www.elsevier.com/locate/jim

Journal of Immunological Methods 284 (2004) 107–118

Immuno-SLM—a combined sample handling

and analytical technique

Madalina Tudorachea, Mariusz Rakb, Piotr P. Wieczorekb,Jan Ake Jonssona, Jenny Emneusa,*

aDepartment of Analytical Chemistry, Lund University, P.O. Box 124, 221 00 Lund, Swedenb Institute of Chemistry, University of Opole, Oleska 48, 45-052, Poland

Received 3 February 2003; received in revised form 3 October 2003; accepted 22 October 2003

Abstract

Immuno-supported liquid membrane (immuno-SLM) extraction is a new technique that makes use of antibody (Ab)–

antigen interactions as the ‘‘extraction force’’ to drive the mass transfer in a selective way. In immuno-SLM, anti-analyte (Ag)

Abs are introduced into the acceptor phase of the SLM unit to trap the Ag that passes from the flowing donor through the SLM

into the stagnant acceptor. The amount of immuno-extracted analyte (AbAg) is quantified by connecting the immuno-SLM unit

on-line with a non-competitive heterogeneous fluorescence flow immunoassay (FFIA) that makes use of a fluorescein-labeled

analyte tracer that titrates the residual excess of Ab present in the acceptor. A restricted access (RA) column is used for the

separation of the two tracer fractions (Ag* and AbAg*) formed, and the eluted AbAg* fraction is measured downstream by a

fluorescence detector.

Factors influencing the optimum immuno-SLM extraction parameters, i.e., donor flow rate, extraction time and type of Ab,

were investigated for immuno extraction of the model analyte atrazine. Immuno-SLM coupled to FFIA (immuno-SLM–FFIA)

and FFIA alone were compared in terms of the assay sensitivities obtained and the sample matrix influence. The concentration

at the mid-point of the calibration curve (IC50) was 16.0F 1.4 and 36F 16 Ag/l, the limit of detection (LOD) was 2.0F 1.1 and

20F 10 Ag/l, and the dynamic range was 2–100 and 20–500 Ag/l atrazine for immuno-SLM–FFIA and FFIA, respectively.

The matrix influence on the FFIA was significant in orange juice and surface water, whereas the influence was minor for

immuno-SLM–FFIA with recoveries between 104% and 115% for 5 Ag/l atrazine in tap water, orange juice and river water.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Immuno extraction; Immunoassay; Supported liquid membrane extraction; Fluorescein; Restricted access; Atrazine; Tap water;

Orange juice; River water

0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.jim.2003.10.014

Abbreviations: SLM, Supported liquid membrane; FFIA, Fluorescence flow immunoassay; SPE, Solid phase extraction; LLE, Liquid–

liquid extraction; LC, Liquid chromatography; PTFE, Polytetrafluoroethylene; RA, Restricted access; LOD, Limit of detection; IC50, Inhibition

concentration at 50%; PBS, Phosphate-buffered saline; EDF, Fluorescein thiocarbamyl ethylene diamine; FITC, Fluorescein isothiocyanate.

* Corresponding author. Tel.: +46-46-222-48-20; fax: +46-46-222-45-44.

E-mail address: jenny.emneus@analykem.lu.se (J. Emneus).

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118108

1. Introduction

The trace-level determination of pollutants in

complex environmental matrices (surface water, soil,

foodstuffs, etc.) is laborious and time-consuming

because sample pre-treatment procedures involve

many steps (Pichon et al., 1998). Many extractions,

based on, e.g., solid phase extraction (SPE) (Battista

et al., 1988; Yook et al., 1994; Sun et al., 1998) and

liquid– liquid extraction (LLE) (Muir and Baker,

1978; Lee and Stokker, 1986; Yrieix et al., 1996),

have been developed and are widely used in this

field. SPE is a modern extraction technique which

involves analyte (Ag) trapping by a solid sorbent

following elution of the analyte with an organic

solvent. In this way, separation of the analyte from

a complex matrix is achieved. Some drawbacks with

the SPE technique can be insufficient clean up of the

sample, insufficient retention of very polar com-

pounds, limited selectivity and high costs of dispos-

able sorbent materials. The SPE selectivity was found

to be greatly enhanced by using antibody–antigen

interactions in the extraction mechanisms, where an

immuno sorbent was used as a selective solid phase

extractor (Rivasseau and Hennion, 1999; Pou et al.,

1994). Immuno sorbents have been used for the

extraction of many different compounds from various

environmental matrices (surface water, wastewater,

soil, sediment, plant and foodstuff) (Thomas et al.,

1994; Pichon et al., 1998).

