Biochimica et Biophysica Acta 1844 (2014) 1960–1969

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Review

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays☆ Yoichi Kumada Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

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Article history: Received 5 April 2014 Received in revised form 5 July 2014 Accepted 11 July 2014 Available online 8 August 2014 Keywords: Immobilization Material-binding peptide Plastics ELISA

a b s t r a c t The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Polyclonal and monoclonal antibodies (whole Abs) produced from animals or hybridoma cells have been utilized as a ligand protein that can be immobilized on the surface of solid supports and used in a variety of research and industrial pursuits such as clinical diagnostics, downstream processes in bioproduction, and studies in molecular biology. Currently, when using methods based on enzyme immunoassay (EIA), whole Abs fill important roles in the detection of antigens such as biomarkers, pathogenic microbes and viruses. In 1969, Engval first developed the “enzyme-linked immunosorbent assay (ELISA)” [1,2], and a number of antigens (or antibodies) have been detected using this method. ELISA utilizes the specific interaction between antigens and antibodies, one of which is immobilized on the surface of a solid support. The sandwich ELISA is a variation wherein antibody-immobilized

☆ This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.bbapap.2014.07.007 1570-9639/© 2014 Elsevier B.V. All rights reserved.

polystyrene (PS) is used for the entrapment of an antigen, and it continues to be the most popular type because target antigen molecules can be detected with high degrees of sensitivity, signal intensity and selectivity [3]. As shown schematically in Fig. 1, the first step in sandwich ELISA is the immobilization of monoclonal or polyclonal antibodies onto the surface of a solid support, such as PS. Immobilization methods for antibodies have been studied for decades, and portions of them have been introduced in several reviews [4–9]. In recent years, technologies for recombinant protein have been developed, and as a consequence, a variety of functional proteins with mutations and/or the genetic fusion of other functional peptides have been produced. In particular, valuable antibody fragments such as scFvs and Fabs with a variety of affinity peptide tags for purification and detection have been designed and produced using recombinant technologies. These antibody fragments have a much smaller molecular size than conventional whole Abs and are also useful as ligands. This paper reviews conventional and alternative methods for the immobilization of antibodies and recombinant antibody fragments onto the surfaces of solid supports, particularly PS, which has been utilized as a support material for the immobilization of antibodies.

Y. Kumada / Biochimica et Biophysica Acta 1844 (2014) 1960–1969

Washing

Immobilization of ligand antibody

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Washing

Capturing of antigen

Blocking

Washing

Washing

Attachment of secondary antibody

Detection of signals

Fig. 1. Schematic image of sandwich ELISA.

2. Conventional antibody immobilization methods Polyclonal and monoclonal antibodies (whole Abs) have been immobilized using the following basic methods: 1) Passive adsorption to plastics; 2) Covalent coupling via activated functional groups; and, 3) Specific interactions between biomolecules. Passive adsorption is the simplest method that is used to immobilize proteins, including whole Abs, onto the surfaces of plastic supports such as PS microtiter plates, latex beads, and porous hydrophobic membranes such as nitrocellulose. Bulter and co-workers have studied the immobilization of immunoreagents such as antibodies and antigens in their adsorptive states [7–9]. Adsorption efficiency of whole Abs is considerably dependent on the surface characteristics of materials as well as on antibody species. In fact, even through the use of the covalent coupling method, 80% of proteins immobilized were initially bound by passive adsorption. In particular, for the use in sandwich ELISA and immuno-chromatography, passive adsorption remains a popular option, although antibodies are immobilized in an uncontrolled orientation and are partially denatured at the attachment points. Butler et al. have reported that less than 3.0% of the binding sites of monoclonal anti-fluorescein antibodies, and approximately 5–10% of polyclonal antibodies in adsorptive states, were capable of capturing antigens [9]. Wang et al. reported the analyses of adsorption states of anti-hCG monoclonal antibody immobilized on the surface of hydrophilic silica. They investigated direct analysis of the antibody by spectroscopic ellipsometry (SE) and atomic force microscopy (AFM). According to the results of SE, adsorption rate was decreased with pH away from isoelectric point of antibody due to repulsion between not only antibody molecules, but also antibody and silica surface. Consequently, it was revealed that pH value for adsorption strongly affected density, remaining activity, and orientation of whole Ab. Furthermore, according to the results from AFM, “flat-on” orientation of adsorbed antibody was observed when the surface density was lower. On the other hand, with increasing surface density of whole Ab, antigen-binding coverage was decreased due to steric hindrance caused by aggregates which was formed on the surface of hydrophilic silica [10]. There are a number of commercially available microtiter plates for the immobilization of proteins, particularly whole Abs. Kenny and co-

