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Chapter 8 Polyclonal

and Monoclonal

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for

Picloram

Immunoassays

Detection

Raymond J. A. Deschamps and J. Christopher Hall Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

A radioimmunoassay (RIA) and two indirect enzyme immunoassays for picloram (4-amino-3,5,6-trichloro-2pyridinecarboxylic acid) were developed for the detection of the herbicide in river water, urine, as well as soil and plant extracts. The RIA method incorporated a rabbit anti-picloram serum as well as a novel radiolabel consisting of [ H]glycine covalently linked to picloram. Using the RIA procedure, picloram concentrations in the range of 50 to 5,000 ng/ml could be detected in fortified river water and urine when a standard curve was prepared in the respective matrix. The I value for picloram by the RIA method was 760 ng/ml. The two indirect enzyme assays were compared in terms of sensitivity, accuracy and precision for detection of picloram in various fortified matrices using standard curves prepared in buffer. The assay using the same rabbit anti-picloram serum employed in the RIA method had a linear working range from 5 to 5000 ng/ml with an I value of 88 ng/ml and a lower detection limit of 5 ng/ml. The assay using a monoclonal antibody obtained from a mouse hybridoma cell line yielded a linear working range from 1 to 200 ng/ml with an I value of 10 ng/ml and a lower detection limit of 1 ng/ml. From the analysis of fortified river water, soil extracts, plant extracts, and urine, the monoclonal antibody-based enzyme immunoassay was shown to be more sensitive, more accurate, and more precise than the polyclonal antiserum-based enzyme immunoassay. 3

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Picloram is used for the control of woody and broadleaf herbaceous plants. It is relatively resistant to breakdown in the environment and has been found to be mobile in the soil (1). Picloram residues

0097-6156/90A)442-O066$06.00A) © 1990 American Chemical Society Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

8. DESCHAMPS AND HALL

Polyclonal and Monodonal Immunoassays

have b e e n f o u n d i n s u r f a c e and groundwater samples (2. 3 ) . The m o b i l i t y i n the environment shown by p i c l o r a m a l o n g w i t h the s u s c e p t i b i l i t y o f c e r t a i n c r o p s t o e x t r e m e l y s m a l l amounts o f t h i s compound (4) make m o n i t o r i n g water f o r p i c l o r a m r e s i d u e s n e c e s s a r y . I n d i s c i p l i n e s such as c l i n i c a l c h e m i s t r y and endrocrinology, immunochemistry i s o f t e n the a n a l y t i c a l method o f c h o i c e b e c a u s e o f i t s s e n s i t i v i t y , s p e c i f i c i t y , speed o f a n a l y s i s , ease o f a u t o m a t i o n , c o s t e f f e c t i v e n e s s , and g e n e r a l a p p l i c a b i l i t y . The p o t e n t i a l o f immunochemical t e c h n o l o g y f o r p e s t i c i d e r e s i d u e a n a l y s i s i n v a r i o u s s u b s t r a t e s s u c h as s o i l , water, p l a n t s , u r i n e , and b l o o d has b e e n examined by s e v e r a l a u t h o r s (5 - 11). Many p e s t i c i d e s , i n c l u d i n g p i c l o r a m , r e q u i r e an e x t e n s i v e sample p r e p a r a t i o n i n c l u d i n g d e r i v a t i z a t i o n b e f o r e t h e y c a n be a n a l y z e d by gas chromatography. As a l t e r n a t i v e methods, immunoassays can be s e n s i t i v e , s p e c i f i c , and p r e c i s e p r o v i d i n g f o r r a p i d , c o s t e f f e c t i v e a n a l y s e s . Immunoassays may be b a s e d on p o l y c l o n a l o r m o n o c l o n a l a n t i b o d i e s . The f o r m e r i s a h e t e r o g e n o u s m i x t u r e o f p r o t e i n s i s o l a t e d from serum t h a t r e p r e s e n t s a v a r i e t y o f a n t i b o d y m o l e c u l e s o f d i f f e r i n g s p e c i f i c i t i e s and affinities. I n c o n t r a s t , the l a t t e r i s a homogenous r e a g e n t p o s s e s s i n g a s i n g l e a n t i b o d y s p e c i f i c i t y and a f f i n i t y . In a v a r i e t y o f a s s a y systems, e i t h e r m o n o c l o n a l o r p o l y c l o n a l a n t i b o d i e s may have c e r t a i n advantages o v e r the o t h e r . F o r a d e t a i l e d d e s c r i p t i o n and c o m p a r i s o n o f p o l y c l o n a l and m o n o c l o n a l a n t i b o d i e s , the r e a d e r i s r e f e r r e d t o the t e x t by Z o l a ( 1 2 ) . The m a j o r i t y o f p u b l i s h e d immunoassay t e c h n i q u e s f o r p e s t i c i d e d e t e c t i o n employ p o l y c l o n a l a n t i b o d i e s . I n a r e v i e w o f immunoassays f o r a g r o c h e m i c a l s , Mumma and Brady (10) c i t e 49 a s s a y s e m p l o y i n g p o l y c l o n a l a n t i b o d i e s and o n l y 12 e m p l o y i n g m o n o c l o n a l a n t i b o d i e s . The r e a s o n f o r t h i s d i s c r e p a n c y i n p o p u l a r i t y may be t h a t p o l y c l o n a l antibody-based assays, a t f i r s t examination, are e a s i e r to develop. However, when u s e d as r e a g e n t s f o r the q u a n t i t a t i o n o f p e s t i c i d e r e s i d u e s , m o n o c l o n a l a n t i b o d i e s have c e r t a i n a d v a n t a g e s o v e r p o l y c l o n a l a n t i b o d i e s w h i c h i n c l u d e : i ) h y b r i d c e l l s c a n be c u l t u r e d i n d e f i n i t e l y , e i t h e r i n v i v o or i n v i t r o to y i e l d a p o t e n t i a l l y u n l i m i t e d s u p p l y o f homogenous, s t a n d a r d i z e d r e a g e n t ; i i ) d u r i n g the h y b r i d o m a s e l e c t i o n p r o c e s s , the i n v e s t i g a t o r c a n s e l e c t a h y b r i d c e l l p r o d u c i n g the d e s i r e d a n t i b o d i e s i n terms o f s p e c i f i c i t y and a f f i n i t y ; i i i ) the m o n o c l o n a l a n t i b o d y w i l l be f r e e o f a n t i b o d i e s t h a t a r e s p e c i f i c f o r i r r e l e v a n t a n t i g e n s which may i n t e r f e r e w i t h the a s s a y ' s p e r f o r m a n c e ; and i v ) c r o s s - r e a c t i v i t y w i t h s t r u c t u r a l l y s i m i l a r m o l e c u l e s (e.g. o t h e r members o f a p e s t i c i d e c l a s s ) c a n be s e l e c t e d f o r o r a g a i n s t d e p e n d i n g upon whether the i n v e s t i g a t o r d e s i r e s an a s s a y t o d e t e c t a s i n g l e p e s t i c i d e o r a c l a s s o f p e s t i c i d e s (13, 14). Despite these i s s u e s which f a v o r monoclonal antibody-based assays, i t i s p o s s i b l e to develop e x c e l l e n t immunoassays b a s e d on p o l y c l o n a l a n t i b o d i e s . I n t h i s p a p e r , we w i l l r e v i e w our p r e v i o u s r e s e a r c h on a radioimmunoassay (RIA) p r o c e d u r e f o r the d e t e c t i o n and q u a n t i t a t i o n of picloram using polyclonal a n t i s e r a (15). F u r t h e r m o r e , we w i l l a l s o d i s c u s s our r e s e a r c h on i n d i r e c t EIA p r o c e d u r e s u s i n g m o n o c l o n a l and p o l y c l o n a l a n t i - p i c l o r a m a n t i b o d i e s w h i c h were compared i n terms o f the c h a r a c t e r i s t i c s o f the s t a n d a r d c u r v e s and p e r f o r m a n c e b a s e d on the d e t e r m i n a t i o n o f p i c l o r a m i n f o r t i f i e d w a t e r , s o i l e x t r a c t s , p l a n t e x t r a c t s , and human u r i n e samples ( 1 6 ) .

Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS

M a t e r i a l s and Methods C h e m i c a l s and M a t e r i a l s . The a n a l y t i c a l s t a n d a r d o f p i c l o r a m c l o p y r a l i d (3,6-dichloro-2-pyridinecarboxylic acid), fluroxypyr ([(4amino-3,5-dichloro-6-fluoro-2-pyridinyl)oxy]acetic acid), triclopyr ([(3,5,6-trichloro-2-pyridinyl)oxy]acetic acid) along with r a d i o l a b e l e d p i c l o r a m ( [ 2 , 6 - ^ C ] p i c l o r a m , sp. a c t . 264 MBq/mmol) were p r o v i d e d b y t h e Dow C h e m i c a l Company, M i d l a n d , MI. Nh y d r o x y s u c c i n i m i d e (NHS), Ν,Ν'-dicyclohexylcarbodiimide (DCC), i s o b u t y l c h l o r o f o r m a t e , t r i e t h y l a m i n e , b o v i n e serum a l b u m i n (BSA), r a b b i t serum a l b u m i n (RSA), Tween 20 ( p o l y o x y e t h y l e n e s o r b i t a n m o n o l a u r a t e ) , Freund's complete a d j u v a n t , and Freund's i n c o m p l e t e a d j u v a n t were o b t a i n e d from Sigma C h e m i c a l Company, S t . L o u i s , MO. A q u a s o l 2 and [ 2 - H ] g l y c i n e ( s p . a c t . 1609.5 GBq/mmol) were o b t a i n e d from New E n g l a n d N u c l e a r R e s e a r c h P r o d u c t s , Boston, MA. D i e t h a n o l a m i n e was o b t a i n e d from F i s h e r S c i e n t i f i c L t d . , Don M i l l s , ON. Female B a l b / c J mice were o b t a i n e d from The J a c k s o n L a b o r a t o r y , Bar Harbor, ME, o r from C h a r l e s R i v e r I n c . , M o n t r e a l , PQ. Cell c u l t u r e media (RPMI and NCTC-109), f e t a l b o v i n e serum, and t h e HAT s e l e c t i v e medium components ( h y p o t h a n t h i n e , a m i n o p t e r i n , and t h y m i d i n e ) were o b t a i n e d from G i b c o I n c . , B u r l i n g t o n , ON. Goat a n t i r a b b i t IgG c o n j u g a t e d and goat anti-mouse IgG c o n j u g a t e d h o r s e r a d i s h p e r o x i d a s e were o b t a i n e d from J a c k s o n Immunoresearch L a b o r a t o r i e s , I n c . , West Grove, PA. 3

P r e p a r a t i o n o f Immunogens. P i c l o r a m was c o n j u g a t e d t o BSA a s d e s c r i b e d b y H a l l e t a l . (15) and F l e e k e r (1Z). E q u i m o l a r amounts o f p i c l o r a m (46 mg, w i t h 45.5 Bq [ C ] p i c l o r a m ) , NHS (22 mg) , and DCC (39 mg) were d i s s o l v e d i n t h e sequence g i v e n i n 2.5 mL o f d i o x a n e . The s o l u t i o n was a l l o w e d t o s t a n d a t room temperature f o r a p p r o x i m a t e l y 18 h a t w h i c h time i t was f i l t e r e d t o remove t h e precipitate. The f i l t r a t e was e v a p o r a t e d t o d r y n e s s on a r o t a r y e v a p o r a t o r under vacuum a t 3 5 C . A s o l u t i o n o f BSA (500 mg) d i s s o l v e d i n 3 mL o f 0.10 M b o r a t e b u f f e r (pH 9, F i s h e r S c i e n t i f i c ) was added t o t h e r e s i d u e and t h e m i x t u r e was a g i t a t e d g e n t l y f o r 1 h a t room t e m p e r a t u r e . The r e s u l t i n g s o l u t i o n was d i a l y z e d a g a i n s t s e v e r a l changes o f d e i o n i z e d water o v e r 36 h a t 4°C and l y o p h i l i z e d . The amount o f h e r b i c i d e bound t o BSA was e s t i m a t e d b y m e a s u r i n g C p r e s e n t i n weighed p o r t i o n s o f p r o d u c t d i s s o l v e d i n PBS (0.01 M p h o s p h a t e , 0.15 M N a C l ) . A p p r o x i m a t e l y 15 t o 20 m o l e c u l e s o f p i c l o r a m were bound p e r BSA m o l e c u l e . 1 4

