Analytical Biochemistry 396 (2010) 257–261

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Analytical Biochemistry

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Bionanocapsule-based enzyme–antibody conjugates for enzyme-linkedimmunosorbent assay

Masumi Iijima a,b, Takashi Matsuzaki a, Hiroyasu Kadoya a, Satoko Hatahira c, Shingo Hiramatsu c,Giman Jung c, Katsuyuki Tanizawa a,*, Shun’ichi Kuroda a,b,*

a Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japanb Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japanc New Frontiers Research Laboratories, Toray Industries, Kamakura, Kanagawa 248-8555, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 July 2009Available online 9 October 2009

Keywords:ELISAIgGProtein ANanoparticlesOriented immobilization

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.10.010

* Corresponding authors. Fax: +81 6 6879 8460 (K.(S. Kuroda).

E-mail addresses: tanizawa@sanken.osaka-u.ac.jpnagoya-u.ac.jp (S. Kuroda).

1 Abbreviations used: EIA, enzyme immunoassay;fluoroimmunoassay; HRP, horseradish peroxidase; Igavidin–biotin complex; BNC, bionanocapsule; HBV, hsurface antigen; ZZ-BNC, IgG Fc-interacting region (Zylococcus aureus protein A; ELISA, enzyme-linkedtransmission electron microscopy; OVA, ovalbumin; PBTMB, 3,30 ,5,50-tetramethylbenzidine; IgE, immunoglobfluoride; EGFR, epidermal growth factor receptor; EGFprotein; LOD, limit of detection; SD, standard deviation

Macromolecules that can assemble a large number of enzyme and antibody molecules have been usedfrequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We gener-ated bionanocapsules (BNCs) of approximately 30 nm displaying immunoglobulin G (IgG) Fc-binding ZZdomains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISAusing primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen onthe solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Westernblot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher sig-nal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody mole-cules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significantenhancement of sensitivity. In combination with the avidin–biotin complex system, biotinylated ZZ-BNCsshowed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC isexpected to increase the performance of various conventional immunoassays.

� 2009 Elsevier Inc. All rights reserved.

Enzyme immunoassay (EIA),1 radioimmunoassay (RIA), and fluo-roimmunoassay (FIA) have been widely used for high-throughputscreening of immunocomplexes. To increase the sensitivity of theseimmunoassays, two types of macromolecules have been used in thereaction with antibody. One type of macromolecule allows clusteringof antibodies and labeling molecules (e.g., enzyme, radioactive mate-rial, fluorescence). For example, polymeric horseradish peroxidase(HRP)–streptavidin conjugate [1], 3DNA dendrimer [2], and immu-noglobulin G (IgG)–poly-D-glutamic acid–(HRP)n conjugate [3] havebeen used previously. However, these macromolecules requirechemical modification of antibodies and do not allow the orientedimmobilization of antibodies [4] that improves the avidity and anti-

ll rights reserved.

Tanizawa), +81 52 789 5227

(K. Tanizawa), skuroda@agr.

RIA, radioimmunoassay; FIA,G, immunoglobulin G; ABC,epatitis B virus; HBsAg, HBVdomain) derived from Staph-

immunosorbent assay; TEM,S, phosphate-buffered saline;ulin E; PVDF, polyvinylidene

P, enhanced green fluorescents; LOQ, limit of quantitation.

gen recognition of antibodies [5]. In general, chemical modificationis considered to reduce the stability or antigen-binding activity ofantibodies. Another type of macromolecule is expected to permitthe oriented immobilization of antibodies. For example, nanoparti-cles have been used for displaying antibodies on their surface (e.g.,streptavidin-conjugated nanobeads [6], biotin-coated liposomes[7]). Because biotinylation occurred randomly at free amino groupson the surface of antibodies, these nanoparticles partially accom-plished oriented immobilization of antibodies using a avidin–biotincomplex (ABC). These results encouraged us to develop macromole-cules that can assemble antibodies and labeling molecules withoutchemical modification in the manner of oriented immobilization.