workers reported the adsorption of human IgG onto 11 types of commercially available microtiter plates provided from 4 different suppliers [11]. Now, PS microtiter plates with a variety of surface modifications are commercially available for the immobilization of proteins with different characteristics. Thermo Fischer Scientific supplies 4 types of PS microplates with different surface hydrophobicity: Polysorp, Medisorp, Maxisorp and Multisorp. With Maxisorp the surface of a PS plate is slightly oxidized, and this state has been exclusively utilized as a solid support for the immobilization of whole Abs. Interactions between antibodies and PS or other plastic supports are complicated and an insufficient lack of understanding persists concerning the mechanisms of the adsorption process and the adsorptive states of antibodies and other proteins on the surfaces of solid supports. For a greater improvement in the sensitivity of a sandwich-type immunoassay, control of the density and the orientation of antigen-binding domains is a necessity. The covalent coupling of whole Abs to solid supports offers advantages that can prevent dissociation from the solid phase and enhance the density of antibodies [12–15], while an over-reaction as well as an inefficient reaction can result in a decrease in the apparent active antibody molecules in immobilized states. Several companies now supply a variety of solid phases containing reactive groups, such as NH2 and COOH, but these are not often utilized for the following reasons. 1) The density of an immobilized antibody is strongly dependent on the functional group introduced on the surface of PS. 2) The passive adsorption of antibodies can be substantial during a coupling reaction; thus, an unfavorable denaturation of antibodies can happen. An amine coupling method is often adopted whereby the primary amine groups of whole Abs are covalently coupled with carboxyl groups introduced on the surface of a solid support in the presence of N-hydroxysuccinimide/1-ethyl-3-(3dimethylaminopropyl) carbodiimide (NHS/EDC) reagents. Residual functional groups that do not react with an antibody must be blocked with 2-iminoethanol because such residual functional groups can cause a non-specific adsorption of serum proteins. Specific bio-molecular interactions such as streptavidin–biotin, IgG– protein A/G, and IgG–Anti-IgG antibody are sometimes useful for the indirect immobilization of whole Abs. In particular, biotin-conjugated antibodies have often been utilized for indirect immobilization onto the surface of a streptavidin-coated support in industrial diagnosis systems, in order to avoid the inactivation caused by the direct attachment of

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Y. Kumada / Biochimica et Biophysica Acta 1844 (2014) 1960–1969

ligand whole Abs with partial denaturation. The biotinylation of antibodies by amine coupling methods sometimes decreases their antigen-binding activities from overreaction and needs the additional preparation costs. Protein A and Protein G, both of which possess a specific binding affinity to a constant region (Fc) of IgG-type antibodies, have also been used as a ligand to orient the immobilization of an antibody [16,17], although the dissociation constant of an IgG against such a substrate is on the order of subnano molar [18]; thus, the dissociation might not be negligible during an immunodiagnostics operation. Jung et al. successfully demonstrated oriented and covalent coupling of whole Ab utilizing specific binding affinity of antibody-binding protein, protein G and photo-reactive cross-linker. They prepared photo-reactive antibody binding proteins that recombinant protein G was sitespecifically conjugated with photo-reactive cross-linker, benzophenone, and also site-specifically biotinylated at its N-terminal region. It could be interacted with Fc region of whole Ab and covalently coupled by irradiation of UV light at 365 nm. Consequently, biotin molecule was indirectly, but covalently introduced at the Fc region of whole Ab. By the use of this photo-reactive antibody binding protein, they successfully demonstrated site-specific and covalent immobilization of whole Ab onto the surfaces of Au and glass flat plates as well as magnetic particles [19]. 3. Immobilization of antibody fragments through affinity peptides 3.1. Preparation of recombinant antibody fragments as a capture ligand The development of phage display systems as well as technologies for recombinant protein production have made it possible to isolate valuable recombinant antibody fragments such as scFv, Fab and VHH [20–29]. Recombinant antibody fragments are expected to be a capture ligand alternative to conventional whole Abs in immunodiagnostics and affinity separation, due to the following reasons: 1) the recombinant antibody fragments can be economically produced by recombinant microorganisms such as Escherichia coli and yeast due to a structure that is simpler than that of conventional whole Abs [30–39]; 2) smaller molecular sizes of recombinant antibody fragments result in an increase in immobilization density, so the binding capacity of antibody-immobilized solid supports will also be increased [40–44]; and, 3) the genetic fusion of various affinity peptide sequences to their N or C-termini will result in the achievement of single-step purification as well as site-specific immobilization onto the solid supports via the introduction of affinity peptide tags [40–44]. Yoshimoto et al. reported the monitoring of adsorption-induced inactivation of antibody fragments, Fab′ immobilized on a gold surface which is used as a sensor chip surface of surface plasmon resonance (SPR) sensor. They directly monitored adsorption behavior of Fab by SPR sensor and atomic force microscopy (AFM). Antigenbinding activity of Fab′, which was directly immobilized on the Au surface through S–Au linkage, was gradually decreased and completely disappeared at 60 min. Furthermore, according to the results of AFM, they clarified that height of immobilized Fab′ was time-dependently decreased due to unfavorable conformational change of Fab′ structure. On the other hand, 70% of antigen-binding activity was successfully retained, when Fab′ and shorter polyethylene glycol (PEG) was coimmobilized. Their findings clearly indicated the density of antibody adsorbed and environment surrounding antibody molecules are considerably related to inactivation of the immobilized antibody [45]. 3.2. Affinity peptide tags for the purification and immobilization of recombinant antibody fragments Hedhammar et al. and Nakanishi et al. have reviewed affinity peptide tags that have been utilized for the purification and detection of recombinant proteins, which include recombinant antibody fragments [46,47]. The hexa-histidine tag (6xHis-tag, HHHHHH) [48–52] is well known to have an affinity for nickel-nitrilotriacetic acid (Ni2+–NTA)immobilized solid supports. Since its binding affinity can be maintained