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P r e p a r a t i o n o f R a d i o l a b e l . The mixed a n h y d r i d e o f p i c l o r a m was p r e p a r e d b y a d d i n g p i c l o r a m (6 mg), t r i e t h y l a m i n e (5 u L ) , and i s o b u t y l c h l o r o f o r m a t e (5 u L ) i n t h e sequence g i v e n t o 500 u L o f dioxane. A p o r t i o n o f t h e mixed a n h y d r i d e s o l u t i o n (100 uL) was added t o a s o l u t i o n o f 100 u L o f [ H ] g l y c i n e (0.1 mCi) , 100 u L o f d i o x a n e , 100 u L o f d i s t i l l e d water, and 2 u L o f 2 M NaOH. A f t e r 1 h, an a d d i t i o n a l 2 u L o f NaOH was added. The r e a c t i o n was a l l o w e d t o p r o c e e d f o r a t o t a l o f 4 h a t room temperature. The p i c l o r a m - [ H ] g l y c i n e c o n j u g a t e was i s o l a t e d and p u r i f i e d by chromatography on s i l i c a g e l TLC as d e s c r i b e d b y H a l l e t a l . (15) u s i n g t h e s o l v e n t system 60:40:2 d i e t h y l e t h e r : p e t r o l e u m e t h e r : formic a c i d (v/v/v). 3

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Polyclonal and Monoclonal Immunoassays

P r e p a r a t i o n o f Coating Conjugates. P i c l o r a m (50 mg) was d i s s o l v e d i n 5 mL t h i o n y l c h l o r i d e (SOClo) i n a s m a l l b o i l i n g f l a s k . The s o l u t i o n was r e f l u x e d f o r 2.5 h a t 85 °C t o form t h e a c i d c h l o r i d e o f picloram. E x c e s s t h i o n y l c h l o r i d e was removed under vacuum a t 60 °C on a r o t a r y e v a p o r a t o r . The r e s i d u e was d i s s o l v e d i n 2 mL o f t e t r a h y d r o f u r a n (THF). The p i c l o r a m a c i d c h l o r i d e s o l u t i o n was added s l o w l y w i t h s t i r r i n g t o 200 mg RSA i n 10 mL o f 0.02 Ν NaOH. Before t h e a d d i t i o n o f t h e a c i d c h l o r i d e s o l u t i o n was completed, p r e c i p i t a t e formed w h i c h d i d n o t r e - s o l u b i l i z e a f t e r s t i r r i n g f o r 18 h a t room temperature. D i l u t i o n o f t h e r e a c t i o n m i x t u r e t o 200 mLs w i t h 0.02 Ν NaOH s u c c e e d e d i n d i s s o l v i n g most o f t h e p r e c i p i t a t e . The r e s u l t i n g s u s p e n s i o n was c e n t r i f u g e d t o remove any p r e c i p i t a t e . The s u p e r n a t a n t was d i a l y z e d a g a i n s t c o l d f l o w i n g t a p w a t e r f o r 24 h and lyophilized. Production o f P o l y c l o n a l A n t i - P i c l o r a m Antibody. Picloram antisera was o b t a i n e d from New Z e a l a n d White r a b b i t s f o l l o w i n g t h e p r o t o c o l d e s c r i b e d b y H a l l e t a l . ( 1 5 ) . The r a b b i t s were i n j e c t e d s u b c u t a n e o u s l y w i t h an e m u l s i o n c o n s i s t i n g o f 0.5 t o 1.0 mg immunogen d i s s o l v e d i n 0.5 mL o f PBS and a n e q u a l volume o f Freund's complete adjuvant. The i n j e c t i o n s were r e p e a t e d 3, 6, and 10 days a f t e r t h e i n i t i a l i n j e c t i o n , s u b s t i t u t i n g Freund's i n c o m p l e t e a d j u v a n t f o r complete a d j u v a n t . A b o o s t e r i n j e c t i o n was g i v e n one month a f t e r t h e i n i t i a l i n j e c t i o n and was r e p e a t e d a t monthly i n t e r v a l s t h e r e a f t e r . The r a b b i t s were b l e d f o r a n t i b o d y t i t e r d e t e r m i n a t i o n s 10 days a f t e r each boost. A n t i s e r a f o r p i c l o r a m immunoassay development were p r e p a r e d from a s i n g l e b l e e d i n each c a s e . P r o d u c t i o n o f Monoclonal A n t i - P i c l o r a m Antibody. T e n 11-week-old mice were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h a t o t a l volume o f 250 u L o f a 1:1 ( v / v ) m i x t u r e o f 70 u g o f immunogen d i s s o l v e d i n PBS and Freund's complete a d j u v a n t ( 1 6 ) . Secondary i n o c u l a t i o n s were g i v e n t h r e e and e l e v e n weeks a f t e r t h e i n i t i a l i m m u n i z a t i o n . One week f o l l o w i n g each s e c o n d a r y i n o c u l a t i o n , t h e mice were b l e d from t h e r e t r o - o r b i t a l p l e x u s and t h e a n t i - p i c l o r a m serum a n t i b o d y t i t e r was d e t e r m i n e d u s i n g t h e RIA p r o c e d u r e d e s c r i b e d b y H a l l e t a l . (IS). A serum sample was c o n s i d e r e d p o s i t i v e f o r a n t i - p i c l o r a m a n t i b o d y a c t i v i t y i f b i n d i n g o f t h e p i c l o r a m r a d i o l a b e l was more t h a n t w i c e the l e v e l o f n o n - s p e c i f i c b i n d i n g . The s p l e e n was removed and c u t i n t o s e v e r a l s m a l l p i e c e s and was g e n t l y f o r c e d t h r o u g h a 400 mesh s t a i n l e s s s t e e l s c r e e n i n t o a P e t r i d i s h w h i c h a l s o c o n t a i n e d RPMI medium. The c e l l s u s p e n s i o n was t r a n s f e r r e d t o a s t e r i l e c e n t r i f u g e tube and any l a r g e t i s s u e a g g r e g a t e s were removed b y t h e s e d i m e n t a t i o n p r o c e d u r e d e s c r i b e d b y Shortman e t a l . (Ifi). The s u s p e n s i o n was c e n t r i f u g e d (200 χ g) f o r 10 m i n u t e s and t h e c e l l p e l l e t was r e s u s p e n d e d i n f r e s h medium. C e l l s i n t r y p a n b l u e v i a b i l i t y s t a i n were enumerated m i c r o s c o p i c a l l y . The s p l e e n c e l l s were mixed w i t h an e q u a l number o f SP/2.0 myeloma c e l l s i n t h e s e m i - l o g growth phase i n RPMI medium. The c e l l m i x t u r e was c e n t r i f u g e d (200 χ g) f o r 10 m i n u t e s and t h e c e l l p e l l e t was suspended i n 1 mL o f p o l y e t h y l e n e g l y c o l (3 000 - 4 000 mol. wt. r a n g e ) a t 37 ° C . The s u s p e n s i o n was mixed c o n t i n u o u s l y f o r one minute, f o l l o w e d b y t h e a d d i t i o n o f 1 mL o f RPMI medium and a n o t h e r one minute o f c o n t i n u o u s m i x i n g . An a d d i t i o n a l 9 mL o f RPMI medium


