Detection of 2,4-Dichlorophenoxyacetic Acid Using a Fluorescence Immunoanalyzer Kim R. Rogers*a, Steven D. Kohla, Lee A. Riddicka and Thomas Glassb a US Environmental Protection Agency, National Exposure Research Laboratory, Las Vegas, NV 89193, USA b Sapidyne Inc., Boise, ID 83706, USA A flow immunoassay method for the measurement of 2,4-dichlorophenoxyacetic acid (2,4-D) was developed. The competitive fluorescence immunoassay relies on the use of antibody- or antigen-coated poly(methyl methacrylate) particles (98 mm diameter) as a renewable solid phase.The assay exhibits a dynamic range of 0.1–100 mg l21 using a monoclonal antibody or alternatively 10 mg l21 to 10 mg l21 using commercially available antiserum. The assay is demonstrated in buffered saline solution as well as in aquatic environmental media. The relative errors for the environmental matrices were similar to those for the buffer control. The precision of concentration values calculated at 1 mg l21 (for the assay using antiserum) were ±0.28, ±0.27 and ±0.43 mg l21 for the buffer, well water and river water matrices, respectively.The method shows cross-reactivity with compounds of closely related structure but little cross-reactivity with compounds dissimilar in structure to 2,4-D. The proposed automated competitive immunoassay method is rapid (between 7 and 15 min per assay), simple and potentially portable. Keywords: 2,4-Dichlorophenoxyacetic acid; fluoroimmunoassay; KinExA immunoanalyzer Methods for the rapid and cost-effective detection of pesticides in environmental settings have gained considerable attention in recent years, primarily due to concerns related to potential human exposure from spray run-off and spills into groundwater sources.1 Classical laboratory-based methods used to measure these compounds are typically time consuming and expensive.These methods usually require extensive extraction, derivatization and separation by GC or HPLC.To meet the need for faster and more cost-effective methods, a variety of techniques have recently been reported and a number of commercial products (methods) have been introduced. These methods are, for the most part, immunochemically based enzyme-linked immunosorbent assays (ELISA) using absorbance, fluorescence or electrochemical detection. These assays have typically been formatted for microtiter plates,2 thick film-based planar microwells, 3 microflow cells,4 test kits,5 or test tube-type optical spectrometers.6 Although automation has been incorporated into a number of these method formats, they typically require manual addition of reagents, several incubation steps, and physical manipulations such as changing tubes, trays or capillaries.Portable automated systems that avoid these physical manipulations and generate (as waste) only spent reagents show potential cost, time and waste disposal advantages for certain applications.Several approaches have been used for the automated immunoassay flow systems. Formats for these systems involve either regeneration of the antibody immobilized to the solid phase (i.e., disruption of antibody– antigen binding)7 or renewal of a mobile solid support (e.g., microspheres which are trapped in a flow cell) which has been coated with either the antibodies or antigens,8,9 or enzymes.10 The use of a mobile solid phase which is renewable for each assay offers certain technical advantages resulting in a fast, versatile and inexpensive assay system. 2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used as a systemic herbicide for broad leaf weeds in a variety of crop and non-crop applications. This pesticide is typically monitored in environmental samples using GC methods which require extensive clean-up and derivatization, however, a number of potential field analytical methods have been reported. These methods include: ELISA3,4,9,11 enzyme immunoassay,1 immunoagglutination, 12 fluorescence polarization,13 and enzyme assays14 as well as several antibody-based biosensor methods. 15–17 Although most of these methods are portable and relatively simple to execute, they typically require multiple steps and generate solid wastes (e.g., plates, tubes and vials). In this paper, we describe the use of the KinExA immunoanalyzer to develop competitive fluorescence immunoassay methods for the detection of 2,4-D using both monoclonal and polyclonal antibodies.These methods use a mobile–renewable solid phase (i.e., microspheres) and can be fully automated. Experimental Instrumentation and KinExA Method The KinExA instrument is an automated fluorescence immunoassay system that uses microspheres as the solid phase (Sapidyne, Boise, ID, USA). For the assay formats described here, these beads [poly(methyl methacrylate), PMMA] were coated with either antibody or antigen and then packed into a capillary flow cell that is integrated into an epi-illumination filter fluorimeter system.For the format using antibody-coated beads, fluorescently labeled probe is introduced into the flow cell in the presence or absence of analyte and continuous interrogation of the bead column for analyte-sensitive accumulation of fluorescently labeled tracer on the solid phase forms the basis of this assay. For the format using antigen-coated beads, monoclonal antibody or anti-2,4-D antiserum is incubated with the analyte (2,4-D), then introduced into the flow cell containing antigencoated beads.This step is followed by fluorescently labeled secondary (species-specific, anti-IgG) antibody. For both formats, the observed fluorescence signal varies inversely with the analyte concentration. The competitive immunoassay format using the antibodycoated beads is described in Fig. 1(a). In this assay, the PMMA particles were coated with anti-sheep IgG which was used to capture and orient the primary antibody from sheep anti-2,4-D serum. Next, inhibition in the accumulation of fluoresceinlabeled 2,4-D tracer resulting from the presence of various concentrations of the analyte 2,4-D allowed the detection and quantification of this analyte.Another competitive immunoassay format described in Fig. 1(b) used beads coated with antigen (2,4-D). The anti- 2,4-D serum or monoclonal antibody was incubated with various amounts of 2,4-D. The