LLE is a classical technique for sample prepara-

tion of liquid samples. It provides a large potential

for tuning the extraction by pH adjustments, select-

ing solvents with specific properties and/or incorpo-

rating different reagents. LLE results in simultaneous

enrichment and clean up of samples, but involves

high consumption of organic solvent, and is difficult

to automate and to connect on-line with analytical

instruments. In order to eliminate the drawbacks of

LLE, a technique based on initial extraction of an

analyte from an aqueous sample (donor) into an

immobilized organic phase (organic membrane) fol-

lowed by re-extraction into a second aqueous phase

(acceptor) was developed, called supported liquid

membrane (SLM) extraction (Jonsson and Mathias-

son, 1999; Jonsson and Mathiasson, 2000). The

advantage of SLM extraction compared to other

LLE techniques consists of its capacity for simulta-

neous selective clean-up, concentration and extrac-

tion of the analyte.

In the present paper, a new combined extraction

and analytical technique called immuno-SLM–FIIA,

previously presented with preliminary results for 4-

nitrophenol (Thordarson et al., 2000), is characterized

for extraction and detection of the model analyte

atrazine. The technique is based on the selective

extraction of the analyte from an aqueous donor phase

over an organic liquid membrane (the SLM) into an

aqueous acceptor phase, containing soluble antibod-

ies. The amount of immuno-extracted analyte in the

acceptor is then quantified on-line by a fluorescence

flow immunoassay (FFIA). To verify the mechanism

of immuno-SLM extraction, the immuno-SLM–FFIA

results obtained have been compared with results

obtained with the FFIA alone, i.e., excluding the

immuno extraction step. Some theoretical aspects of

immuno-SLM are discussed and its selectivity and

applicability to measure the model analyte atrazine in

tap and river waters as well as in orange juice are

demonstrated.

2. Materials and methods

2.1. Materials and solutions

2-Chloro-4-ethylamino-6-isopropylamino-1,3,5-

triazine (atrazine) of 99.2% purity was purchased

from the Institute of Organic Industrial Chemistry

(Warsaw, Poland). A stock solution of atrazine (5

mg/l) was prepared in water (by stirring for 1

week) purified with a Milli-Q/RO4 unit (Milli-

pore, Milford, MA, USA) and kept at room

temperature.

Phosphate-buffered saline (PBS) was prepared by

mixing a basic solution (0.15 M Na2HPO4 and 0.15

M NaCl) with an acidic solution (0.15 M NaH2PO4

and 0.15 M NaCl) until reaching a pH of 7.4. All

buffer reagents were purchased from Merck (Darm-

stadt, Germany). Di-n-hexyl ether (97%, Sigma-

Aldrich, Steinheim, Germany) was used to impregnate

the polymeric membrane (PTFE). 0.5 M H2SO4 (95–

97% Merck, Darmstadt, Germany) was used as the

regeneration solution of the organic membrane (to

avoid any memory effect of analyte not extracted from

the organic membrane).

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118 109

Three different antibodies were obtained from two

different sources: Ab I (poly-IgG-anti-atrazine, from

rabbit, lot A9) and Ab II (poly-IgG-anti-atrazine, from

rabbit, lot ARINA P) were kindly provided by Dr.

Sergei Eremin (Lomonosov Moscow State University,

Russia) and Ab III (affinity purified anti-atrazine,

from sheep) was kindly provided by Dr. Ram Abu-

knesha (Kings College, London, UK). The antibody

stock solutions (1 mg/ml), prepared in 0.15 M PBS

(pH 7.4), were kept at + 4 jC and were diluted daily

with 0.15 M PBS (pH 7.4). A s-triazine hapten

derivative iPr/Cl/(CH2)5COOH, kindly provided by

Dr. Sergei Eremin, was used to prepare a fluorescein-

labeled tracer.

Fluorescein thiocarbamyl ethylene diamine (EDF)

was synthesized from fluorescein isothiocyanate

(FITC, isomer I, lot 18H2603, Sigma, St. Louis,

MO, USA) as described by Pourfazaneh et al.

(1980). The carboxylic group of the iPr/Cl/(CH2)5COOH hapten was reacted with EDF, forming a

fluorescent tracer, as described by Onnerfjord et al.

(1998a). The tracer stock solution in methanol was

kept at � 20 jC and the working tracer solutions were

prepared daily by diluting with 0.15 M PBS (pH 7.4).

