Monoclonal Immunoassay of Triazine Herbicides - ACS Symposium

Chapter 6

Monoclonal Immunoassay of Triazine Herbicides Development and Implementation 1

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A. E. Karu , Robert O. Harrison , D. J. Schmidt , C. E. Clarkson , J. Grassman , M. H. Goodrow, A. Lucas , B. D. Hammock , J. M. Van Emon , and R. J. White 1

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Downloaded by SUFFOLK UNIV on January 21, 2018 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch006

Department of Plant Pathology, University of California, Berkeley, CA 94720 Department of Entomology and Environmental Toxicology, University of California, Davis, CA 95616 Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, NV 89193

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This paper summarizes a three-laboratory effort to develop a sensitive, reliable enzyme immunoassay (EIA) for triazine herbicides using monoclonal antibodies (MAbs). Simazine and atrazine haptens with mercaptopropionic acid and aminohexanoic acid spacers were synthesized and conjugated to proteins via N-hydroxysuccinimide active esters. MAbs derived from mice immunized with these conjugates had I values of 3 ppb to 4 ppm for various triazines in standard and simazine-enzyme conjugate competition EIAs. The EIAs are compatible with simplified methods for triazine extraction and concentration from soil and water. The limit of detection for atrazine was approximately 0.05 to 0.1 ppb, similar to that obtained with gas chromatography. EIA and GC results agreed closely for 75 groundwater samples, with no "false negatives." Gas-liquid chromatography and EIA data for simazine in 48 soil extracts had a correlation of 0.97. The EIA has also been used to monitor groundwater from beneath a toxic waste pit and water from agricultural evaporation ponds. 50

The s-triazines, first developed in the early 1950s (1), are among the most effective and widely used herbicides known. They are of 3 major types, based on the substituent at Rl (Figure 1): the chlorotriazines, of which simazine and atrazine are the most-used, methoxytriazines, such as prometon, and the methylthio triazines, of which ametryne and prometryne are representative. Atrazine has been cited as the second most-used pesticide in the United States, with an estimated annual usage on the order of 79 million lbs (2). Roughly 3 million lbs. of triazines — mostly atrazine, simazine, and prometon — were applied in California from 1983 through 1987, with the largest percentages used in non-agricultural applications, such as industrial soil sterilization, landscape maintenance, and clearing of rights-of-way (2). Current address: ImmunoSystems, Inc., 4 Washington Ave., Scarborough, ME 04074 0097-6156/91/0451-O059$O6.00/0 © 1991 American Chemical Society

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Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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IMMUNOASSAYS F O R T R A C E C H E M I C A L ANALYSIS

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H c' Downloaded by SUFFOLK UNIV on January 21, 2018 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch006

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Figure 1. Atrazine (D, simazine ODD, and haptens and conjugates used in this work. H I — atrazine-mercaptopropionic acid hapten, and IV — atrazine aminohexanoic acid hapten, which were conjugated to BSA, CON, or KLH, and used as immunizing and EIA coating antigens. V — simazine-alkaline phosphatase "haptenated enzyme," used as the detector in competition EIAs where the monoclonal antibody was immobilized on the solid phase. Atoms on the triazine ring are numbered clockwise from Ni shown in structure I



6.

KARUETAL.

