ANALYTICAL

Analytical Biochemistry 322 (2003) 156–163

BIOCHEMISTRY

www.elsevier.com/locate/yabio

Monitoring of the heat-shock response in Escherichia coli usingan optical biosensor

Igor Vostiar,1 Jan Tkac,2 and Carl-Fredrik Mandenius*

Division of Biotechnology, Department of Physics and Measurement Technology, Link€ooping University, 581 83 Link€ooping, Sweden

Received 29 April 2003

Abstract

A surface plasmon resonance (SPR) method for monitoring the concentration of the chaperone DnaK and its relation to

physiological stress response in a recombinant Escherichia coli strain subjected to heat shock is described. The DnaK protein, an

abundantly occurring representative of the heat-shock proteins, was used as a marker of physiological stress. The SPR biosensor

instrument was used for label-free immunoaffinity detection directly in cell culture lysates using an anti-DnaK monoclonal IgG

antibody immobilized on the sensor surface. The SPR method provides a fast response (<8min) and a reproducible (RSD < 2%),

accurate (comparison to the direct enzyme-linked immunosorbent assay), and sensitive (LOD < 1 nM) assay for determination of

the DnaK level in cell culture lysates. The operational stability of the method was high compared to that of other SPR assays; the

sensitivity decreased at only 2.7%/h. This allowed measurement of more than 220 samples per sensor surface. Storage stability was

determined at 25 �C (100% after 17 h) and 10 �C (101% after 1month). The method was validated by standard additions of DnaK

(30, 60, and 120 nM) with recovery indices in the range 95.7–103.7%.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Surface plasmon resonance; Heat shock response analysis; Stress response sensor; DnaK analysis; Escherichia coli

Bacterial cells have developed efficient protection sys-tems to copewith a variety of physiologically unfavorable

conditions. One of the best studied is the heat-shock re-

sponse, used by the cell to adapt to different physiological

stresses [1,2]. The effect of the response is a fast transient

increase of the cell�s biosynthesis of ATP-dependent

proteases and molecular chaperones [3], which facilitate

appropriate folding or cleavage of misfolded proteins. Of

these, the DnaK–DnaJ–GrpE and GroEL–GroES chap-erone systems in the cytoplasm of Escherichia coli and

other bacteria are considered the most important [4,5].

Although the heat-shock response in bacterial cells is

intensively studied, mainly semiquantitative immuno-

electroblottingmethods have beenused for determination

of the intracellular concentration of the key heat-shock

protein DnaK. Recently, methods based on two-dimen-

* Corresponding author. Fax: +46-13-122587.

E-mail address: cfm@ifm.liu.se (C.-F. Mandenius).1 Present address: Department of Biochemical Technology, Faculty

of Chemical and Food Technology, Slovak University of Technology,

Radlinskeho 9, 812 37 Bratislava, Slovak Republic.2 Present address: Institute of Chemistry, Slovak Academy of

Sciences, Dubravska cesta 9, 842 38 Bratislava, Slovak Republic.

0003-2697/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2003.07.019

sional electrophoresis with matrix-assisted laser desorp-tion ionization time-of-flightmass spectrometry detection

[6], N-terminal protein sequencing [8], or detection of

[L-35S]methionine-labeled proteins have been described

in the literature [7–9]. Also use of the green fluorescent

protein gene [10] and the lux gene [11,12] fused to heat-

shock promoters are reported to allowmonitoring of gene

expression under the control of this promoter. In addi-

tion, DNA-microarray technology has successfully beenused for expression profiling of heat-shock genes in bac-

terial cell cultures subjected to physiological stress [13,14].

Here we describe a label-free method for quantitative

detection ofDnaK directly in the bacterial cell lysate. The

method is based on the use of a surface plasmon resonance

(SPR)3 biosensor system (Biacore). The SPR technology

has previously successfully been used to study the

3 Abbreviations used: SPR, surface plasmon resonance; ELSIA,

enzyme-linked immunosorbent assay; EA, ethanolamine; NHS,

N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-car-

bodiimide; HBS, Hepes buffer-solution; TMB, tetramethylbenzidine;

TES, trace element solution; HRP, horseradish peroxidase; BSA,

bovine serum albumin; PBS, phosphate-buffered saline.