solution was then drawn across the antigen-coated beads and anti-2,4-D antibodies with free Analyst, October 1997, Vol. 122 (1107–1111) 1107binding sites were bound to the beads. The amount of antibody bound to the beads was quantified by flowing a fluoresceinlabeled, species-specific antibody through the bead pack. This fluorescein-labeled antibody bound to the 2,4-D-specific antibody immobilized in the bead pack. The observed fluorescence was again inversely related to the concentration of analyte.The instrument flow system was operated under negative pressures using a syringe pump. An in-line vacuum de-gasser was used to prevent the formation of bubbles. All of the pumps and valves were operated through the KinExA hardware interface and PC-based software using a Windows environment. The instrument operations can be divided into two areas: bead handling and sample handling. For bead handling, the coated beads were drawn into the flow cell and removed to waste after the assay had been completed using a back-flush peristaltic pump.For sample handling, samples containing various concentrations of 2,4-D were drawn through channels 2–6 of a rotary valve, then over the packed bead column at a rate of 0.50 ml min21. The KinExA instrument software monitored the fluorescence signal once every second for a total of 420 s and displayed the data on the PC monitor. The fluorescence data were stored as voltage values in Excel and macros (included with the KinExA system) were used for analysis of the data.Signal intensities at specific time points in the instrument cycle were used as an indicator of the accumulation of fluorescent probe on the coated beads. The system was programmed to cycle automatically through the five samples, each using a new bead pack in the capillary. For the antibody-coated bead format, each cycle consisted of (1) bead packing, (2) analyte tracer accumulation and data acquisition, (3) buffer wash and (4) removal of the used beads to waste.For the antigen-coated bead format, step 2 was replaced by the accumulation of anti-2,4-D serum (incubated with various amounts of 2,4-D) followed by the accumulation of fluorescein-labeled anti-IgG antibody. Chemicals PMMA beads (98 mm) were purchased from Bang’s Laboratories (Carmel, IN, USA). Photopolymeric N-oxysuccinimde (PNOS) modified PMMA beads were purchased from BSI (Eden Prairie, MN, USA). Rabbit anti-sheep IgG and sheep anti-2,4-D-serum were obtained from The Binding Site (San Diego, CA, USA).Fluorescein-conjugated rabbit anti-sheep IgG was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The anti-2,4-D monoclonal antibodies (clone E2/G2) were generated by Dr. Milan Franek at the Veterinary Research Institute (Brno, Czech Republic) and kindly provided by Dr. Sergei Erernin (Moscow State University, Russia). Fluorescein isothiocyanate (FITC), 2,4-D, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC), N-hydroxysuccimide and dimethylformamide were purchased from Sigma (St.Louis, MO, USA). Phenol and benzoic acid were purchased from Mallinckrodt (Paris, KY, USA). Atrazine was purchased from ChemService (West Chester, PA, USA). 2,4-Dichlorophenol was purchased from Aldrich (Milwaukee, WI, USA). Chlordane was obtained from the Environmental Protection Agency (Research Triangle Park, NC, USA).All other compounds and solvents used were of analytical-reagent grade. Environmental Samples River water samples were obtained from the Virgin River in Southern Utah. The water was passed through a coarse filter to remove any insoluble matter, and stored at 4 °C until use. The pH of the water at the time of use was 7.0. Well water was obtained from Well HM-1, Salmon site, Louisiana, it was stored at 4 °C until use, at which time its pH was 7.5. Preparation of Fluorescein-labeled 2,4-D Fluorescein thiocarbamylethylenediamine was synthesized from FITC and ethylenediamine as described by Pourfarzaneh et al.18 The amine-derivatized FITC was then conjugated to 2,4-D as reported by Eremin.19 Briefly, the N-hydroxysuccinimide ester of 2,4-D was synthesized and conjugated to fluorescein thiocarbamylethylenediamine in the presence of EDC in dimethylformamide.The tracer was purified by thinlayer chromatography [silica gel plates were developed in ethyl acetate–methanol–acetic acid (90 + 8 + 2, v/v/v)] and the concentration was determined spectrophotometrically using previously reported molar absorption coefficients.18 Preparation of Antibody-coated Beads Antibody-coated PMMA beads were prepared by immersing 200 mg of dry beads into 1 ml of anti-sheep IgG (20 mg ml21) in PBS (phosphate-buffered saline: 10 mm NaHPO4; 100 mm NaCl; pH 7.4).The mixture was then agitated for 2 h at 37 °C, after which the beads were centrifuged (5000g, 2 min) and the supernatant was discarded.The IgG-coated particles were rinsed twice by centrifugation with PBS (1 ml). The sheep anti- 2,4-D serum (diluted 1 + 199 into PBS) was immobilized onto the anti-sheep IgG-coated beads by incubation (with gentle rocking) for 2 h at 37 °C. The beads were again rinsed as described above, suspended in 1 ml of PBS and stored at 4 °C until the day of use, at which time they were diluted with 19 ml, of PBS and placed in the instrument reservoir.Preparation of Antigen-coated Beads Antigen-coated beads were prepared using amine reactive PNOS-coated beads. An amine derivative of 2,4-D was prepared by conjugating ethylenediamine to the N-hydroxysuccinimide ester of 2,4-D. The amine-derivatized 2,4-D was purified by thin-layer chromatography (silica gel plates developed in ethanol). Coupling of the ligand to the beads was carried out in 0.10 m borate buffer of pH 8.5, as suggested by the supplier (BSI). The mixture containing 200 mg of (PNOS) coated beads and 0.5 mm derivatized 2,4-D in 1 ml of the same buffer was agitated for 2 h at room temperature.The beads were then washed sequentially with 0.10 m borate buffer containing 1 m NaCl, followed by 0.10 m acetate buffer (pH 4) containing 1 m NaCl and finally with PBS. The beads were diluted with 19 ml of PBS for use in the instrument. Fig. 1 Diagrammatic representation of the immunoassay format for measurement of 2,4-D. (A) Competitive immunoassay format using antibody-coated beads, (B) competitive immunoassay format using antigencoated beads. 