even in the presence of a high concentration of a denaturant such as 8 M of urea or 6 M of guanidine–HCl, it is useful for the recovery of recombinant proteins from an inclusion body [53,54]. Hexa–His-tagged recombinant proteins were indirectly and site-specifically immobilized through Ni2+-immobilized or anti-6xHis antibody-immobilized solid supports. Lo et al. reported single-walled carbon nanotube field effect transistors (SWNT-FET) on which Ni was decorated in order to sitespecific immobilization of 6xHis-tagged anti-CEA scFv. The oriented immobilization through the 6xHis-tag could increase signal for antigenbinding, while randomly oriented scFv did not import an enough amount of conductance for detection [55]. Anti-6xHis antibody showed a much higher selectivity for capturing 6xHis-tagged proteins than Ni2+-NTA, while the density of anti-6xHis antibody immobilized was generally much lower than that of Ni2+-NTA. C-myc tag (EQKLISEEDL) is an epitope peptide of monoclonal antibody (9E10) [56], and it is useful for the detection of recombinant proteins by anti-c-myc monoclonal antibody with high specificity. Strep tag (SAWRHPQEGG [57]), Strep II tag (WSHPQFEK [57]), and Strep tag II (SNWSHPQFEK [58]) have been isolated as peptides that strongly bind to streptavidin, which is a highly stable tetramer and can interact with biotin as well as biotin-conjugated molecules with a dissociation constant (Ka) of 10− 14 M [59]. Streptagged antibody fragments have been successfully produced and recovered with high purity by streptavidin-coupled resin [60,61]. These streptavidin-binding peptides also are useful for site-directed immobilization onto streptavidin-coated plastic surfaces, while improving the binding affinity for streptavidin and/or streptactin will be the next object for long-term retention of antibody fragments. The productivity of recombinant antibody fragments as well as the solubility and stability of target antibody fragments is significantly changed by the host strain, and also by the characteristics of the affinity peptide tags that are introduced, because the genetic fusion of affinity peptide tags may significantly change the target-protein characteristics such as solubility, stability and even folding efficiency [25,62]. Therefore, the optimization of the production system for each tagged antibody fragment is important for the efficient and economical preparation of a ligand antibody. The periplasmic secretion of tagged recombinant antibody fragments in E. coli has been investigated, while production levels, types of peptide tags, and cultivation strategies have differed considerably between scFv species [25,30–32,37,62]. Pichia pastoris is another host strain candidate for large-scale production by fed-batch cultivation [33,63–69]. However, target recombinant antibody fragments were often produced with unfavorable glycosylation [70]. Therefore, antigen-binding activities and the selectivity of target antibody fragments might be drastically changed. Genetic fusion of the affinity peptide tags described above, such as 6xHis tag and Strep tag, is the realization of the indirect immobilization of recombinant antibody fragments via ligand molecules introduced on a solid surface. However, site-directed immobilization of the antibody fragments sometimes did not significantly improve the performance of immune-solid supports, probably due to one of the several reasons described below. First, when the antibody fragments were indirectly immobilized by specific interaction between the affinity peptide tags and the pre-immobilized partner ligands, the densities of the antibody fragments were strongly related to that of the ligand molecules that were introduced and to the strength of the interaction between the peptide-tags and the ligand molecules. If the binding affinity of the interaction is not enough for retention of the antibody fragments, with time, they will be dissociated from the solid supports. Furthermore, in indirect immobilization, the maximum density of the antibody fragments is less than that of a ligand molecule pre-immobilized on the surface of a solid support. Therefore, the performance of immuno-sorbents will be strongly changed not only by the binding affinity and specificity of the recombinant antibody fragment themselves, but also by other factors that include the ligand density and/or the dissociation rate of the peptide tag towards the ligand molecule. Furthermore, even in the indirect immobilization of an antibody, the non-specific adsorption of