69

70


was added s l o w l y w i t h m i x i n g . The f u s i o n p r o d u c t s were c e n t r i f u g e d a t 200 χ g f o r 10 min, the s u p e r n a t a n t d i s c a r d e d , and the c e l l p e l l e t r e s u s p e n d e d i n RPMI medium supplemented w i t h 10% f e t a l b o v i n e serum, 10% NCTC-109 medium and 1% HAT. The c e l l s u s p e n s i o n was dispensed (100 u L / w e l l ) i n t o s i x s t e r i l e 9 6 - w e l l m i c r o t i t r a t i o n p l a t e s . The p l a t e s were i n c u b a t e d a t 37 °C i n an atmosphere o f 5% C 0 i n a i r . F u s i o n p r o d u c t c u l t u r e s u p e r n a t a n t s were s c r e e n e d by enzyme immunoassay i n a m u l t i - s t a g e manner t o i d e n t i f y h y b r i d c e l l c o l o n i e s producing anti-picloram antibodies. The v a r i o u s s t a g e s o f the s c r e e n i n g p r o c e s s were as f o l l o w s : ( i ) i d e n t i f y w e l l s c o n t a i n i n g c o l o n i e s p r o d u c i n g mouse IgG, ( i i ) i n d e n t i f y w e l l s c o n t a i n i n g a n t i b o d i e s w i t h a c t i v i t y a g a i n s t picloram-RSA c o a t i n g conjugate, ( i i i ) i n d e n t i f y w e l l s c o n t a i n i n g a n t i b o d i e s s p e c i f i c f o r p i c l o r a m as opposed t o the c a r r i e r p r o t e i n p o r t i o n o f the c o a t i n g c o n j u g a t e (RSA) by c o n d u c t i n g a d o u b l e s c r e e n u s i n g RSA and p i c l o r a m - R S A c o a t i n g c o n j u g a t e , ( i v ) c o n f i r m s p e c i f i c i t y f o r p i c l o r a m by a t t e m p t i n g t o i n h i b i t b i n d i n g o f a n t i b o d i e s to picloram-RSA c o a t i n g conjugate with p i c l o r a m i n s o l u t i o n , (v) i d e n t i f y w e l l s c o n t a i n i n g a n t i b o d i e s s p e c i f i c f o r p i c l o r a m as opposed t o o t h e r p y r i d i n e h e r b i c i d e s by conducting c r o s s - r e a c i t i v i t y experiments with c l o p y r a l i d , t r i c l o p y r , and f l u r o x y p y r ( F i g u r e 1 ) . O n l y the w e l l s showing p o s i t i v e r e s u l t s were c a r r i e d on t o the n e x t s t a g e o f s c r e e n i n g . Out o f 1152 c u l t u r e s , o n l y 5 r e m a i n e d a t the c o m p l e t i o n o f the s c r e e n i n g p r o c e s s . 2

The c u l t u r e showing the b e s t r e s u l t s from an EIA assessment d e s i g n e d t o d e t e c t the p r e s e n c e o f p i c l o r a m s p e c i f i c a n t i b o d i e s was s e l e c t e d f o r the l i m i t i n g d i l u t i o n p r o c e d u r e t o a c h e i v e the c l o n a l i t y o f the h y b r i d o m a c e l l s . A d i l u t i o n was c a l c u l a t e d t o y i e l d one c e l l per w e l l o f a 96 w e l l m i c r o t i t r a t i o n p l a t e . The w e l l s o f the p l a t e were c h e c k e d d a i l y f o r the p r e s e n c e o f a s i n g l e c o l o n y . Once a c o l o n y was v i s i b l e , i t was f e d w i t h 125 uL o f RPMI medium. S u p e r n a t a n t (125 uL) was removed from the w e l l f o r s c r e e n i n g by EIA when the c e l l s o f the c o l o n y were o n e - q u a r t e r t o o n e - h a l f c o n f l u e n t . C e l l s from c o l o n i e s t e s t i n g p o s i t i v e f o r a n t i - p i c l o r a m a n t i b o d y a c t i v i t y were t r a n s f e r e d t o 24 w e l l p l a t e s , r e s c r e e n e d by EIA and t r a n s f e r e d a g a i n i n t o 25 cm^ f l a s k s i f they remained p o s i t i v e f o r picloram antibodies. The l i m i t i n g d i l u t i o n p r o c e d u r e was r e p e a t e d t o ensure monoclonality. A f t e r a f i n a l assessment by EIA, the c e l l s p r o d u c i n g the m o n o c l o n a l a n t i b o d i e s s p e c i f i c f o r p i c l o r a m were c o l l e c t e d f o r the p r o d u c t i o n o f a s c i t i c f l u i d i n mice. M i c e were g i v e n an i n j e c t i o n o f 0.5 mL p r i s t a n e (2,6,10,14tetramethylpentadecane), a hybridoma growth p r o m o t i n g compound. Seven days l a t e r , the mice were i n j e c t e d w i t h 3 χ 10° h y b r i d o m a c e l l s i n 200 uL o f PBS supplemented w i t h 5% f e t a l b o v i n e serum. A p p r o x i m a t e l y two weeks f o l l o w i n g the i n j e c t i o n o f c e l l s , a s c i t e s f l u i d was withdrawn, c e n t r i f u g e d t o remove r e d b l o o d c e l l s , and f r o z e n a t -20 °C u n t i l u s e d . Sample P r e p a r a t i o n . Water was c o l l e c t e d from the Speed R i v e r , Guelph, O n t a r i o and s t o r e d a t 4 °C. The w a t e r was f o r t i f i e d w i t h an acetone s o l u t i o n o f p i c l o r a m . S o i l (40 g) was shaken 15 min w i t h 200 mL o f a 1:1 methanol/water s o l u t i o n . The m i x t u r e was filtered t h r o u g h a g l a s s f i b r e f i l t e r and the methanol was removed under vacuum a t 50 °C. The volume o f the r e s u l t i n g aqueous s o l u t i o n was r e t u r n e d t o 100.00 mL w i t h P i b u f f e r (0.1 M p h o s p h a t e , 1 mM M g C l , pH 2