The concentration of the tracer stock solution (5 AM)

was estimated spectrophotometrically at 492 nm,

assuming that the absorptivity in sodium bicarbonate

buffer (0.05 M, pH 9) was the same as for fluorescein

(8.78� 104 M� 1 cm� 1) Onnerfjord et al. (1998a). All

vials containing tracer were covered with aluminium

foil to protect the tracer against light.

Samples were tap water (Chemical Center, Lund

University, Lund, Sweden), river water (Hoje River, 2

km south of Lund, Sweden) and orange juice with

fruit parts (Monte Carlo, Arla, Stockholm, Sweden).

To remove particle matter from the samples, tap and

river waters were filtered (Millipore, 0.45 Am), where-

Fig. 1. The immuno-SLM–FFIA system: (1) SLM unit; (2, 5, 6) automatic

mixing coil; (8) restricted access column (C8); (9) fluorescence detector.

as the juice samples were first centrifuged and then

filtered. The pH of all samples was adjusted with

NaOH solution to pH 7.4.

2.2. System set-up and method

2.2.1. SLM unit

The SLM unit (Chemical Center Workshop, Lund

University, Lund, Sweden) consisted of two blocks of

inert material (one of PEEK and one of PTFE), each

with a machined groove (2.5� 0.1� 40 mm). When

the blocks were clamped together, a porous membrane

(PTFE, TE 35, Schleicher and Schuell, Dassel, Ger-

many, length 7.10 cm, width 0.60 cm with a 180-Am-

thick supporting polyester backing, 0.2 Am i.d. pore

size and 60–80% porosity) impregnated with the

organic solvent di-n-hexyl ether was held between

them. The membrane separated two identical channels

serving as donor and acceptor, respectively, with a

volume of approximately 10 Al each. The SLM

membrane was prepared by immersing the porous

polymer support membrane in the organic solvent

for 30 min (immobilization time), after which the

membrane was placed in the SLM unit as described

above. By pumping 3 ml water through each channel

(acceptor and donor), the excess of organic solvent

was removed from the SLM membrane surfaces,

making the unit ready for analyte extraction.

2.2.2. Instrumentation

The immuno-SLM–FFIA system is shown in

Fig. 1 and contained the following components:

SLM unit (1), a peristaltic pump (Minipuls 3,

Gilson, Villiers-le-Bel, France) (3), three syringe

pumps (P/N 50300 with six-port valve, Kloehn,

Las Vegas, NV, USA) (2, 5, 6), a 50-Al loop placed

on a manual six-port injection valve (Rheodyne,

syringe pumps; (3) peristaltic pump; (4) manual injection valve; (7)

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118110

California, USA) (4), a 250-Al mixing coil (1 mm

i.d. and 32 cm length) (7), a restricted access (RA)

column (PEEK, 2 mm i.d. and 2 cm length, packed

with LiChrospher 60, RP-8 ADS, 25 Am porosity

(Merck)) (8), and a fluorescence detector (L-7480,

Hitachi, Tokyo, Japan) (9), set at 515 nm excitation

wavelength and 490 nm emission wavelength. The

different parts of the flow set-up were connected

using PEEK tubing (0.25 mm i.d.) and finger tight

screw fittings.

2.2.3. Immuno-SLM–FFIA procedure

Fig. 1 shows the immuno-SLM system used. The

Ab solution (in 0.15M PBS pH 7.4) was dispensed into

the acceptor channel of the SLM unit (1) by a syringe

pump (2) after which the flow was stopped. The

sample, containing analyte (Ag = atrazine), was pump-

ed for 15 min by a peristaltic pump (3) with a constant

flow rate through the donor channel of the SLM unit

(1). The Ag molecules diffused from the donor over

the SLM membrane to the acceptor, in which they

formed antibody–analyte complexes (AbAg). Since

the Ab was present in excess, both free Ab and AbAg

complex were found in the acceptor after extraction.

The donor flow was then stopped and the acceptor

content (Ab and AbAg) was dispensed into the loop

of a manual injection valve (4) by a second syringe

pump (5). The injection valve (4) was switched and the

loop content was dispensed and mixed with a tracer

(Ag*) solution in excess, pumped with a third syringe

pump (6), in a mixing coil (7). Pumps (5) and (6) were

stopped for 5 min to allow efficient incubation between

the residual excess of Ab with excess of Ag*. After

incubation, the content of the mixing coil, now contain-

ing Ag +AbAg +AbAg* +Ag*, was passed through a

RA column (8) where the Ag and Ag* fractions were

trapped and theAbAgandAbAg* fractionswere eluted.