On the other hand, we previously developed a yeast-derivedhollow nanoparticle applicable for pinpoint delivery of drugs andgenes [8]. The nanoparticle, bionanocapsule (BNC), has a diameterof approximately 30 nm and is composed of hepatitis B virus (HBV)surface antigen (HBsAg) L proteins embedded in a liposome [9].The L protein is a three-membrane-spanning protein possessing apre-S region at the N terminal of the S region (see Fig. 1A) [10].BNCs can incorporate various therapeutic materials (e.g., drugs,genes) by electroporation [8] and liposome fusion [11] and candeliver them specifically to the human liver [8] based on the li-ver-specific recognition ability of the pre-S region [12]. Recently,to alter the tissue specificity of BNC, our collaborators made a

A

IgG

ZZ-BNC

Lipid bilayer

Hepatitis-B virus surface antigen

(HBsAg)

ZZ domain

S region

B

ZZ-BNC + Goat total IgG-gold

Fig. 1. (A) Schematic structure of a ZZ-BNC. (B) Transmission electron microscopy (TEM) images of ZZ-BNC conjugated with 10-nm gold particle-labeled goat total IgG.Bar = 40 nm.

258 Bionanocapsule-based enzyme–antibody conjugates / M. Iijima et al. / Anal. Biochem. 396 (2010) 257–261

derivative of BNC by replacing the pre-S region with a tandemsequence of the IgG Fc-interacting region (Z domain) derived fromStaphylococcus aureus protein A [13] and designated it ZZ-BNC [14](Fig. 1A). ZZ-BNC allowed us to display antibodies on its surfaceand to deliver various therapeutic materials to tissues of interestin an antibody-dependent manner [14].

These properties led us to imagine that ZZ-BNC spontaneouslyadsorbs antibodies in the manner of oriented immobilization. Inthis study, we examined whether ZZ-BNCs could contribute to sig-nal enhancement of enzyme-linked immunosorbent assay (ELISA)through formation of IgG–ZZ-BNC complexes.

Materials and methods

ZZ-BNCs

ZZ-BNCs were overexpressed in Saccharomyces cerevisiaeAH22R� carrying the ZZ-BNC expression plasmid pGLD-ZZ50[14]. According to the preparation method for BNCs [8], ZZ-BNCswere extracted by the disruption with glass beads and purifiedusing an AKTA chromatography system (GE Healthcare, Amer-sham, UK) by affinity chromatography on porcine IgG and gelfiltration.

Transmission electron microscopy

ZZ-BNCs (1 lg as protein) were mixed with 10-nm gold parti-cle-labeled goat total IgG (50 ll, Sigma–Aldrich, St. Louis, MO,USA), adsorbed onto a carbon-coated copper grid (JEOL, Tokyo,Japan), negatively stained using 2% (w/v) phosphotungstic acid(pH 7.0), and subjected to transmission electron microscopy(TEM) using a model JEM1011 (Jeol).

ELISA for ovalbumin on solid phase

Ovalbumin (OVA, 100 ll, 0–6.25 ng/ml, Sigma–Aldrich) was ad-sorbed to each well of a Nunc–Immuno Plate II (96 wells, NalgeNunc International, Rochester, NY, USA). The plate was kept at4 �C overnight and washed three times with PBST (200 ll of phos-phate-buffered saline [PBS], 137 mM NaCl, 2.7 mM KCl, 10 mMNa2PO4, and 2 mM KH2PO4 [pH 7.4] containing 0.05% [v/v] Tween20). Antibodies were diluted with PBST containing 5% (w/v)skimmed milk (Nacalai Tesque, Kyoto, Japan). A primary antibody,a mouse anti-OVA IgG1 antibody (100 ll, 0.4 lg/ml, Abcam, Cam-bridge, UK), was added to each well, incubated at room tempera-