Y. Kumada / Biochimica et Biophysica Acta 1844 (2014) 1960–1969

antibody fragments to the solid supports must be considered. Compared with the whole antibody, the recombinant antibody fragments have small portions that are not related to antigen binding. That fact indicates that the non-specific attachment of an antigen-binding domain will induce unfavorable conformational changes; thus, the binding activity and capacity of an immobilized antibody must be drastically decreased. Here, we introduce alternative affinity peptides that directly recognize the surface structure of a solid material. 4. Immobilization of protein through material-binding peptides 4.1. Material-binding peptides Material-binding peptides with the ability to recognize, and associate strongly with the surface of a certain material have recently isolated. Oligo-peptides binding to inorganic substance such as Au, Pd, Pt, and ZnO are well-known in the research areas of bio-mineralization and crystallization, and their characteristics were reviewed by Sarikaya et al. [71]. Also, Shiba and co-workers successfully isolated binding peptides specific to TiO2, ZrO2 and carbon nanohorns [72–75]. A silicabinding domain (Si-tag) has been isolated from the sequence of E. coli ribosomal L1 protein. Si-tagged recombinant proteins such as GFP, luciferase and the ZZ domain of protein A have been successfully prepared, and site-directly immobilized with high biological activity [76–80]. Especially, Si-tagged protein A was useful for oriented immobilization of whole Ab on the surface of silicon oxide which will be an important material for fabrication of semiconductor. Peptides that bind with organic polymers also have been isolated. Serizawa et al. reported the successful isolation of affinity peptides for isotactic poly(methylmethacrylate) (iso-PMMA), syndiotactic PS (synPS), and poly-(L-lactide), all of which are from the peptide-displayed phage library [81–83]. Also, Feng et al. reported a dodecapeptide of PS-binders for the immobilization of an antigen in the diagnosis of the HIV virus [84]. The author also successfully isolated peptides binding to the surface of hydrophilic PS (phi-PS) that a bare PS plate was highly oxidized by O2 plasma irradiation [85–87]. We further identified peptide motifs binding to the surfaces of polycarbonate (PC), poly(methylmethacrylate) (PMMA), and silicon nitrite (SiN) from the amino acid sequences of E. coli proteins that had preferentially adsorbed onto the surfaces of these substrates [88,89]. Fig. 2 summarizes the method for the screening of material-binding peptides (especially for PC-tag, PMMA-tag and SiNtag) utilizing proteome analysis technologies. With this method, proteins that preferentially adsorbed onto the target substance were first isolated and identified from a protein mixture of E. coli cell lysate. The proteins that adsorbed onto the surfaces of solid supports were eluted with a buffer for isoelectronic focusing (IEF) that contained a high concentration of chaotropic agent, urea,

and zwitterionic surfactant, CHAPS. The isolated proteins were directly separated as protein spots on the acrylamide gel by two-dimensional electrophoresis (2DE), and they were identified by peptide mass fingerprinting (PMF) method. The genes of the identified proteins were cloned from E. coli genome (or chemically synthesized on the basis of amino acid sequences) and the material-binding proteins were prepared with high purity by recombinant protein technologies. The binding properties of the peptide components that were prepared by the digestion of material-binding proteins with endo-protease, such as trypsin, were analyzed by RP-HPLC. Peptide fragments that bound to the target surface, were isolated and MALDI-TOF MS/MS and/or Nterminal protein sequencing was used to identify the sequences. Synthesized peptides and fusion proteins were used to further investigate the binding characteristics of peptide candidates from the materialbinding proteins. The material-binding peptides described above have the potential to directly immobilize target proteins on the surface of a target material. The genetic fusion or chemical conjugation of material-binding peptides will attain direct and site-specific attachment of target proteins onto the surface of target materials with minimum inactivation of biological activity. Here, the characteristics of two types of material-binding peptides, PS-tag and PMMA-tag, both of which were developed by this research group, are introduced and their application to the immobilization of recombinant antibody fragments onto the surface of plastics is described.

4.2. Polystyrene-binding peptides (PS tags) Polystyrene-binding peptides (PS tags) have been identified by screening from the library of randomized peptides, which displayed the pili proteins of the E. coli library [85]. After the 10th round of biopanning using a hydrophilic PS dish as a target surface, 15 candidates with completely different amino acid sequences were identified. All of the candidates were introduced at the C-terminus of glutathione Stransferase (GST), and then the adsorption characteristics of the fusion proteins against PS latex beads as well as hydrophobic PS (pho-PS) and hydrophilic PS (phi-PS) plates were investigated. Consequently, it was revealed that 4 kinds of peptides, PS16 (SRVHRAVLNGVS), PS17 (RPPGVVRRYALG), PS19 (RAFIASRRIKRP), and PS23 (AGLRLKKAAIHR) shown in Table 1, possessed a strong binding affinity towards the PS surfaces. In particular, GST fused with PS19 peptide maintained a 10fold higher specific activity in the adsorptive state, compared with wild-type GST. The exposure of the N-terminus of GST in the adsorptive state was also analyzed using a polyclonal antibody that specifically recognizes the N-terminal region of GST. The N-terminal region of GSTPS19 in the adsorptive state was highly exposed towards the solution side, suggesting that the PS19 peptide located on the opposing site,

E. coli cell proteins

Adsorption Analysis and isolation by HPLC Sequence determination by MALDI TOF MS

Adsorption and recovery Isolation and identification by 2DE and PMF Cloning, expression and purification

Peptide candidates

Affinity protein candidates

Evaluation using peptidefused protein

Digestion with proteases (trypsin, chymotrypsin etc.) Peptide fragments

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Material-binding peptides Sequence optimization Fig. 2. Strategy for isolation of material-binding peptides.