Monoclonal Immunoassay of Triazine Herbicides

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Because of their different solubilities and modes of action, the triazines are selective to varying degrees in their effects on weeds and agriculturally important crops (4). Resistant plants dealkylate these compounds. Corn, sugarcane, and many other crops are naturally resistant, making triazines ideal for weed control on these crops. Various triazines can be used for pre- or post-emergence weed control, alone or in combination with other pesticides. Persistence of these compounds varies, and is a function of the soil properties and microbial ecology, and the climate. The triazines vary widely in their retention in various soils and their potential for leaching, and their mobility in groundwater is a good index of movement of other pesticides. We undertook development of monoclonal antibodies and an immunoassay for triazines with sponsorship from the Environmental Monitoring and Pest Management Branch of the California Department of Food and Agriculture (CDFA), as part of a long-term plan to augment or replace more costly analytical methods with immunoassays, for regulatory purposes. The primary concern of the Environmental Monitoring and Pest Management Branch is groundwater. There are on the order of 40,000 domestic and municipal wells in California, and the state regulatory agencies analyze about 2,000 groundwater samples annually — primarily by gas chromatography (GC, fi). The number of wells CDFA must monitor will continue to increase, due to to recent legislation and increased public interest in water quality. This report describes the initial results of a cooperative effort, in which haptens and conjugates were synthesized at UCD, monoclonal antibodies were derived and characterized at UCB, sample recovery methods were developed and initial feasibility tests with various types of field samples were conducted at UCD, UCB, and EMSL. The antibodies and assay methods were provided to the CDFA Analytical Laboratory in Sacramento, CA, in August 1989. Staff of that laboratory are in the process of validating the assay and acquiring data and experience that will be used to integrate the triazine EIA into their repertoire of tests for regulatory monitoring. Methods Details of the synthesis of haptens and conjugates, the production and characteristics of the MAbs, and optimization of the immunoassays, will be published separately (g; Schmidt et al., in preparation; Jung, et al., in preparation). Synthesis of triazine haptens and hapten-protein conjugates. Simazine and atrazine were derivatized with mercaptopropionic acid (mpa) at Rl, or aminohexanoic acid (aha) at R2, and these haptens were covalently linked to keyhole limpet hemocyanin (KLH), conalbumin (CON), or bovine serum albumin (BSA), by forming active esters with N-hydroxysuccinimide (S) (Figure 1, structures III and IV). This technique was also used to couple simazineaminohexanoic acid to calf intestine alkaline phosphatase (Figure 1, structure V), for use as the "haptenated enzyme" in the EIA format described below.



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IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS

Preparation of triazine-specific M A b s . P a i r s of S w i s s Webster, B i o z z i , and B 1 0 . Q mice were i m m u n i z e d w i t h 4 doses of one of the triazine-protein conjugates i n R i b i adjuvant ( M P L + T D M E m u l s i o n , R i b i Immunochem Research, H a m i l t o n , Montana) over 3 months. T h e sera showed wide variations i n triazine-specific serum titers, l i m i t i n g detectable dose a n d I50 (the dose g i v i n g half-maximal inhibition) i n a competition E I A , u s i n g conjugates w i t h a carrier and l i n k e r different from those of the i m m u n i z i n g antigen. Splenocytes from the four best-responding mice (two S w i s s Webster and two B10.Q) were fused w i t h P 3 X 6 3 A G 8 . 6 5 3 myelomas, essentially as described by Fazekas de St. G r o t h and Scheidegger (7). 15,936 cultures were seeded (166 96-well culture plates), from w h i c h 3,156 colonies developed, and were screened for triazine-directed antibodies, again u s i n g conjugates w i t h a carrier and l i n k e r different from those of the i m m u n i z i n g antigen. O f 232 triazine-specific antibodies, 74 were inhibited by free atrazine or simazine, and 36 of these proved to be genetically stable after several passages i n culture. T h e 15 most sensitive M A b s h a d I50 values of 3 to 15 ppb for atrazine a n d 35 to 60 ppb for simazine, and a l l were of the IgGx subclass. B y contrast, the sera from the mice used to derive the hybridomas had I50 values of 100 to 200 ppb for atrazine a n d simazine. T h e 5 most sensitive M A b s were subcloned b y l i m i t i n g d i l u t i o n , and at least 12 clones of each cell l i n e were frozen. C u l t u r e s were expanded to produce pools of 500 to 750 m l of antibody-containing culture fluid, w h i c h were used without purification i n the assays. (Figure 2) E n z v m e Immunoassays. We carried out these studies w i t h 3 variations of the competition E I A . I n i t i a l surveys of the responses i n mice, screening a n d i n i t i a l characterization of the hybridomas, a n d some of the method development studies were performed u s i n g a "classical" competition E I A , i n w h i c h t r i a z i n e i n solution competed w i t h atrazine-protein conjugate immobilized on the E I A plates, for b i n d i n g a l i m i t i n g amount of antibody, w h i c h was i n solution. M o s t of the studies to optimize the q u a n t i t a t i v e E I A w i t h soil a n d water extracts, a n d m a n y of the specificity studies were carried out u s i n g a "haptenated enzyme" format, i n which the M A b was immobilized on the E I A plate by t r a p p i n g i t w i t h a goat anti-mouse antibody, and t r i a z i n e i n solution competed w i t h a simazinea l k a l i n e phosphatase conjugate for b i n d i n g to the M A b . We recently perfected a more r a p i d and convenient version of t h i s format, which was done as follows: E I A wells (Immulon 2, Dynatech) were coated overnight at 4 ° C w i t h 0.1 m l (approx. 200 ng) of affinity-purified goat anti-mouse I g G + I g M (BoehringerM a n n h e i m no. 605 24) 1:1,000 i n "coating buffer" (0.015 M N a 2 C 0 3 — 0.035 M N a H C 0 — 0.003 M N a N , p H 9.6). T h e wells were washed 3 times w i t h " P B S Tween" (0.01 M p H 7.4 — 0.15 M N a C l — 0.02% N a N — 0.05% Tween 20), 0.1 m l of t r i a z i n e M A b A M 7 B 2 (hybridoma culture fluid, diluted 1:400 w i t h PBS-Tween c o n t a i n i n g 0.5% bovine serum albumin) was then added to each well, the plates were incubated for 1 h r at room temperature, a n d then stored (with the fluid left i n the wells) at -20°C i n a sealed container to prevent evaporation u n t i l they were needed. A t the time of assay, the E I A plates were thawed and washed 3 times w i t h PBS-Tween. Standards a n d u n k n o w n s were d i l u t e d i n PBS-Tween i n microplates or polypropylene tubes, a n d m i x e d 3

