homogeneous enzyme immunoassay for pyrethroid pesticides and their derivatives using bacillary...

ANALYTICA CHIMICA ACTA

ELSEVIER Analytica Chimica Acta 347 (1997) 13 l-1 38

Homogeneous enzyme immunoassay for pyrethroid pesticides and their derivatives using bacillary alpha-amylase as label

Anatoliy V. Zherdev*, Boris B. Dzantiev, Janna N. Trubaceva

Received 12 September 1996; received in revised form 5 March 1997; accepted 13 March 1997

Abstract

A new enzyme immunoassay for detection of some pyrethroid pesticides (permethrin, phenothrin) and their derivatives

containing 3-phenoxybenzoic group has been developed. It is based on enzyme multiplied immunoassay technique (EMIT), modulation of catalytic activity of hapten-enzyme conjugate by anti-hapten antibodies and restoring the initial activity level by free hapten in the sample tested. Alpha-amylase from Bacillus subtilis is proposed as a new enzyme label for EMIT. Inhibition of the 3-phenoxybenzoic acid-amylase conjugate by specific antibodies was revealed at the systems detecting non-

splinted starch or released aldehyde groups. EMIT conditions for pyrethroid pesticides and their derivatives have been optimized. The assay time is not more than 45 min, the detection limits reach 2-5 ng ml _I_ Photometric measurements can be carried out by means of standard ELBA equipment. The proposed label was also used to develop EMIT for pesticide 2,4- dichlorophenbxyacetic acid.

Keywords: Alpha-amylase; Immunoassay; Peslicides; Pyrethroids

1. Introduction

Pesticides have become an indispensable part of

modern agricultural technologies, but their high toxi- city generates a need to control pesticides’ content in

different substances: soil, water, agricultural products, etc. Enzyme immunoassay is one of the most acceptable techniques for quantitative determination of pesticides. Being based on the use of specific antibodies and high-active enzyme labels, it ensures specificity and sensitivity corresponding to the current

*Corresponding author. Fax: +7 95 954 2732: e-mail:

[email protected].

practical requirements. This is a matter of its intensive elaboration for different pesticides nowadays [l-7].

The vast majority of investigators propose solid

phase technique of the enzyme immunoassay (ELISA). The alternate approach is homogeneous

technique that permits to exclude diffusion-dependent processes, separation and washing steps and this way to accelerate and simplify the assay. Principle of homogeneous enzyme multiplied immunoassay technique (EMIT) consists in modulation of catalytic activity of enzyme-antigen conjugates after their binding with antibodies 181. However, special choice of enzyme and conjugation chemistry is necessary so that significant changes in the catalytic activity are attained. Known approaches for solving

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132 A.V Zherdev er al./Analyka Chimica Acla 347 (1997) 131-138

of this problem [9-141 are not sufficiently universal.

Therefore the choice of appropriate labels is an extre- mely important problem for further EMIT development.

In the present investigation we propose and char- acterize alpha-amylase from BacilEus subtilis (E.C. 3.2.1.1) as a new label for EMIT. Synthetic pyre-

throids were chosen as model antigens because of intensive implementation of these insecticides

[15-171.

2. Experimental

2. I. Materials

Purified preparations of pesticides permethrin and phenothrin were obtained and provided by Dr. E.A.

Shapiro from N.D. Zelinsky Institute of Organic Chemistry, Moscow. Their derivatives 3-phenoxyben-

zoic acid (3-PBAc), permethrinic acid, chrysanthemic

acid and 3-phenoxybenzalcohol were provided by Dr. S.A. Eremin from Moscow State University. (Struc- tural formulas of the pesticides are given in Fig. 1.)

0 Phenothrin

Permethkic acid

H3C\ c ,“*5 3-Phenoxybenzoic acid (3-PBAc) -

HaC / H

‘d

hapten for conjugates preparation

Chtysanthemic acid

Fig. 1. Structural formulas of the pyrethroid pesticides and their derivatives used.