7.5) and f i l t e r e d t h r o u g h a 0.45 urn n y l o n f i l t e r . The f i l t e r e d e x t r a c t s o l u t i o n was f o r t i f i e d w i t h a n a c e t o n e s o l u t i o n o f p i c l o r a m . G r a s s c l i p p i n g s (20 g) were homogenized i n 100 mL o f 0.1 Ν KOH w i t h 10% KC1. The homogenate was shaken f o r 30 m i n a n d f i l t e r e d t h r o u g h a glass fibre f i l t e r . The f i l t r a t e was a c i d i f i e d t o pH 2 w i t h 3 Ν H S 0 , r e f r i g e r a t e d a t 4 °C f o r 30 m i n and c e n t r i f u g e d a t 3000 χ g f o r 10 min. The volume o f t h e s u p e r n a t a n t was made up t o 100.00 mL w i t h P i b u f f e r a n d a l i q u o t s were f o r t i f i e d w i t h an a c e t o n e s o l u t i o n o f picloram. P r i o r t o a n a l y s i s , 10.00 mL o f t h e f o r t i f i e d s o l u t i o n was f o r c e d t h r o u g h a C^g r e v e r s e d phase l i q u i d chromatography column. The column was washed w i t h 5 mL o f w a t e r and d r i e d w i t h a g e n t l e s t r e a m o f f o r c e d a i r f o r 1 min. The column was e l u t e d w i t h 9 mL o f methanol. The e l u a t e was e v a p o r a t e d t o d r y n e s s a n d t h e r e s i d u e was r e d i s s o l v e d i n 10.00 mL o f P i b u f f e r . Human u r i n e was f o r t i f i e d w i t h an a c e t o n e s o l u t i o n o f p i c l o r a m . The u r i n e was a n a l y z e d w i t h o u t f u r t h e r p r o c e s s i n g b y t h e RIA p r o c e d u r e . F o r t h e enzyme immunoassays, 10.00 mL a l i q u o t s were a c i d i f i e d t o pH 2 w i t h 3 Ν H S0^. The p i c l o r a m was e x t r a c t e d t h r e e times w i t h 3 mL p o r t i o n s o f d i e t h y l e t h e r . The e t h e r f r a c t i o n s were p o o l e d a n d e v a p o r a t e d t o d r y n e s s . The r e s i d u e was r e d i s s o l v e d i n 10.00 mL P i b u f f e r , a n d c e t r i f u g e d a t 12 000 χ g f o r 10 min. R e c o v e r i e s f o r t h e e x t r a c t i o n s d e s c r i b e d above were d e t e r m i n e d u s i n g [ ^ C ] p i c l o r a m added t o s o i l , g r a s s c l i p p i n g s , a n d u r i n e , respectively. R e c o v e r i e s were 95% f o r t h e s o i l e x t r a c t i o n , 90% f o r the p l a n t e x t r a c t i o n a n d 90% f o r t h e u r i n e e x t r a c t i o n . 2

4

2

I n d i r e c t Enzyme Immunoassay. The f o l l o w i n g p r o c e d u r e i s a m o d i f i e d v e r s i o n o f t h a t d e s c r i b e d b y Deschamps e t a l . ( 1 6 ) . M i c r o t i t r a t i o n p l a t e s were c o a t e d b y a d d i n g t o each w e l l 200 u L o f c o a t i n g c o n j u g a t e d i s s o l v e d i n P i b u f f e r (0.1 ug c o a t i n g a n t i g e n p e r mL). The p l a t e s were i n c u b a t e d o v e r n i g h t a t 4 °C. The p l a t e s were e m p t i e d a n d washed t h r e e t i m e s w i t h w a s h i n g s o l u t i o n ( P i b u f f e r supplemented w i t h 0.1% Tween 2 0 ) . I f t h e p l a t e s were n o t t o be u s e d i m m e d i a t e l y , t h e y were wrapped w i t h p l a s t i c and and s t o r e d a t 4 °C f o r up t o 24 h . S i t e s on the p o l y s t y r e n e w e l l s u r f a c e unoccupied by c o a t i n g c o n j u g a t e were b l o c k e d b y a d d i n g 200 u L o f 0.1% (w/v) g e l a t i n s o l u t i o n i n P i b u f f e r a n d i n c u b a t e d f o r 20 m i n a t 4 °C. The p l a t e s were e m p t i e d a n d washed as d e s c r i b e d above. A n t i s e r u m d i l u t e d 1 t o 20 000 o r a s c i t e s f l u i d d i l u t e d 1 t o 10 000 i n P i b u f f e r supplemented w i t h 0.05% Tween 20 s u r f a c t a n t (5) were p r e i n c u b a t e d 1:1 ( v / v ) w i t h p i c l o r a m s t a n d a r d o r sample s o l u t i o n s . A l i q u o t s o f t h e p r e i n c u b a t e d m i x t u r e were t r a n s f e r e d t o t h e w e l l s o f the m i c r o t i t r a t i o n p l a t e (200 u L p e r w e l l ) . The p l a t e s were incubated f o r 1 h a t 4 °C. A f t e r w a s h i n g t h e p l a t e s as b e f o r e , 200 u L o f g o a t a n t i - r a b b i t o r g o a t anti-mouse IgG h o r s e r a d i s h p e r o x i d a s e c o n j u g a t e d i l u t e d 1 t o 5000 i n P i b u f f e r was added t o each w e l l and t h e p l a t e s were i n c u b a t e d f o r 1 h a t 4 °C, emptied a n d washed. S u b s t r a t e (1 mg/mL ABTS, 1 mg/mL u r e a h y d r o g e n p e r o x i d e i n c i t r a t e b u f f e r : 0.024 M c i t r a t e , 0.047 M p h o s p h a t e , pH 5.0) was added and c o l o r was a l l o w e d t o d e v e l o p f o r 30 min. The c o l o r r e a c t i o n was s t o p p e d b y t h e a d d i t i o n o f 100 u L 0.5 M c i t r i c a c i d . Absorbance o f e a c h w e l l was measured a t 405 nm w i t h a m i c r o t i t r e p l a t e r e a d e r . A b s o r b a n c e v a l u e s o f t h e s t a n d a r d s a n d t h e samples (A) were d i v i d e d


71


72

by t h e maximum a b s o r b a n c e v a l u e ( A ) r e p r e s e n t i n g t h o s e w e l l s i n w h i c h b i n d i n g o f a n t i b o d y t o t h e c o a t i n g c o n j u g a t e was n o t c h a l l e n g e d w i t h f r e e p i c l o r a m i n s o l u t i o n . The A / A v a l u e s f o r s t a n d a r d s were p l o t t e d against the l o g o f picloram concentration t o construct a standard curve. C o n c e n t r a t i o n s o f samples were c a l c u l a t e d on t h e b a s i s o f the standard curve. Q