3

KH2PO4-K2HPO4,


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KARU ET AL.


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w i t h a l i m i t i n g amount of simazine-N(C2)- a l k a l i n e phosphatase i n P B S - T w e e n to give a final volume of 0.24 m l per w e l l . A l i q u o t s of 0.05 m l of these m i x t u r e s were then transferred to the E I A plates. T h e competition reaction was complete after 30 m i n at room temperature (Figure 3), at w h i c h t i m e the plates were washed 3 times w i t h P B S - T w e e n , a n d d r i e d by r a p p i n g on lint-free paper towels. Substrate solution (1 mg/ml p-nitrophenyl phosphate i n 10% (w/v) dietha n o l a m i n e - H C l , p H 9.8 — 0.4 m M M g C ^ — 3 m M N a N 3 ) was then added, a n d color development at 405 n m was monitored on an E I A reader. D a t a A n a l y s i s . S t a n d a r d curves (generally 11 d i l u t i o n s i n t r i p l i c a t e from a spectrophotometrically standardized stock solution) were fitted b y iterative regression to the 4-parameter logistic equation (1Q) u s i n g Passage II™ (Passage Software, Inc., F o r t C o l l i n s , C O ) on a M a c i n t o s h computer, or Softmax™ software (Molecular Devices, M e n l o P a r k , C A ) on an I B M P C . Sample concentrations were determined by interpolation from the best-fit curves. Values that fell outside of the " w o r k i n g range," defined as 20% to 70% of the m a x i m u m normalized response, were not used. Solid-Phase E x t r a c t i o n of A t r a z i n e from Water. Water samples of 100 to 220 m l were divided i n two aliquots, one of w h i c h was s p i k e d w i t h atrazine s t a n d a r d to 0.2 ppb. C i s solid-phase extraction ( S P E ) columns (Analytichem "Bond-Elut") c o n t a i n i n g 100 m g or 300 m g r e s i n were conditioned successively w i t h 2 column volumes of pesticide analysis grade hexane, e t h y l acetate, methanol, a n d glassd i s t i l l e d water. T h e water samples were filtered through two layers of W h a t m a n N o . 4 paper to remove solids, a n d the filtrates were applied to the columns at 8 to 15 m l / m i n , followed by a wash w i t h 2 column volumes of glass-distilled water. T r i a z i n e s were eluted into glass tubes w i t h a total of 2 m l of e t h y l acetate. T h e eluates were evaporated to near-dryness under nitrogen, a n d dissolved i n 1 m l of P B S - T w e e n . Generally, 5 d i l u t i o n s of each sample were assayed i n t r i p l i c a t e on each of two E I A plates, w h i c h also included atrazine standards i n triplicate. T h i s procedure shown schematically i n F i g u r e 4. Solvent E x t r a c t i o n of S i m a z i n e from S o i l . S o i l samples of 10 grams (sandy loam w i t h low organic carbon content) were d r i e d at 80 °C, suspended i n 10 m l of e t h y l acetate, and shaken or sonicated at low power for 30 m i n . Solids were allowed to settle, a n d the extract was decanted. T h e soil was resuspended i n 10 m l of e t h y l acetate, and t h i s second extract was added to the first one, a n d filtered through N a 2 S 0 4 . These extracts were used directly for gas chromatography. F o r E I A , the e t h y l acetate was evaporated to dryness, the eluate was reconstituted i n 1 m l P B S - T w e e n , a n d aliquots were t a k e n directly into the E I A . Solid-phase E x t r a c t i o n of A t r a z i n e from S o i l . T h i s procedure w a s modified from the method described by H i l l a n d Stobbe (g). F o r studies i n v o l v i n g s p i k e d samples, atrazine standards i n m e t h a n o l were added to give the desired n g of atrazine per g r a m of d r y soil, a n d the samples were d r i e d again before extraction. Samples of 5 grams of " U . S . A r m y S t a n d a r d S o i l " were suspended i n 10 m l of acetonitrile: water :: 9:1, a n d the s l u r r y was sonicated (30 m i n ,


