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Enzyme and Microbial Technology 46 (2010) 309–314

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

ypersensitive immunoassay by using Escherichia coli outer membrane withutodisplayed Z-domains

in Parka, Joachim Joseb, Jae-Chul Pyuna,∗

School of Materials and Sciences, College of Engineering, Yonsei University, 134 Shin-chon-dong, Seo-dae-mun-gu, Seoul 120-749, Republic of KoreaInstitute of Pharmaceutical Chemistry, Heinrich Heine University, Duesseldorf, Germany

r t i c l e i n f o

rticle history:eceived 24 September 2009eceived in revised form 7 December 2009ccepted 7 December 2009

a b s t r a c t

In this work, the Z-domain of protein A was autodisplayed on the surface of the Escherichia coli outer mem-brane as a fusion protein of AIDA-1, and then layered on a microplate in order to develop a hypersensitiveimmunoassay. By using microplates with controlled hydrophilicity the formation of outer membranelayer was carried out, and the driving force for the layer formation was proven to be a hydrophobic

eywords:mmunoassay-domainrientation control-reactive protein

interactions. Through the orientation control of the immobilized antibodies by using autodisplayed Z-domains, the limit of detection (LOD) and the sensitivity of this new immunoassay were determinedto have improved as much as 30-fold compared to the conventional ELISA. The applicability of the newimmunoassay for medical diagnosis was demonstrated by the detection of C-reactive protein (CRP) whichis known to be a biomarker protein for inflammatory diseases.

utodisplayuter membrane

. Introduction

The autodisplay technology is a method to express target pro-eins on the surface of the outer membrane of Escherichia coli. Sincehe target protein is expressed as a fusion protein of an alreadyxisting outer membrane protein, the autodisplayed protein shouldave a conserved structure and orientation [1–5]. Recently, weeported the autodisplay of the Z-domain on the surface of the E.oli outer membrane [6]. The Z-domain fusion protein was ana-yzed to be one of the major proteins on the E. coli outer membranerom SDS-PAGE analysis among the outer membrane proteins. ThegG-binding activity of the autodisplayed Z-domain was confirmedy treating IgG labeled with Cy3 in comparison to the intact E. coliithout Z-domain on the E. coli surface. As shown in Fig. 1, the E.

oli with autodisplayed Z-domain showed intensive fluorescenceignal in comparison to the intact E. coli without the Z-domain. Theatio of E. coli with the active Z-domains was evaluated to be morehan 95% by fluorescence labeled cell sorting (FACS) analysis.

In this work, the Z-domain of protein A was autodisplayedn the surface of the E. coli outer membrane as a fusion pro-

ein of an adhesin involved in diffuse adherence (AIDA-1) [5], andhen layered on a microplate in order to develop a hypersensi-ive immunoassay. The immunoassays have been widely used for

edical diagnosis, environmental monitoring, forensic tests, and so

∗ Corresponding author. Tel.: +82 2 2123 5851; fax: +82 2 365 5882.E-mail address: jcpyun@yonsei.ac.kr (J.-C. Pyun).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.12.004

© 2009 Elsevier Inc. All rights reserved.

on [7]. Based on the highly specific antigen–antibody interactions,the immunoassays can detect target analytes in complex mixturesamples, such as human blood. The conventional immunoassayshave exploited solid supports such as microplates and immunos-ticks, to immobilize antibodies (or antigens) [8]. To detect a targetanalyte more sensitively, the immobilized antibodies should be ori-ented to expose the binding pockets at Fab regions of each antibodymolecule towards the target analytes. The portion of such well-oriented antibodies was reported to be less than 20% by physicaladsorption of antibodies [9]. Until recently, various methods havebeen applied for the orientation control of antibodies in order toimprove the sensitivity of immunoassays. The protein A has beenmost frequently used for the orientation control of antibodies byusing the affinity of protein A towards the Fc region of the antibodies(IgG). For immunoassays using protein A, protein A was first coatedon a microplate, and then the antibodies were immobilized to themicroplate with a controlled-orientation [10]. Similarly, the highaffinity of avidin or streptavidin toward the biotin molecule wasalso used for the orientation control of antibodies. For immunoas-says using those proteins, avidin or streptavidin was first coatedon a microplate, and then antibodies conjugated with biotin werebound to the avidin or streptavidin on the microplate [11]. As theamino groups used for the biotinylation were known to be located

at the Fc region of the antibodies, the biotinylated antibodies couldbe immobilized with a controlled-orientation [12]. In both cases,the sensitivity of each immunoassay was reported to be improvedin comparison to the conventional immunoassays by the orienta-tion control effects.