Alpha-amylase was obtained from Amylosubtilin

Gl Ox- 1 crude preparation of Bacillus subtiEs cell-free extracts (Ferment, Lithuania) through the purification technique [ 181, that included precipitation by Ca- phosphate gel, ion-exchange chromatography on CM-cellulose column with elution by NaCl gradient, and gel-filtration on Sephadex G-75 (Pharmacia) column.

The bacillary alpha-amylase, bovine serum albumin

(BSA, Sigma), haemocyanine from Paralithodes

camtschatica (HC, provided by Dr. I.Yu. Sakharov, Moscow State University), ovalbumin (OA, Serva)

and soybean trypsin inhibitor (STI, Reanal) were used for syntheses of protein-hapten conjugates.

1-cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CalBiochem), 2,4,6-trini- trobenzensulfonic acid (Chemapol), N-hydroxysucci- nimide, 3,5-dinitrosalicylic acid, sodium

dodecylsulfate-o-dianisidine (all from Sigma), dimethylformamide, polyethylene glycol (Mw 6

kDa), Tween-20, 2,4-dichlorophenoxyacetic acid (2,4-D) (all from Serva), benzoic acid, 3-hydroxybenzoic acid, 2-naphthoic acid (all from Aldrich), horse-

radish peroxidase (Reanal), 2,3,5-triphenyl-2H- tetrazolium bromide, glucose oxidase and potato starch (all from Reakhim, Russia) were also used in the experiments. Constituents of buffer solutions and other chemicals were of analytical grade.

Optically transparent polystyrene microELISA plates (Dynatech) were used to carry out immunoas- says.x

2.2. Preparation of hapten-protein conjugates

The 3-PBAc-protein conjugates were synthesized according to [19], with modifications.

15 mg 1-cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and 4 mg N-hydro- xysuccinimide were added to 4 mg 3-PBAc dissolved in 0.5 ml of dimethylformamide and incubated for 2 h at room temperature with stirring. Then a 1% protein water solution was mixed with the activated hapten, ensuring the initial hapten : protein molar ratio equal to 200 : 1 (for HC), 50 : 1 (for BSA) or 20 : 1 (for OA, ST1 and alpha-amylase). The reaction mixture was incubated for 1 h at room temperature with stirring and then at 4°C overnight.

A.I! Zherdev et al./Analytica Chimica Acta 347 f 1997) 131-138 133

The conjugates were separated from low molecular weight compounds by dialysis and/or gel-filtration on Sephadex G-25 (Pharmacia) column in 0.05 M K-phosphate buffer with 0.1 M NaCl, pH 7.4

(PBS).

2.3. Determination of the conjugates composition

To calculate hapten : protein ratios for the obtained

conjugates, the numbers of surface amino groups in the native protein and in conjugated one were compared. They were detected according to the following

procedure [20,21]. Water solutions of native protein and hapten-pro-

tein conjugates (1 mg ml-‘) were prepared. 50 ~1 aliquots of these solutions and water (blank sample) were dispensed on a plate; 50 ~1 of 2,4,6-trinitroben- zenesulfonic acid solution (2 mg ml-‘) and 50 ~1 of saturated NaHC03 solution in water were then added. The mixture was incubated for 2 h at 37°C. Then 25 pl of 10% sodium dodecylsulfate water solution and

25 ~1 of 0.5 M HCl were added. The amino group concentrations are proportional to increase of optical

density at 405 nm, being measured by a vertical

ELISA photometer MR-580 (Dynatech). The titration results were compared with the

calculations based on changes in ultraviolet spectra of the proteins after incorporation of the hapten groups.

2.4. Immuniz.ation

Chinchilla rabbits weighting 3-4 kg were immu- nized with 3-PBAc-HC and 3-PBAc-BSA according

to the following procedure [19]. Immunogen dissolved in PBS (1 mg ml-‘) was

emulsified with equal volume of Freund’s complete

adjuvant (Difco). On days 1, 15 and 29, 1 ml of the prepared mixture was injected intracutaneously at multiple sites on the back from scapula to sacrum.

On the 89th day the first cycle of reimmunization was carried out as follows: the rabbits were boosted intra- venously with 0.3 ml of the immunogen dissolved in PBS (1 mg ml-‘), and were bled 8 days later. This boosting/bleeding procedure was repeated 3-4 times on a monthly basis.