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'ClassicalCompetition

EIA

Coat wells with Goat anti-mouse Ig Incubate limiting MAb + std. or unknown


Add mixture to wells coated with atr-protein conjugate

±

"Haptenated - enzvme" ElAf Coat wells with Goat anti-mouse Ig Incubate MAb + triazine-enzyme + std. or unknown

Add mixture to wells coated with goat anti-mouse Ig

Coat wells with goat anti-mouse Ig Bind MAb in PBS-Tween Store plate at-20°

Add triazine-enzyme + std. or unknown to coated wells

Add goat anti-mouse Ig "detecting Ab"

I

Add substrate Develop color



Figure 2. Flow diagrams of 3 competition EIA procedures. The "classical" competition EIA (left panel) was used to monitor the immunizations, select the hybridomas, and for several of the demonstration projects described in this paper. The haptenated-enzyme EIA in the center panel was used for most of the method development. This is the assay that is presently being evaluated by the CDFA analytical laboratory. The simplified haptenatedenzyme EIA in the right panel is described in Methods.

Atrazine (ppb)

Figure 3. Kinetics of the competition step in the simplified "haptenatedenzyme" EIA. The assay diagrammed in the rightmost panel of Fig. 2 was conducted at room temperature as described in Methods. Mixtures of atrazine standards and simazine-alkaline phosphatase conjugate in PBSTween were added to rows of EIA wells coated with MAb AM7B2, which was "trapped" on the wells by affinity-purified goat anti-mouse IgG. At the times indicated, the wells were rinsed, substrate solution was added, and the absorbance was read 50 min later. Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


KARU ET AL.


SAMPLE (220 ML)|

Filter (Whatman #1) 110 ml no spike C-18SPE column

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B r a n s o n B 1 2 sonic bath). T h e extracts were decanted, centrifuged (10 m i n , 10,000 x g) to remove particulate m a t e r i a l , and 0.01 volume of glacial acetic acid was added. These solutions were applied to S C X aromatic sulfonic acid S P E columns (Analytichem) c o n t a i n i n g 300 m g of resin. T h e columns were washed w i t h 5 m l of 1 M K2HPO4, a n d atrazine was then eluted w i t h 2 m l of acetonitrile: 0.1 M K2HPO4 :: 1:1. T h e eluates were d i l u t e d to 5 m l w i t h P B S Tween, and dilutions were analyzed by E I A .


Results D e r i v a t i o n of M A b s W e used 3 strategies to obtain M A b s w i t h the greatest sensitivity and specificity: F i r s t , to m a x i m i z e the chances of evoking different repertoires of antibodies, we tested s i m a z i n e and atrazine haptens w i t h two different l i n k e r groups (aha or mpa) on each of 3 different carriers ( B S A , C O N , and K L H ) as i m m u n i z i n g antigens i n pairs of 3 strains of mice (Swiss Webster, B i o z z i , and B10.Q). T h e responses to the t r i a z i n e were quantified by E I A on wells coated w i t h a conjugate t h a t h a d a l i n k e r and carrier different from the i m m u n i z i n g antigen. Second, for hybridoma production we selected only the best responding mice, w i t h respect to serum titer, lowest detectable dose, and I50 for atrazine a n d simazine. T h i r d , we prepared and screened a large number of hybridomas. A l t h o u g h a l l of the i m m u n i z i n g antigens evoked good triazinedirected responses i n most of the mice, the statistics cited i n the Methods section demonstrate t h a t the most sensitive M A b s were only a s m a l l percentage of a l l of the triazine-directed M A b s . Specificity of the M A b s . A t U C B we compared the specificity of the M A b s u s i n g the "classical" competition E I A , w h i c h measured the a b i l i t y of various t r i a z i n e s to compete w i t h atrazine conjugates (immobilized on the E I A wells) for b i n d i n g the M A b s . A s i m i l a r set of experiments at U C D was done u s i n g the "haptenated enzyme" E I A format, w i t h s i m a z i n e - N ( C 2 ) - a l k a l i n e phosphatase as the competitor. Table I summarizes the relative recognition of 37 t r i a z i n e analogs and haptens, by M A b s A M 7 B 2 and A M 5 D 1 . T h e results u s i n g the two different E I A formats and simazine-N(C2)-alkaline phosphatase were essentially the same for 7 of the most-used triazines. These results can be s u m m a r i z e d as follows: (a) Propazine, procyazine, and cyanazine were recognized better t h a n atrazine. Atrazine-mercaptopropionic acid, w h i c h was the hapten used to elicit the antibodies, was also recognized better t h a n atrazine b y both M A b s . T h i s indicated that the M A b s bound better to analogs w i t h isopropyl, cyclopropyl, or cyanoisopropyl groups at R 2 or R 3 . (b) B o t h M A b s were m u c h less reactive w i t h prometon, w h i c h is used i n s u b s t a n t i a l amounts i n C a l i f o r n i a and elsewhere, t h a n they were for atrazine a n d s i m a z i n e . (c) H y d r o x y a t r a z i n e and hydroxysimazine reacted only 1% to 5% as w e l l as atrazine. (d) T h e monodealkylated triazines reacted 0.1% to 0.2% as well as atrazine, and (e) these M A b s d i d not measurably (< 0.2% ) recognize di-dealkylated triazines. T h u s , the M A b s are not effective probes for these t r i a z i n e metabolites.


