Polarization Fluoroimmunoassay for Rapid, Specific Detection of

Chapter 16

Polarization Fluoroimmunoassay for Rapid, Specific Detection of Pesticides Sergei A. Eremin

Downloaded by UNIV LAVAL on May 3, 2016 | http://pubs.acs.org Publication Date: March 23, 1995 | doi: 10.1021/bk-1995-0586.ch016

Division of Chemical Enzymology, Department of Chemistry, M. V. Lomonosov State University, Moscow 119899, Russia Polarization fluoroimmunoassay (PFIA) is a simple, inexpensive, easily automated screening method for pesticide residues in large numbers of environmental samples. PFIA measures the increased polarization of fluorescence when a fluorophore-labeled hapten (tracer) is bound by specific antibody, and the decreased signal when free analyte competes with the tracer for binding. No separation of free and bound analyte is required. PFIAs for 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5trichlorophenoxyacetic acid (2.4,5-T), simazine, and atrazine were automated on an Abbott TDx Analyzer. Ten water samples of 0.05 mL can be analyzed in 7 minutes, with detection limits of 100 ng/mL for 2,4-D and 5 ng/mL for simazine, and coefficients of variation < 5%. The monitoring of pesticide residues in ground water, surface water, soil, and other environmental samples has gained increasing importance worldwide. Established procedures for detecting pesticides include high-pressure liquid chromatography (HPLC) and gas chromatography (GC). These require extraction of the samples to concentrate the residues and remove interfering matrix materials. Over the last ten years immunochemical methods such as enzyme-linked immunosorbent assay (ELIS A) have been increasingly used for detection of pesticides (7). ELIS A and related methods have several advantages and facilitate analysis of large numbers of samples. ELISAs are much less expensive to run and their detection limits can be as good or better than those of instrumental methods. However, ELISAs are difficult to automate and standardize. They generally require several washing steps, a step in which the free and bound analyte is separated, and the approach to equilibrium binding may be relatively slow (30 to 120 min). From our point of view, simplifying the assay and minimizing the analysis time per sample are the primary goals in developing screening methods for large numbers of samples. Polarization fluoroimmunoassay (PFIA) is a "homogeneous" immuno-chemical method, i.e., it does not require washing or separation of the free and bound analyte. The principle and some of the critical factors in design and automation of PFIA for pesticides are summarized below. Details of the theory and application of PFIA may be found in recent reviews (2,3). PFIA originated from experiments in the early 1960s by Dandliker and his colleagues, in which antigen-antibody reactions were monitored by changes in fluorescence polarization (4). Subsequently, PFIA became widely used in clinical chemistry because of its simplicity and precision. There are now clinically accepted PFIAs for monitoring the administration and effects of about 100 therapeutic drugs (5). The first application of PFIA to pesticide detection was reported by Colbert and Coxon, who developed a PFIA for paraquat in serum samples and adapted it to run on 0097-6156/95/0586-0223$12.00/0 © 1995 American Chemical Society Nelson et al.; Immunoanalysis of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

224

IMMUNOANALYSIS OF AGROCHEMICALS


the Abbott TDx Analyzer, an instrument that was specially designed to automate PFIA (6). The TDx Analyzer can be set up to perform sample pre-treatment, add fluoresceinlabeled hapten and antiserum, measure the signals, and calculate and report the results. A major advantage of PFIA on the Abbott TDx Analyzer is that it is generally not necessary to run standards every time an assay is repeated over periods of one week to a few months. This substantially reduces costs and workload (3). The reproducibility is due primarily to the stability of the small molecular weight fluorophore-labeled hapten tracers in solution, and the way that the response is measured. Fluorescence polarization units are a ratio of intensities of different polarized components of the fluorescence, so they are relatively independent of time and nonspecific fluorescence caused by the sample matrix. Subsequently we published preliminary results of a PFIA for 2,4-D and other pesticides ( 7,8). Principles of PFIA. PFIA is a competition method based on detection of the difference of fluorescence polarization between a small fluorescent-labeled antigen and its immuno-complex with specific antibody (2). PFIA depends upon the difference in the signal given by a relatively small fluorescein-labeled hapten when it is in the free form as compared with the much higher polarization value when it has been bound by its specific antibody. Eliminating the need to separate the free and bound tracer is a considerable advantage, as it simplifies the assay, often improves its precision, and makes it much easier to fully automate. The polarization of fluorescence (P) is determined by exciting the mixture of antibody, sample, and tracer with vertically polarized light and measuring the intensity of both the vertically (I ) and horizontally (Ih) polarized components of the emitted fluorescence. The Ρ value is defined as the ratio of difference and sum of these components: v

