On-Line Immunochemical Detection System for Pesticide Residue

Chapter 11

On-Line Immunochemical Detection System for Pesticide Residue Analysis Development and Optimization

Downloaded by STANFORD UNIV GREEN LIBR on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch011

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Petra M . Krämer , Renate Kast , Ursula Bilitewski , Stephan Bannierink , and Ulrich Brüss 2

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GesellschaftfürBiotechnologische Forschung mbH, Mascheroder Weg 1, 38124 Braunschweig, Germany Meta GmbH, Oststrasse 7, 48341 Altenberge, Germany

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The prospect of an automated immunochemical detection system for on-line analysis of pesticide residues has important implications for environmental monitoring and control. Over the past seven years a flow injection immunoanalysis (FIIA) system was developed and optimized in our laboratories; this led to the development of a prototype of a flow injection immunoaffinity analysis (FIIAA) device. The strategy followed during this development is described here. First results of the newest version of this system are presented. The advantages and limitations of FIIAA compared with conventional analytical techniques, such as L C and GC/MS, microtiter plate ELISA, and the commercially available test-kits are also discussed. The currently undergoing demonstration for real water samples, and the potential of this FIIAA system for water monitoring will be outlined.

Immunochemical detection methods for environmental analysis have gained acceptance over the past few years (7). This is true for the commercially available test-kits, such as the magnetic particle-based and microtiter plate based assays. Both formats are very useful tools for fast screening of samples at contaminated sites (2, 3), and also for quantitative analysis in the laboratory (4, 5). However, most commercial immunoassay kits are not applicable for automated analysis. Our goal was to develop an additional format for immunochemical determination which would allow on-line pesticide monitoring in aqueous solutions, e.g. in groundwater and surface water. This technique is based on flow injection immunoanalysis (FIIA), where the antibodies are immobilized on solid supports (6-8). The triazine herbicides, which are used intensively worldwide were selected as the target analytes for this study. Conventional analysis of these compounds by liquid

© 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

chromatography (LC) and gas chromatography (GC) has been well established thus allowing easy confirmation of results. In addition, many sensitive immunoassays for s-triazines are available for comparison (e.g., 9-11). Although the most widely used herbicide atrazine has been banned in some European countries, e.g. in Germany and in Denmark, its usage is still permitted in most European countries, e.g. in France, Greece, Italy, Portugal, Spain, The Netherlands, etc. It has been detected in groundwaters (12-14), and it is expected that atrazine and its metabolites (deethylatrazine, deisopropylatrazine) will be found in ground- and surface water supplies for many years (e.g., 12, 14). Atrazine can also act as an indicator for the presence of other pesticide residues, such as alachlor, cyanazine, metolachlor, and metribuzin. These herbicides are also used in large quantities (75), and all except metribuzin are classified as probable or transient leachers (14). Immobilization and Assay Strategies A number of different assay and immobilization techniques were explored during the development of the FIIA. The following gives an account of the overriding strategy behind this development. For detailed information regarding the early research, readers are directed to the literature cited. Immobilization of Polyclonal Antibodies on Membranes. Initial studies involved the immobilization of polyclonal antibodies on immunoaffinity membranes (Immunodyne or Fluorotrans transfer membrane, P A L L Corp. Glen Cove, NY), either directly or via protein A (6, 7). This procedure was combined with the exchange of the membrane after each assay, and a special membrane exchanging mechanism was designed for this purpose (7). The main target analyte was atrazine, together with propazine and simazine, which also cross-reacted with these polyclonal antibodies. The system worked in the sequential saturation technique: analyte and enzyme-tracer were incubated sequentially with the antibodies (16). This implies, that the analyte and enzyme-tracer are not incubated together and therefore do not compete simultaneously for the limited binding sites of the antibodies. This method is different from the usual technique used in microtiter plate formats. The enzyme used was horseradish peroxidase (HRP), with H 0 2 and 3-(phydroxyphenyl) propionic acid (HPPA) as fluorescent substrate (17). The fluorescence of the product was determined downstream in a fluorometer flow through cell (A, 320 nm, A , 404 nm). This procedure allowed the measurement of atrazine and propazine at the very low ppb level (0.1 pg/L), which is relevant for the European drinking water directive (18). However, this technique required large amounts of antibodies, that were about 130 times higher than in the corresponding enzyme-linked immunosorbent assay (ELISA); (7)), and the reproducibility of immobilization was not comparable with that achieved with the microtiter plate format. Furthermore, it was critical to exchange the membrane without the presence of the operator, which made it difficult for unattended operation. These problems needed to be overcome before the development of a prototype instrument as an automated, unattended control system was initiated. 2

