after reduction with 2-MEA anaverage of 3.2 thiol groups were present per Fab′ fragmentproduced from goat antibodies (the theoretical value is 1 as goatantibodies posses one inter-heavy disulfide bridge) and 4.7 thiolgroups for mouse antibodies (theoretical value is 3), which couldbe explained by reduction of intrachain disulfide bonds (Desilvaand Wilson, 1995). Meanwhile, although recombinant antibodyfragments can be used to deflect the problems in reduction,recombinant immunoglobulins exhibit non-consistent affinitiesand stabilities; e.g., scFv fragments have been reported to be lessstable than Fab′ fragments (Quintero-Hernandez et al., 2007).

2.3. Site-directed immobilization via oxidized oligosaccharidemoieties

Immunoglobulins are glycoproteins, having a branched oligosac-charide N-linked to asparagine 297 present in the Fc region. Theoligosaccharide moiety is in a considerate distance from the antigenbinding sites and can be used for immobilization without affectingantibody reactivity with antigen (Fig. 3C) (Sutton and Phillips, 1983).IgG class antibodies normally have approximately two oligosaccharidechains per molecule in the CH2 domain. The chains mainly consist ofsialic acid, mannose, fucose, N-acetylglucoseamine and galactose(Mizuochi et al., 1982). Site-directed antibody immobilization viaoxidized oligosaccharide moieties was firstly applied to preparationof affinity chromatography immunosorbents. In order to achieveantibody immobilization on the solid surface the carbohydrate moietywas oxidized by an excess of sodium m-periodate (O'Shannessy andQuarles, 1985; Hoffman and O'Shannessy, 1988). Reactive aldehydegroups can be generated from oxidation of sialic acid exocyclic diolsor endocyclic diols within the monosaccharides (Rothfus and Smith,1963). Oxidized antibodies can be immobilized on various supportsand subsequently exhibit different antigen binding characteristics.The main functional groups suitable for this type of immobilizationare amines, hydrazines, hydrazides and semicarbazides (Quash et al.,1978; Melnyk et al., 2002; Batalla et al., 2008). Oxidized antibodycoupling to amino groups is achieved at a slightly basic pH, which

enables deprotonation of the amino groups, via the formation of aSchiff base. However, this method might end up in inter- andintramolecular coupling within the amino groups present in theantibody molecules. In addition, borohydride, which is used forstabilization in this reaction, might affect the structure of the anti-body, more precisely, reduce its disulfide bonds, especially if theantibody is monoclonal (Peng et al., 1987). For these reasons,hydrazide terminated supports are used more often as the couplingreaction of hydrazone formation requires a slightly acidic pH (pH 5.0–5.5), so the probability of cross-linking is reduced and the stable bonddoes not require additional treatment with a reducing agent. Affinitychromatography sorbent preparation experiments have shown thateven though a higher amount of antibodies are bound to aminoterminated supports, the antigen binding response is very low incomparison to hydrazide bound antibodies possibly because ofinterlinked antibody molecules (Kugel et al., 1992).

Although antibody oxidation with sodium m-periodate is themost common technique, it has significant disadvantages. Firstly, itis important to control the extent of antibody oxidation. Differentreactions conditions, such as temperature, pH and periodateconcentration, strongly affect the reaction rate and can producea different number of oxidized sites (Wolfe and Hage, 1995).A smaller amount of reactive aldehyde groups (e.g., a couple) areneeded for antibody immobilization in comparison with antibodylabeling where as much as possible reactive aldehyde groups arepreferred. It is also crucial not to destroy the oligosaccharidemoiety completely if the reaction conditions are too harsh(Hudson and Barker, 1967). Excessive oxidation might not onlyaffect the carbohydrate moiety but rapidly and extensively oxidizeamino acids too, particularly cysteine, cystine, methionine, trypto-phan, tyrosine, and histidine (Clamp and Hough, 1965).