1108 Analyst, October 1997, Vol. 122Assay Procedure for Antibody-coated Beads 2,4-D stock solutions were prepared in ethanol and diluted to final concentrations as specified under Results and Discussion in PBS or an environmental water sample containing the fluorescein-labeled 2,4-D (1 nm or as specified under Results and Discussion). The final assay sample contained less than 1% ethanol which was included in the control and had no effect on the assay. The 420 s cycle (i.e., bead packing, tracer accumulation, buffer washing and bead expulsion) was then initiated through the KinExA software.Fluorescence signal data were collected, displayed and end-point measurements calculated from the difference in the average fluorescence signal of the initial five and last five data points. Assay Procedure for Antigen-coated Beads 2,4-D stock solutions were prepared in ethanol and diluted to final concentrations in PBS.Sheep anti-2,4-D serum diluted 1 + 39 999 in PBS or monoclonal antibody (E2/G2) at a final concentration of 12 ng ml21 was reacted with various concentrations of 2,4-D. The timing cycle was modified to include a step for the addition of the fluorescently labeled secondary antibody (either anti-rabbit or anti-mouse diluted 1 + 4999 in PBS containing 1 mg ml21 BSA). Fluorescence data were handled as above.Curve Fitting and Statistical Analysis The four parameter logistic equation as defined below was used to fit the immunoassay data.20 y a d x c d b = - + æ è ç ö ø ÷ + ( ) 1 where y = instrument response; a = response at high asymptote, b = slope factor; c = concentration corresponding to 50% specific binding (EC-50); d = response at low asymptote; and x = calibrator concentration. Calculations were performed using SOFTmax software (Molecular Devices, Menlo Park, CA, USA).Because the response error over the concentration range of the assay was heteroscedastic, an estimate of error for the precision of concentration was determined at a concentration near the EC-50 using the standard deviation/slope of the response curve at that point.20 Results and Discussion Shown in Fig. 2 are representative fluorescence signal tracings. There are three representative portions of the fluorescence tracing. First, the horizontal portion to the left shows the baseline fluorescence response for the packed beads prior to addition of the fluorescent tracer.Second, the steepest portion of the fluorescence curve represents the introduction of the bulk tracer into the capillary, filling the interstitial spaces between the beads (approximately the first 1–2 min), followed by the accumulation of the tracer on the coated bead surfaces. The third segment shows the removal of the free tracer from the bead pack. Tracings A, B and C represent control experiments for the antibody-coated bead format [shown in Fig. 1(a)]. Tracing A (Fig. 2) shows the non-specific signal when non-analyte specific sheep IgG was substituted for anti-2,4-D serum on the antibody-coated beads. Tracings B and C show the analytedependent response and result from the presence and absence, respectively, of 2,4-D (10 mg l21) in the assay buffer. The concentration-dependent signal response to 2,4-D was similar when determined from either the accumulation portion of the curve at 300 s or from the tracer remaining on the beads after a buffer wash measured by the fluorescence signal at 420 s.Readings at 420 s were routinely used to generate competition response curves. For most competitive immunoassay methods, the lowest potential detection limit of the assay is primarily determined by the affinity of the antibody for the antigen and, to a lesser extent, by the relative experimental error. However, for non-equilibrium methods such as is reported here, the relative concentrations of immobilized antibody and analyte probe may also impact the assay characteristics.Consequently, the effects of analyte tracer concentration and the polyclonal antibody immobilized on the solid phase were determined. For the antibody-coated bead format, analyte tracer concentrations were varied over the range 0.1–50 nm. A tracer concentration of 0.1 nm yielded no response to the analyte (2,4-D) and 50 nm tracer resulted in unacceptable levels of variability in the fluorescence response. A probe concentration of 1 nm yielded an acceptable signal and minimal variability.The antibody concentration used for immobilization was varied over the range of 0.1–2.0 mg protein per mg dry beads, and had no effect on the competition curve for 2,4-D (data not shown). Shown in Fig. 3 is the signal response curve for 2,4-D using the polyclonal antibody-coated bead format with fluoresceinlabeled 2,4-D as the fluorescent tracer.The mean fluorescence response data were fitted using a four parameter mode.20 The dynamic range extended from 10 mg l21 to 10 mg l21. The inflection point of the curve (EC-50) was 0.87 mg l21 and the multiple correlation coefficient (R) was 0.999. The precision of concentration was calculated at 1 ± 0.28 mg l21. Also shown in Fig. 3 is the signal response curve for 2,4-D, using the antigen-coated bead format for the polyclonal antiserum with fluorescein labeled anti-species antibody as the fluorescent tracer.The dynamic range for this format extended from 2 mg l21 to 20 mg l21. The EC-50 was 1.0 mg l21 and R was 0.998. The precision of concentration was calculated at 1 ± 0.12 mg l21. The competition curves for 2,4-D were similar for both assay formats suggesting that both assay formats were reporting the unoccupied (antibody) binding sites (in the presence of 2,4-D) and that the measuring process did not interfere with the steadystate association of the analyte with the antiserum.In addition, there are several advantages which may be realized by the versatility of this assay system. For example, the ability to link Fig. 2 Typical assay response and control experiments for antibodycoated bead format (A) non-specific binding signal; fluorescein-labeled 2,4-D (1 nm) was used with PMMA beads that were coated with nonanalyte- specific IgG. (B/C) IgG from anti-2,4-D serum was immobilized onto the PMMA beads (as described under Experimental).Antibody-coated beads were