P = (Iv-Ih)/dv + Ih) It is convenient to use "milliunits" of ρ (mP values) such that mP = 1,000(P). Several factors influence the ρ values. The most significant of these is the size of the fluorescein-labeled tracer. Because it is present in limiting amounts in the PFIA, much more of the tracer is bound than is free. Other important variables are the length and type of bridge between the fluorescein and the analyte moiety of the tracer, and the temperature and viscosity of the reaction mixture. As with any analytical method, PFIA has disadvantages. These include poorer detection limits than obtained with the best ELISAs, and the cost and limited availability of instrumentation to detect polarized fluorescence. In addition, PFIAs do not work for high molecular weight (> 1000 dalton) analytes. PFIAs are susceptible to interference from substances in some sample matrices such as plant extracts, or serum. However, ground water and surface water samples are generally free of interfering compounds. PFIAs are particularly suitable for routine pesticide contamination tests where the most sensitive limit of detection is not needed, or where it is possible to extract and concentrate the analyte prior to assay. In Russia 2,4-dichlorophenoxyacetic acid (2,4D) and simazine are two of the most widely used herbicides. The regulatory action levels in surface water are 100 ng/mL for 2,4-D and 2.4 ng/mL for simazine. For preliminary screening of surface water it is only necessary to semi-quantitatively detect these amounts, but the assays must be highly reliable. To monitor these two herbicides in water samples throughout the agricultural areas of Russia we would need at least one million assays. Even semi-quantitative screening at this level of sensitivity would be of great value in detecting dioxin, which is a trace contaminant of 2,4,5trichlorophenoxyacetic acid (2,4,5-T). In Russia, many water samples positive for dioxin are also contaminated with 2,4,5-T. Here we briefly report our most recent results adapting pesticide PFIA for 2,4-D, 2,4,5-T, simazine, atrazine and related compounds so that they could be run on the

Nelson et al.; Immunoanalysis of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

16. EREMIN

Polarization Fluoroimmunoassay

225

Abbott TDx Analyzer. Other polarization fluorimeters such as the Perkin-Elmer LS50, Merck VITALAB, and the Roche COBAS FARA II can be adapted to run PFIA. In the process of developing our PFIAs, we studied how the structure of the labeled antigen affects sensitivity. The sensitivity was greatest using the shortest chemical "bridge" between the antigen and the fluorescent label. Labeled antigens that were structurally homologous or heterologous to the primary target analytes were investigated. Our results indicated that competitive-binding PFIAs are more sensitive when structurally heterologous tracers are used.


Materials and Methods 2,4-D. Antiserum was raised in a rabbit immunized with 2,4-D conjugated via the carboxyl group to bovine serum albumin (BSA) (9). The mouse monoclonal antibody designated E2/G2 to 2,4-D was derived using the same immunogen at the Veterinary Research Institute (Brno, Czech Republic), and provided as unpurified ascites fluid (10). Fluorescent tracers were prepared by synthesizing an N-hydroxysuccinimide ester of 2,4-D and conjugating it to fluoresceinthiocarbamyl derivatives of 1,2diaminoethane (n=2), 1,4-diaminobutane (n=4), and 1,6-diaminohexane (n=6) (Figure 1) (9). These derivatives were synthesized by a direct reaction of NH2(CH2) NH2 2HCl with fluorescein isothiocyanate, according to established procedures (77). The tracers were purified by thin-layer chromatography and their concentrations were estimated spectrophotometrically using the published molar extinction coefficient forfluorescein(77). e