ture for 1.5 h, and washed three times with PBST. A secondaryantibody, HRP-conjugated rabbit anti-mouse IgG (100 ll, 2 lg/ml,Sigma–Aldrich), was added to each well, incubated at room tem-perature for 1.5 h, and washed three times with PBST. When bio-tinylated rabbit anti-mouse IgG (100 ll, 2 lg/ml, Sigma–Aldrich)was used as the secondary antibody, the ABC system (ABC peroxi-dase staining kit, Pierce, Rockford, IL, USA) was used for the label-ing with HRP. The colorimetric reaction was carried out at roomtemperature for 15 min in 100 ll per well of a 3,30,5,50-tetrameth-ylbenzidine (TMB) substrate kit (Pierce). The reaction was stoppedwith 100 ll of 2 N H2SO4. Absorbance at 450 nm was measured ona Varioskan microplate reader (Thermo Fisher Scientific, Waltham,MA, USA) using absorbance at 690 nm as the reference. When thecalibration curve was generated using 0–6.25 ng/ml OVA (n = 8),the wells containing 0 ng/ml OVA were defined as blank wells forsubtracting background.

ELISA for anti-OVA immunoglobulin E antibodies in aqueous phase

OVA (100 ll, 15 lg/ml) was adsorbed onto each well of a Nunc–Immuno Plate II. The plate was kept at 4 �C overnight and washedthree times with 200 ll of PBST. Mouse anti-OVA immunoglobulinE (IgE) antibody (0–6.25 ng/ml, 100 ll, AbD Serotec, Oxford, UK)diluted with PBST containing 5% skimmed milk was added to eachwell, incubated at room temperature for 1.5 h, and washed threetimes with PBST. An HRP-conjugated rabbit anti-mouse IgE Fc anti-body (100 ll, 5 lg/ml, Nordic Immunological Laboratories, Tilburg,Netherlands) was added to each well, incubated at room tempera-ture for 1.5 h, and washed three times with PBST. When biotin-la-beled rabbit anti-mouse IgE Fc antibody (100 ll, 5 lg/ml, NordicImmunological Laboratories) was used as the secondary antibody,the ABC peroxidase staining kit was used for the labeling with HRP.The colorimetric reaction was carried out at room temperature for15 min in 100 ll per well of a TMB substrate kit. The reaction wasstopped with 100 ll of 2 N H2SO4. Absorbance at 450 nm was mea-sured on a Varioskan microplate reader using absorbance at690 nm as the reference. When the calibration curve was gener-ated using 0–6.25 ng/ml anti-OVA IgE (n = 8), the wells containing0 ng/ml anti-OVA IgE were defined as blank wells for subtractingbackground.

Western blot analysis

Here 1 ll of OVA (0.001–10 mg/ml) was blotted onto a polyvi-nylidene fluoride (PVDF) membrane (Millipore, Billerica, MA,

Bionanocapsule-based enzyme–antibody conjugates / M. Iijima et al. / Anal. Biochem. 396 (2010) 257–261 259

USA) and dried at room temperature for 30 min. The membranewas blocked with 5% skimmed milk in TBST (20 mM Tris–HCl,140 mM NaCl, and 0.05% Tween 20 [pH 7.4]) at room temperaturefor 30 min and then incubated at room temperature for 1 h withthe mouse anti-OVA IgG1 antibody (1 lg/ml) diluted with TBST.The membrane was washed three times with TBST and incubatedwith the HRP-conjugated rabbit anti-mouse IgG (1 lg/ml) at roomtemperature for 1 h. When biotinylated rabbit anti-mouse IgG(1 lg/ml) was used as the secondary antibody, the ABC systemwas used for the labeling with HRP. After washing three times withTBST, the membrane was treated with ECL Western blotting detec-tion reagents (GE Healthcare) and then the immunoreactive spotswere visualized under a luminescence image analyzer (LAS-4000mini, Fujifilm, Tokyo, Japan).