The AbAg* fraction was detected by a fluorescence

detector (9) where the analytical signal obtained was

inversely proportional to the Ag concentration in the

sample.

2.2.4. FFIA procedure

The FIIA procedure was identical to the immuno-

SLM–FFIA procedure, excluding the SLM extraction

step. Suitable concentrations of Ab and Ag were pre-

mixed and incubated for 15 min and then introduced

into the acceptor by the syringe pump (2), while the

donor channel contained pure buffer solution. The

subsequent assay steps were identical to those de-

scribed above for the immuno-SLM–FFIA procedure.

3. Results and discussion

Considering the basic principle of SLM extraction,

involving antigen–antibody interactions as the extrac-

tion driving force, the immuno-SLM technique was

introduced (Thordarson et al., 2000) in order to

improve SLM extraction selectivity. The SLM is a

nonporous membrane(Jonsson and Mathiasson,

2000), which contains an organic solvent (e.g., di-n-

hexyl ether, n-undecane or a combination of both

solvents) (Trocewicz, 1996; Chimuka et al., 1997;

Megersa et al., 2000) immobilized in the pores of the

support material (PTFE) by capillary forces. In

immuno-SLM, a continuous flow of sample, contain-

ing the Ag, is passed through the donor channel while

a stagnant solution of anti-analyte antibodies is pres-

ent in the acceptor. Uncharged analyte molecules are

transported by diffusion from the donor through the

SLM into the acceptor where the formation of strong

AbAg complexes takes place, thus preventing the Ag

from re-entering the membrane. Since the analyte is

found as AbAg in the acceptor and as Ag in the donor,

the mass transfer of the diffusing species (Ag) will be

unaffected by the total concentration of extracted Ag

in the acceptor channel, allowing a high degree of

analyte enrichment.

An on-line non-competitive FFIA system was de-

veloped to quantify the degree of immuno-SLM ex-

traction. The acceptor content, after extraction

(AbAg + Ab + Ag), was mixed with excess of a

fluorescein-labeled tracer to titrate the unbound

residual Ab. The resulting reagent mixture (AbAg +

AbAg* +Ag* +Ag) was then introduced into a restrict-

ed access column to separate the free and bound

fractions, as described in the experimental section.

3.1. Immuno-SLM–FFIA

The extraction of analytes from the first aqueous

phase (the donor) to the second aqueous phase (the

acceptor) through the SLM membrane can be de-

scribed by two quantitative parameters: the extraction

efficiency (E) and the enrichment factor (Ee). E is

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118 111

defined as the fraction of analyte that is transported

from the donor phase through the SLM membrane to

the acceptor phase, see Eq. (1). Ee is defined as the

ratio between the analyte concentration in the acceptor

and donor, respectively, as seen in Eq. (2) (Knutsson

et al., 1996).

E ¼ VACA=VSCS ð1Þ

Ee ¼ CA=CS ¼ EVS=VA ð2Þ

where VA and VS are the acceptor channel volume

and the total volume of extracted sample, respec-

tively, and CA and CS are the analyte concentra-

tion in the acceptor and in the extracted sample,

respectively.

The rate of mass transfer over the membrane is

proportional to the difference between the analyte

concentration in the donor and acceptor (DC), which

can be described by Eq. (3).

DC ¼ CS � aACA ð3Þ

It is assumed that the analyte is in its extractable

form in the donor, and aA represents the fraction of

analyte that is in its extractable form in the acceptor

(Jonsson et al., 1993).

The analyte concentration in the acceptor increases

as the extraction proceeds and thus DC approaches

zero. When the enrichment of the analyte in the

acceptor reaches the maximum level (Ee =Ee(max)),

then DC = 0, mass transfer stops, and Eq. (3) can be

re-written to Eq. (4) (Chimuka et al., 1998).

EeðmaxÞ ¼ ðCA=CSÞmax ¼ 1=aA ð4Þ

The equilibrium reaction between the antibody and

the analyte, taking place in the acceptor, and the affinity

constant (K) that characterizes the corresponding equi-

librium may be simply described by the law of mass

action, given by Eq. (5):

K ¼ ½AbAg�=½Ab�½Ag� ð5Þ

where [AbAg] is the concentration of the immuno

complex, [Ab] is the concentration of free antibody

and [Ag] is the concentration of extractable analyte

in the acceptor, and the fraction of extractable

analyte in the acceptor channel (aA) can thus be

written as Eq. (6):

aA ¼ ½Ag�=ð½Ag� þ ½AbAg�Þ ð6Þ

The maximum enrichment factor (Ee(max)) for

immuno-SLM extraction is obtained by combining

Eqs. (4), (5) and (6), giving Eq. (7) (Thordarson et al.,

2000):

EeðmaxÞ ¼ 1þ K½Ab� ð7Þ

Eq. (7) shows that the affinity of the antibody for

the antigen (K) as well as the antibody concentration

in the acceptor influence the value of Ee(max) of the

immuno-SLM extraction. To obtain a high degree of

immuno extraction, an antibody with a high affinity

for the analyte must therefore be used.