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Y. Kumada / Biochimica et Biophysica Acta 1844 (2014) 1960–1969

4.4. Application of PS-tag-linked proteins

Table 1 Amino acid sequences of PS-tag derivatives. Name of PS-tag

Amino acid sequence

Reference

PS19 PS23 PS16 PS17 KPS19R10 PS19-1 PS19-2 PS19-3 PS19-4 PS19-5 PS19-6 PS19-6K PS19-6H PS19-6G PA19-6A PS19-6V PS19-6L

RAFIASRRIKRP AGLRLKKAAIHR SRVHRAVLNGVS RPPGVVRRYALG KRAFIASRRIRRP RAFIASRRIRRP RAFIAS RRIRRP RAIARRIRR (RAFIASRRIRRP)2 RIIIRRIRR KIIIKKIKK HIIIHHIHH RGGGRRGRR RAAARRARR RVVVRRVRR RLLLRRLRR

[81] [81] [81] [81] [89] [82] [82] [82] [82] [82] [82] [83] [83] [83] [83] [83] [83]

the C-terminus of GST, was site-specifically attached; thus, the orientation of GST-PS19 was controlled to a greater extent [85]. These results indicated that the site-specific attachment of a material-binding peptide improves the stability as well as the orientation of a target protein in the adsorptive state.

4.3. Recognition of PS-tag to the surface of a phi-PS plate The adsorption characteristics of GSTs fused with a PS19 peptide and its derivatives are shown in Table 1 and were prepared in order to clarify the mechanisms of the interaction between a PS-tag and a phi-PS plate [86,87]. Among the derivatives prepared, the genetic fusion of PS19-6 (RIIIRRIRR) resulted in the highest enzymatic activity after the immobilization of GST. Furthermore, the adsorption of PS19-6 peptide-fused GST (GST-PS19-6) was not inhibited even in the presence of 10 mg/ml bovine serum albumin (BSA) as a competitor which was 2000-fold higher in concentration than GST-PS19-6 [86]. Adsorption and remaining activity levels of wild-type GST, GSTPS19, and GST-PS19-6 were investigated using a commercially available PS plate with a different hydrophobicity. GSTs fused with both PS19 and PS19-6 peptides were preferentially adsorbed onto the surfaces of PS plates with a greater hydrophilic nature. In particular, Multisorp (Nunc) was strongly recognized by PS19-6 peptide and its fusion protein, as were other hydrophilic PS plates that are commercially supplied for animal cell culture from each company [86]. It is noteworthy that the binding affinity levels of PS tags were not changed even in the presence of up to 10% of a nonionic surfactant such as Tween 20 as a competitor, while they were gradually decreased with an increase in the concentrations of NaCl and urea. That result indicated that electrostatic interaction as well as hydrogen bonds formed between oxidized plastic surface and a PS-tag containing 5 arginine residues might maintain a strong binding affinity even in the presence of competitors such as Tween 20 and BSA. On the other hand, the adsorption ability of GST fused with either PS19-6K (KIIIKKIKK) or PS19-6H (HIIIHHIHH) was drastically decreased, while replacement of isoleucine (I) with shorter aliphatic amino acids such as valine (V), alanine (A), and glycine (G) resulted in a gradual decrease in the activity of GST in the adsorptive state. These results suggested that the PS19-6 peptide recognizes the surface structure of oxidized functional groups of PS, such as carboxyl, carbonyl and hydroxyl groups that are produced by O2 plasma or UV–O3 irradiation, rather than a hydrophobic phenyl group itself [87]. Therefore, although PS tags such as the PS19-6 peptide are known to bind strongly to an oxidized PS surface, they might also interact with the surfaces of other oxidized plastics.