Results and discussion

Display of antibodies on ZZ-BNC surface

BNC is a yeast-derived hollow nanocapsule consisting ofapproximately 110 HBsAg L proteins embedded in a liposome[8]. Recently, we replaced the N-terminal region of L protein(pre-S region) with a tandem sequence of protein A-derived IgGFc-binding domain (Z domain) and designated it ZZ-BNC [14](Fig. 1A). When mixed with anti-EGFR (epidermal growth factorreceptor) antibody, the ZZ-BNCs labeled with enhanced green fluo-rescent protein (EGFP) accumulated on the surface of human cervi-cal carcinoma HeLa cells that express EGFR abundantly [14].Therefore, we mixed ZZ-BNC with 10-nm gold particle-labeled goattotal IgG and observed it under TEM. As shown in Fig. 1B, approx-imately 30-nm ZZ-BNCs were surrounded by several molecules of10-nm gold particle-labeled goat total IgG. This result strongly sug-gested that ZZ domains surrounding the ZZ-BNC surface tether the

0

1

2

3

*

A

OVA

Mouse anti-OVA Ab

HRP-conjugated anti-mouse Ab

Biotinylatedanti-mouse A

Avidin

BiotinylHRP

ZZ-BNC

1 2

B

* * *

*

**

*

Plastic plate

10 2 3 4 5 6 7

OVA (ng/ml)

Ab

sorb

ance

(A

450–

A69

0)

13

24

5

C

2.5

1.5

0.5

Fig. 2. (A) Schematic representations of various ELISA systems for OVA detection. Panel 1IgG antibody; panel 2: conventional ELISA in the presence of ZZ-BNC; panel 3: conventiothe presence of ZZ-BNCs; panel 5: conventional ELISA with the ABC system in the presencELISA systems. Results are means ± SD (n = 8). Numbers correspond to the methods shsystems. Results are means ± SD (n = 8). Numbers correspond to the methods shown in

IgG Fc domains to display all IgG Fv regions outward for effectiveantigen binding. Thus, ZZ-BNC was expected to cluster antibodieson its surface in the manner of oriented immobilization, whichmight improve the avidity and antigen recognition of antibodies[5].

ELISA for antigens on solid phase

The enhancement of the sensitivity was examined in the detec-tion of antigen (OVA) by ELISA with or without ZZ-BNCs (seeFig. 2A, panels 1 and 2). As described in Materials and methods,OVA (0–6.25 ng/ml) was adsorbed onto each well of the immuno-plate, contacted with primary antibody, and then contacted withsecondary antibody (see Fig. 2A, panel 1). When the purified ZZ-BNCs (2 lg/ml as protein) were preincubated with the secondaryantibody (2 lg/ml) at room temperature for 30 min and thenadded to each well (see Fig. 2A, panel 2), the signal at 3.125 ng/ml OVA was approximately 10-fold higher than that without ZZ-BNCs (Fig. 2B, lines 1 and 2). The ABC system has been widely usedfor the enhancement of ELISA signals because it forms a tetramericavidin-based complex of biotinylated antibodies and biotinylatedHRP (see Fig. 2A, panel 3). The signal at 3.125 ng/ml OVA withthe ABC system was approximately 4-fold higher than that withthe conventional ELISA (Fig. 2B, lines 1 and 3). When ZZ-BNC(2 lg/ml as protein) was preincubated with the biotinylated sec-ondary antibody (2 lg/ml) at room temperature for 30 min andthen added to each well (100 ll) (see Fig. 2A, panel 4), the signalat 3.125 ng/ml OVA was approximately 13-fold higher than thatwithout ZZ-BNCs or the ABC system (Fig. 2B, lines 1 and 4). Next,biotinylated ZZ-BNCs (2 lg/ml as protein) were prepared withEZ-Link Sulfo-NHS-Biotin (Pierce) according to the manufacturer’sprotocol and added to the secondary antibody instead of ZZ-BNCs(see Fig. 2A, panel 5). The signal at 3.125 ng/ml OVA was approxi-mately 19-fold higher than that without ZZ-BNCs or the ABC sys-

b

ated

BiotinylatedZZ-BNC

3 4 5

*

*

*

*

*

*

*

*

**

*

*

**

*

*

*

**

*

Ab

sorb

ance

(A

450–

A69

0)

anti-OVA IgE (ng/ml)

