Immuno-SLM—a combined sample handling and analytical technique

Journal of Immunological Methods 284 (2004) 107 – 118 www.elsevier.com/locate/jim

Immuno-SLM—a combined sample handling and analytical technique Madalina Tudorache a, Mariusz Rak b, Piotr P. Wieczorek b, ˚ ke Jo¨nsson a, Jenny Emne´us a,* Jan A a

Department of Analytical Chemistry, Lund University, P.O. Box 124, 221 00 Lund, Sweden b 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 16.0 F 1.4 and 36 F 16 Ag/l, the limit of detection (LOD) was 2.0 F 1.1 and 20 F 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

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: [email protected] (J. Emne´us). 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2003.10.014

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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 compounds, limited selectivity and high costs of disposable 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 preparation of liquid samples. It provides a large potential for tuning the extraction by pH adjustments, selecting solvents with specific properties and/or incorporating 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) followed by re-extraction into a second aqueous phase (acceptor) was developed, called supported liquid membrane (SLM) extraction (Jo¨nsson and Mathiasson, 1999; Jo¨nsson 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 extraction of the analyte. In the present paper, a new combined extraction and analytical technique called immuno-SLM –FIIA, previously presented with preliminary results for 4nitrophenol (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 antibodies. 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-1,3,5triazine (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 (Millipore, 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 (Darmstadt, Germany). Di-n-hexyl ether (97%, SigmaAldrich, 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).

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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 Abuknesha (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 fluoresceinlabeled 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)5 COOH hapten was reacted with EDF, forming a ¨ nnerfjord et al. fluorescent tracer, as described by O (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 ¨ nnerfjord et al. (1998a). All (8.78  104 M
1 cm
1) O 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 (Ho¨je 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-

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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, Germany, length 7.10 cm, width 0.60 cm with a 180-Amthick 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,

Fig. 1. The immuno-SLM – FFIA system: (1) SLM unit; (2, 5, 6) automatic syringe pumps; (3) peristaltic pump; (4) manual injection valve; (7) mixing coil; (8) restricted access column (C8); (9) fluorescence detector.

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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 0.15 M 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 pumped 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 containing Ag + AbAg + AbAg* + Ag*, was passed through a RA column (8) where the Ag and Ag* fractions were trapped and the AbAg and AbAg* fractions were 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.

donor channel contained pure buffer solution. The subsequent assay steps were identical to those described above for the immuno-SLM –FFIA procedure.

3. Results and discussion Considering the basic principle of SLM extraction, involving antigen –antibody interactions as the extraction 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(Jo¨ nsson and Mathiasson, 2000), which contains an organic solvent (e.g., di-nhexyl 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, containing the Ag, is passed through the donor channel while a stagnant solution of anti-analyte antibodies is present 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 developed to quantify the degree of immuno-SLM extraction. 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 restricted access column to separate the free and bound fractions, as described in the experimental section. 3.1. Immuno-SLM – FFIA

2.2.4. FFIA procedure The FIIA procedure was identical to the immunoSLM –FFIA procedure, excluding the SLM extraction step. Suitable concentrations of Ab and Ag were premixed and incubated for 15 min and then introduced into the acceptor by the syringe pump (2), while the

The extraction of analytes from the first aqueous phase (the donor) to the second aqueous phase (the acceptor) through the SLM membrane can be described by two quantitative parameters: the extraction efficiency (E) and the enrichment factor (Ee). E is

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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 VA CA =VS CS

ð1Þ

Ee ¼ CA =CS ¼ EVS =VA

ð2Þ

DC ¼ CS
aA CA

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 (Jo¨nsson 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 =CS Þmax 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 equilibrium may be simply described by the law of mass action, given by Eq. (5): K ¼ ½AbAg=½Ab½Ag

analyte in the acceptor channel (aA) can thus be written as Eq. (6): aA ¼ ½Ag=ð½Ag þ ½AbAgÞ

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

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 1 þ K½Ab

where VA and VS are the acceptor channel volume and the total volume of extracted sample, respectively, and CA and CS are the analyte concentration 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).

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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 (Eapp e ) 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 striazines, 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-

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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)5COEDF 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 B*0.25 = signal at 25% binding of the tracer.