2.5. Antibody preparation

Antisera were emitted by placing blood samples for 12 h at 4°C. Upper layers were collected, divided into aliquots and stored at -20°C.

Antisera titres were determined by the standard technique of indirect ELISA for antibodies. 3- PBAc-ST1 or 3-PBAc-OA conjugate was immobilized

in wells of a microELISA plate. After this antiserum

dilution, goat-anti-rabbit peroxidase conjugate and peroxidase substrate were added to the plate

sequentially. Optical densities of the peroxidase products were measured using the vertical photometer

MR-580. IgG was precipitated twice from the antisera by

equal volume of 20% polyethylene glycol solution

WI.

2.6. Analysis of products for amylolytic reaction

Cleavage products for Amylose- 17 (glucose oligo- mer, n=17) were analyzed by reverse phase high

pressure liquid chromatography [23]. The enzyme or the enzyme-antibody complexes was incubated

with substrate solution (10 mg ml-‘) in 67 mM K- phosphate buffer, pH 6.5. Then products were separated on Silasorb-NH? column in the system

acetonitrile : water =7 : 3. The obtained peaks were compared with standard samples of glucose and its oligomers.

2.7. Activity measurements

Potato starch (1% in 67 mM K-phosphate buffer,

pH 6.5) was used as substrate solution for amylase and its conjugates. Hydrolysis was carried out at 37°C. The following techniques were used for the activity

measurement [24-271.

Glucose formed was detected through coupled glucose oxidaselperoxidase system: glucose was oxidized to glucuronic acid and H202, and then o- dianisidine was oxidized by the H202 obtained.

Non-splinted starch chains were determined by iodine probe. KI (0.5 mg ml-‘) and I2 (0.05 mg ml-‘) were dissolved sequentially in

0.1 M HCl. 7.5 ~1 of hydrolysate was added to 150 pl of this solution and stirred.

134 A.V Zherdev et al./Armlytica Chimica Acta 347 (1997) 131-138

3. Released aldehydes were detected as described below. 50 pl of the following solution was added to the equal volume of hydrolysate: 3,5-dinitrosalicylic acid (5 mg ml-‘) and sodium tartrate

(150 mg ml-‘) dissolved in 0.2 M NaOH. The obtained mixture was boiled for 5 min, and then aliquots were transferred to another plate for opti-

cal measurements.

4. By other technique of the released aldehydes detection 2,3,5-triphenyl-2H-tetrazolium bromide solution (7 mg ml-‘) in 0.2 M NaOH was added to the

equal volume of the hydrolysate (50 p1+50 ~1). The mixture was boiled for 5 min, and then aliquots were transferred to another plate.

The colored products were measured by the vertical

photometer MR-580 at 405 nm (technique I), 630 nm (technique II) or 570 nm (techniques III and IV).

2.8. EMIT of pyrethroid pesticides

3-PBAc-amylase conjugate and anti-3-PBAc anti-

bodies were diluted in 67 mM K-phosphate buffer, pH 6.5, containing 0.05% Tween-20. Then 25 pl of the conjugate and 50 pl of antibodies were incubated together in plate wells with the pesticide sample to

be analyzed (25 pl) at 37°C with stirring. 100 ~1 of 2% starch solution in the same buffer was added, and hydrolysis was carried out at 37°C. Techniques II-IV

(see above) were used for the activity measurements.

Concentrations of the immunoreagents and durations of the steps were optimized by special experiments. The chosen EMIT conditions are shown in

Section 3 (see Table 2).

2.9. EMT of 2,4-dichlorophenoxyacetic acid

(2,4-D)

Obtaining of anti-2,4-D antibodies was described in

[ 191. The 2,4-D-amylase conjugates were prepared and characterized in the same manner as the 3-PBAc- amylase ones, controlling the same molar ratios of the

pesticide and other compounds. The 2,4-D-amylase conjugate was used for the

EMIT in concentration 0.5 pg ml-‘, and anti-2,4-D IgG in concentration 2 pg ml-‘. Immune reaction and starch hydrolysis continued 15 min each. Technique II (see above) was applied for the final measurements.