Although not based on oxidation, another interesting techniquethat targets the oligosaccharide moiety is boronic acid, which can beused to form reversible cyclic covalent complexes with adjacent 1,2 or1,3 diols (Springsteen and Wang, 2002). It was reported that anti-biotin antibodies immobilized on a preformed 3-aminophenylboronicacid layer exhibited appropriate limit of detection (LOD) in anelectrochemical signal transducer and liposome biolabel enhancedimmunosensor model (Ho et al., 2010). The advantages of thistechnique are simplicity and no need of oxidation, which mightsubsequently damage the antibodies. This technique has not beenextensively investigated, so it will be interesting to observe its furtherdevelopment.

Matson and Little (1988) reported a case, in which polyclonalantibodies immobilized via oxidized oligosaccharide moiety yieldeda considerate enhancement in antigen binding. However, certaintypes of monoclonal antibodies immobilized following the sameprocedure exhibited no such improvement. The authors offeredseveral possible explanations. One of them was that the antigenbinding site and oligosaccharide moiety of monoclonal antibodiescould have been damaged during affinity purification and the otherthat oxidation conditions might have been too harsh. A study byAbraham et al. (1991) revealed that the effect of periodate oxidationon immunoreactivity depended on the type of monoclonal antibody:while some antibodies could be deactivated even at mild oxidationconditions, the others were quite robust. For example, in the study ofFleminger et al. (1990) the molar ratio of antigen to one mole ofimmobilized oxidized monoclonal antibodies was reported to be 1.8,which is close to the theoretical value. An advancement of oligosac-charide moiety oxidation, although more expensive and complex, isselective oxidation by neuraminidase-galactosidase. Neuraminidaseis used for removal of sialic acid and the exposed galactose residuesare oxidized by galactose oxidase. Solomon et al. (1990) showed thatenzymatically oxidized monoclonal antibodies fully retain theirimmunoreactivity binding 1.9 M of antigen per 1 M of antibody incomparison to 0.8 M of the chemically oxidized system.

Table 2Comparison of performance of site-directed and random immobilization techniques in different antibody-antigen systems.

Immobilization methods chosen as the most efficient by the authors are in bold. Four types of antibody immobilization techniques are marked in different colors: yellow—viaFc binding proteins, green—via antibody fragments, blue—via an oxidized oligosaccharide moiety, white—random immobilization. Ab—antibody, Ag—antigen. ND—notdetermined.

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Despite being an apparently convenient and effective immobi-lization technique widely used in chromatography, there are fewpublications reporting immunosensing applications of antibodyimmobilization via an oxidized carbohydrate moiety. A flowthrough optical detection cabaryl analysis model system has beenreported (Gonzalez-Martinez et al., 1997). Another immunosen-sing model system for human chorionic gonadotropin (hCG)detection was developed in the lateral flow assay where anti-hCG were immobilized via an oligosaccharide moiety on magneticnanoparticles to serve as secondary antibodies in a sandwich assayshowing advantages over the randomly oriented antibody immo-bilization methods (Puertas et al., 2010). Coupling antibodies tonanostructures via the polysaccharide moieties has also beenapplied in electrochemical immunosensing for the detection ofhuman IgG (hIgG). Anti-hIgG were immobilized on amino termi-nated multiwall carbon nanotubes (Lotfabadi et al., 2011). Recently

this immobilization method has been proposed for scFv frag-ments in a competitive enzyme immunoassay, so an logical nextstep would be its application for immunosensor platforms(Hu et al., 2012).

3. Comparison of different antibody immobilization strategies

Since all site-directed antibody immobilization methodsdescribed above seem promising, it is interesting to investigatewhat is the relation between the randomly oriented antibodiesand immobilized in a site-directed manner and to compare theresults obtained by different site-directed immobilization strate-gies. However, not only different approaches to evaluating antigenbinding of the immobilized ligands are presented in publicationsreviewed in this chapter, e.g., sensitivity, kinetic parameters or

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direct comparison of signal transducer responses, but even theexpressions of analyte concentration units differ. Subsequentlywhile trying to look at a broader picture we approximatelyappraised the analyte concentrations in molar units where it waspossible hoping for a less complicated comparison between thereviewed studies. The more compact comparison of works dis-cussed in this chapter is presented in Table 2.