then exposed to the fluorescein-labeled 2,4-D (1 nm) in the presence (B) or absence (C) of target analyte 2,4-D (10 mg l21). Analyst, October 1997, Vol. 122 1109the antigen either to a fluorescent tag or to the derivatized beads (each with a different chemical linker) allows one to avoid similarities (and subsequent cross-reactivity) to the linker chain used to form the immunogen. This is particularly important for the use of commercially available antibodies for which the specific immunogen structure is not known.Shown in Fig. 4 are the signal response curves generated by spiking 2,4-D into various environmentally significant media, using polyclonal antiserum with the antibody-coated bead format. The signal response from the 2,4-D spiked well water significantly overlapped the signal response using buffer. The response for 2,4-D spiked into river water appeared slightly lower than that for the buffer or well water matrices.This bias was evidenced by the mean values for seven of the eight data points being lower than for the buffer control as well as the standard deviation values for four of the 2,4-D levels not overlapping those for the spiked buffer. The reason for the tendency of the river water matrix to result in an overestimation of the 2,4-D is not clear. 2,4-D contamination of the water was ruled out by HPLC analysis using standard methods.It is possible that compounds such as humic substances may have interfered with the assay. The relative errors for the environmental matrices were similar to those for the buffer control. The precision of concentration values calculated at l mg l21 were 1 ± 0.28 mg l21 for the buffer, 1 ± 0.27 mg l21 for the well water and 1 ± 0.43 mg l21 for the river water. Shown in Table 1 is the cross-reactivity profile for several structurally related compounds and structurally unrelated pesticides.As expected from previous reports of immunoassays using polyclonal anti-2,4-D serum, compounds similar in structure to 2,4-D, such as 2,4,5- and 2,4-dichlorophenol, elicit a response from this method.19 Compounds with structures unlike 2,4-D showed little if any response. The calibration plot for 2,4-D in Fig. 5 shows the response of the monoclonal antibody (E2/G2) using the antigen-coated bead format. The data were again fitted using the four parameter model.The EC-50 was 2.8 mg l21, R was 1.00 and the assay response curve showed a dynamic range of 0.1–100 mg l21. In comparison with the other reported bioanalytical methods for 2,4-D, the described KinExA method showed a higher response to the analyte than a reported enzyme assay,14 and fluorescence polarization immunoassay.13 The displacement curve and dynamic range for this method using the monoclonal antibody (E2/G2) are similar to those typically reported for ELISAs11 or ELISA-type assays4,17 and are about an order of magnitude less sensitive than microformat ELISAs using chemiluminescence detection.3 Nevertheless, given the profound influence of the affinity characteristics of any particular antibody on the assay response, comparison of immunoassay techniques that use different antibodies is tenuous.Conclusions A variety of immunoassay methods have been reported for the detection and measurement of 2,4-D, each with distinct analytical and assay format characteristics.The potential advantages of the proposed method in its application to environmental monitoring relates primarily to its ease of use (i.e., the degree of automation), the limited waste generated Fig. 3 Assay response as a function of 2,4-D concentration for the antibody-coated bead format (-), or the antigen-coated bead format (5). Error bars represent standard deviation, n = 3. Fig. 4 Effect of environmental water samples as assay matrix using the antibody-coated bead format. 2,4-D standard curves were prepared in PBS (-), spiked into well water (5) or river water (:) samples. Multiple regression coefficients were 0.999, 0.999 and 0.998 for the PBS, well water and river water, respectively. Error bars represent standard deviation, n = 3. Table 1 Cross-reactivity profile Compound Relative response* 2,4-D 100 2,4,5-T† 92 2,4-Dichlorophenol 86 Atrazine 20 Chlordane 14 Benzoic acid 10 Phenol 0 * Relative response to 2,4-D at 1 mg l21 determined using the following relationship: (response for test compound/response for 2,4-D) 3 100.† 2,4,5-Trichlorophenoxyacetic acid. Fig. 5 Assay response as a function of 2,4-D concentration using monoclonal antibody (E2/G2) in the antigen-coated bead format. 1110 Analyst, October 1997, Vol. 122(i.e., the absence of contaminated plates, tubes or vials), and the versatility allowed by the instrument and software in the development of immunoassays. The ability to use either the antibody or antigen as the immobilized phase allows the method to be tailored to the particular characteristics of the immunochemicals and requirements of the potential application (e.g., optimization of assay time and reagents per assay).The US Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded the work involved in preparing this paper. It has been subject to the Agency’s peer review and has been approved for publication.Mention of trade names or commercial products does not constitute endorsement or recommendation by the US EPA. References 1 Meulenberg, E. P., Mulder, W. H., and Stoks, P.G., Environ. Sci. Technol., 1995, 29, 553. 2 Hall, J. C., Deschamps, R. J. A., and Krieg, K. K., J. Agric. Food Chem., 1989, 37, 981. 3 Dzgoev, A., Mecklenburg, M., Larson, P.-O., and Danielsson, B., Anal. Chem., 1996, 68, 3364. 4 Bauer, C. G., Eremenko, A. V., Ehrentreich-Forster, E., Bier, F. F., Makowwer, A., Halsall, H.B., Heineman, W. R., and Scheller, F. W., Anal. Chem., 1996, 68, 24S3. 5 Dohrman, C., Anal. Environ. Lab., 1991, October, 31. 6 Fleeker, J., J. Assoc. Off. Anal. Chem., 1994, 70, 874. 7 Ogert, R. A., Kusterbeck, A. W., Wemhoff, G. A., Burke, R., and Ligler, F. S., Anal. Lett., 1992, 25, 1999. 8 Alarie, J. P., Bowyer, J. R., Sepaniac, M. J., Hoyt, A. M., and Vo- Dinh, T., Anal. Chim. Acta, 1990, 236, 237. 9 Pollema, C. H., and Ruzicka, J., Anal. Chem., 1994, 66, 1825. 10 Mayer, M., and Ruzicka, J., Anal. Chem., 1996, 68, 3808. 