3. Results and discussion

3.1. Immunoreagents preparation and

characterization

Permethrin, phenothrin and related insecticides are known to be the esters composed from the 3-phenoxybenzoic residue and a variable group. So it is impor-

tant to control the content of not only the native

pesticides but 3-phenoxybenzoic group at different stages of destruction as well.

That is why we have obtained antibodies against 3- PBAc. Antisera from different animals and different immunization cycles were compared using indirect

ELISA of antibodies. The limiting dilution for that the optical density of the peroxidase reaction product had been reliably @=0.95) higher comparing to non-specific binding was regarded as the titre of the antiserum tested. For further investigations the best preparations

were chosen, having titres in the reaction with 3- PBAc-ST1 and 3-PBAc-OA conjugates equal to

1 : 15000 and 1 : 20000. These levels were achieved after first and second reimmunizations by 3-PBAc- BSA.

The synthesized conjugates of alpha-amylase with 3-phenoxybenzoic acid retain 60-70% of the initial enzymatic activity, see Fig. 2. Calculations of their composition by surface amino groups titration and by

change in ultraviolet adsorption spectra gave good agreement of the results. In accordance to them, the

conjugate preparations contained from 5 to 10 resi- dues of 3-PBAc per one enzyme molecule.

3.2. Catalytic properties of free amylase and its

complexes with antibodies

The active center of the bacillary alpha-amylase includes 8 binding sites, each interacting with one next glucose residue of the substrate chain [28-301. Because of this the immune complexes formation may influence not only on the total level of the enzymatic activity, but on the ratio of the reaction

products as well. To study this effect in more details, we have determined relative shares of the hydrolytic products for Amylose-17. Amylase bound to antibodies was demonstrated to be rather exoenzyme that endoenzyme: share of short hydrolytic fragments increased, see Table 1. Analogous data were obtained

A.K Zherdev et al./Analytica Chimica Acta 347 (1997) 131-138 Ii5

A570

2 1

I 1.5

1

li

J 0.5

B- E OL I “,,I, I / I I’ll, T-

0.01 0.1 1

Concentration by amylase, yg/mL

Fig. 2. Comparison of catalytic activity for native alpha-amylase

(1) and 3-PBAc-amylase conjugate (2). The time of starch

hydrolysis was 15 min; the released aldehydes were detected by

2,3,5-triphenyl-2H-tetrazolium bromide.

in [ 181. The phenomenon can be interpreted as a result

of partial steric hindrances for the interaction with long starch fragments.

3.3. Influence of antibodies to 3-PBAc-amylase

catalytic activity for different detection

techniques

The data shown in Table 1 demonstrates that glu-

cose concentration in the hydrolysate indicates the conjugate interaction with the specific antibodies. The

released glucose can be measured by glucose oxidasel peroxidase coupled system [24]. But glucose is not the main product for starch hydrolysis by alpha-amylases,

and so its reliable detection is impeded. The changes

of the products share evoke also more deep starch hydrolysis by immune complexes as compared with free enzyme. We demonstrated, however, that reliable detection of these changes requires practically full conversion of the substrate. Such equilibrium, when the formed short oligomers are not cleaved further, is

reached only after 1 h and more. This long duration of the assay is a significant obstacle for its analytical

applications.

Two ways of activity detection at non-equilibrium conditions were found to be more acceptable for the revealing of antibodies influence onto the enzyme and

the development of analytical systems. The first one is based on determination of non-splinted starch fragments by iodine probe, and the second one on interaction of 2,3,5-triphenyl-2H-tetrazolium or 3,S-

dinitrosalicylic acid with the aldehyde groups, that are released after breaking of the starch bonds. The both techniques have advantages and disadvantages.

From one hand, the iodine reaction is over almost instantaneously, whereas the interaction of aldehydex with a specific reagent increases the assay duration.

From other hand, the second approach is more sensitive (it permits to detect lower enzyme concentrations)

and gives prolonged linear interval on the calibration curve (see Fig. 3). 2,3,5-triphenyl-2H-tetrazolium and 3,5-dinitrosalicylic acid demonstrate equal sensitivities of the activity detection.