Apparent extraction efficiency (Eapp) and apparent

enrichment factor (Eeapp) are now introduced to point

out that the experimental values obtained using the

above equations with the present system are not true

values since the sensitivity of the FFIA used directly

influences the extraction parameters, i.e., any change

in antibody concentration in the acceptor will be

followed by a change in the FFIA sensitivity and a

change in antibody affinity will change the FFIA

sensitivity due to the complex interplay between

different antibody affinity ratios for analyte versus

tracer (Wild, 1994; Diamandos and Chistopoulos,

1996). These apparent values should thus be looked

upon as comparative constants describing the whole

immuno-SLM–FIIA system.

3.1.1. Mechanism of the immuno-SLM process

In order to test the mechanism of the immuno-SLM

process, an antibody–antigen system that we knew

worked well in the simple FFIA format was used. To

minimize analyte trapping by the ‘‘conventional’’

SLM process (Chimuka et al., 1997), the pH of the

donor and the acceptor should be the same and the

analyte be neutral at this pH. For this purpose the s-

triazines, and, in particular, atrazine was found to be

the ideal analyte being a weak base (pKa = 1.68) and

thus a pH of 7.4 could be used in both acceptor and

donor.

Three different experiments were performed as

shown in Fig. 2. In Experiment I, atrazine concen-

Fig. 2. Visualizing the immuno-SLM process: (o) Experiment I—immuno-SLM–extraction of analyte from a continuous flowing donor into an

antibody containing acceptor; (5) Experiment II—pre-incubation of antibodies and analyte off-line, then injection into the system (Fig. 1),

briefly passing over the acceptor, and with a stagnant donor flow (no analyte present in donor); (x) Experiment III—pre-incubation of antibody

and analyte offline as in Experiment II, but then kept in the acceptor for 15 min and with a continuously pumped donor (no analyte present in

donor). Conditions: SLM: PTFE support (7.1 cm length and 0.6 cm width) impregnated with di-n-hexyl ether, [Ag*]w = 5 nM iPr/Cl/(CH2)5CO-

EDF I, [Ab III]0.25 = 1.3 Ag/ml, donor flow rate = 300 Al min� 1 and extraction time = 15 min. B*= signal of the antibody bound tracer and

B0.25* = signal at 25% binding of the tracer.

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118112

trations in the range 0–1000 Ag/l were extracted for

15 min from a continuous donor flow into the

stagnant Ab-containing acceptor, i.e., the immuno-

SLM procedure. In Experiment II, the Ab was pre-

incubated off-line with atrazine and then introduced

into the acceptor while the donor flow, containing no

analyte, was kept stagnant. In this experiment, the

acceptor content was immediately transferred into the

FFIA system, before any free atrazine molecules

could diffuse from the acceptor to the donor. Thus,

no analyte extraction was performed and an experi-

ment corresponding to a simple FFIA was attempted

with E = 0. In Experiment III, the Ab was pre-

incubated offline with the analyte and introduced

into the acceptor channel, as in Experiment II, but

then kept there for 15 min. The donor flow, con-

taining only buffer solution, was continuously

pumped through the channel, thus testing the degree

of extraction of the analyte from the acceptor to the

donor. By comparing Experiments I and II in Fig. 2,

it can be seen that extraction and enrichment of

atrazine was achieved with Experiment I, since a

shift of the calibration curve to lower concentrations

was observed and the sensitivity for the analysis was

improved (limit of detection LOD10% = 2 Ag/l and

IC50 = 16 Ag/l) compared with Experiment II

(LOD10% = 20 Ag/l and IC50 = 36 Ag/l). If Experi-

ments II and III are compared, a shift of the

calibration curve to even higher concentrations and

thus lower sensitivity was observed for Experiment

III (LOD10% = 40 Ag/l and IC50 = 74 Ag/l), indicatingthat about half of the atrazine was lost from the

acceptor by diffusion through the membrane to the

donor. Here, the continuous pumping of the donor

creates an additional driving force for the diffusion

of the analyte from the acceptor into the donor.