310 M. Park et al. / Enzyme and Microbial Technology 46 (2010) 309–314

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ig. 1. IgG-binding activity test of Escherichia coli by treatment of Cy3 labeled IgGithout Z-domain on the surface of outer membrane.

In this work, the formation of the outer membrane layer was car-ied out by using microplates with controlled hydrophilicity. Theriving force for the layer formation was proven to be a hydropho-ic interaction. The limit of detection (LOD) and the sensitivityf this new immunoassay were determined to improve as muchs 30-fold compared to the conventional ELISA, and the effectf orientation control was evaluated by using a model analyte.he applicability of the new immunoassay for medical diagnosisas demonstrated by the detection of C-reactive protein (CRP)hich is known to be a biomarker protein for inflammatory dis-

ases.

. Materials and methods

.1. Materials

Purified C-reactive protein (CRP), Anti-CRP antibodies (polyclonal), anti-hIgGntibodies labeled with Cy3 (polyclonal), anti-HRP antibodies (polyclonal), anti-IgG antibodies labeled with HRP (polyclonal), anti-CRP antibodies conjugatedith HRP (polyclonal) were bought from AbCam (Cambridge, UK). Aprotininas purchased from Roche Korea (Seoul, Korea). Phenylmethanesulfonyl flu-

ride, bovine serum albumin (BSA), lysozyme, DNase, horseradish peroxidaseHRP) and all of the other chemicals (of analytical grade) were purchased fromigma–Aldrich Korea (Seoul, Korea). The microplates with controlled hydrophilic-ty called Maxisorp, Medisorp, Multisorp, Polysorp were purchased from Nunc Co.USA).

.2. Preparation of E. coli outer membrane with autodisplayed Z-domain

The autodisplay was performed by transformation using an autodisplay vectoronstructed by the cloning of the antibody binding Z-domain from Staphylococcusureus as described in the previous work [6]. The outer membrane (OM) of E. coli withutodisplayed Z-domain was prepared by using lysozyme reaction and subsequentsolation procedures as described in the previous work [13,14]. The OM of intact E.oli was also prepared by the same procedure.

E. coli with Z-domain on the surface of the outer membrane and (b) intact E. coli

2.3. Preparation of an OM layered microplate for ELISA

The OM solution was prepared to have the total protein concentration of300 �g/ml in PBS. For each well of a 96-well microplate, 100 �l of the OM solu-tion was incubated for 2 h at room temperature. Then, the microplate was washedwith 0.1% Tween 20 in PBS for three times. To immobilize the detection antibod-ies, the corresponding antibody solution (100 �l) was dispensed at each well ofthe microplate and then incubated for 2 h at room temperature. The HRP assaywas reported by the chromogenic reaction of the TMB solution (Pierce, USA). Afterquenching with 2 M sulfuric acid solution, the optical density was measured at thewavelength of 450 nm by using an ELISA reader (Vesamax, Molecular Devices, USA).

2.4. ELISA with an OM layered microplate

For the immobilization of antibodies to the OM layered microplate with autodis-played Z-domains, the antibody solution prepared in PBS was incubated for 2 h at37 ◦C. After incubation step, the microplate was washed with 0.1% Tween 20 in PBSby using an automated washing machine from Molecular Devices (USA). For thesandwich-type ELISA, secondary antibodies coupled with HRP were reacted afterthe analyte incubation. To quantify the bound analyte, TMB solution was treatedfor 10 min, and the reaction was quenched with 2 M sulfuric acid and the opticaldensity was measured at the wavelength of 450 nm with a reference wavelength of650 nm by using an ELISA reader.