Q

RIA P r o c e d u r e . The RIA p r o c e d u r e d e s c r i b e d b y H a l l e t a l . (11) was u s e d . A l i q u o t s (100 u L ) o f s t a n d a r d o r sample were t r a n s f e r r e d i n t o 1.5 mL m i c r o c e n t r i f u g e t u b e s . I n c u b a t i o n mix (300 u L p e r t u b e ) c o n s i s t i n g o f one p a r t d e i o n i z e d water, one p a r t i n e r t serum, 12 p a r t s PBS, and s u f f i c i e n t r a d i o l a b e l t o y i e l d 10,000 cpm p e r a s s a y was added t o e a c h t u b e . A n t i s e r a d i l u t e d i n PBS was added t o t h e t u b e s (100 u L p e r t u b e ) . One s e t o f c o n t r o l tubes d i d n o t r e c e i v e a n t i s e r a f o r d e t e r m i n a t i o n o f n o n - s p e c i f i c b i n d i n g and a s e c o n d s e t o f c o n t r o l tubes r e c e i v e d a n t i s e r a o n l y f o r maximun b i n d i n g o f r a d i o l a b e l ( B ) . The c o n t e n t s o f t h e t u b e s were mixed t h o r o u g h l y , incubated a t 4 C, the antibody-bound r a d i o l a b e l f r a c t i o n p r e c i p i t a t e d w i t h ( N H ^ ) S 0 ^ , c e n t r i f u g e d , and t h e p e l l e t d i s s o l v e d i n w a t e r p r i o r to assaying f o r r a d i o a c t i v i t y . Q

e

2

A l l r e s u l t s were c o r r e c t e d f o r n o n - s p e c i f i c b i n d i n g . Values f o r s t a n d a r d s were d i v i d e d b y B and were p l o t t e d a g a i n s t t h e l o g o f t h e h e r b i c i d e c o n c e n t r a t i o n (ng/mL). The q u a n t i t y o f t h e h e r b i c i d e i n the unknown sample was c a l c u l a t e d b a s e d on t h e s t a n d a r d c u r v e . Q

Results

and D i s c u s s i o n

Radioimmunoassay. A l i n e a r r e l a t i o n between t h e l o g o f p i c l o r a m c o n c e n t r a t i o n and r e l a t i v e b i n d i n g ( B / B ) was f o u n d i n t h e range o f 50 t o 5,000 ng/mL o f p i c l o r a m f o r t h e RIA p r o c e d u r e ( F i g u r e 2 ) . The c o e f f i c i e n t o f v a r i a t i o n w i t h i n a r u n was 3% o r l e s s f o r p i c l o r a m d e t e r m i n e d b y t h e RIA method. The a c c u r a c y o f d e t e r m i n a t i o n s o f p i c l o r a m i n f o r t i f i e d r i v e r w a t e r and human u r i n e samples d e t e r m i n e d b y t h e RIA was good w i t h t h e mean o v e r a l l amounts d e t e c t e d v a r y i n g from 82% t o 110% o f t h e amount o f p i c l o r a m added ( T a b l e I ) . The range o f c o n c e n t r a t i o n s o v e r w h i c h p i c l o r a m was a c c u r a t e l y q u a n t i t a t e d w i t h no sample c l e a n - u p correspond w i t h l e v e l s found i n u r i n e i n a p p l i c a t o r exposure s t u d i e s c o n d u c t e d b y L i b i c h e t a l . (19). I t must be emphasized t h a t d e t e r m i n a t i o n o f unknown c o n c e n t r a t i o n s o f p i c l o r a m i n r i v e r w a t e r and u r i n e was p e r f o r m e d b y u s i n g s t a n d a r d c u r v e s p r e p a r e d i n t h e respective matrix. Q

Table

I . P i c l o r a m i n F o r t i f i e d R i v e r Water and Human U r i n e Samples as D e t e r m i n e d b y RIA

Amount o f p i c l o r a m added. ug/mL 0.25 2.50 a

Amount o f p i c l o r a m R i v e r water 0.25 2.60

± 0.03 (6) ± 0.19 (6)

M e a n amount d e t e r m i n e d : ug/mL + SE (number o f

0

determined Human u r i n e 0.19 ± 0.04 (6) 2.22 ± 0.35 (6) determinations)


73 Polyclonal and Monoclonal Immunoassays


(b)

OCHjCOOH CI

COOH

(d)

ÇCKjCOOH -CI

F i g u r e 1. C h e m i c a l s t r u c t u r e s o f (a) p i c l o r a m , ( c ) f l u r o x y p y r , and (d) t r i c l o p y r .

0.000

(b) c l o p y r a l i d ,

10 000

1.000 b. 0.800 +

0.600 ^

0.400

0.200

A

\

PcAb EIA

McAb EIA \

0.000 0.1

1

10 100 1000 Picloram Concentration (ng/ml)

° 10 000

F i g u r e 2. S t a n d a r d c u r v e s f o r : a. p o l y c l o n a l a n t i s e r u m - b a s e d radioimmunoassay and b. p o l y c l o n a l a n t i s e r u m - b a s e d and m o n o c l o n a l a n t i b o d y - b a s e d enzyme immunoassays (PcAb EIA and McAb EIA, respectively).



74

The RIA method r e p o r t e d h e r e i n c o r p o r a t e s a n o v e l r a d i o l a b e l . Herbicides l a b e l l e d with a r e e a s i l y o b t a i n e d b u t do n o t l e n d t h e m s e l v e s t o s e n s i t i v e and a c c u r a t e immunoassay work b e c a u s e o f low s p e c i f i c a c t i v i t i e s ( 8 ) . Radioimmunoassays u t i l i z i n g h i g h s p e c i f i c a c t i v i t y r a d i o l a b e l s such as [ H]2,4-D (20) o r an [ I ] 2 , 4 - D d e r i v a t i v e (21,) have g i v e n good r e s u l t s . Covalently linking picloram with [^H]glycine y i e l d s a r a d i o l a b e l with h i g h s p e c i f i c a c t i v i t y w i t h o u t t h e expense o f p u r c h a s i n g a custom s y n t h e s i z e d t r i t i a t e d h e r b i c i d e o r the h e a l t h hazards connected with i o d a t e d r a d i o l a b e l s . 3