11 Lawruk, T. S., Hottenstein, C. S., Fleeker, J. R., Hall, J. C., Herzog, D., and Rubio, F. M., Bull. Environ. Contam. Toxicol., 1994, 52, 538. 12 Lukin, Y. V., Dokuchaer, I. M., Polyak, I. M., and Eremin. S. A., Anal. Lett., 1994, 27, 2973. 13 Eremin, S. A., Landon, J., Smith, D. S., and Jackman, R., in Food Safety and Quality Assurance: Applications of Immunoassay Systems, ed. Morgan, M. R. A., Smith, C.J., and Williams, P. A., Elsevier Applied Science, New York, 1992. 14 Gu, Y., Knaebel, D. B., Korus, R. A., and Crawford, R. L., Environ. Sci. Technol., 1995, 29, 1622. 15 Suleiman, A. A., and Guilbault, G. G., Analyst, l994, 119, 2279. 16 Minunni, M., Skladal, P., and Mascini, M., Anal. Lett., 1994, 27, 1475. 17 Skladal, P., and Kalab, T., Anal. Chim. Acta, 1995, 316, 73. 18 Pourfarzaneh, M., White, G. W., Landon, J., and Smith, D. S., Clin. Chem., 1980, 26, 730. 19 Eremin, S.A., in Immunoanalysis of Agrochemicals, Emerging Technologies, ACS Symposium Series 586, ed. Nelson, J. O., Karu, A. E., and Wong, R. B., American Chemical Society, Washington, DC, 1995. 20 Howes, I., in Immunoassays Essential Data, ed. Edwards, R., Wiley, New York, 1996. Paper 7/01511I Received March 4, 1997 Accepted June 13, 1997 Analyst, October 1997, Vol. 122 1111 Detection of 2,4-Dichlorophenoxyacetic Acid Using a Fluorescence Immunoanalyzer Kim R. Rogers*a, Steven D.Kohla, Lee A. Riddicka and Thomas Glassb a US Environmental Protection Agency, National Exposure Research Laboratory, Las Vegas, NV 89193, USA b Sapidyne Inc., Boise, ID 83706, USA A flow immunoassay method for the measurement of 2,4-dichlorophenoxyacetic acid (2,4-D) was developed. The competitive fluorescence immunoassay relies on the use of antibody- or antigen-coated poly(methyl methacrylate) particles (98 mm diameter) as a renewable solid phase. The assay exhibits a dynamic range of 0.1–100 mg l21 using a monoclonal antibody or alternatively 10 mg l21 to 10 mg l21 using commercially available antiserum.The assay is demonstrated in buffered saline solution as well as in aquatic environmental media. The relative errors for the environmental matrices were similar to those for the buffer control. The precision of concentration values calculated at 1 mg l21 (for the assay using antiserum) were ±0.28, ±0.27 and ±0.43 mg l21 for the buffer, well water and river water matrices, respectively.The method shows cross-reactivity with compounds of closely related structure but little cross-reactivity with compounds dissimilar in structure to 2,4-D. The proposed automated competitive immunoassay method is rapid (between 7 and 15 min per assay), simple and potentially portable. Keywords: 2,4-Dichlorophenoxyacetic acid; fluoroimmunoassay; KinExA immunoanalyzer Methods for the rapid and cost-effective detection of pesticides in environmental settings have gained considerable attention in recent years, primarily due to concerns related to potential human exposure from spray run-off and spills into groundwater sources.1 Classical laboratory-based methods used to measure these compounds are typically time consuming and expensive.These methods usually require extensive extraction, derivatization and separation by GC or HPLC. To meet the need for faster and more cost-effective methods, a variety of techniques have recently been reported and a number of commercial products (methods) have been introduced.These methods are, for the most part, immunochemically based enzyme-linked immunosorbent assays (ELISA) using absorbance, fluorescence or electrochemical detection. These assays have typically been formatted for microtiter plates,2 thick film-based planar microwells, 3 microflow cells,4 test kits,5 or test tube-type optical spectrometers.6 Although automation has been incorporated into a number of these method formats, they typically require manual addition of reagents, several incubation steps, and physical manipulations such as changing tubes, trays or capillaries. Portable automated systems that avoid these physical manipulations and generate (as waste) only spent reagents show potential cost, time and waste disposal advantages for certain applications.Several approaches have been used for the automated immunoassay flow systems. Formats for these systems involve either regeneration of the antibody immobilized to the solid phase (i.e., disruption of antibody– antigen binding)7 or renewal of a mobile solid support (e.g., microspheres which are trapped in a flow cell) which has been coated with either the antibodies or antigens,8,9 or enzymes.10 The use of a mobile solid phase which is renewable for each assay offers certain technical advantages resulting in a fast, versatile and inexpensive assay system. 2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used as a systemic herbicide for broad leaf weeds in a variety of crop and non-crop applications.This pesticide is typically monitored in environmental samples using GC methods which require extensive clean-up and derivatization, however, a number of potential field analytical methods have been reported. These methods include: ELISA3,4,9,11 enzyme immunoassay,1 immunoagglutination, 12 fluorescence polarization,13 and enzyme assays14 as well as several antibody-based biosensor methods. 15–17 Although most of these methods are portable and relatively simple to execute, they typically require multiple steps and generate solid wastes (e.g., plates, tubes and vials). In this paper, we describe the use of the KinExA immunoanalyzer to develop competitive fluorescence immunoassay methods for the detection of 2,4-D using both monoclonal and polyclonal antibodies. These methods use a mobile–renewable solid phase (i.e., microspheres) and can be fully automated.Experimental Instrumentation and KinExA Method The KinExA instrument is an automated fluorescence immunoassay system that uses microspheres as the solid phase (Sapidyne, Boise, ID, USA). For the assay formats described here, these beads [poly(methyl methacrylate), PMMA] were coated with either antibody or antigen and then packed into a capillary flow cell that is integrated into an epi-illumination filter fluorimeter system. For the format using antibody-coated beads, fluorescently labeled probe is introduced into the flow cell in the presence or absence