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Table I. Relative reactivity of triazine MAbs AM7B2 and AM5D1 with various triazines and triazine haptens.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Compound

R1

procyazine atrazine-mpa propazine cyanazine atrazine dipropetryne simazine-mpa simazine prometryne tertbutylazine terbutryne atr-N(C5)-COOH sim-N(C5)-COOH ametryne sim-N(C4)-COOH cyanazine amide hydroxy atrazine prometon terbumeton simetryne sim-N(C3)-COOH atratone trietazine atr-N(C2)-COOH hydroxysimazine desmetryne sim-N(C2)-COOH desethyl simazine desethyl atrazine desethyl simetryne atr-N(C1)-COOH sim-N(C1)-COOH didesethyl simazine ammelide ammeline meiamine cyanuric acid

CI S(CH ) COOH CI CI CI SCH2CH3 S(CH )2COOH CI SCH CI SCH3 CI CI SCH CI CI OH OCH3 OCH3 SCH CI OCH3 CI CI OH SCH CI CI CI SCH CI CI CI NH NH NH OH 2 2

2

3

3

3

3

3

2

2

2

R3

R2 a

NHCH(CH ) NHCH CH NHCH(CH ) NHCH CH NHCH CH NHCH(CH ) NHCH2CH3 NHCH CH NHCH(CH ) NHCH CH NHCH CH NH(CH ) COOH NH(CH ) COOH NHCH CH NH(CH )4C00H NHCH2CH3 NHCH CH NHCH(CH ) NHCH CH NHCH CH NH(CH ) COOH NHCH2CH3 NHCH2CH3 NH(CH ) COOH NHCH CH NHCH3 NH(CH ) C00H NH NH NH NHCH COOH NHCH COOH NH OH NH NH OH 2

2

3

2

3

2

3

3 2

3 2

2

3

2

3

3 2

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2 5

2 5

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3 2

2 3

2 2

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2 2

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NHCCN(CH ) NHCH(CH ) NHCH(CH ) NHCCN(CH ) NHCH(CH )2 NHCH(CH )2 NHCH2CH3 NHCH2CH3 NHCH(CH ) NHC(CH ) NHC(CH ) NHCH(CH ) NHCH2CH3 NHCH(CH ) NHCH CH NHCC0NH (CH )2 NHCH(CH3)2 NHCH(CH3)2 NHC(CH ) NHCH2CH3 NHCH2CH3 NHCH(CH ) N(CH CH )2 NHCH(CH ) NHCH2CH3 NHCH(CH ) NHCH CH NHCH2CH3 NHCH(CH ) NHCH2CH3 NHCH(CH ) NHCH2CH3 NH OH OH NH OH 3 2

3 2

3 2

3 2

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3 3

3 2

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% cross-reactivitv AM7B2 AM5D1 583 526 261 181 161 196 116 106 100 100 95 68 66 76 31 31 30 16 23 22 17 21 24 21 19 16 14 14 8.2 12 6.5 6.2 4.1 5.7 35 5.1 4 5 4.4 4.7 4 3.8 2.3 2.3 1.7 1.8 1.1 1.5 1.3 1.1 1.1 1.2 1.5 1.2 1 0.9 0.8 0.7 0.3 0.2

Monoclonal Immunoassay of Triazine Herbicides - ACS Symposium

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