1 2 5

I n d i r e c t Enzvme Immunoassays. P i c l o r a m s t a n d a r d s i n P i b u f f e r were u s e d t o g e n e r a t e s t a n d a r d c u r v e s f o r c o m p a r i s o n o f two i n d i r e c t EIA p r o c e d u r e s i n w h i c h p o l y c o n a l and m o n o c l o n a l a n t i b o d i e s were used, respectively. A l i n e a r r e l a t i o n between t h e l o g o f p i c l o r a m c o n c e n t r a t i o n and r e l a t i v e a b s o r b a n c e ( A / A ) was f o u n d i n t h e range 5 t o 5000 ng/mL f o r t h e p o l y c l o n a l a s s a y and 1 t o 200 ng/mL f o r t h e m o n o c l o n a l a s s a y ( F i g u r e 2 ) . The m o n o c l o n a l a s s a y , t h e r e f o r e , h a d a s t a n d a r d c u r v e w i t h a much s t e e p e r s l o p e compared t o t h e p o l y c l o n a l assay. T y p i c a l c o e f f i c i e n t o f d e t e r m i n a t i o n v a l u e s ( r ^ ) were 0.97 f o r t h e m o n o c l o n a l a s s a y and 0.95 f o r t h e p o l y c l o n a l a s s a y . Q

The p o l y c l o n a l a s s a y h a d an I ^ Q v a l u e o f 88 ng/mL w i t h a lower d e t e c t i o n l i m i t o f 5 ng/mL. The m o n o c l o n a l a s s a y was more s e n s i t i v e w i t h an I ^ Q v a l u e o f 10 ng/mL and a lower d e t e c t i o n l i m i t o f 1 ng/mL. B o t h a s s a y s were more s e n s i t i v e t h a n t h e RIA f o r p i c l o r a m w h i c h h a d an I5Q v a l u e o f 760 ng/mL and a lower d e t e c t i o n l i m i t o f 50 ng/mL (15). U s i n g the absorbance v a l u e s o f the p i c l o r a m standards, the i n t e r w e l l v a r i a b i l i t y was d e t e r m i n e d f o r t h e two E I A p r o c e d u r e s . The p o l y c l o n a l a s s a y showed a mean i n t e r w e l l c o e f f i c i e n t o f v a r i a t i o n (CV) o f 6.4% o v e r t h e s t a n d a r d c u r v e . The mean i n t e r w e l l CV o v e r the s t a n d a r d c u r v e f o r t h e m o n o c l o n a l a s s a y was s l i g h t l y lower a t 5.3%. I n t e r a s s a y CV o f t h e p i c l o r a m s t a n d a r d A / A v a l u e s d e t e r m i n e d on f o u r s e p a r a t e runs f o r t h e p o l y c l o n a l a s s a y r a n g e d from 2.1 t o 23% w i t h a mean o f 12.8%. F o r t h e m o n o c l o n a l a s s a y , t h e i n t e r a s s a y CV o f A / A v a l u e s d e t e r m i n e d on s e v e n s e p a r a t e o c c a s i o n s r a n g e d from 5.1 t o 26% w i t h a mean o f 16%. I n b o t h c a s e s , CV v a l u e s i n c r e a s e d w i t h an i n c r e a s e i n p i c l o r a m s t a n d a r d c o n c e n t r a t i o n due t o d e c r e a s i n g A / A values. S i n g h e t a l . (22) showed s i m i l a r r e s u l t s f o r t h e i r enzyme immunoassay i n t e n d e d f o r r o u t i n e s l a u g h t e r h o u s e d e t e r m i n a t i o n s o f t h e a n t i b i o t i c s u l f a m e t h a z i n e i n swine plasma. I n t r a s s a y CV v a l u e s were o b t a i n e d on p i c l o r a m d e t e r m i n a t i o n s i n f o u r f o r t i f i e d p l a n t e x t r a c t samples. The p o l y c l o n a l a s s a y showed a much h i g h e r v a r i a b i l i t y w i t h a mean CV v a l u e o f 80% o v e r t h e f o u r p l a n t e x t r a c t samples compared t o o n l y 19% f o r t h e m o n o c l o n a l a s s a y o v e r t h e same samples ( T a b l e II). Q

Q

Q

Three s t r u c t u r a l l y r e l a t e d p y r i d i n e h e r b i c i d e s , c l o p y r a l i d , f l u r o x y p y r , and t r i c l o p y r ( F i g u r e 1) were t e s t e d f o r c r o s s - r e a c t i v i t y w i t h t h e p o l y c l o n a l and m o n o c l o n a l a n t i - p i c l o r a m a n t i b o d i e s . Neither antibody cross-reacted appreciably with the other p y r i d i n e h e r b i c i d e s as t h e I ^ Q v a l u e s i n a l l c a s e s were g r e a t e r t h a n t h e h i g h e s t c o n c e n t r a t i o n o f h e r b i c i d e t e s t e d (50 000 ng/mL f o r t h e p o l y c l o n a l a n t i b o d y , 10 000 ng/mL f o r t h e m o n o c l o n a l a n t i b o d y ) . D e t e r m i n a t i o n s o f p i c l o r a m i n f o r t i f i e d water, s o i l e x t r a c t s , p l a n t e x t r a c t s , and u r i n e i n d i c a t e d t h a t t h e m o n o c l o n a l a s s a y was f a r superior f o r q u a n t i t a t i v e determinations (Tables I I I , I V ) .




T a b l e I I . I n t r a a s s a y V a r i a b i l i t y o f P i c l o r a m i n Four F o r t i f i e d P l a n t E x t r a c t Samples from Enzyme Immunoassay S t a n d a r d Curve u s i n g P o l y c l o n a l o r Monoclonal A n t i b o d i e s

P i c l o r a m added, ng/mL

0 4 20 40 400

Picloram determined McAb E I A CV, % mean, ng/mL

a

b

PcAb E I A mean, ng/mL

3.6 9.9 39 99 780

P o l y c l o n a l a n t i b o d y enzyme 'Monoclonal a n t i b o d y enzyme

65 87 89 84 59

CV, %

1.1 3.9 24 52 450

32 29 15 21 10

immunoassay. immunoassay.