of analyte and continuous interrogation of the bead column for analyte-sensitive accumulation of fluorescently labeled tracer on the solid phase forms the basis of this assay.For the format using antigen-coated beads, monoclonal antibody or anti-2,4-D antiserum is incubated with the analyte (2,4-D), then introduced into the flow cell containing antigencoated beads.This step is followed by fluorescently labeled secondary (species-specific, anti-IgG) antibody. For both formats, the observed fluorescence signal varies inversely with the analyte concentration. The competitive immunoassay format using the antibodycoated beads is described in Fig. 1(a). In this assay, the PMMA particles were coated with anti-sheep IgG which was used to capture and orient the primary antibody from sheep anti-2,4-D serum. Next, inhibition in the accumulation of fluoresceinlabeled 2,4-D tracer resulting from the presence of various concentrations of the analyte 2,4-D allowed the detection and quantification of this analyte. Another competitive immunoassay format described in Fig. 1(b) used beads coated with antigen (2,4-D). The anti- 2,4-D serum or monoclonal antibody was incubated with various amounts of 2,4-D. The solution was then drawn across the antigen-coated beads and anti-2,4-D antibodies with free Analyst, October 1997, Vol. 122 (1107–1111) 1107binding sites were bound to the beads.The amount of antibody bound to the beads was quantified by flowing a fluoresceinlabeled, species-specific antibody through the bead pack. This fluorescein-labeled antibody bound to the 2,4-D-specific antibody immobilized in the bead pack. The observed fluorescence was again inversely related to the concentration of analyte. The instrument flow system was operated under negative pressures using a syringe pump.An in-line vacuum de-gasser was used to prevent the formation of bubbles. All of the pumps and valves were operated through the KinExA hardware interface and PC-based software using a Windows environment. The instrument operations can be divided into two areas: bead handling and sample handling. For bead handling, the coated beads were drawn into the flow cell and removed to waste after the assay had been completed using a back-flush peristaltic pump. For sample handling, samples containing various concentrations of 2,4-D were drawn through channels 2–6 of a rotary valve, then over the packed bead column at a rate of 0.50 ml min21.The KinExA instrument software monitored the fluorescence signal once every second for a total of 420 s and displayed the data on the PC monitor. The fluorescence data were stored as voltage values in Excel and macros (included with the KinExA system) were used for analysis of the data. Signal intensities at specific time points in the instrument cycle were used as an indicator of the accumulation of fluorescent probe on the coated beads. The system was programmed to cycle automatically through the five samples, each using a new bead pack in the capillary.For the antibody-coated bead format, each cycle consisted of (1) bead packing, (2) analyte tracer accumulation and data acquisition, (3) buffer wash and (4) removal of the used beads to waste. For the antigen-coated bead format, step 2 was replaced by the accumulation of anti-2,4-D serum (incubated with various amounts of 2,4-D) followed by the accumulation of fluorescein-labeled anti-IgG antibody.Chemicals PMMA beads (98 mm) were purchased from Bang’s Laboratories (Carmel, IN, USA). Photopolymeric N-oxysuccinimde (PNOS) modified PMMA beads were purchased from BSI (Eden Prairie, MN, USA). Rabbit anti-sheep IgG and sheep anti-2,4-D-serum were obtained from The Binding Site (San Diego, CA, USA). Fluorescein-conjugated rabbit anti-sheep IgG was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The anti-2,4-D monoclonal antibodies (clone E2/G2) were generated by Dr.Milan Franek at the Veterinary Research Institute (Brno, Czech Republic) and kindly provided by Dr. Sergei Erernin (Moscow State University, Russia). Fluorescein isothiocyanate (FITC), 2,4-D, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC), N-hydroxysuccimide and dimethylformamide were purchased from Sigma (St.Louis, MO, USA). Phenol and benzoic acid were purchased from Mallinckrodt (Paris, KY, USA). Atrazine was purchased from ChemService (West Chester, PA, USA). 2,4-Dichlorophenol was purchased from Aldrich (Milwaukee, WI, USA). Chlordane was obtained from the Environmental Protection Agency (Research Triangle Park, NC, USA). All other compounds and solvents used were of analytical-reagent grade. Environmental Samples River water samples were obtained from the Virgin River in Southern Utah.The water was passed through a coarse filter to remove any insoluble matter, and stored at 4 °C until use. The pH of the water at the time of use was 7.0. Well water was obtained from Well HM-1, Salmon site, Louisiana, it was stored at 4 °C until use, at which time its pH was 7.5. Preparation of Fluorescein-labeled 2,4-D Fluorescein thiocarbamylethylenediamine was synthesized from FITC and ethylenediamine as described by Pourfarzaneh et al.18 The amine-derivatized FITC was then conjugated to 2,4-D as reported by Eremin.19 Briefly, the N-hydroxysuccinimide ester of 2,4-D was synthesized and conjugated to fluorescein thiocarbamylethylenediamine in the presence of EDC in dimethylformamide. The tracer was purified by thinlayer chromatography [silica gel plates were developed in ethyl acetate–methanol–acetic acid (90 + 8 + 2, v/v/v)] and the concentration was determined spectrophotometrically using previously reported molar absorption coefficients.18 Preparation of Antibody-coated Beads Antibody-coated PMMA beads were prepared by immersing 200 mg of dry beads into 1 ml of anti-sheep IgG (20 mg ml21) in PBS (phosphate-buffered saline: 10 mm NaHPO4; 100 mm NaCl; pH 7.4).The mixture was then agitated for 2 h at 37 °C, after which the beads were centrifuged (5000g, 2 min) and the supernatant was discarded. The IgG-coated particles were rinsed twice by centrifugation with PBS (1 ml).The sheep anti- 2,4-D serum (diluted 1 + 199 into PBS) was immobilized onto the anti-sheep IgG-coated beads by incubation (with gentle rocking) for 2 h at 37 °C. The beads were again rinsed as described above, suspended in 1 ml of PBS and stored at 4 °C until the day of use, at which time they were diluted with 19 ml, of PBS and placed in the instrument reservoir. Preparation of Antigen-coated Beads Antigen-coated beads were prepared using amine reactive PNOS-coated beads.An amine derivative of 2,4-D was prepared by conjugating ethylenediamine to the N-hydroxysuccinimide ester of 2,4-D. The amine-derivatized 2,4-D was purified by thin-layer chromatography (silica gel plates developed in ethanol). Coupling of the ligand to the beads was carried out in 0.10 m borate buffer of pH 8.5, as suggested by the supplier (BSI). The mixture containing 200 mg of (PNOS) coated beads and 0.5 mm derivatized 2,4-D in 1 ml of the same buffer was agitated for 2 h at room temperature.The beads were then washed sequentially with 0.10 m borate buffer containing 1 m NaCl, followed by 0.10 m acetate buffer (pH 4) containing 1 m NaCl and finally with PBS. The beads were diluted with 19 ml of PBS for use in the instrument. Fig. 1 Diagrammatic representation of the immunoassay format for measurement of 2,4-D. (A) Competitive immunoassay format using antibody-coated beads, (B) competitive immunoassay format using antigencoated beads. 1108 Analyst, October 1997, Vol. 122Assay Procedure for Antibody-coated Beads 2,4-D stock solutions were prepared in ethanol and diluted to final concentrations as specified under Results and Discussion in PBS or an environmental water sample containing the fluorescein-labeled 2,4-D (1 nm or as specified under Results and Discussion). The final assay sample contained less than 1% ethanol which was included in the control and had no effect on the assay. The 420 s cycle (i.e., bead packing, tracer accumulation, buffer washing and bead expulsion) was then initiated through the KinExA software.Fluorescence signal data were collected, displayed and end-point measurements calculated from the difference in the average fluorescence signal of the initial five and last five data points. Assay Procedure for Antigen-coated Beads 2,4-D stock solutions were prepared in ethanol and diluted to final concentrations in PBS. Sheep anti-2,4-D serum diluted 1 + 39 999 in PBS or monoclonal antibody (E2/G2) at a final concentration of 12 ng ml21 was reacted with various concentrations of 2,4-D.The timing cycle was modified to include a step for the addition of the fluorescently labeled secondary antibody (either anti-rabbit or anti-mouse diluted 1 + 4999 in PBS containing 1 mg ml21 BSA). Fluorescence data were handled as above. Curve Fitting and Statistical Analysis The four parameter logistic equation as defined below was used to fit the immunoassay data.20 y a d x c d b = - + æ è ç ö ø ÷ + ( ) 1 where y = instrument response; a = response at high asymptote, b = slope factor; c = concentration corresponding to 50% specific binding (EC-50); d = response at low asymptote; and x = calibrator concentration.Calculations were performed using SOFTmax software (Molecular Devices, Menlo Park, CA, USA). Because the response error over the concentration range of the assay was heteroscedastic, an estimate of error for the precision of concentration was determined at a concentration near the EC-50 using the standard deviation/slope of the response curve at that point.20 Results and Discussion Shown in Fig. 2 are representative fluorescence signal tracings. There are three representative portions of the fluorescence tracing. First, the horizontal portion to the left shows the baseline fluorescence response for the packed beads prior to addition of the fluorescent tracer. Second, the steepest portion of the fluorescence curve represents the introduction of the bulk tracer into the capillary, filling the interstitial spaces between the beads (approximately the first 1–2 min), followed by the accumulation of the tracer on the coated bead surfaces.The third segment shows the removal of the free tracer from the bead pack. Tracings A, B and C represent control experiments for the antibody-coated bead format [shown in Fig. 1(a)]. Tracing A (Fig. 2) shows the non-specific signal when non-analyte specific sheep IgG was substituted for anti-2,4-D serum on the antibody-coated beads.Tracings B and C show the analytedependent response and result from the presence and absence, respectively, of 2,4-D (10 mg l21) in the assay buffer. The concentration-dependent signal response to 2,4-D was similar when determined from either the accumulation portion of the curve at 300 s or from the tracer remaining on the beads after a buffer wash measured by the fluorescence signal at 420 s.Readings at 420 s were routinely used to generate competition response curves. For most competitive immunoassay methods, the lowest potential detection limit of the assay is primarily determined by the affinity of the antibody for the antigen and, to a lesser extent, by the relative experimental error. However, for non-equilibrium methods such as is reported here, the relative concentrations of immobilized antibody and analyte probe may also impact the assay characteristics. Consequently, the effects of analyte tracer concentration and the polyclonal antibody immobilized on the solid phase were determined.For the antibody-coated bead format, analyte tracer concentrations were varied over the range 0.1–50 nm. A tracer concentration of 0.1 nm yielded no response to the analyte (2,4-D) and 50 nm tracer resulted in unacceptable levels of variability in the fluorescence response. A probe concentration of 1 nm yielded an acceptable signal and minimal variability.The antibody concentration used for immobilization was varied over the range of 0.1–2.0 mg protein per mg dry beads, and had no effect on the competition curve for 2,4-D (data not shown). Shown in Fig. 3 is the signal response curve for 2,4-D using the polyclonal antibody-coated bead format with fluoresceinlabeled 2,4-D as the fluorescent tracer. The mean fluorescence response data were fitted using a four parameter mode.20 The dynamic range extended from 10 mg l21 to 10 mg l21.The inflection point of the curve (EC-50) was 0.87 mg l21 and the multiple correlation coefficient (R) was 0.999. The precision of