However, due to the high partition coefficient

(Kp>1) of atrazine between the organic and aqueous

phases it is very likely that some of the analyte was

lost to the membrane phase also in Experiment II.

The experiments illustrated in Fig. 2 suggest that the

diffusion of atrazine through the SLM membrane

was a reversible process and that the presence of

antibodies in the acceptor resulted in extraction and

enrichment of the analyte in the acceptor.

An apparent low dose hook effect was seen in both

Experiments II and III, i.e., when no immuno extrac-

tion was performed. Based on the literature on this

M. Tudorache et al. / Journal of Immunolo

topic (Bachas et al., 1984; Barbarakis et al., 1993), we

have no reasonable scientific explanation for this

effect in our experiments based on e.g., the type of

tracer label, size of analyte, size of antibody). There

was, however, a significant difference between Ex-

periment I (no low dose hook effect) and Experiments

II and III (with a low dose hook effect). The analyte

concentrations given in Fig. 2 for Experiment I are the

concentrations in the donor, whereas the ones given

for Experiments II and III are the analyte concentra-

tions in the acceptor, since no extraction was per-

formed. This means that the analyte concentration in

the acceptor after extraction/enrichment was always

higher in Experiment I than the analyte concentrations

present in the acceptor during Experiments II and III.

3.1.2. Optimization of immuno-SLM extraction

The theory of traditional SLM extraction describes

the quantitative parameters (E and Ee) of the extrac-

tion as depending on donor flow rate and extraction

time, as well as on the polarity of the organic

membrane (Jonsson et al., 1993). Thus, high efficien-

cy of immuno-SLM extraction should be obtained by

optimizing conditions such as donor flow rate, extrac-

tion time, stability of organic membrane and the type

and concentration of the antibody in the acceptor.

These factors will be discussed in the following

sections.

Fig. 3. Dependence of the apparent enrichment factor (Eeapp) and appare

[atrazine] = 5 Ag/l, otherwise, as in Fig. 2.

3.1.2.1. Donor flow rate, extraction time and stability

of organic liquid membrane. The SLM theory states

that the enrichment factor (Ee) increases and the

extraction efficiency (E) decreases with increasing

donor flow rate (Jonsson and Mathiasson, 1999). This

theory was confirmed also for immuno-SLM, as

shown in Fig. 3 for the extraction of 5 Ag/l atrazineat different flow rates. It can be seen that Ee

app

increased with donor flow rate up to approximately

450 Al min� 1, while Eapp decreased within the tested

range of flow rates.

Since the Kp (partition coefficient between organic

and aqueous phases) for atrazine was high, the ex-

traction of atrazine is limited by mass transfer in the

donor (donor-controlled extraction) (Jonsson and

Mathiasson, 1999), suggesting that the highest donor

flow rate should be used (Knutsson et al., 1996).

However, a high donor flow rate reduces the lifetime

of the organic membrane, which is why a compromise

was made, and a flow rate of 300 Al min� 1 was

chosen for all further experiments.

Another factor influencing the enrichment factor is

the extraction time, i.e., the time that the analyte is

passed through the donor at a constant flow rate (300

Ag/ml). Eeapp increases with the extraction time until a

sufficient concentration of atrazine in the acceptor

channel has been reached and equilibrium between

the analyte concentrations of all the three phases has

gical Methods 284 (2004) 107–118 113

nt extraction efficiency (E app) on the donor flow rate. Conditions:

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118114

been established (acceptor–organic–donor). This

occurs after 15 min and in all further experiments,

an extraction time of 15 min was used.

The nature of the organic solvent has an important

influence on the rate of mass transfer and selectivity in

SLM extraction. Low viscosity, low volatility and low

solubility in water are physical properties that must

characterize the organic solvent immobilized in the

SLM membrane (Audunsson, 1986). Di-n-hexyl ether

was previously found to be the best organic solvent

for s-triazine extraction by traditional SLM (Chimuka

et al., 1997) and was thus also used in this work. The

stability of the SLM membrane (PTFE impregnated

with di-n-hexyl ether) was tested by immuno-SLM

extractions of 10 and 100 Ag/l atrazine. After about 50extractions, the Eapp decreased with about 25% for

each concentration. This is mainly due to the fact that

the organic solvent is lost from the SLM membrane

during the washing step (the donor channel was

washed with 300 Al of 0.5 M H2SO4 after each

extraction), which is performed to eliminate the mem-

ory effect in the membrane. It was thus found that one

liquid membrane could be used for approximately

thirty extractions before the membrane had to be

exchanged.