Kogat et al. synthesized a PS-tag-linked peptide aptamer and applied it to the detection of protective antigens of Bacillus anthracis [90]. Koyanaga et al. prepared an engineered fusion protein of epidermal growth factor (EGF) with a PS-tag, and applied it to the differentiation of neural stem cells [91]. Tang et al. reported that the PS-tag-fused ZZ domain of protein A was successfully prepared and used for the indirect and oriented immobilization of a whole antibody [92]. This research group developed the KPS19R10 peptide (KRAFIASRRIRRP) wherein the 10th lysine (K) of the PS19 peptide was replaced with Arginine (R) and another lysine was additionally introduced at the N-terminus of the PS19 peptide. It was designed for conjugation with whole Abs. A KPS19R10 peptide was conjugated with polyclonal or monoclonal whole antibodies, and, consequently, PS tagconjugated whole Abs were successfully immobilized on the surface of a phi-PS plate even in the presence of Tween 20 and BSA [93]. Since these contaminants were not adsorbed strongly onto the surface of a phi-PS plate, the site-specific immobilization of a PS tag-conjugated whole Ab as well as its complexation with an antigen could be possible. The higher antigen-binding activity of a PS tag-conjugated whole Ab in the adsorptive state was detected when it was immobilized onto the surface of a phi-PS plate in the presence of Tween 20 [94]. The specific antigen-binding activity of an antibody in the adsorptive state is generally known to be drastically changed with immobilization density, probably because the attachment area of immobilized antibody molecules might be increased with a decrease in density [94,95]. Furthermore, the authors successfully developed and demonstrated one-step and two-step ELISAs using PS-tag-conjugated whole Abs [93]. In the one-step ELISA shown in Fig. 3, an immune-complex composed of a PS-tag-conjugated whole Ab, antigen, and horseradish peroxidase (HRP)-conjugated whole Ab was formed in a solution. Then the immune complex and residual PS-tag-conjugated whole Ab were directly collected onto the surface of a phi-PS plate through the strong binding affinity of the PS-tag. Consequently, the one-step detection of an antigen (insulin) with high sensitivity was possible. The one-step ELISA method developed previously was considerably useful for the rapid and sensitive detection of a target antigen, particularly when the binding affinity of an antibody towards its target antigen was relatively low, because dissociation of the antigen from the ligand antibody could be suppressed to a greater extent due to the single incubation and washing steps. With the two-step ELISA, an immune-complex without an HRPlabeled antibody was immobilized in the first step, and the HRPconjugated whole Ab that was subsequently introduced in the second step, was also advantageous in promoting rapid detection as well as a broad detection range. Other PS-tag-linked proteins could be utilized as a ligand in a onestep ELISA method for the detection of specific antibodies as well as protein–protein interactions. The author prepared PS-tag-conjugated or PS-tag-fused o-acethylserine sulhydrylase A (OASS-A), and successfully demonstrated the one-step detection of a cysteine synthase complex that consisted of OASS-A and serine acetyltransferase (SAT) [96]. Consequently, the sensitive detection of a cysteine synthase complex was attained using either a one-step or a two-step ELISA method, compared with conventional multi-step detection. 4.5. Production of recombinant antibody fragments genetically fused with PS-tags Recombinant protein fused with material-binding peptides strongly may change the solubility and stability of the original protein; thus, the host strain that is to be utilized for the efficient production of a fusion protein should be considered. Here, we introduce the characteristics of PS-binding peptides (PS-tags) for the efficient production and immobilization of recombinant antibody fragments. A variety of host strains such as E. coli [25,30–32,37,62], yeast [33,63–70], insect cells [97–100],

Y. Kumada / Biochimica et Biophysica Acta 1844 (2014) 1960–1969

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One-step ELISA PS-tag-linked ligand anbody

Two-step ELISA

Convenonal ELISA

Fig. 3. One-step and two-step ELISA using PS-tag-linked ligand antibody.

and plant cells [101–104], have been investigated for the production of recombinant antibody fragments. Using E. coli cells as a host, the secretion of recombinant antibody fragments to periplasmic space by the genetic fusion of a signal peptide such as a pel B reader at its N-terminus was a common strategy that was used to obtain soluble and active forms of antibody fragments, although the expressed production levels of the scFvs were relatively low and drastically changed with their amino acid sequences [62]. Furthermore, the genetic fusion of material-binding peptides such as a PS-tag and a PMMA-tag at their Cterminus mostly resulted in a decrease in the productivity of the soluble form, and as a consequence, they were mainly located in an insoluble fraction as an inclusion body [105]. Therefore, depending on the characteristics of the material-binding peptides, an appropriate refolding operation would be different. Here, we introduce two types of refolding strategies for the preparation of scFv-immobilized hydrophilic PS (phiPS) and PMMA plates. 4.6. Development of solid-phase refolding for the immobilization and activation of PS tag-fused scFvs PS-tag-fused antibody fragments were scarcely recovered as a soluble fraction by the conventional refolding methods of dialysis and rapid dilution probably due to an increase in the isoelectric point (pI) by genetic fusion of the PS tag, which included 5 basic amino acid residues. Although we investigated the production of PS-tag-fused scFv in bacteria (E. coli) as well as in yeast (P. pastoris), it could not be recovered as a soluble form in either strain, which suggested difficulties in the folding process as well as lower stability and solubility under physiological conditions. Therefore, in order to obtain a soluble and active form of scFv-PS for immobilization onto a phi-PS plate, the refolding of scFv-PS was necessary. Although we also investigated conventional refolding methods for scFv, the recovery of scFv-PS was considerably less than 10%. Refolded ScFv-PSs were efficiently immobilized onto the surface of a phi-PS plate [105]. Even in the presence of a nonionic surfactant such as Tween 20, they were efficiently adsorbed onto the surface of a phiPS plate, and showed high antigen-binding activity. The antigenbinding activity of immobilized scFv-PS was highly maintained despite a decrease in density, while that of whole Abs was drastically decreased, suggesting that the site-specific attachment of a PS-tag introduced at the C-terminus resulted in the prevention of unfavorable adsorption to the surface of plastics by the antigen-binding domain. Furthermore, both the one-step and two-step ELISAs described above were also