3. Results and discussion

3.1. Formation of OM layer on microplate

The outer membrane of E. coli was known to have a lay-ered structure composed of lipopolysaccharide (LPS), phospholipid,

lipoprotein, and peptidoglycan on the plasma membrane [15]. Forthe isolation of E. coli outer membrane, the peptidoglycan layer ofthe outer membrane was hydrolysed by the reaction of lysozyme.Then, the isolated outer membrane was solubilized by using sar-cosine [14]. According to the subsequent centrifugations, the outer

robial Technology 46 (2010) 309–314 311

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Fig. 2. Formation of the outer membrane layer by hydrophobic interactions. (a)IgG-binding activity of the outer membrane layer formed on various microplatesurfaces with controlled hydrophobicity. As the surface, the following microplates(with the contact angle value at parenthesis) were used: Maxisorp (64.8◦), Medis-orp (66.7◦), Multisorp (44.7◦), Polysorp (77.6◦), parylene A coated microplate (63.7◦),

M. Park et al. / Enzyme and Mic

embrane was isolated to be an outer membrane particle, whichas analyzed to have negatively charged surface with a diameter

f 100 nm by using a particle analyzer [16]. Actually, the surfaceharge of an intact E. coli was measured to be −25.5 mV usinghe zeta-potential measurement. The zeta-potentials of E. coli withutodisplayed Z-domain and its outer membrane particle wereeasured to be −25.1 and −22.5 mV, respectively. These results

ndicated that the layered structure of the E. coli outer membraneas maintained for E. coli with autodisplayed Z-domain as well as

he outer membrane particle. Such negative charge of the E. coli wasenerated from the LPS layer at the surface of the E. coli cell, andhe core side of the outer membrane particle consisted relativelyydrophobic layers [15]. When such particles with a hydrophilicurface and a hydrophobic core were treated to a hydrophobic sub-trate, the particles were known to make an ordered layer throughydrophobic interactions [17]. When the outer membrane layeras formed on the hydrophobic substrate by using the prepared

uter membrane particles, the relatively hydrophobic lipoproteinayer was expected to make a direct hydrophobic interaction withhe substrate, and the negatively charged LPS layer was expectedo be located at the surface. As the Z-domain was expressed as ausion protein located at the lipopolysaccharide, the Z-domain wasxpected to be located at the surface of the outer membrane layer.

In order to explain the role of the hydrophobic interactionuring the formation of the outer membrane layer, the outer mem-rane layer was prepared on several microplates having controlledydrophobicity. The density of the outer membrane layer was eval-ated by the IgG-binding activity. To measure the IgG-bindingctivity, the outer membrane layer was formed on the microplatesy treating the outer membrane particles at the same concentra-ion of 300 �g/ml by incubation. Then, the antibodies (IgG) labeledith HRP were treated and the amount of antibodies bound to

he Z-domain was estimated by the chromogenic reaction betweenRP and TMB. As shown in Fig. 2(a), the IgG-binding activity wasbserved to correlate linearly to the hydrophobicity of the sur-ace. These results show that the outer membrane layer was moreense according to the hydrophobicity of surfaces, and also thathe outer membrane layer was expected to have a larger numberf autodisplayed Z-domains on the surface. Therefore, the surfacehowing the highest hydrophobicity presented the highest IgG-inding activity. These results illustrates that the formation of theuter membrane layer was derived by the hydrophobic interactionetween the outer membrane particle and the microplate surface.

The outer membrane particle from the intact E. coli cell withouthe autodisplayed Z-domain was also treated to the microplatess a negative control to determine the non-specific binding of theRP labeled antibodies (IgG). As shown in Fig. 2(a), the non-specificinding of IgG labeled with HRP was reciprocal to the hydropho-icity of surfaces. As the outer membrane layer was more densely

ayered due to the hydrophobicity of surfaces, the outer mem-rane layer was expected to have condensed negative charges onhe surface. In other words, the most hydrophobic surface wasxpected to have the most hydrophilic surface, which could gener-lly reduce the non-specific binding of proteins. From these results,he non-specific binding of proteins was expected to be effectivelyrevented by the outer membrane layer prepared on the hydropho-ic surfaces. As shown in Fig. 2(a), the polystyrene microplateresented the highest Z-domain activity and this microplate wassed for the immunoassays in this work.