A good reproducibility of the immuno-SLM ex-

traction was obtained when testing four different

Fig. 4. Reproducibility of immuno-SLM—FFIA extraction for atrazine

Conditions: as in Fig. 2.

membranes (see Fig. 4), considering that each new

SLM membrane differed from the old one not only in

terms of a fresh organic liquid, but also with a new

PTFE support. Therefore, immuno-SLM extraction

can be reproduced in a satisfactory way even when

the SLM membrane is replaced.

3.1.2.2. Type of antibody and antibody concentra-

tion. The efficiency of the immuno-SLM extraction

is governed by two factors: the affinity of the antibody

for the analyte and the antibody concentration (Eq.

(7)). As stated above, a conflict appears since the

sensitivity of the FFIA used to quantify the immuno

extraction will change as soon as the Ab concentration

or the Ab affinity changes (Wild, 1994; Diamandos

and Chistopoulos, 1996). A compromise between

high efficiency of the immuno extraction and high

sensitivity of the FFIA must thus be made in order to

attain the maximum performance of immuno-SLM–

FFIA system, as will be discussed below.

Three different antibodies (Ab I–III) were com-

pared for extraction of atrazine, using the same tracer

at a fixed concentration. Ab III and Ab I–II differ

with respect to their source (see experimental section)

and in the fact that Ab III is affinity purified using an

analyte affinity column, so the antiserum contains

antibody clones which to different degrees all have

using four different SLM membranes with the same composition.

Table 1

The influence of the type of antibody on the immuno-SLM

extraction parameters

Type of

antibody

[Ab]25%(Ag/ml)

Eapp

(%)

Eeapp LOD

(Ag/l)

Ab I 150 2.6 12 1

Ab II 300 1.0 4.6 4

Ab III 1.3 4.3 19 2

Conditions: as in Fig. 2. (The values in the table are the mean values

of duplicate measurements.)

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118 115

affinity for the analyte. The other two are original

antisera simply purified by ammonium sulfate precip-

itation, meaning that only a small fraction of the

antibody clones have affinity for the analyte.

Working antibody concentrations, leading to 25%

binding of the tracer ([Ab]25), was chosen for all three

cases, followed by analyte calibrations at the fixed

antibody concentrations. In Table 1, the working

antibody concentrations, apparent extraction parame-

ters (Eapp and Eeapp) and sensitivities (LOD) are shown

for the three antibody systems.

As seen in Table 1, significantly lower [Ab]25 was

needed for Ab III compared to the other two, and this,

in turn, led to the highest Eapp and Eeapp. On the other

hand, when looking at the sensitivities of the three

assays, Ab I led to the lowest LOD. Due to the large

Fig. 5. Determination of atrazine by FFIA (.) and immuno-SLM–FFIA (noff-line with the Ab before injection into the acceptor in the system in Fi

amount of Ab I needed to perform each assay all

further experiments were performed with Ab III, an

antiserum that performed well and could be used in

minimum amounts.

3.2. Immuno-SLM–FFIA versus FFIA

Keeping the optimum conditions as specified

above, the immuno-SLM–FFIA and the FFIA were

compared (see Fig. 5) in terms of sensitivity, preci-

sion of measurements and the possibility of applica-

tions in different sample matrixes. The LOD and IC50

for atrazine decreased from 20F 10 to 2.0F 1.1 Ag/l and 36F 16 to 16.0F 1.4 Ag/l, respectively, moving

from the FFIA to the immuno-SLM–FFIA format.

The higher LOD and IC50 obtained for the present

FFIA system compared to a similar system presented

previously (Onnerfjord et al., 1998b) is due to the

fact that the amount of antibody is substantially

higher and the tracer is added in a secondary flow

and mixed in a mixing coil with the eluting acceptor

(Fig. 1).

The ‘‘within-assays’’ and ‘‘between-assays’’ var-

iances were calculated for immuno-SLM–FFIA, con-

sidering the assays in Fig. 5. The ‘‘between-assays’’

variance was higher than the ‘‘within-assays’’ vari-

). In FFIA, the analyte is not enriched, i.e., the analyte is incubated

g. 1. Conditions: as in Fig. 2.

Table 2

[Atrazine]

(Ag/l)Tap

water (%)

Orange

juice (%)

River

water (%)

(a) Recoveries of atrazine from real samples using FFIA

50 99 nd 34

100 99 nd 36

500 95 nd 23

(b) Recoveries for atrazine from real samples using

immuno-SLM–FFIA

5 115 104 111

10 137 153 127

50 145 78 102

100 100 97 96

nd: not detectable.