I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163 157

interaction of the DnaK chaperone with its cochaperoneDnaJ [15,16] and to elucide the molecular mechanism of

the chaperone GroEL [17]. However, a selective method

for detection of DnaK directly in the cell lysate with SPR

is here described for the first time. The method was tested

on an Escherichia coli culture under heat stress. The SPR

data were compared with data acquired using conven-

tional bioanalytical methods (Western blotting, ELISA).

The SPRmethod proved to be sensitive and fast, allowingreproducible off-line monitoring of the intracellular

DnaK level.

Experimental

Materials

DnaK solution (0.9 gL�1) was obtained from Stress-

Gen Biotechnologies (Victoria, Canada) and anti-

DnaK monoclonal antibody solution (1.4 gL�1) from

Calbiochem (CA, USA). Ethanolamine (EA), N-hy-

droxysuccinimide (NHS), 1-ethyl-3-(3-dimethylamino-

propyl)-carbodiimide (EDC), sensor chip CM5 (research

grade), Hepes buffer solution (HBS; 10mMHepes (N-[2-

hydroxyethyl]piperazine-N 0-[2-ethane sulfonic acid]),150mM NaCl, 3.4mM EDTA, 0.005% Tween 20, pH

7.4), coupling buffers (10mMacetate buffers, pH4.5–5.5),

and regeneration buffers (10mMglycine–HCl buffers, pH

1.5–3.0) were purchased from Biacore International AB

(Uppsala, Sweden). Protease inhibitor cocktail tablets

(Complete Mini EDTA–free) were obtained from Roche

(Mannheim, Germany) and bacterial extraction reagent

CelLytic was provided by Sigma (MO, USA).Tris(hydroxymethyl)aminoethane (Tris), Hepes, hy-

drochloric acid, sodium hydroxide, sulfuric acid, and

acetic acid were purchased from Merck (Germany).

Immunoblot (Western blot) and ELISA assays were

performed using goat anti-mouse IgG labeled with

horseradish peroxidase (HRP). 4-Chloro-1-naphthol and

hydrogen peroxide (Bio-Rad, CA, USA) were used as

substrates for immunoblot assay while tetramethylbenz-idine (TMB) in combination with hydrogen peroxide

(RD, MN, USA) was used for ELISA. Prestained mo-

lecular standards (broad range; 6.4–198 kDa) and all

other reagents used for Western blot were obtained from

Bio-Rad. Polystyrene microtiter plates were purchased

from Nunc (Denmark).

E. coli cultivation

Recombinant E. coli strain HMS174(DE3) (pET11a/

rhSOD) [18] was used as a model culture to establish a

method for determination of DnaK protein in the cell

lysate. The medium consisted of (g L�1): glucose 16.0,

KH2PO4 3.0, K2HPO4 � 3H2O 6.0, yeast extract 0.05,

trypticase 1.0, MgSO4 � 7H2O 0.5, CaCl2 � 2H2O 0.05,

sodium citrate � 2H2O 1.25, (NH4)2SO4 2.25, NH4Cl 1.85,TES 250 ll, CuCl2 � 2H2O 0.02, ZnSO4 � 7H2O 0.017. The

TES consisted of (g L�1) FeSO4 � 7H2O 40.0, MnSO4 �H2O 10.0, AlCl3 � 6H2O 10.0, CoCl2 4.0, ZnSO4 � 7H2O

2.0, Na2MoO2 � 2H2O 2.0, CuCl2 � 2H2O, 1.0, andH3BO3

0.5 in 5M HCl.

The cells were grown overnight at 30 �C in 500-ml

flasks containing 100mlmedium to reach adesired optical

density (OD600 ¼ 1:0). Subsequently, the cultivationtemperature was shifted to 42 �C. Samples were taken out

through a sampling device that allowed aseptic sampling

during the cultivation. Samples were immediately pre-

treated as follows: 1ml of sample was centrifuged for

3min at 10,000g and 4 �C. Cell sediment was resuspended

in 150 ll of lysing buffer containing 100 ll of TEN buffer

(50mMTris buffer, pH7.4, 3mMEDTA, 0.9%w/vNaCl,

and 1 tablet of protease inhibitor per 10ml of the buffer)and 50 ll of detergent. The suspension was allowed to

react for 15min at 22 �C with intermittent mixing. Sub-

sequently, the mixture was centrifuged for 10min at

10,000g and 4 �C and the supernatant was collected and

stored at 4 �C until measured.