The optimal concentration of the outer membrane particle wasn order to form a layer by using the polystyrene microplate. The

ncubation of the outer membrane particle was performed for 2 h atoom temperature. As shown in Fig. 2(b), different concentrationsf the outer membrane particles were treated to the microplatend then the IgG-binding activity was determined by using HRPabeled antibodies. The outer membrane particle from intact E. coli

parylene N coated microplate (72.6◦), and polystyrene microplate (78.2◦). (b) Opti-mization of concentration for plate coating by using outer membrane from E. coliwith autodisplayed Z-domain (�) and intact E. coli (�).

was also treated to the microplate as a negative control. As shownin Fig. 2(b), the IgG-binding activity of the outer membrane layerwith autodisplayed Z-domains was increased as the concentrationof outer membrane particle treated to the microplate increased.These results could also be explained by the increase in number ofthe autodisplayed Z-domains according to the dense layer of theouter membrane particles. The saturated response was observed atthe concentration of more than 1000 �g/ml with an optical density(OD) value of 0.82 (AU). When the concentration of the outer mem-brane particle was more than 300 �g/ml, the IgG-binding activitywas calculated to be more than 90% of the saturated response withan OD value of 0.78 (AU). In this work, this concentration was usedfor outer membrane layer formation. In the case of the negative con-trol, the outer membrane particles at a lower concentration than1000 �g/ml showed that the adsorption of the antibodies was esti-mated to reach a stable baseline with an OD value of less than 0.01

(AU). When the concentration of the outer membrane particle washigher than 300 �g/ml, the IgG-binding activity was calculated tobe approximately 90% of the baseline with an OD value of 0.04 (AU).This result means that the outer membrane layer could prevent the

312 M. Park et al. / Enzyme and Microbial Technology 46 (2010) 309–314

Fig. 3. HRP detection with an orientation-controlled antibody layer (�) and a randomly oriented antibody layer as the conventional ELISA (�). (a) Schematics for assayc rationd n andw oncen

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onfiguration, (b) comparison of assay response by treatment of HRP at the concentifference of the assay response between antibody layer with controlled-orientatioith controlled-orientation and randomly oriented antibody layer at the antibody c

on-specific adsorption of proteins when the concentration of thereated outer membrane particle was higher than 300 �g/ml. Basedn these results derived from the outer membrane layer formation,he concentration of the outer membrane particle at 300 �g/ml wasptimal for the antibody immobilization as well as enough for therevention of the non-specific binding of proteins.

.2. ELISA with OM layered microplates

As previously mentioned, the Z-domain is one of the IgG-bindingomains of protein A [18]. Due to its specific binding activity tohe Fc region of the antibodies (IgG), the outer membrane layerith the autodisplayed Z-domain could be effectively used for the

rientation control of antibodies. In this work, the effect of suchrientation control using autodisplayed Z-domain was estimatedy the analyte-binding activity of the immobilized antibodies inomparison to the conventional ELISA based on a randomly ori-nted antibody layer. When the same concentration of antibodiesas treated to immobilize the antibodies, the randomly oriented

ntibody layer would have a smaller number of well-oriented anti-odies at a unit area than the orientation-controlled antibody layer.herefore, the antibody solution should be more concentrated toetect the analyte for the randomly oriented antibody layer thanhe orientation-controlled antibody layer. In this work, HRP wasxploited as a model analyte as shown in Fig. 3(a) and the con-entration of antibodies required for the detection of HRP waseasured by using a randomly oriented antibody layer (�) and an

rientation-controlled antibody layer (�). The randomly orientedntibody layer was prepared by physical adsorption of antibod-

es as in the case of the conventional ELISA. The outer membraneayer of the intact E. coli was prepared as a negative control layern order to estimate the non-specific binding (�) of the antibod-es. The amount of bound analyte (HRP) was evaluated through thehromogenic reaction of TMB and the OD value at the wavelength

of 1 �g/ml according to the concentration of immobilized anti-HRP antibodies, (c)randomly oriented antibody layer and (d) assay response between antibody layertration of the maximum assay response difference (600 ng/ml).