13

24

5

0

1

2

2.5

*

**

*

*

*

*

**

*

*

**

*

**

*

*

10 2 3 4 5 6 7

1.5

0.5

: conventional ELISA using an anti-OVA antibody and an HRP-conjugated anti-mousenal ELISA with the ABC system; panel 4: conventional ELISA with the ABC system ine of biotinylated ZZ-BNC. Ab, antibody. (B) Detection of OVA (solid phase) by variousown in panel (A). (C) Detection of anti-OVA IgE (aqueous phase) by various ELISApanel (A).

10 µg

5 µg

1 µg

500 ng

OVA (weight/spot)

ZZ-BNC

Bio-ZZ-BNC

ABC

+- -+-- +- --- +- ++

260 Bionanocapsule-based enzyme–antibody conjugates / M. Iijima et al. / Anal. Biochem. 396 (2010) 257–261

tem (Fig. 2B, lines 1 and 5). The sensitivity for ELISA is defined bythe limit of detection (LOD, mean ± 3SD [standard deviations]) andthe limit of quantitation (LOQ, mean ± 10SD) [15] at the lowest endof the quantifiable concentration range of OVA (n = 20). As shownin Table 1, the LOD and LOQ of ELISA (see Fig. 2A, panel 1, 750and 1500 pg/ml, respectively) were decreased by 47% using ZZ-BNCs (see Fig. 2A, panel 2, 350 and 700 pg/ml, respectively), indi-cating that the two sensitivities (LOD and LOQ) were increased2.1-fold. The ABC system enhanced the two sensitivities 2.5- and20-fold in the presence of ZZ-BNCs (see Fig. 2A, panel 4) and biotin-ylated ZZ-BNCs (see Fig. 2A, panel 5), respectively.

100 ng

50 ng

10 ng

5 ng

1 ng

1 2 3 4 5

Fig. 3. Western blot analyses of spotted OVA by various detection methods. Lanenumbers correspond to the methods shown in Fig. 2A. The minimum OVA amountsdetectable by each method are indicated with arrowheads.

ELISA for antibodies in aqueous phase

We investigated the enhancement of the sensitivity of detectionof the primary antibody by ELISA using ZZ-BNCs and biotinylatedZZ-BNCs. OVA adsorbed onto each well was contacted with pri-mary antibody (0–6.25 ng/ml) and then contacted with secondaryantibody (see Materials and methods and Fig. 2A, panel 1). WhenZZ-BNC (1.25 lg/ml as protein) was preincubated with the second-ary antibody (5 lg/ml) at room temperature for 30 min and thenadded to each well (see Fig. 2A, panel 2), the signal at 3.125 ng/ml mouse anti-OVA IgE antibody was 2.3-fold higher than thatwithout ZZ-BNCs (Fig. 2C, lines 1 and 2). Next, with the concurrentuse of biotinylated secondary antibody (5 lg/ml), biotinylated ZZ-BNCs (2.5 lg/ml as protein), and the ABC system, the signal at3.125 ng/ml mouse anti-OVA IgE antibody was 4.4-fold higher thanthat of conventional ELISA (Fig. 2C, lines 1 and 5). Furthermore, thesensitivity for ELISA is defined by the LOD and LOQ [15] at the low-est end of the quantifiable concentration range of anti-OVA IgE(n = 20). The LOD was decreased by 50% and 8% using ZZ-BNCsand biotinylated ZZ-BNCs, respectively (Table 1).