SPR measurements

All experiments were performed using a Biacore 2000

instrument. For optimization of the immobilization

procedure, a preconcentration test using 10mM acetate

coupling buffers with pH in the range from 4.5 to 5.5

was performed. Running buffer (HBS) was used at a

flow rate of 10 ll min�1, when not stated otherwise.

Other buffers were used after filtration through a 0.2-llfilter and degassing; 10mM glycine buffers (pH 1.5–3.0)were tested for regeneration.

The Biacore evaluation software (Version 3.0) was

applied for estimation of the concentration of DnaK.

The response from the reference channel with inacti-

vated anti-DnaK or rabbit anti-myoglobin was sub-

tracted from the sensor response.

Surface immobilization

Anti-DnaK was immobilized on the CM5 (carbo-

xymethylated dextran-modified gold-surface) chip using

a standard amino coupling kit. A 35-ll aliquot of a mix-

ture of EDC (200mM) and NHS (50mM) (1:1) was in-

jected at a flow rate of 5 ll min�1. After the surface

activation, 70 ll of anti-DnaK (45 lgml�1) solution was

injected and followed by a deactivation of the sensorsurface using an ethanolamine solution (1M; pH 8.5;

35 ll). After immobilization, several 5-ll injections (usu-ally 10) of the regeneration buffer (pH 2.0) were used to

wash out noncovalently bound antibody until a stable

baseline was obtained. A reference surface immobilized

with an irrelevant antibody to compensate for nonspecific

binding effects was used as detailed below.

158 I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163

Western blot assays

Electrophoresis of samples was run according to

Laemmli [19]. Proteins were transferred to nitrocellulose

membrane for 45min (0.8mA cm�2) using a semidry

electroblotter (GelmanSciences, Denmark), blocked

with gelatin (3% w/v gelatin in TBS (20mM Tris,

500mM NaCl, pH 7.5)), and washed with TTBS

washing buffer (20mM Tris, 500mM NaCl, 0.05%Tween 20, pH 7.5). In the next step the nitrocellulose

membrane was incubated with primary antibody (dilu-

tion 1/1000 in 1% w/v gelatin solution in TTBS) for 1 h,

washed with TTBS washing buffer, and incubated with

secondary antibody–HRP conjugate (diluted 1/3000 in

1% w/v gelatin solution in TTBS) for 1 h. Membrane

was washed with distilled water and developed in the

substrate solution, and densitometric data were evalu-ated using the Image processing toolbox in MATLAB

6.1 software (The MathWorks, USA).

ELISA

A direct ELISA protocol for DnaK was developed

for use as a reference method. The microtiter plates were

precoated with appropriately diluted samples and stan-dards (up to 1.5 nM) (50 ll per well) in sodium bicar-

bonate buffer (50mM, pH 9.6), incubated at laboratory

temperature for 2 h, and blocked with BSA (1% w/v in

PBS (100mM phosphate buffer, 500mM NaCl, pH 7.4))

(50 ll per well) for 30min. In the next step wells were

filled with anti-DnaK monoclonal antibody solution

(dilution 1/1000 in PBS–Tween antibody buffer (100mM

phosphate buffer, 500mM NaCl, 0.1% w/v BSA, 0.05%v/v Tween 20, pH 7.4)) (30 ll per well) and incubated at

22 �C for 2 h. Antibody–HRP conjugate solution (dilu-

Fig. 1. Effect of extraction time (A) and extraction volume (B) on the efficiency

centrifuged and resuspended in 100ll HBS containing protease inhibitor. (A

mixed with 50–200ll detergent incubated 15min. DnaK content in cell lysa

tion 1/2 000 in PBS–Tween antibody buffer) (50 ll perwell) was then added and incubation continued at lab-

oratory temperature for 1 h. Between all the mentioned

steps, a five-cycle washing procedure using PBS–Tween

washing buffer (100mM phosphate buffer, 500mM

NaCl, 0.05% v/v Tween 20, pH 7.4, 5� 200 ll per well)was adopted. Finally, substrate solution (0.41mM TMB

and 1.8mM H2O2; 100 ll per well) in acetate buffer

(50mM; pH 5.2) was added into each well, and plateswere incubated for 30min in the dark at 22 �C. The

enzymatic reaction was stopped after 10–30min by ad-

dition of H2SO4 solution (1M; 50 ll per well). The

plates were read and recorded at 450 nm after 25min.