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In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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On-Line Detection of Pesticide Residues

Immobilization of Monoclonal Antibodies on Polystyrene or Glass Beads. Following literature studies, the immobilization of antibodies on beads was explored as an alternative to the use of membranes to obtain better performance and decrease consumption of antibodies (79). Monoclonal anti-terbutryne antibodies (mab K1F4; (10)) were immobilized either on polystyrene or glass beads, with diameters of about 220 pm or from 250-500 pm, respectively, and the antibodies were regenerated after each assay with glycine/HCl (pH 2.1; (8)). This system offered several advantages over the membrane method. The antibody consumption was reduced, because at least 60 regenerations of the antibodies were possible. Secondly, the unattended operation time was extended, because no exchange mechanism was needed for approximately one day. The reproducibility also improved as a zero standard concentration could be determined prior to each sample analysis, providing a control value. Although the absolute signals [mV] of the zero standards showed much variation (Figure 1), the coefficient of variation (CV) of the corresponding %control-values for the pesticide standards was only about 10%. The result of this %control-value was independent of the decline in signal due to high analyte concentration of the preceding sample, regeneration of the antibodies, or a decrease in enzyme-tracer activity. These promising results prompted us to begin the prototype development of this device for on-line monitoring of pesticide residues in aqueous solutions. At this point it was noted that the use of another monoclonal antibody, namely the antiatrazine antibody (K4E7), caused problems. Each antibody-hapten system has different stability and regeneration characteristics, therefore each individual system must be optimized, which could present difficulties to an untrained user. A new strategy was therefore investigated with the aim of producing a universal system. Immunoaffinity Column with Protein A or G . As an alternative to the regeneration of the antigen-binding sites of the antibodies, a more universal system is the regeneration of the specific binding site of protein A or G to the Fc region of IgG. This system is very well established and utilized for affinity columns such as for antibody purification. The binding of a specific class or subclass of IgG to protein G or A is well characterized, with an affinity binding that is high enough (approximately 10 M ' ; (20)) for measurements in a flow system. Protein G was immobilized on glass beads using the glutardialdehyde immobilization method described earlier for antibody immobilization (8). Briefly, silanized glass beads (150-212 pm) were activated with glutardialdehyde (2.5% v/v), the beads were washed and protein G (2 mg/mL) was incubated for 4 h at room temperature. These beads were then incubated with 1.5% bovine serum albumin (BSA) to avoid nonspecific binding of protein. A column reactor was filled with this material, and then anti-atrazine antibodies (mab K4E7; (77)) were pumped into this protein G column, where they were incubated for 2 min. The atrazine and then the enzyme-tracer were pumped sequentially through the column, followed by the substrate for the enzyme-tracer. Again, the product formation was determined downstream in a flow through cell of a fluorometer (A, 320 nm, A , 415 nm; Figures 2, 3). With this system, the independence of the regeneration of the antibodyantigen system was gained, because the antibodies were replaced after each assay, and protein G was regenerated. It was possible to regenerate the protein G column over 8

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150 200 Time [min] Figure 1. Flow injection immunoanalysis with regeneration of the antigenbinding site. Peak profile of a complete standard curve for terbutryne. Arrows show the peak for the corresponding concentration of terbutryne. Prior to each standard a zero concentration (*) was determined.