n

2,4,5-T. Rabbit polyclonal antiserum was raised using 2,4,5-T conjugated via its carboxyl group to BSA or keyhole limpet hemocyanin (KLH) (72). The tracer was prepared from 2,4,5-T and afluoresceinthiocarbamylderivative of 1,2-diaminoethane (n=2) by the same method used for the 2,4-D tracer. Simazine. Reaction of 2,4-dichloro-6-(ethylamino)-l,3,5-triazine with 6aminohexanoic acid yielded simazine derivatives which were conjugated to KLH using the carbodiimide method (13). The resulting immunogen was used to raise antiserum in rabbits (Figure 4). The tracer was prepared from the same simazine derivatives andfluoresceinthiocarbamylderivatives of 1,2-diaminoethane (n=2) (Figure 4) (14). Atrazine. A derivative of atrazine with thiopropionic acid substituted in place of the chlorine atom (13) was conjugated to KLH. This conjugate was used to raise antiserum in sheep (Figure 5). A tracer with the homologous structure was synthesized from the same triazine hapten andfluoresceinthiocarbamylderivatives of 1,2diaminoethane (n=2). Tracers with heterologous structure were prepared from 2,4dichloro-6-(isopropyl)-l,3,5-triazine by condensation with fluoresceinthiocarbamyl derivatives of NH (CH )nNH2 (n=2,4,6) (Figure 5) (75). 2

2

PFIA Analyzer. A TDx Analyzer (Abbott Laboratories, USA) was used to measure fluorescence polarization in milli-units (mP). To perform the measurements, up to ten TDx glass cuvettes were loaded into the special "Photo Check" carousel. Measurement and calculations were performed automatically and printed by the instrument. The total time for measurement of 10 samples was about 7 min. PFIA Procedures. Sodium borate buffer (0.05M, pH 8.6) was used as the diluent in all experiments. To determine antibody concentrations usable for competition PFIA, 0.5 mL of various dilutions of antiserum was mixed with 0.5 mL of tracer at 10



226


Figure 1. Structures of 2,4-D (top), the hapten conjugate used for antibody production (center), and fluorescent tracers of differing bridge length (n=2,4, or 6; bottom).

300

0

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ι ι ι 11

I

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T-~

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i

θ" τ-

Dilution of antiserum Figure 2. Rabbit anti-2,4-D serum dilution curves obtained using tracers of differing bridge length. Dilutions of the antisera were incubated with each tracer and fluorescence polarization was measured. (O) 2 carbon; ( · ) 4 carbon; (A) 6 carbon. The dotted line (--• -) shows nonspecific binding with normal rabbit serum (similar for all tracers).


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227

nmol/L in TDx glass cuvettes at room temperature, and fluorescence polarization was measured immediately. The competition PFIA was performed in TDx cuvettes by sequentially adding 50 μ ι of standard or sample, 0.5 mL of tracer solution (10 nmol/L), and 0.5 mL of antibody at a dilution that gave about 70% of maximum binding of tracer (determined from the antibody dilution curve). After the measurement offluorescencepolarization in mP as described above, the standard curves were plotted as mP vs. logarithm of the concentration of analyte.