Quinn et al. (1999) compared monoclonal antibody againstglutathione-S-transferase (anti-GST) site-directed immobilizationvia (i) protein A, (ii) protein G and (iii) goat polyclonal anti-Fccoated CM-dextran surfaces by employing the SPR technique.When exposed to 100 nM concentration of antigen, all threetechniques compared relatively well in the association phase butdeviated in the dissociation phase. The antigen response of captureligands immobilized via protein A could be best described byLangmuir association isotherm, while antibodies immobilized viaprotein G and anti-Fc coated surfaces showed higher residualsfrom the ideal behavior. Authors explained this by differentcapture protein–antibody binding characteristics: according tothem protein A binds the Fc region of antibody and protein Gbinds between the Fab and Fc regions possibly causing sterichindrance for antigen access. Additionally, dissociation could beaffected by interference caused by steric hindrance due to matrixeffects and mass transport limitations. As a result, antibodyimmobilization via protein A was determined to be best suitedfor kinetic analysis. In contrast, Patel and Andrien (2010) havereported that the response to antigen monoclonal antibodyimmobilized via anti-Fc coated surface could be described byLangmuir kinetics with residuals indicating a good fit. Apparently,for kinetic analysis each antibody–antigen pair has to be testedempirically to evaluate the most suitable site-directed immobili-zation method for kinetic analysis. It is important to note that bothstudies were performed on a three dimensional dextran matrix incomparisons to planar surfaces discussed below.

Nakanishi et al. (1996) employed QCM to compare anti-humanserum albumin antibody (anti-HSA) immobilization based onethylenediamine plasma polymerized film matrix. Three immobi-lization strategies were tested: via (i) amine coupling (randomimmobilization); (ii) protein A with cross-linking and (iii) sulfhy-dryl groups of the reduced Fab′ fragments (site-directed immobi-lization). While fewer antibodies were immobilized via protein Athan random immobilization, the QCM frequency change after theantibody-antigen interaction of the site-directed antibodiesexceeded the frequency change of the randomly oriented ones.However, the immobilized Fab′ fragments exhibited the lowestfrequency response after antigen interaction despite the largestamount of immobilized molecules. The authors explained this byeither the possibility that the amount of immobilized antibodiesplayed a more important role than the orientation or Fab′fragments had lower activity than the intact antibodies. Covalentlyimmobilized randomly oriented system and cross-linked protein Asystem resulted in good regenerability.

Pulido-Tofino et al. (2000) compared antibody immobilizationvia (i) protein A with cross-linking, (ii) without cross-linking and(iii) an oxidized oligosaccharide moiety on a controlled pore glassof a flow-through fluoroimmunosensor. By using antibodiesagainst isoproturon it has been shown that in a competitiveimmunoassay format the best performance was exhibited by thesystem based on antibody immobilization via protein Awith cross-linking. This system allowed reaching LOD of 3 mg L�1 (14.5 nM), adynamic range 3–100 mg L�1 (14.5–485 nM) and more than 1000regeneration cycles without losing sensitivity. The same LOD wasexhibited by the immunosensing surface based on antibodyimmobilization via an oxidized oligosaccharide moiety with adynamic range of 3–200 mg L�1 (14.5–970 nM), however, for thistechnique an approximately 10-fold decrease in regeneration

cycles was observed. Antibodies immobilized via protein A with-out cross-linking showed the highest LOD of 4.5 mg L�1 (21.8 nM),a dynamic range of 4.5–150 mg L�1 (21.8–727 nM) and could beonly used for a single assay.