Results and discussion

Culture sample pretreatment

The DnaK assay included a pretreatment procedure

for the culture samples. Samples from the E. coli culti-

vations were disintegrated using a nonionic surfactant

detergent. This approach has advantages compared to

typical disintegration procedures in that since the mild

nonionic detergent efficiently disaggregates the cellcomponents at room temperature, this approach does

not require expensive disintegration equipment and has

the potential to be used with microfluidic systems for

on-line application of intracellular analysis [20].

To improve the performance of the pretreatment

procedure, the extraction time and detergent volume

were first optimized. The concentration of DnaK pro-

tein released from the cells reached a steady state valueafter 15min of extraction, implying that an extraction

time of 15min would be sufficient (Fig. 1A). The rate of

of DnaK release from E. coli cells. One milliliter of cell suspension was

) Samples mixed with 50ll detergent incubated 5–60min. (B) Samples

tes was measured with SPR.

Fig. 2. Influence of the detergent concentration on DnaK binding to

anti-DnaK as analyzed by SPR. Samples of 30 nM DnaK were diluted

in HBS with detergent in the range 0–10% (v/v).

Fig. 3. Influence of pH on the affinity binding of DnaK to immobilized

anti-DnaK. Samples of 30 nM DnaK in HBS at pH 6.0–8.5 were an-

alyzed by SPR.

I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163 159

release of DnaK paralleled the release of other intra-cellular E. coli proteins.

The significance of the amount of detergent added to

the cells was evaluated by addition of varying volumes

of detergent (50–200 ll) to a wet cell mass of 1ml of an

E. coli suspension (OD600 ¼ 1:0). The efficiency of ex-

traction of DnaK was almost linearly proportional to

the detergent volume (Fig. 1B). Despite this, only 50 llof detergent was used in the subsequent work; this wasnecessary to allow higher concentrations of DnaK in

samples to be injected into the SPR instrument which

required extensive dilution to minimize the negative ef-

fect of the detergent on the operational stability (see

below). The pretreatment of the E. coli culture samples

showed high reproducibility (RSD¼ 1.8%; from five

independent extractions of a sample measured in tripli-

cate). No significant changes in DnaK content werefound (RSD < 5%) after storage of samples at 10 �C for

5 days.

Immobilization of anti-DnaK

The ability of the anti-DnaK ligand to diffuse into the

dextran matrix on the SPR chip surface (preconcentra-

tion) was investigated in the pH range 4.5–5.5. The ex-tent of preconcentration is an effect of the balance

between the negatively charged carboxy groups of the

dextran matrix and the positively charged groups of the

protein. The pH suitable for immobilization is that value

at which the degree of ionization of the dextran matrix

optimally attracts the charged protein molecule. The

best preconcentration effect for anti-DnaK was obtained

at pH 5.0 when using a 10mM acetate buffer. Underthese conditions anti-DnaK coupled to a level of

�15,000 resonance units (RU; where 1000 RU corre-

sponds to 1.0 ngmm�2) which was sufficient for attain-

ing sensitive determination of DnaK.

Effect of buffer composition on DnaK binding

The influence of the pretreatment detergent on theaffinity binding of the DnaK–anti-DnaK complex at the

SPR surface was studied in detail. In this study we found

that lower concentrations of the detergent increased the

SPR response of DnaK in standard solutions, whereas

higher concentrations had a reverse effect (Fig. 2). The

corresponding concentration of detergent that was

present in the culture samples was added to pure DnaK

solutions to calibrate the content in the culture samples.Reproducibility of the DnaK standard measurements in

the presence of detergent resulted in an RSD of 0.9%

(n ¼ 12).