For further experiments the conjugate concentrations in the range 1.5-5 pg ml -’ (by protein) were chosen. They correspond approximately to

half of the full substrate hydrolysis. At these condi-

tions techniques II-IV demonstrate inhibition of the 3-PBAc-amylase activity by anti-3-PBAc anti-

bodies (see Fig. 4): the hydrolysate obtained contains more starch chains and less released aldehyde groups.

Table 1 Relative share of Amylose- 17 hydrolysis products for free alpha-amylase and amylase-antibody complexes

Glucose Maltose Maltotriose Other carbohydrates

Without antibodies: 30 min hydrolysis 3 17 28 52

With antibodies: 30 min hydrolysis 4.5 1s 18 62.5

Without antibodies; 60 min hydrolysis 4 25 31 33

With antibodies; 60 min hydrolysis 8.5 18 23 50.5

136 A.V Zherdev et al./Analytica Chimica Acta 347 (1997) 131-138

1.5

1

0.5

0

A570 1 4,

1 , 11111111 I / lllllli I 1111111 !

0.1 1 10

Concentration by amylase, pg/mL

$30

1.5

1

0.5

0

Fig. 3. Detection of 3-PBAc-amylase catalytic activity by iodine

probe (1) and by 2,3,5-triphenyl-2H-tetrazolium bromide (2). The

time of starch hydrolysis was 15 min.

0.4 -

0.3 -

0.2 -

0.1 -

Dec:Fng activity

0.1 1 10 100

IlecY, pem-

Fig. 4. Dependences of 3-PBAc-amylase catalytic activity (in

concentration 1 pg ml-‘, by protein) from the anti-3-PBAc IgG

concentration for two antisera preparations. The times of antigen-

antibody interaction and starch hydrolysis were equal to 15 min

each. The data are the average of four measurements with variation

coefficients below 15%. Curves were plotted by a four-parameter curve fit.

3.4. Development of EMIT for pyrethroid pesticides

and their derivatives

The discovered modulation of the activity was used as the basis for homogeneous immunodetection of

pyrethroid pesticides and their derivatives. The assay is based on competitive interaction of pesticides with antibodies followed by restoration of the initial level

of conjugate activity (i.e. activity of the conjugate without antibodies). The proposed technique includes three steps:

1. Incubation of the pesticide-containing sample and

pesticide-amylase conjugate with anti-pesticide antibodies.

2. Starch hydrolysis. 3. Detection of products of the hydrolysis.

Choice of reagent concentrations and stage dura-

tions for the EMIT was carried out. Concentrations of 3-PBAc-amylase conjugate and anti-3-PBAc IgG were varied from 0.5 to 20 pg ml-‘; durations of the assay stages from 5 to 60 min. Maximal sensitivity of the assay was a criterium of the optimization. Therefore, the chosen concentrations do not ensured full inhibition of 3-PBAc-amylase conjugate by anti-

bodies. In other case, excess of antibody molecules impedes detection of competitive binding with native

pesticides. Moreover, some antibody preparations (see curve 1 in Fig. 4) cannot inhibit hydrolytic activity of

the conjugate to completion. This phenomenon is probably stipulated for their specificity to auxiliary glucose-binding sites of the enzyme active center rather than to the site of hydrolysis.

The optimized parameters of EMIT are shown in Table 2. Competitive curves obtained at these conditions are given in Fig. 5. As curve 1 suggests, 3-PBAc

can be detected at concentrations as low as 5 ng ml-’ (range of quantitative detection is from 5 to 50 ng ml-‘). Native pesticides permethrin and phenothrin, both containing 3-phenoxybenzoic group, also

compete for binding with the antibodies (Fig. 5, curves 3, 4). Sensitivities of their detection reach 2 ng ml-’ (ICsn S-10 ng ml-‘), being even higher that in the 3-PBAc case.