Tedeschi et al. (2003) evaluated different antibody immobiliza-tion methods in the total internal reflection fluorescence (TIRF)immunosensor developed on quartz and fiber optic surfaces. Theycompared systems based on anti-HSA immobilized on SiO2 sur-faces: (i) random immobilization of antibodies on dextran coating(3D support), (ii) site-directed immobilization via the oxidizedoligosaccharide moiety, (iii) sulfhydryl mediated Fab′ immobiliza-tion with and (iv) without a spacer molecule. The highest densityof active antigen binding sites (Γ) on the sensor surface wasregistered after random antibody immobilization on dextran andwas equal to 0.5 pmol/cm2, followed by Fab′ fragments immobi-lized without a spacer (Γ¼0.45 pmol/cm2). These two systemswere chosen for further studies and optimization. Subsequentlythey gave satisfactory responses to changes in analyte concentra-tions of the order of 10�8 M. The remaining techniques exhibitedslightly lesser densities of active sites on the sensor surface.

In a SPR study by Peluso et al. (2003) immobilization of intactmonoclonal antibodies against interleukin 8 (anti-IL 8), antibodiesagainst interleukin 2 (anti-IL 2) and their Fab′ fragments wasinvestigated by comparing random and site-directed immobiliza-tion techniques on streptavidin coated monolayer surfaces. Overall,four immobilization strategies were investigated. In a randomimmobilization approach (i) intact antibodies and (ii) their Fab′fragments were biotinylated via amino groups and immobilized onstreptavidin coated supports. To achieve site-directed intact anti-body immobilization, (iii) the oligosaccharide moiety was oxidizedand the resulting aldehyde biotinylated. Site-directed immobiliza-tion of (iv) Fab′ fragments was carried out by biotinylating freesulfhydryl groups. Three different monoclonal antibody types wereimmobilized via the aforementioned four methods and the antigenbinding activities were compared by dividing the density of boundanalyte by the binding site density. The highest antigen bindingactivity with at least 70% of binding sites active was achieved bysite-directed Fab′ fragments independent of the monoclonal anti-body type used. However, further results deviated for differentmonoclonal antibody types. Analyte binding and specific activityvaried highly for different systems, oriented Fab′ fragment immo-bilization being the most reproducible capture agent immobiliza-tion technique. Despite the variations, oriented immobilizationtechniques consistently outperformed the random techniques.

In an often-cited SPR study by Bonroy et al. (2006) four sensingsurfaces modified with a self-assembled monolayer were com-pared: random immobilization of (i) polyclonal intact antibodiesagainst human IgG (anti-human IgG), (ii) their F(ab')2 and (iii) Fab′fragments and (iv) site-directed immobilization of Fab′ fragmentsvia their sulfhydryl groups. Despite the fact that in the testedconcentration range of 100 ng mL�1 to 100 mg mL�1 (0.667–667 nM) the highest absolute antigen binding responses wereshown by site-directed Fab′ fragments (more than two-foldincrease in signal in comparison to randomly oriented system),the authors also evaluated the binding efficiency for 100 mg mL�1

(667 nM) of analyte, i. e., the percent of immobilized antibodies(with their specific number of antigen binding sites), which areable to bind the antigen. The calculated value for site-directed Fab′fragments was equal to 16% in comparison to 21% of the randomlyoriented fragments. This could be explained by possible sterichindrance due to close to monolayer coverage of the site-directedfragments. The remaining surface modification techniques ofintact antibodies and their F(ab′)2 fragments exhibited smallerantigen binding efficiencies of 9% and 7% respectively. The authorsalso tested monoclonal antibodies against human holo-transferrinand the behavior of the system was completely different.

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Monoclonal F(ab′)2 reduction to Fab′ resulted in cleavage ofdisulfide bonds between the heavy and light chain independentlyof the reduction method used, so no comparisons between therandomly oriented and site-directed systems could be made.