Western blot analysis

Signal enhancement of ELISA for antigens on solid phase by ZZ-BNCs led us to examine whether they are applicable for Westernblot analysis. The dot blot containing OVA (from 1 ng/spot to10 lg/spot) was incubated with the primary antibody at roomtemperature for 1 h, incubated with the secondary antibody atroom temperature for 1 h, and then subjected to chemilumines-cence detection (see Materials and methods). When ZZ-BNCs(1 lg/ml as protein) were preincubated with the secondaryantibody (1 lg/ml) at room temperature for 30 min, the spot con-taining 10 ng of OVA could be detected, which was approximately50-fold higher than that without ZZ-BNCs (Fig. 3, lanes 1 and 2).Next, the use of the ABC system and biotinylated secondary anti-body facilitated us to detect the spot containing 50 ng of OVA(Fig. 3, lane 3). The adaptation of ZZ-BNCs (1 lg/ml as protein)

Table 1Sensitivities of various ELISA systems for OVA.

Method LOD (pg/ml)(mean ± 3SD)

CV (%) LOQ (pg/ml)(mean ± 10SD)

CV (%)

For detection of antigen (OVA)Conventional ELISA 750 4.5 1500 3.4+ ZZ-BNC 350 6.4 700 6.9+ ABC 500 3.0 1000 3.1+ ABC + ZZ-BNC 300 5.3 600 11.8+ ABC + bio-ZZ-BNC 40 3.5 80 4.3

For detection of primary antibody (anti-OVA IgE)Conventional ELISA 500 5.0 ND ND+ ZZ-BNC 250 5.4 500 4.6+ ABC + bio-ZZ-BNC 40 4.2 130 3.6

Note. bio-, biotinylated; ND, not detected.

and biotinylated ZZ-BNCs (1 lg/ml as protein) to this systemachieved approximately 5- and 10-fold higher sensitivity (Fig. 3,lanes 4 and 5, respectively), comparable to 50- and 100-foldenhancements from the conventional method (lane 1). It was con-firmed that the combined use of the ABC system and biotinylatedZZ-BNCs significantly enhanced the sensitivity of not only ELISAbut also Western blot analysis. Furthermore, ZZ-BNCs might beapplicable for the signal enhancement of immunohistochemicalanalysis.

Conclusions

It has been demonstrated that ZZ-BNCs could assemble anti-bodies on their surface even without chemical modification. ZZ-BNCs could then enhance the signals in the detection of antigensand antibodies when added to the aqueous phase of conventionalELISA and Western blot analysis. A combination of the ABC systemand biotinylated ZZ-BNCs significantly improved the sensitivity ofconventional ELISA and Western blot analysis. In Fig. 1B, ZZ-BNCsare shown to tether the Fc regions of IgG on the ZZ domain so thatall of the Fv regions are displayed outward for effective formationof immunocomplexes. Thus, oriented immobilization of antibodieshas presumably been achieved on the surface of ZZ-BNCs. As men-tioned above, BNCs (including ZZ-BNCs) can incorporate variousmaterials by electroporation and liposome fusion. This indicatedthat ZZ-BNCs may be applicable for RIA and FIA. BNCs could be ad-sorbed onto a mica surface without the disruption of its particlestructure [16]. Therefore, ZZ-BNCs would also be applicable forthe oriented immobilization of antibodies in the solid phase ofvarious immunoassay systems.

Acknowledgments

We thank Profs. A. Kondo (Kobe University), M. Seno (OkayamaUniversity), and M. Ueda (Keio University) for helpful advice. Thiswork was supported by the Research and Development Programfor New Bio-industry Initiatives from the Bio-oriented TechnologyResearch Advancement Institution (BRAIN) of Japan; the Researchon Advanced Medical Technology from the Ministry of Health,

Bionanocapsule-based enzyme–antibody conjugates / M. Iijima et al. / Anal. Biochem. 396 (2010) 257–261 261

Labor, and Welfare of Japan; and the Regional Research and Devel-opment Resources Utilization Program of the Japan Science andTechnology Agency (JST).

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