A possible cause of this effect is a negative influence of COOH group on the immune recognition. Really, this group was masked in the immunogen after

Table 2

A.% Zherdev et al./Analytica Chimica Acta 347 (1997) 131-138 137

Optimal conditions of the developed EMIT for pyrethroids

Detection of starch by iodine probe Detection of released aldehydes

Concentration of 3.PBAc-amylase by protein (ug ml-‘) 4

Concentration of anti-3-PBAc IgG (Pg ml-‘) 5

Time of immune reaction (min) 15

Time of hydrolysis (min) 15

Color development (min) 1

Total duration (including handling procedures) (min) 40

A 630

0.6 j-

0.4 1

I 0.2 1

0.1 Increasing

of activity ii

(J 1 ~~.. , , , , ,,,, , / , , ,,,, , , / / , ,,/, , , , 0.1 1 10 100

IPesticide]. ng/mL

Fig. 5. Competitive curves of the developed EMIT for 3-PBAc (I),

3-phenoxybenzalcohol (2). phenothrin (3). permethrin (4). perme-

thrinic acid (5) and chrysanthemic acid (6). The assay conditions

accord to Table 2. The data are the average of four measurements.

Curves were plotted by a four-parameter curve fit.

the conjugation. The advanced assumption is sup-

ported by curve 2, that demonstrate high EMIT sensitivity for 3-phenoxybenzalcohol (limit of revealing,

2.5 ng ml-‘, I&,, 20 ng ml-‘). Thus, the technique

may be used both for the native pyrethroids and the products of their degradation. Testings of the influence of benzoic acid, 3-hydroxybenzoic acid and 2- naphthoic acid (in the range of concentrations from 0.1 ng ml-’ to 0.5 pg ml-‘) on the competitive curves

of the developed EMIT have demonstrated that the studied analogs of 3-PBAc do not bind with anti-3- PBAc antibodies. The same effects have been obtained for permethrinic acid and chrysanthemic acid, products of hydrolysis for permethrin and phenothrin, accordingly (Fig. 5, curves 5, 6).

I .s IS

15

5

45

The assay time does not exceed 45 min, being substantially less than the one of pyrethroid ELISAs, the later usually is in the range l-2 h [31-361. The reached sensitivities are comparable with known ELISA systems. Techniques II-IVof activity detection

do not differ trustworthy in the obtained EMIT sensi-

tivities. Inter-assay coefficients of variation (n=4) are 10.3% for 5 ng ml-’ of 3-PBAc and 7.4% for

20 ng ml-’ of 3-PBAc; intra-assay C.V. (n=4) 14.8% and 11.6%, accordingly.

It should be noted that the color products obtained can be measured by standard photometers for ELISA.

3.5. EMIT,for 2,4-dichlorophenoxycetic acid

It is important to demonstrate that the described

modulation of the amylase activity by antibodies is not

A 630

0.5

0.4

0.3

0.2

0.1 Increesh(

Of activity

0 7T’vr- --- -y-- --T7

10 100 1000

12,4-D], ng/mL

Fig. 6. Competitive curves of the developed EMIT for 2.4-D

obtained through two preparations of anti-2.4-D antisera (l,2). The

data are the average of four measurements. Curves were plotted by

a four-parameter curve fit.

138 A.V Zherdev et al./Analytica Chin&a Acta 347 (1997) 131-138

the unique property of the pair: 3-phenoxybenzoic acid plus the antibodies obtained. With this in mind analogous EMIT for the pesticide 2,4-dichlorophe-

noxyacetic acid (2,4-D) has been developed. This

system also turns to be appropriate for reliable and sensitive measurements, see Fig. 6.

4. Conclusions

Bacillary alpha-amylase was proposed as a new label for homogeneous enzyme immunoassay. With its use the assay for pyrethroids and their derivatives

containing 3-phenoxybenzoic group was developed. The assay is more rapid as compared to the solid phase

ones and can be carried out by standard ELISA

equipment. The proposed approach is suitable for determination of other low molecular weight com-

pounds.

Acknowledgements

The authors are grateful to A.S. Glukhikh from Moscow State University for the help in reverse phase liquid chromatography experiments and to N.A.

Bizova from A.N. Bach Institute of Biochemistry (Moscow), who characterized the 2,4-D-amylase con-

jugates.

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homogeneous enzyme immunoassay for pyrethroid pesticides and their derivatives using bacillary...

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