concentration was calculated at 1 ± 0.28 mg l21. Also shown in Fig. 3 is the signal response curve for 2,4-D, using the antigen-coated bead format for the polyclonal antiserum with fluorescein labeled anti-species antibody as the fluorescent tracer. The dynamic range for this format extended from 2 mg l21 to 20 mg l21.The EC-50 was 1.0 mg l21 and R was 0.998. The precision of concentration was calculated at 1 ± 0.12 mg l21. The competition curves for 2,4-D were similar for both assay formats suggesting that both assay formats were reporting the unoccupied (antibody) binding sites (in the presence of 2,4-D) and that the measuring process did not interfere with the steadystate association of the analyte with the antiserum. In addition, there are several advantages which may be realized by the versatility of this assay system.For example, the ability to link Fig. 2 Typical assay response and control experiments for antibodycoated bead format (A) non-specific binding signal; fluorescein-labeled 2,4-D (1 nm) was used with PMMA beads that were coated with nonanalyte- specific IgG. (B/C) IgG from anti-2,4-D serum was immobilized onto the PMMA beads (as described under Experimental). Antibody-coated beads were then exposed to the fluorescein-labeled 2,4-D (1 nm) in the presence (B) or absence (C) of target analyte 2,4-D (10 mg l21).Analyst, October 1997, Vol. 122 1109the antigen either to a fluorescent tag or to the derivatized beads (each with a different chemical linker) allows one to avoid similarities (and subsequent cross-reactivity) to the linker chain used to form the immunogen. This is particularly important for the use of commercially available antibodies for which the specific immunogen structure is not known.Shown in Fig. 4 are the signal response curves generated by spiking 2,4-D into various environmentally significant media, using polyclonal antiserum with the antibody-coated bead format. The signal response from the 2,4-D spiked well water significantly overlapped the signal response using buffer. The response for 2,4-D spiked into river water appeared slightly lower than that for the buffer or well water matrices. This bias was evidenced by the mean values for seven of the eight data points being lower than for the buffer control as well as the standard deviation values for four of the 2,4-D levels not overlapping those for the spiked buffer.The reason for the tendency of the river water matrix to result in an overestimation of the 2,4-D is not clear. 2,4-D contamination of the water was ruled out by HPLC analysis using standard methods. It is possible that compounds such as humic substances may have interfered with the assay.The relative errors for the environmental matrices were similar to those for the buffer control. The precision of concentration values calculated at l mg l21 were 1 ± 0.28 mg l21 for the buffer, 1 ± 0.27 mg l21 for the well water and 1 ± 0.43 mg l21 for the river water. Shown in Table 1 is the cross-reactivity profile for several structurally related compounds and structurally unrelated pesticides. As expected from previous reports of immunoassays using polyclonal anti-2,4-D serum, compounds similar in structure to 2,4-D, such as 2,4,5- and 2,4-dichlorophenol, elicit a response from this method.19 Compounds with structures unlike 2,4-D showed little if any response.The calibration plot for 2,4-D in Fig. 5 shows the response of the monoclonal antibody (E2/G2) using the antigen-coated bead format. The data were again fitted using the four parameter model. The EC-50 was 2.8 mg l21, R was 1.00 and the assay response curve showed a dynamic range of 0.1–100 mg l21.In comparison with the other reported bioanalytical methods for 2,4-D, the described KinExA method showed a higher response to the analyte than a reported enzyme assay,14 and fluorescence polarization immunoassay.13 The displacement curve and dynamic range for this method using the monoclonal antibody (E2/G2) are similar to those typically reported for ELISAs11 or ELISA-type assays4,17 and are about an order of magnitude less sensitive than microformat ELISAs using chemiluminescence detection.3 Nevertheless, given the profound influence of the affinity characteristics of any particular antibody on the assay response, comparison of immunoassay techniques that use different antibodies is tenuous.Conclusions A variety of immunoassay methods have been reported for the detection and measurement of 2,4-D, each with distinct analytical and assay format characteristics. The potential advantages of the proposed method in its application to environmental monitoring relates primarily to its ease of use (i.e., the degree of automation), the limited waste generated Fig. 3 Assay response as a function of 2,4-D concentration for the antibody-coated bead format (-), or the antigen-coated bead format (5). Error bars represent standard deviation, n = 3. Fig. 4 Effect of environmental water samples as assay matrix using the antibody-coated bead format. 2,4-D standard curves were prepared in PBS (-), spiked into well water (5) or river water (:) samples.Multiple regression coefficients were 0.999, 0.999 and 0.998 for the PBS, well water and river water, respectively. Error bars represent standard deviation, n = 3. Table 1 Cross-reactivity profile Compound Relative response* 2,4-D 100 2,4,5-T† 92 2,4-Dichlorophenol 86 Atrazine 20 Chlordane 14 Benzoic acid 10 Phenol 0 * Relative response to 2,4-D at 1 mg l21 determined using the following relationship: (response for test compound/response for 2,4-D) 3 100. † 2,4,5-Trichlorophenoxyacetic acid.Fig. 5 Assay response as a function of 2,4-D concentration using monoclonal antibody (E2/G2) in the antigen-coated bead format. 1110 Analyst, October 1997, Vol. 122(i.e., the absence of contaminated plates, tubes or vials), and the versatility allowed by the instrument and software in the development of immunoassays. The ability to use either the antibody or antigen as the immobilized phase allows the method to be tailored to the particular characteristics of the immunochemicals and requirements of the potential application (e.g., optimization of assay time and reagents per assay). 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