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 immunoSLM procedure. In Experiment II, the Ab was preincubated 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 experiment corresponding to a simple FFIA was attempted with E = 0. In Experiment III, the Ab was preincubated offline with the analyte and introduced into the acceptor channel, as in Experiment II, but then kept there for 15 min. The donor flow, containing 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 IC 50 = 16 Ag/l) compared with Experiment II (LOD10% = 20 Ag/l and IC50 = 36 Ag/l). If Experiments 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), indicating that 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 extraction was performed. Based on the literature on this

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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 Experiment 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 concentrations in the acceptor, since no extraction was performed. 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 extraction as depending on donor flow rate and extraction time, as well as on the polarity of the organic membrane (Jo¨nsson et al., 1993). Thus, high efficiency of immuno-SLM extraction should be obtained by optimizing conditions such as donor flow rate, extraction 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.

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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 (Jo¨nsson and Mathiasson, 1999). This theory was confirmed also for immuno-SLM, as shown in Fig. 3 for the extraction of 5 Ag/l atrazine at different flow rates. It can be seen that Eapp e 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 extraction of atrazine is limited by mass transfer in the donor (donor-controlled extraction) (Jo¨nsson 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). Eapp increases with the extraction time until a e sufficient concentration of atrazine in the acceptor channel has been reached and equilibrium between the analyte concentrations of all the three phases has

Fig. 3. Dependence of the apparent enrichment factor (Eeapp) and apparent extraction efficiency (E app) on the donor flow rate. Conditions: [atrazine] = 5 Ag/l, otherwise, as in Fig. 2.

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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 50 extractions, 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 memory 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 extraction was obtained when testing four different

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 concentration. 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 compared 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

Fig. 4. Reproducibility of immuno-SLM—FFIA extraction for atrazine using four different SLM membranes with the same composition. Conditions: as in Fig. 2.

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

150 300 1.3

2.6 1.0 4.3

12 4.6 19

1 4 2

Conditions: as in Fig. 2. (The values in the table are the mean values of duplicate measurements.)

affinity for the analyte. The other two are original antisera simply purified by ammonium sulfate precipitation, 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 parameters (Eapp and Eapp e ) 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 Eapp e . On the other hand, when looking at the sensitivities of the three assays, Ab I led to the lowest LOD. Due to the large

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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, precision of measurements and the possibility of applications in different sample matrixes. The LOD and IC50 for atrazine decreased from 20 F 10 to 2.0 F 1.1 Ag/ l and 36 F 16 to 16.0 F 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 ¨ nnerfjord et al., 1998b) is due to the previously (O 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’’ variances were calculated for immuno-SLM – FFIA, considering the assays in Fig. 5. The ‘‘between-assays’’ variance was higher than the ‘‘within-assays’’ vari-

Fig. 5. Determination of atrazine by FFIA (.) and immuno-SLM – FFIA (n). In FFIA, the analyte is not enriched, i.e., the analyte is incubated off-line with the Ab before injection into the acceptor in the system in Fig. 1. Conditions: as in Fig. 2.

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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.)

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

of atrazine), as presented in Table 2a and b, respectively. 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 recoveries were in general much better (Table 2b) in almost all cases within the acceptable limits (between 70% and 120%) as recommended by the guidelines published 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 concentrations 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

Fig. 6. Comparison of immuno-SLM – FFIA of atrazine in spiked orange juice with the real values. Conditions: as in Fig. 2.

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(a = 0.59 F 18.42 and b = 0.78 F 0.10, 95% confidence 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 concentration 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 selectivity in different sample matrices. The immunoSLM – 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 analysis of atrazine). The presented immuno-SLM – FFIA system (unlabelled 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-CT970476 (INExSPORT), IC15-CT98-0138 (BIOTOOLS), IC15CT98-0910 (MEBFOOD), ICA2-CT-200010033 (BIOFEED) and INTAS contract 99-0995), the Swedish Council for Forestry and Agricultural Research (SJFR), the Swedish Research Council

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(Vetenskapsra˚det) and the Swedish Environmental Protection Agency (NVV-Naturva˚rdsverket). 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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