Results The PFIA can be used to rapidly measure the relative binding of different antibodies to a particular tracer, and to compare tracers of different structure using a particular antibody or serum. Figure 2 shows the antibody dilution curves for polyclonal anti2,4-D serum with tracers that have different bridge lengths between the 2,4-D and fluorescein, as shown in Figure 1. This antiserum had a higher titer for the tracer with the longest bridge, but displacement of 2,4-D was significantly greater with the tracer that had the shortest bridge (Figure 3). The most sensitive assay, in terms of the minimal detectable concentration at the 95% confidence level, was 0.1 μg/mL (5 ng of 2.4-D in the 50 μΐ. sample) using tracer with the shortest bridge. The detection limit using monoclonal antibodies (MAbs) with the same tracer was also 0.1 μg/mL, but the specificity with the MAbs was greater (Table I). A very similar PFIA was developed for 2,4,5-T. Polyclonal antisera to 2,4,5-T were raised in three rabbits using KLH conjugates as immunogen, and in three other rabbits using BSA conjugates of the same hapten. The rabbits immunized with the KLH conjugates developed higher titer sera against 2,4,5-T than those immunized with the BSA conjugate. The specificity of the 2,4,5-T PFIA, using the best tracer and polyclonal anti-2,4,5-T-KLH serum, was comparable to that of the monoclonal 2,4-D PFIA (Table I). The detection limit was 0.1 μg/mL, equivalent to 5 ng of 2,4,5-T in the sample of 50 μL. This is comparable to the detection limit for GC of 2,4,5-T, making PFIA potentially suitable for screening water samples. Table II shows 50% inhibition of tracer binding and percent cross-reactivity for PFIA of simazine using polyclonal antiserum and tracer with a structure homologous to the immunogen (Figure 4). The sensitivity for simazine was greater than for 2,4-D (5 ng/mL in a 50 μ ΐ sample or 250 pg per test), making the simazine PFIA one of the best we have tested. However, this serum cross-reacted about equally with simazine and atrazine (Table Π). To develop a more specific PFIA for atrazine we designed an immunogen with a thio-group analog of s-triazines. The polyclonal antiserum to atrazine bound very well (titer 1/2000) with an atrazine tracer structurally homologous to the immunogen (Figure 5). However, use of this tracer resulted in a poor competitive PFIA (results not shown). Accordingly we synthesized and tested heterologous tracers (Figure 5) for better competition with atrazine using this polyclonal antiserum. A sensitive competition PFIA for atrazine was developed using the heterologous tracers (Table III). As in the assay for 2,4-D, the most sensitive competition PFIA for atrazine - a detection limit of 10 ng/mL in a 50 μΙ, sample - was obtained using the tracer with the shortest bridge (Figure 6). Herbicides structurally related to atrazine were tested and the cross-reactivities are given in Table III. This PFIA was about 100-fold more sensitive to atrazine than to simazine. This means that our polyclonal antibodies recognize the isopropyl group in s-triazines. The antisera actually detected propazine, ametryne, and prometryne better than atrazine in this assay. Ametryne and prometryne, which are structurally similar to the immunogen, could be detected at 5 ng/mL with this assay. However, propazine, ametryne, and prometryne are not commonly used in Russia.



228


1 20-J

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2,4-D, pg/mL

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Figure 3. Standard curves for PFIA of 2,4-D using tracers with differing bridge length: (O) 2 carbon; ( · ) 4 carbon; (A) 6 carbon.

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Figure 4. Structures of simazine (top), the immunizing conjugate (center), and the tracer for PFIA (bottom).


16.

EREMIN

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Table I. Relative cross-reactivity of some compounds structurally related to 2,4-D in PFIAs with polyclonal or monoclonal antibodies, and in PFIA of 2,4,5-T with polyclonal antibodies.


Cross-reactivity (%) No.

R2

R3

R4

R5

R6

2,4-D

2,4-D

1

CI

H

Cl

H

H

OCH2COOH

(poly)

(mono) (poly)

100

100

5.6

2

CI

H

Cl

H

H

OCH(CH3)COOH

2.5

.b

5.4

3

CI

H

Cl

H

H

0(CH2)3COOH

41

3.6

15

4

CH

3

H

Cl

H

H

OCH2COOH

7.2

2.1

9.3

5

CH3

H

Cl

H

H

OCH(CH3)COOH

1.0

-

5.0

6

CH3

H

Cl

H

H

0(CH2) COOH

12

-

21

7

CH

H

Cl

H

Cl

OCH2COOH

2.6

-

4.0

8

CH3

H

H

H

H

OCH2COOH

1.0

-

4.0

9

CH3

H

H

H

Cl

OCH2COOH

1.0

-

3.6

10

CI

H

H

H

H

OCH2COOH

7.7

2.8

2.4

11

CI

Cl

H

H

H

OCH2COOH

34

-

-

12

Η

Cl

Cl

H

H

OCH2COOH

8

-

-

13

Cl

H

Cl

Cl

H

OCH2COOH

59

5.0

100

14

Cl

H

Cl

H

H

Œ2COOH

0.1

-

-

3

Ζ

a

3

2,4,5-T

a Percent cross-reactivity (% CR) is defined as the ratio of mP units at 10 ppm μg/mL) of 2,4-D to mP units for the indicated analyte. - Not tested Compounds tested: 1. 2,4-Dichlorophenoxyacetic acid 8. o-Tolyloxyacetic acid 2. 2-(2,4-Dichlorophenoxypropionic acid 9. 6-Chloro-otolyloxyacetic acid 3. 4-(2,4-Dichlorophenoxy)butyric acid 10. 0-Chlorophenoxy acetic acid 4. 4-Chloro-o-tolyloxyacetic acid 11. 2,3-Dichlorophenoxyacetic acid 5. 2-(4-Chloro-

Polarization Fluoroimmunoassay for Rapid, Specific Detection of

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