The affinity binding of DnaK to the immobilized

anti-DnaK was investigated in the pH range 6.0–8.5,

and pH 6.5 was found to be optimal (Fig. 3). Buffers of

10mM Hepes containing 3mM EDTA with a NaCl

concentration ranging from 0 to 200mM were use to

study the effect of the ionic strength on the binding(Fig. 4). Increasing ionic strength had a positive effect on

the binding efficiency of DnaK in both sample and

standard solutions. This was ascribed to hydrophobic

interaction effects between DnaK and anti-DnaK, which

normally are promoted by higher salt concentration.

However, high ionic strength of the buffer may also re-

sult in a high incidence of nonspecific interaction be-

tween cell lysate components. To minimize this, a buffercontaining 150mM NaCl and Tween 20 (0.005% v/v)

was tested. Surprisingly, the response to a real sample

diluted in the Tween 20-containing buffer was signifi-

cantly higher than that to a sample diluted in buffer

without the detergent (Fig. 4). A possible explanation

Fig. 4. Influence of the ionic strength on the binding of DnaK to im-

mobilized anti-DnaK. DnaK standards and cell lysate samples were

diluted in HBS at pH 7.4 with NaCl concentrations 0–200mM ( , cell

lysate; , 30 nM DnaK).

160 I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163

might be that Tween 20 diminished the interaction of

DnaK with the other components of lysate. Such an

interaction could hinder DnaK binding to anti-DnaK

and reduce the response with a buffer without Tween 20.The DnaK binding was unaffected by the pH in the

range 6.5–8.0 with HBS (pH 7.4; containing 150mM

NaCl, 0.005% v/v Tween 20) which was used for further

study described below.

Assay of DnaK

After immobilization of anti-DnaK, the sensor sur-face was washed by several injections of buffer to desorb

noncovalently bound antibody. The sensor was cali-

brated with DnaK standard solutions ranging from 2 to

Fig. 5. Sensorgram (A) and calibration curve (B) for DnaK (2–320 nM) from S

surface with immobilized anti-DnaK with a consecutive regeneration step u

beginning and the end of a standard injection. Graph insert represents linea

320 nM (Fig. 5) and exhibited a linear dependence in therange from 2 to 60 nM (sensitivity 2.60RUnM�1).

Taking into account the noise of measurement (�1RU),

the limit of detection (S=N ¼ 3) is lower than 1 nM.

Only an efficient regeneration procedure of the chip

surface allows repeatable, reproducible, and highly

precise measurements. An optimal regeneration proce-

dure should efficiently wash away affinity-bound DnaK

while the binding capacity of the surface should bepreserved to ensure sensitivity and reproducibility of the

assay.

Four 10mM glycine buffers of different pH (pH 1.5,

2.0, 2.5, and 3.0) were used for evaluating the regen-

eration of the surface. The regeneration buffers with pH

2.5 and 3.0 had not sufficient capacity to restore the

baseline which increased after repeated injections of

DnaK. The degree of regeneration and remaining sur-face binding activity were significantly below 100%.

However, by using a glycine buffer of pH 2.0, regen-

eration and surface activity were close to 100%. The

regeneration buffer per se had a marginal effect on the

surface activity. After 12 injections of DnaK standard

with consecutive regeneration, no significant decrease in

response was observed and the baseline remained stable

after regeneration (Fig. 6). When 10mM glycine buffer(pH 1.5) was used, the baseline response after regen-

eration decreased to a negative value, probably due to

the leakage of bound anti-DnaK, and resulted in a

gradual decrease of sensitivity. For further measure-

ments, glycine regeneration buffer of pH 2.0 was

therefore used.

It is important to decide whether the objective for the

assay is precision or speed of analysis. The measurementsensitivity of DnaK standard (and thus precision) and

the analysis time were proportional to the amount

(volume) of standard injected (Table 1). The SPR system

PR analysis. DnaK standards (30-ll pulses) were injected over the chip

sing 5-ll pulses of 10mM glycine buffer (pH 2.0). Arrows indicate the

r region of the calibration curve.