of 450 nm was measured. As shown in Fig. 3(b), the minimum anti-body concentration required for the detection of a HRP sample(10 ng/ml) was estimated to be 3 and 45 ng/ml for the antibodylayers with the controlled-orientation by Z-domain (�) and therandom-orientation (�), respectively. This result represents thata 15-fold lower concentration of the antibody was required for thedetection of the analyte (HRP) at the same concentration by usingthe orientation-controlled layer than the conventional ELISA.

Comparing the assay results from antibody layers, the maximumdifference was observed at the antibody concentration of 600 ng/mlas shown in Fig. 3(c). When an orientation-controlled antibodylayer and a randomly oriented antibody layer were prepared at thisconcentration, the orientation-controlled antibody layer showedthe limit of detection improved by 30-fold as shown in Fig. 3(d).This result means that the maximum effect of the orientation con-trol could be estimated to be as much as 30-fold in comparison tothe randomly oriented antibody layer. Additionally, the saturatedassay response from the orientation-controlled antibody layer (�)was observed to be far higher than the randomly oriented one (�) asshown in Fig. 3(d). Such difference in response could be explainedfrom the difference in the number of analyte-binding sites. Sincethe same concentration of antibodies was used for the immobiliza-tion, such difference could be considered to have resulted from theorientation control effect.

The low non-specific binding of proteins to the outer membraneof E. coli was analyzed to contribute in improving the sensitivity.As the non-specific binding of proteins usually results in a falsepositive signal in immunoassays, it should be reduced as low as pos-sible [8]. In this work, the response by the non-specific binding to

the outer membrane layer was evaluated by using the outer mem-brane layer (from intact E. coli) without autodisplayed Z-domain asa negative control. When the same concentration of anti-HRP anti-bodies were treated, the response to the intact outer membrane(�) was observed to be insignificant in comparison to the outer

M. Park et al. / Enzyme and Microbial Technology 46 (2010) 309–314 313

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ig. 4. CRP detection with an orientation-controlled antibody layer (�) and a ranonfiguration, (b) comparison of assay response by treatment of CRP at the concentifference of the assay response between antibody layer with controlled-orientatioith controlled-orientation and randomly oriented antibody layer at the antibody c

embrane layer with autodisplayed Z-domain (�) at the wholenalyte concentration range as shown in Fig. 3(d). These resultshow that the non-specific binding to the outer membrane layers insignificant. Usually, the hydrophilic surfaces were known toeduce the non-specific binding of proteins for immunoassay [19].s mentioned previously, the outer membrane of E. coli was known

o be negatively charged [16], and the zeta-potential of the intact E.oli (negative control) and the E. coli with autodisplayed Z-domainere actually measured to be −22.5 and −25.1 mV, respectively.

herefore, the significantly low non-specific binding of E. coli wasetermined to have resulted from the hydrophilicity of the E. coli’suter membrane.

.3. Application to the detection of C-reactive protein

The new immunoassay based on the microplate with the outerembrane layer was applied to the medical diagnosis by the detec-

ion of C-reactive protein (CRP) which was known to be a biomarkerrotein of the inflammatory diseases such as rheumatoid arthri-is. As shown in Fig. 4(a), anti-CRP antibodies were immobilizedo the microplate with the outer membrane layer. The randomlyriented antibody layer was prepared by physical adsorption ofntibodies as the conventional ELISA. The outer membrane layerf the intact E. coli was prepared as a negative control to estimatehe non-specific binding (�) of antibodies. For the evaluation of therientation control effect, the concentration of antibodies requiredor the detection of CRP at a concentration of 25 ng/ml was mea-ured by using the orientation-controlled antibody layer (�) andhe randomly oriented antibody layer (�). As shown in Fig. 4(b),

he minimum antibody concentration required for the detection ofhe CRP (10 ng/ml) was estimated to be 30 and 300 ng/ml for thentibody layers with the controlled-orientation by Z-domains (�)nd the random-orientation (�), respectively. These results showhat a 10-fold lower antibody concentration was required for the

oriented antibody layer as the conventional ELISA (�). (a) Schematics for assayof 1 �g/ml according to the concentration of immobilized anti-HRP antibodies, (c)randomly oriented antibody layer and (d) assay response between antibody layertration of the maximum assay response difference (450 ng/ml).