Tsai and Pai (2009) proposed an SPR immunosensor model on aSAM modified gold surface. In the immunosensor development(i) intact randomly immobilized antibodies against a staphylococcalenterotoxin B (anti-SEB), and (ii) their F(ab′)2 and (iii) Fab′ fragmentswere compared with (iv) Fab′ fragments immobilized in a site-directed manner. In randomly oriented systems the best antigenbinding response was exhibited by Fab′ fragments, followed by intactantibodies and F(ab′)2 fragments. When comparing random and site-directed immobilization of Fab′ fragments, site-directed systemshowed a 26% increase in the absolute antigen-binding response.The antigen binding efficiency of Fab′ fragments was 90% for the site-directed and 60% for the random immobilization modes. Site directedFab′ immobilization was applied in SPR immunosensor design. Astandard curve with a linear relationship between 0.01–1 mg mL�1

(0.35–35 nM) of SEB was obtained in buffer and 0.1–1 mg mL�1 (3.5–35 nM) in spiked milk samples. In comparison, a standard curveusing randomly oriented anti-SEB antibodies and different concen-trations of SEB also exhibited linear relationship in buffer in the sameconcentration range; however, the curve had a smaller slope thanthat of site-directed Fab′ fragments correlating to a smaller sensitivityof the randomly oriented system.

Kausaite-Minkstimiene et al. (2010) performed a comparativeSPR study of monoclonal antibody against human growth hor-mone (anti-hGH) immobilization techniques. They compared ran-domly immobilized intact antibodies on the (i) self-assembledmonolayer and (ii) carboxymethyldextran coatings, and site-directed immobilized antibodies (iii) via protein G or (iv) half-antibody fragments. The most intensive SPR response followingantigen binding was registered by the antibodies immobilized viaprotein G and was equal to 184.75 m1. Half-antibody fragmentsimmobilization yielded approx. 1.3 times lower signal, while bothrandom immobilization techniques resulted in approx. 10 timeslower response. However, when comparing the binding efficiency(the percentage of immobilized antibody molecules capable ofbinding antigen) of antibodies immobilized via protein G and theirfragments using the same antigen concentration, it can be con-cluded that both immobilization techniques yield very similarresults.

Patel and Andrien (2010) published an SPR study comparingthe kinetic behavior of humanized monoclonal antibodies andscFvs immobilized on a carboxymethylated dextran (CM5) goldsurface via different techniques. Despite the fact that this studywas aimed for evaluating different antibody immobilization tech-niques for potential drug design applications and performed on athree dimensional support, it undoubtedly provides valuableinsights for immunosensor design. The best kinetic parameters, i.e., the largest ka and the smallest KD, were exhibited by the systembased on monoclonal antibodies immobilized (i) via Fc bindingantibodies, followed by (ii) randomly immobilized monoclonalantibodies and lastly (iii) the scFvs.

SPR and dual polarization interferometry (DPI) served in aninvestigation by Song et al. (2011). In a sandwich immunoassay format(i) antibodies against prostate specific antigen (anti-PSA) were immo-bilized randomly on sulfo-N-succinimidyl 4-maleimidobutyrate (sulfo-GMBS) modified surface of a DPI chip and on streptavidin modifiedSPR gold sensor surfaces in comparison to site-directed immobilization(ii) via protein G with and (iii) without cross-linking. For enhancementof analytical signal secondary antibodies were applied. Protein Gmediated antibody immobilization without cross-linking yielded thehighest antigen binding response allowing to detect 10 pg mL�1

(0.33 pM) antigen by both techniques. The absolute response of thissystemwas slightly lower after the cross-linking but antigen could still

be detected at the same concentration. In contrast, randomly modifiedsystems showed no signal increase up to 10 ngmL�1 of antigen(4330 pM).

In an electrochemical measurements based study Ho et al.(2010) compared the responses of (i) half-antibody fragmentsagainst biotin (anti-biotin) immobilized via thiolate bonds on goldnanoparticle layer pre-formed on screen printed graphite electro-des and (ii) intact antibodies immobilized via boronic acid–oligosaccharide interactions using liposomal biolabels for signalamplification. Both methods allowed achieving impressive sensi-tivity. LOD of the half-antibody fragments based system was20.5 pg (6 mL of 1.4�10�8 M). Site-directed intact antibody basedsystem proved to be better by yielding LOD of 0.19 pg (5 mL of1.6�10�10 M).