Fig. 6. Stability of the sensor response after repeated 30-ll DnaK

standard (60 nM) injections with a consecutive regeneration step using

5-ll pulses of 10mM glycine buffer (pH 2.0); (–s–) baseline level re-

generation; (–d–) sensor response after repeated DnaK injections.

Table 1

Influence of injected volume of DnaK (60 nM) on sensitivity of mea-

surement and time of analysis

Injected volume

(ll)Response (RRU) Time of analysis

(min)

5 9.0� 1.3 5.3

10 19.6� 0.6 5.8

20 38.3� 0.3 6.8

30 55.0� 0.9 7.8

40 70.2� 0.1 8.8

50 84.4� 0.2 9.8

The surface was regenerated by injection of 5ll of 10mM glycine

buffer (pH 2.0); flow rate¼ 10ll min�1.

Fig. 7. Operational stability of the SPR assay of DnaK.

I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163 161

has a dead time of 4.8min (time needed for sample and

regeneration buffer injection by the autosampler to

reach the detector). An injection volume of 30 ll ofstandards and culture samples was used for further work

(a compromise between sensitivity and analysis time).

Operational and storage stability

Operational stability was evaluated for the DnaK

standards and culture samples (Fig. 7). When DnaK

standards were injected in the absence of detergent thesensitivity decrease was only 0.4%h�1, but in its pres-

ence more rapid decrease in the sensitivity (2.7% h�1)

was observed. During measurement of culture samples,

an intermittent DnaK standard solution was injected

after every 11 samples for recalibration (eight samples/

h). Immediately after immobilization the sensitivity

dropped sharply (7.1%h�1) but within 15 h of mea-

surement the decrease was stabilized (2.3%h�1) and re-mained so for more than 35 h. The initial sharp

sensitivity decrease was ascribed to desorption of non-

covalently bound antibody. The subsequent sensitivity

decrease (2.3%h�1) is close to the one measured withdetergent-containing DnaK standards of the same

amount as in the culture samples (2.7%h�1). This indi-

cated that the detergent contributed significantly to

surface inactivation, while the cell lysate had only a

marginal effect, perhaps due to efficient inhibition of its

proteolytic activity.

Storage stability of the anti-DnaK-immobilized sur-

faces was high when the chip was stored at 25 �C inHBS. The sensitivity remained at 100% of the initial

value for 17 h. When the chip was stored at 10 �C, thesensitivity after 1 month of storage was 101% compared

to initial. The good stability properties allowed analysis

of more than 220 samples.

Measurement of culture samples

Culture samples were diluted in HBS (1:10) and im-

mediately analyzed by SPR, Western blot, or ELISA.

Measurements using the SPR biosensor were highly re-

producible (n ¼ 3; 22 samples; average RSD¼ 3.4%).

Samples taken 30min before and 90 min after induction

of stress were spiked after primary extraction with three

concentrations of DnaK standard (30, 60, and 120 nM).

Recovery index of added DnaK was not dependent onthe amount of standard DnaK added to the sample and

was close to 100% (Table 2).

The SPR method was validated by a comparison with

Western blotting and ELISA. Western blotting has

previously been used for assaying concentration of heat-

shock proteins in bacterial lysates [10]. Despite its

advantages, results are often erroneous due to low re-

producibility, partly because of electroblotting effi-ciency. Repeated assays of DnaK in culture samples

were not reproducible (RSD6 50%; n ¼ 4), thus pro-

viding only semiquantitative information, and were not

suited to use as a reference. The cross-reactivity of the

Table 2

Recovery index of samples taken from fermentation stressed by tem-

perature up-shift

Concentration of internal

standard

Recovery index (%)a

)30minb 90minb

30 nM DnaK 103.7� 2.7 99.8� 3.0

60 nM DnaK 97.6� 1.4 97.9� 2.2

120 nM DnaK 97.8� 1.9 95.7� 0.9

aRecovery index ð%Þ ¼ ½ðR1� R2Þ=R3� � 100; R1, response for

cell lysate sample spiked with DnaK standard solution; R2, responsefor cell lysate sample; R3, response for DnaK standard containing

detergent amount equivalent to the content in the cell lysate samples.b Time relative to the stress induction.