detection of the analyte (HRP) at the same concentration using theorientation control in comparison to the conventional ELISA.

The maximum difference of the assay result was observed at theantibody concentration of 450 ng/ml as shown in Fig. 4(c). When themicroplate was coated with anti-CRP antibodies at this concentra-tion, the limit of detection was estimated to be 1.5 and 25 ng/ml forthe orientation-controlled antibody layer and the randomly ori-ented antibody layer, respectively. As shown in Fig. 4(d), the limitof detection with controlled-orientation was estimated far higherthan the microplate with randomly oriented antibody layer. Suchimprovement in the sensitivities represents the increase of analyte-binding sites by the orientation control effect of the autodisplayedZ-domains. From the negative control experiment by using outerthe membrane layer from the intact E. coli, the non-specific bind-ing of proteins was maintained to be insignificant at the wholedetection range. These results represent that the new immunoassaybased on the outer membrane layer with autodisplayed Z-domaincould be applied for immunoassays requiring far lower limit ofdetection as well as far higher sensitivity than the conventionalELISA without orientation control of antibodies.

4. Conclusions

The Z-domain of protein A was autodisplayed on the surfaceof the E. coli outer membrane as a fusion protein of AIDA-1, andthe E. coli outer membrane with autodisplayed Z-domains werelayered on a microplate for the development of a hypersensitiveimmunoassay. In order to explain the role of hydrophobic inter-action during the formation of the outer membrane layer, the

outer membrane layer was prepared on several microplates with acontrolled hydrophobicity. From this experiment, the outer mem-brane layers were observed to be much dense, proportional tothe hydrophobicity of surfaces. The most hydrophobic surface pre-sented the highest IgG-binding activity. From this work, the driving

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[18] Nilsson B, Moks T, Jansson B, Abrahmsen L, Elmblad A, Holmgren E, et al. Asynthetic IgG-binding domain based on staphyloccal protein A. Protein Eng

14 M. Park et al. / Enzyme and Mic

orce for the layer formation was proven to be a hydrophobicnteraction. The effect of such orientation control by the autodis-layed Z-domain was estimated by the analyte-binding activity ofhe immobilized antibodies compared to the conventional ELISAased on randomly oriented antibody layer. In this work, the min-

mum antibody concentration required to detect a HRP sample10 ng/ml) was estimated to be 3 and 45 ng/ml for the antibodyayers with the controlled-orientation by Z-domain (�) and theandom-orientation (�), respectively. At the optimal antibody con-entration which was evaluated using this new immunoassay, theimit of detection (LOD) and the sensitivity were determined toave improved as much as 30-fold compared to the conventionalLISA. The applicability of the new immunoassay for medical diag-osis was demonstrated by the detection of C-reactive proteinCRP), which is known to be a biomarker protein for inflammatoryiseases. From this work, the limit of detection was improved toe as much as 15-fold by using the orientation-controlled antibody

ayer. These results represented that the new immunoassay basedn the outer membrane layer with autodisplayed Z-domain coulde applied for immunoassays requiring far lower limit of detections well as far higher sensitivity than the conventional ELISA withoutrientation control of antibodies.

cknowledgements

This research was supported by the National Research Foun-ation of Korea (NRF) funded by the Ministry of Education,cience and Technology (2009-0062890, R15-2004-024-00000-0nd 2009-008-1529) and by “System IC 2010” project of Ministryf Knowledge Economy and by the Seoul Research and Businessevelopment Program (10816).

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