Conditions: as in Fig. 2. (The values in the table are the mean values

of triplicate measurements.)

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118116

ance giving a calculated F = 19.2 compared with the

one tailed critical F (3.587) for 95% confidence,

suggesting that the repeatability of the extraction

was better than the reproducibility.

3.2.1. Application to real samples

Different sample matrices such as tap water, river

water and orange juice were spiked with atrazine and

the recoveries obtained with FFIA alone and with

immuno-SLM–FFIA were compared with those for

reagent water spiked with atrazine (standard solutions

Fig. 6. Comparison of immuno-SLM–FFIA of atrazine in spiked

of atrazine), as presented in Table 2a and b, respec-

tively. Different spiking levels for the two systems had

to be chosen due to the difference in sensitivity of the

systems, although, all were within the dynamic range

of the assays. For the FFIA (Table 2a), the recovery of

atrazine at different concentrations in the case of tap

water was close to 100%, while the recovery of

atrazine in river water and orange juice was less

satisfactory, which means that the sample matrices

had a strong impact on the FFIA under the conditions

used. For the immuno-SLM–FFIA system, the recov-

eries were in general much better (Table 2b) in almost

all cases within the acceptable limits (between 70%

and 120%) as recommended by the guidelines pub-

lished by US EPA for analysis of environmental

samples (Krotzky and Zeeh, 1995), demonstrating

the reduction of matrix effects and the enrichment of

the analyte by immuno extraction of the analyte

through the SLM membrane.

Finally, the analysis of atrazine from orange juice

was the topic of three inter-laboratory experiments.

Juice samples spiked with unknown atrazine concen-

trations were analysed by immuno-SLM–FIIA. The

values experimentally determined by immuno-SLM–

FIIA were compared with the expected values, as

shown in Fig. 6, in which a relatively high correlation

coefficient was obtained (R = 0.94). Comparing the

constants in the equation of the regression line

orange juice with the real values. Conditions: as in Fig. 2.

M. Tudorache et al. / Journal of Immunological Methods 284 (2004) 107–118 117

(a = 0.59F 18.42 and b = 0.78F 0.10, 95% confi-

dence interval) with the ‘‘ideal’’ situation (a = 0 and

b = 1), it can be seen that the effect of immuno-SLM–

FFIA led to an underestimation of the real concentra-

tion value of the analyte by approximately 20%.

3.3. Conclusions

A new immuno extraction–detection technique,

immuno-SLM–FFIA, was investigated for the analyte

atrazine in terms of extraction efficiency, enrichment

factor, flow rate, extraction time, sensitivity and selec-

tivity in different sample matrices. The immuno-

SLM–FFIA results were compared with a FFIA

system without extraction, demonstrating that this

new technique results in automatic and simultaneous

enrichment and sample clean up of the analyte in

sample matrices such as tap and river water and

orange juice with a total analysis time of 27 min

(compared to 60 min for a conventional SLM-LC ana-

lysis of atrazine).

The presented immuno-SLM–FFIA system (unla-

belled antibodies in the acceptor for analyte extraction

and titration of residual free antibodies of the acceptor

with an analyte tracer) is at this initial stage, a

relatively complex assay system in which an increase

in antibody concentration leads to increased extraction

efficiency and enrichment in the immuno-SLM part of

the system, but where the sensitivity of the FFIA used

for quantifying the extraction is counteracted by this

parameter, thus making it difficult to optimize the

conditions of the immuno SLM extraction. A more

suitable flow immunoassay format in which some of

these counteracting effects can be eliminated is a

system that makes use of labeled antibodies in the

acceptor and an antigen support to trap the residual

excess of labeled antibodies.

Acknowledgements

Financial support is kindly acknowledged from the

European Commission (EC contracts: ENV4-CT97-

0476 (INExSPORT), IC15-CT98-0138 (BIOTOOLS),

IC15CT98-0910 (MEBFOOD), ICA2-CT-2000-

10033 (BIOFEED) and INTAS contract 99-0995),

the Swedish Council for Forestry and Agricultural

Research (SJFR), the Swedish Research Council

(Vetenskapsradet) and the Swedish Environmental

Protection Agency (NVV-Naturvardsverket). The

authors are also very grateful for the kind supply of

antibodies and analyte hapten derivatives from Dr. R.

Abuknesha (Kings College, London, UK), M.-P.

Marco (CID-CSIC, Barcelona, Spain) and Dr. S.

Eremin (M.V. Lomonosov Moscow State University,

Russia) as well as restricted access material from Dr.

D. Lubda (Merck) and Prof. K.-S. Boos.

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