Quang Huy et al. (2012) electrochemically compared fourimmobilization techniques of serum antibodies against the Japa-nese encephalitis virus (anti-JEV) on silanized electrodes using asandwich assay approach. Three random immobilization techni-ques, (i) direct adsorption, (ii) covalent binding with glutaralde-hyde, and (iii) anti-IgG antibody layer mediated immobilization,were compared with (iv) a protein A mediated immobilization,which gave the highest signal for detection of antigen in a linearrange of 25 ng mL�1 to 1 mg mL�1 and lowest LOD of 10 ng mL�1.

Although generally it can be summed up that oriented antibodyimmobilization outweighs the random immobilization, from theliterature overview it seems that it is not possible to exclude onetechnique as better than others. Each antibody–antigen pairrequires testing immobilization methods and tailoring the optimalassay conditions. In addition, although in some cases more anti-bodies on the sensing surface equals better antigen response, theresults in this area have been ambiguous. It is not clear at whatamount of the immobilized ligand is sufficient to bind mostantigens without steric hindrance that negatively affects theimmunosensor sensitivity.

4. Conclusions

Despite constantly appearing new trends in oriented antibodyimmobilization, most published immunosensor applications arestill based on the more conventional techniques, i.e., the Fcbinding proteins, antibody fragments and immobilization via theoxidized oligosaccharide moiety in some cases additionallyimproved by certain modifications (e.g., biotin and avidin/strepta-vidin or signal molecule tagging). From the overview of literatureit seems that neither of these methods is significantly advanta-geous than others. Although all of them show improvements overthe random antibody immobilization approaches, the method ofchoice for a particular system should be determined and tailoredempirically according to the goals and recourses of the researcher.While Fc binding proteins are very popular because of the affinitybased nature of bonds with the antibody and site-directed surfacesmodified in this way usually exhibit high sensitivity to analyte anddo not require the modification of antibodies, it is not applicable tocertain antibody types and there is no possibility of regenerationof antibody layer (all affinity based interactions are disruptedincluding the ones between the Fc binding proteins and anti-bodies), which is essential in the development of a multiple useimmunosensor. Although cross-linking of antibodies to Fc bindingproteins partially solves the regeneration problem, as a rule it isalso the cause of the loss of activity of the immobilized antibodies.On the other hand, some studies imply that antibody fragmentsare more efficient as capture ligands in immunosensing; however,it is crucial to determine the precise reduction conditions as it isvery easy to reduce the disulfide bonds holding the heavy andlight chains together this way resulting in a denatured antibody

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with a reduced antigen binding efficiency. Single-chain and otherforms of recombinant antibodies offer a solution to this problem,yet often show unsatisfactory stability. Immobilization via anoxidized oligosaccharide moiety offers an attractive possibility toimmobilize antibodies at a region far from the antigen bindingcenters, however, this technique can cause problems by over-oxidising and this way denaturing an antibody and also might notalways yield good results with monoclonal antibodies becausethey sometimes have their oligosaccharide moiety damaged dur-ing the purification procedure.

All in all, although antibody orientation usually improves theefficacy of the immunosensor, it is closely related and evendependent on the other aspects of the system, such as the natureof the used antibody–antigen system, the influence of sterichindrance and non-specific interactions, the choice of regenera-tion conditions and the assay format, not forgetting the stabilityand, last but not the least, the price. While publications exploitingother capture ligands, such as bacteriophages (Balasubramanianet al., 2007; Tawil et al., 2012), aptamers or other nucleic acidbased ligands (Li et al., 2012; Wu et al., 2012b) and syntheticreceptors, e.g., molecularly imprinted polymers (Ramanavicieneand Ramanavicius, 2004; Chen et al., 2012; Prasad et al., 2013;Reddy et al., 2013) or recombinant peptides (Qi et al., 2012; Wuet al., 2012a) report very good sensor characteristics and it is verylikely that the use of these ligands will expand in the future and atleast partially replace antibodies in biosensor applications, todayantibodies still remain the most popular recognition molecules.

Acknowledgements

This research is funded by a grant (No. MIP-059/2012) from theResearch Council of Lithuania.

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