Fig. 9. Comparison of the SPR signal during typical injection cycles of

E. coli cell lysate. Arrows indicate start (1) and end (2) of the injections

of sample and regeneration buffer (3) (- - - response channel with im-

mobilized anti-DnaK; – � – � – response reference channel with an ir-

relevant immobilized antibody; ––––– differential response).

Table 3

Comparison of ELISA and SPR method for DnaK assay

ELISAa SPR

Limit of detection (nM) 0.05 1.0

Dynamic range of method (nM) 1.5 5000

Average RSD of method (%) 8.5 3.4

a ELISA method was not fully optimized.

162 I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163

monoclonal antibody was tested with Western blot as-

says for DnaK in total cell lysates. Cell lysate samples

resulted in one sharp band, with the same retention on

the gel as that of the DnaK standard (Fig. 8A). The

molecular weight was estimated at 69 kDa based onrelative mobility. This value is in a good agreement with

the published data [4]. No significant cross-reactivity

with other components in the cell lysate, which would

negate ELISA and SPR detection, was noted, although

cross-reactivity with the components in native confor-

mation could not be completely excluded. Furthermore,

bacterial proteins can also interact with conserved IgG

regions, resulting in erroneous positive results. The IgGcross-reactivity with a monoclonal rabbit anti-myoglo-

bin antibody was also tested by SPR as a reference. No

binding of cell lysate to anti-myoglobin immobilized in

Fig. 8. DnaK determination in the cell lysate of E. coli exposed to heat shock. (A) Comparison of densitometric curves of electrophoreograms of the

lysate stained with Coomassie blue (2), Western blot assay of DnaK in cell lysate samples taken at )30, 0, 30, and 60min relative to heat shock (3–6)

and molecular weight standards (1). (B) Correlation of DnaK content in the lysate measured with SPR and the ELISA reference method (stressed

culture: –s– SPR, –�– ELISA; reference culture: –d– SPR, –j– ELISA).

I. Vostiar et al. / Analytical Biochemistry 322 (2003) 156–163 163

the reference channel of the chip was observed, whereasthe anti-DnaK antibody immobilized on the measure-

ment channel bound significantly (Fig. 9). As an alter-

native approach to eliminate nonspecific responses, anti-

DnaK immobilized in the reference channel was dena-

tured with 7M guanidine chloride. However repeated

injections of cell lysates gradually increased the SPR

response, as did also injections of DnaK standards. This

may be explained by refolding of denatured antibodyrestoring the DnaK binding activity.

The cross-reactivity test verified the specificity of the

interaction of anti-DnaK with DnaK in complex ma-

trices and showed that nonspecific effects will not sig-

nificantly influence the detection of DnaK in complex

culture samples.

The ELISA method quantitated DnaK in cell lysate

samples with a relative standard deviation of 8.5%,which was significantly better than that with Western

blotting (Table 3). The method�s limit of detection was

0.05 nM and the dynamic range reached up to 1.5 nM.

The DnaK concentration roughly correlated with Wes-

tern blot assays in E. coli culture exposed to heat stress

(Fig. 8A). The ELISA results were compared with SPR

in culture samples and showed good agreement (Fig. 8B).

The average relative error of SPR compared to ELISAwas 7.1% (22 culture samples analyzed in triplicate).

The amount of DnaK after heat-shock treatment of

the culture by a temperature increase (30–42 �C) was

detected in the cell lysate. The DnaK raised by 80%

compared to the levels at 30 �C and rapid increase of the

DnaK concentration reached a new steady state value

approximately 30min after the temperature shift to

42 �C. A nonstressed control culture, grown at 30 �C,showed no detectable concentration increase.

The observed good correlation of the SPR assay for

analysis of DnaK in the cell culture lysates with ELISA

and Western blotting suggests that SPR monitoring is a

preferable alternative for analyzing intracellular con-

centrations due to its practical convenience. In addition,

the SPR assay is easily automated, more precise, and less

laborious than Western blotting or ELISA.

Acknowledgments

This work was partly supported by an EU Marie

Curie Fellowship (HPMT-CT-2000-00130) (I.V.).

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