successfully performed by the use of scFv-PS with sensitivity that compared favorably to a conventional sandwich ELISA. On the other hand, a PS-tag has the potential to interact with the surface of a phi-PS plate even in the presence of a nonionic surfactant as well as chaotropic agents such as urea [87]. According to the adsorption isotherm of a FITC-labeled PS19-6 peptide to a phi-PS plate in the presence of different concentrations of urea, the adsorptive amount of the PS-tag was gradually decreased with increases in the urea concentration, while high adsorption levels were maintained even in the presence of 0.5–2 M of urea that the antibody fragments including scFv were only partially refolded. Therefore, a solid-phase refolding method whereby refolding and immobilization is simultaneously performed is effective for the preparation of scFv-immobilized phi-PS plate. PS-tag-fused antibody fragments could be immobilized even under semi-denatured conditions in the presence of 0.5–2 M of urea. The effect of urea concentration during the adsorption of anti-c-reactive protein (CRP) and anti-ribonuclease A (RNase A) scFv-PSs on antigen-binding activities after solid-phase refolding was investigated, and consequently, the antigen-binding activities of both scFv-PSs were efficiently recovered by solid-phase refolding in the presence of 0.5–2 M of urea during adsorption. By using the solid-phase refolding method, scFv-(PS) and scFv-PSII both of which had a PS tag inside the scFv molecule, as shown in Fig. 4, were successfully immobilized and refolded on the surface of a phi-PS plate, compared with the conventional liquid-phase refolding methods whereby both PS tag-fused scFvs were scarcely obtained. Among the 12 types of scFvs tested thus far, approximately 60% of them could be successfully immobilized with high antigenbinding activity using the solid-phase refolding method. 4.7. Using lectin ELISA for the structural analysis of an oligo-saccharide conjugated with a biomarker via scFv-immobilized phi-PS plates In addition to the sensitive detection of an antigen using sandwich ELISA, scFv-immobilized phi-PS plates prepared by solidphase refolding were also useful for the structural analysis of oligosaccharide conjugated with antigen [106]. As shown in Fig. 5, conventional ligand antibody, whole Abs, have complex-type N-linked oligosaccharides; thus, it would be difficult to detect oligo-saccharides conjugated with antigen specifically, after capturing it on the surface of a solid support immobilized with whole Abs. However, since scFvPS, which is produced in E. coli, is not glycosylated, it is useful as a ligand in lectin ELISA whereby the oligosaccharide of antigens can be

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Removal of denaturant by washing with buffer

Solubilizationof scFv-PS by denaturant

Immobilization of semidenatured scFv through PS-tag

Activated scFv in adsorption state

Fig. 4. Solid-phase refolding of PS-tag-fused scFv.

specifically detected by lectins. ScFv-PS, which specifically binds to carcinoembryonic antigen (CEA), was immobilized onto the surface of a phi-PS plate using the solid-phase refolding method. Compared with whole Ab-immobilized maxisorp, a higher degree of sensitivity was detected by the use of a scFv-PS-immobilized phi-PS plate. Furthermore, oligo-saccharides of CEA were also detected by 12 types of HRPconjugated lectins with different oligo-saccharide recognition properties. In particular, scFv-PS-immobilized phi-PS plates detected antigen-dependent signals with a much higher S/N ratio, while some lectins bound to the N-lined oligo-saccharides of whole Abs, and as a consequence, the background signals became high. Thus far, lectin ELISA formats for CEA, CA19-9, α-antitrypsin, human IgG, human IgA, transferrin, and α-fetoprotein have been successfully constructed.

4.8. Reconstitution of a Fab antibody on a phi-PS plate by solid-phase refolding The solid-phase refolding method was also available for the preparation of a Fab antibody-immobilized phi-PS plate. A PS-tag-fused heavy chain partial fragment composed of VH and CH1 domains (Fab H), and a full-length of light chain (Fab L) were separately produced, and then the formation of Fab antibody with antigen-binding activity was performed via one-step or two-step solid-phase refolding using PS-tagfused heavy chain (Fab H-PS) and light-chain (Fab L-PS) fragments, as shown in Fig. 6. The antigen-binding activity of anti-RNase Fab antibody was efficiently recovered when solid-phase refolding was performed in the presence of 0.5–2 M of urea. PS-tag-fused fragments of both heavy and light chains were necessary in order to achieve the highest level of antigen-binding activity, which was scarcely detectable when using a phi-PS plate where only heavy or light chains had been individually immobilized. A two-step, solid-phase refolding method whereby Fab H-PS and Fab L-PS were subsequently refolded resulted in a detection of antigen-binding activity that was comparable to that by a one-step method [107].