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On-Line Detection ofPesticide Residues

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Downstream Measurement of Fluorescence of Product in Row-Through Cell of Fluorometer


(tex 320 nm, Xem 415 nm)

Addition of Substrate for Enzyme Reaction and Incubation _ r-|

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Addition of Enzyme-Tracer and Incubation

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Addition of Pesticide (Standard/Sample) and Incubation A A

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Addition of Pesticide-specific Antibody and Incubation Protein A (G) Support in Reactor

Figure 2. Schematic illustration of sequential saturation analysis with flow injection immunoaffinity analysis (FIIAA). Steps (1-5) are performed automatically and controlled via computer. A regeneration step follows after step 5, and the protein A (G) column is then ready for the next incubation with antibodies (step 1).



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Waste-

6 - Port Selection Valve

10 - Port Injection Valve

PBS BufferRegenerationSolution

-Waste Pump

Ethanol— Sample 1 — Sample 2 — StandardKZero)— Standard2 — Standard3—

Detector

Enzyme Pesticide Tracer Antibody

Affinity Column with Protein A bstrate

6 - Port Selection Valve

Figure 3. Most recent flow chart of the automated on-line monitoring system for one analyte, using a temperature-controlled (15°C) immunoaffinity column with protein A, where the immunochemical reaction takes place. Two sample inlets (1 and 2) are included in this system.



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On-Line Detection of Pesticide Residues

200 times with only little decrease in the binding capacity of the column, and with good reproducibility of the protein G regeneration. This system, with immobilized protein G (A) on the column, was designated FIIAA (flow injection immunoaffinity analysis; Figure 2). Recently, we used a commercially available protein A affinity material. In the acidic pH range (< 5.5), the affinity interaction of IgG to immobilized protein A is weaker compared to immobilized protein G (20), which makes it more suitable for regeneration (antibody elution from the column). Using protein A on this affinity material, at least 1000 regenerations with 0.1 M sodium citrate (pH 2.5) are possible. High antibody and enzyme-tracer dilutions (1:20,000 to 1:80,000) can be used, which are comparable to the conventional microtiter plate ELISA. Therefore, the consumption of antibodies and enzyme-tracer is no longer a critical issue. This system was tested in an alternating mode of a zero standard and a 0.1 pg/L atrazine standard (Figure 4), which is a simulation of the on-line operation, where only minor concentration changes will usually occur. Four measurements of a zero standard were necessary before the system showed stable signals (Figure 4). After the system was stabilized, the reproducibility of the signal was very good (CV 2.4%). In addition, analysis of alternating atrazine concentrations was performed, again with a prior determination of zero concentration (Figure 5). This performance illustrates the system as in the automated laboratory version, where different concentrations will be measured in a random sequence. It should be noted though, that the concentration difference of the standards in this example was up to 500 times (0.02 pg/L was determined after 10 pg/L). Although the absolute signals (mV) of the zero concentration values and of the different atrazine concentrations showed great variations during the runs, the standard deviation (SD) was acceptable, when the atrazine standard was expressed as %control-value of the preceding zero standard (Table I).

Concentration [Ug/L] 0.5 0.05 10 0.02 1 0.1

Table I. % Control-Values of Figure 5 First Run Second Run %Controi 42.2 77.6 10.4 83.2 34.6 74.3

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%Controi 37,9 79.2 12.6 79.5 34.0 67.7

Average a

%Control ±SD

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40.1 ± 3 . 0 78.4 ± 1.1 11.5 ± 1.6 81.4 ± 2 . 6 34.3 ± 0.4 71.0 ± 4 . 7

signal [mV] of standard/sample a

%Control =

x 100 signal [mV] of preceding zero standard


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200 mV

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< 0.1

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0.L

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0.1

Time [h]

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Figure 4. Scan of an original flow chart of a sequence of zero concentrations (*) and an atrazine standard of 0.1 ug/L in flow injection immunoanalysis. A run of about 17 h is shown (right corner, bar represents 2 h). The maximal height of the zero signal corresponds to 400 mV.

200 mV

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On-Line Immunochemical Detection System for Pesticide Residue

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