4.9. PMMA-binding peptide A PMMA-binding peptide (PMMA-tag, DVEGIGDVDLVNYFEVGA TYTFNK) was isolated and identified using the method described above, as shown in Fig. 2. It was one of the peptide fragments that were produced by the digestion of E. coli outer membrane protein F (OMP F), which was preferentially adsorbed onto the surface of a PMMA substrate [88]. The genetic fusion of a PMMA-tag to the Cterminus resulted in the quick attachment of GST onto the surface of a PMMA plate, when the adsorption phenomena was directly monitored by a quartz crystal microbalance (QCM) sensor equipped with a PMMAcoated sensor chip. Furthermore, the specific activity of PMMA tagfused GST in its adsorptive state was 3-fold higher than that of wildtype GST. These results indicate that the attachment of the PMMA tag that was introduced was site-specific to the surface of the PMMA substrate. 4.10. Efficient refolding of PMMA tag-fused recombinant antibody fragments The genetic fusion of a PMMA-tag to the C-terminus of scFvs considerably suppressed aggregation formation during refolding, compared with conventional scFvs and scFv-PSs. In contrast to a positivelycharged PS tag, a PMMA-tag contains 5 acidic amino acids (D and E), and as a consequence, it is negatively charged at a neutral pH. Thus, the genetic fusion of a PMMA tag to the C-termini of scFvs (scFv-PM) considerably improved their solubility as well as refolding yields at neutral or weak-alkaline pH due to a decrease in the apparent isoelectric point (pI) of scFv-PM [108]. We introduced a PMMA tag to 12 types of scFvs with different amino acid sequences, and investigated the effects of isoelectric points of scFvPM on refolding yields. As a consequence, most of the scFv-PMs were efficiently refolded with more than 90% recovery, when dialysis refolding was performed at a pH of 8.5. Furthermore, they were successfully immobilized on the surface of not only PMMA plates but also

(a)

(b) HRP-labeled lecns

Glyco-chain

scFv-PS

Biomarker

Whole Ab

Fig. 5. Lectin ELISA for structural analysis of oligo-saccharides conjugated with biomarker. (a) Whole Ab-immobilized PS plate, (b) scFv-PS-immobilized phi-PS plate.

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One-step solid-phase refolding washing

Light chain (Fab L-PS) Heavy chain (Fab H-PS)

Two-step solid-phase refolding washing

washing

Fig. 6. One-step or two-step solid-phase refolding for preparation of Fab-immobilized phi-PS plate.

polycarbonate (PC) and phi-PS plates with a higher degree of antigenbinding activity in the adsorptive state, compared with scFv without material-binding peptides. The amino acid contribution and recognition mechanism of a PMMA-tag towards various plastic surfaces remain unknown and will be the next subject to be solved in the near future. The tags of PMMA, and others, also might have functions such as those of the PS tags described above: one-step ELISA, solid-phase refolding, and lectin ELISA. 5. Summary This review summarizes the immobilization technologies for use with antibodies and recombinant antibody fragments. In particular, several material-binding peptides and their potential attachment abilities towards a target substance were introduced. Considering the diverse characteristics of peptide fragments such as length, components, and sequences, more material-binding peptides against a variety of materials will be successfully isolated in the near future. Clarification of the binding mechanisms of material-binding peptides, along with the computational design of target fusion proteins that will genetically fuse with a material-binding peptide, will be necessary for the preparation and immobilization of target proteins that retain the original function, activity, stability and even solubility. Furthermore, both the preparation (production) and immobilization processes of fusion proteins must be considered for industrial use. In particular, engineered antibody fragments (VHH, scFv and Fab) are expected to be the next generation of ligand antibodies, and, as such, the selection of efficient production and immobilization strategies will further reduce the costs and improve the sensitivity of the reagents used for diagnoses. References [1] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G, Immunochemistry 8 (1971) 871–874. [2] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigencoated tubes, J. Immunol. 109 (1972) 129–135. [3] K. Itoh, T. Suzuki, Antibody-guided selection using capture-sandwich ELISA, Methods Mol. Biol. 178 (2002) 195–199. [4] B. Lu, M.R. Smyth, R. O'Kennedy, Oriented immobilization of antibodies and its applications in immunoassays and immunosensors, Analyst 121 (1996) 29R–32R. [5] A. Kondo, S. Oku, K. Higashitani, Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles, Biotechnol. Bioeng. 37 (1991) 537–543. [6] M. Nisnevitch, M.A. Firer, The solid phase in affinity chromatography: strategies for antibody attachment, J. Biochem. Biophys. Methods 49 (2001) 467–480.

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