T a b l e I I I . R e c o v e r y o f P i c l o r a m from F o r t i f i e d Water, S o i l and P l a n t Samples D e t e r m i n e d by Enzyme Immunoassay u s i n g P o l y c l o n a l o r Monoclonal A n t i b o d i e s P i c l o r a m added, ng/mL Fortified 20 200 2000

water

Fortified 4 20 40 400

soil

PcAb

P i c l o r a m d e t e r m i n e d . ng/mL McAb E I A EIA

27 ± 5.1 (18) 569 + 79 (18) 3590 ± 550 (18)

F o r t i f i e d plant extract 4 20 40 400

C

b

11 ± 0.98 (12) 165 + 7.4 (12) 1920 ± 80 (12)

23 90 110 1010

± ± ± ±

4.4 23 23 280

(24) (24) (24) (24)

2.1 13 33 480

± ± ± ±

0.15 (36) 0.53 (36) 1.1 (36) 13 (12)

9.9 39 99 780

± ± ± ±

2.5 10 24 130

(12) (12) (12) (12)

3.5 24 51 450

± ± ± ±

0.23 (24) 1.0 (24) 2.4 (24) 13 (24)

fMean + SE (number o f d e t e r m i n a t i o n s ) . P o l y c l o n a l a n t i b o d y enzyme immunoassay. M o n o c l o n a l a n t i b o d y enzyme immunoassay. c


75


76

T a b l e IV. R e c o v e r y o f P i c l o r a m from F o r t i f i e d Human U r i n e Samples D e t e r m i n e d by Enzyme Immunoassay u s i n g a M o n o c l o n a l A n t i b o d y a

P i c l o r a m added^

P i c l o r a m recovered,ng/mL

KcAfr E*4

ng/mL 4 20 40 400 a

11 30 50 450

+ ± ± ±

D

C

0.58 (12) 2.2 (12) 2.1 (12) 13 (12)

P o l y c l o n a l a s s a y was n o t s u c c e s s f u l due t o unknown ^Mean + SE (number o f d e t e r m i n a t i o n s ) . M o n o c l o n a l a n t i b o d y enzyme immunoassay.

contaminant'.

c

O v e r a l l mean v a l u e s o f p i c l o r a m d e t e r m i n e d f o r the m o n o c l o n a l a s s a y were 78, 73, 112, and 167% o f the amount added f o r w a t e r , s o i l e x t r a c t , p l a n t e x t r a c t , and u r i n e , r e s p e c t i v e l y . For the p o l y c l o n a l a s s a y , o v e r a l l mean v a l u e s o f p i c l o r a m d e t e r m i n e d were 200, 388, and 221% o f t h e amount added f o r w a t e r , s o i l e x t r a c t , and p l a n t e x t r a c t . The p o l y c l o n a l a s s a y f o r d e t e r m i n a t i o n o f p i c l o r a m i n u r i n e was n o t s u c c e s s f u l b e c a u s e o f u n a c c e p t a b l e i n t e r f e r e n c e from an unknown contaminant. P i c l o r a m c o n c e n t r a t i o n e s t i m a t e s were t a k e n from a s t a n d a r d c u r v e made i n P i b u f f e r . I n t e r f e r e n c e from components o f the sample m a t r i x l i k e l y a c c o u n t s f o r much o f the e r r o r i n t h e concentration estimates. Such i n t e r f e r e n c e s from sample components have b e e n r e p o r t e d by Wie and Hammock ( 2 3 ) . In p r e l i m i n a r y s t u d i e s , we have f o u n d t h a t the i o n i c s t r e n g t h o f t h e m a t r i x s o l u t i o n and p o s s i b l y t h e p r e s e n c e o f o r g a n i c c o - e x t r a c t i v e s i n f l u e n c e the amount o f sample m a t r i x i n t e r f e r e n c e . These e f f e c t s were m i n i m i z e d i f the a n t i b o d i e s were d i l u t e d i n P i b u f f e r supplemented w i t h 0.05% (v/v) Tween 20 as d e s c r i b e d by H u n t e r and Lenz ( 9 ) . The RIA p r o c e d u r e f o r p i c l o r a m i n water and u r i n e u s i n g the same p o l y c l o n a l a n t i s e r a as i n t h e EIA d e s c r i b e d h e r e showed a h i g h degree o f a c c u r a c y (82 t o 110% r e c o v e r y ) when the s t a n d a r d c u r v e s were c o n s t r u c t e d u s i n g b l a n k w a t e r or u r i n e . S i n g h e t a l . (22) u s e d swine p l a s m a as the r e f e r e n c e m a t r i x i n t h e i r EIA t o d e t e r m i n e the a n t i b i o t i c s u l f a m e t h a z i n e i n a swine p l a s m a sample m a t r i x w i t h e x c e l l e n t a c c u r a c y . We chose n o t t o do t h i s f o r the i n d i r e c t m o n o c l o n a l and p o l y c l o n a l p r o c e d u r e s f o r t h e f o l l o w i n g reason. S o i l o r w a t e r samples from d i f f e r e n t g e o g r a p h i c a l r e g i o n s o r u r i n e samples from d i f f e r e n t s u b j e c t s w i l l v a r y w i d e l y i n composition. S e l e c t i n g one b l a n k w a t e r o r s o i l sample t o use f o r the s t a n d a r d c u r v e would n o t be a p p r o p r i a t e and attempts t o o b t a i n a r e p r e s e n t a t i v e w a t e r o r s o i l sample w o u l d be d i f f i c u l t . Upon e x a m i n a t i o n o f t h e d e t e r m i n a t i o n s o f p i c l o r a m i n t h e v a r i o u s sample m a t r i c e s ( T a b l e s I I I and I V ) , i t i s e v i d e n t t h a t t h i s c h o i c e had more s e v e r e consequences w i t h r e s p e c t t o the p o l y c l o n a l a n t i b o d y - b a s e d assay than w i t h the monoclonal antibody-based assay. The advantage of a standard curve with a steep slope i s t h a t small e r r o r s i n absorbance v a l u e s w i l l not t r a n s l a t e to l a r g e e r r o r s i n c o n c e n t r a t i o n estimates. One d i s a d v a n t a g e o f a s t a n d a r d c u r v e w i t h a s t e e p s l o p e i s the narrow l i n e a r w o r k i n g range. R a t h e r t h a n making s e v e r a l d i l u t i o n s o f a sample i n the hope o f o b t a i n i n g one d i l u t i o n i n the p r o p e r range,




77

it may be more efficient to conduct a separate assay with a wide working range to rank samples so that appropriate dilutions can be made with certainty for accurate quantitation by a second assay. The polyclonal system described here would be adequate for the role of ranking samples. Alternatively, one could modify the parameters of the monoclonal assay (e.g., increase the antibody concentration) to achieve a standard curve with a flatter slope and a wider working range. For polyclonal antibody production, the design and the preparation of the immunogen are most critical (7. 8. 24). Several studies have illustrated the influence of hapten structure, bridging groups, immunogen structure, and coating conjugate structure on immunoassay performance (25 - 27) . The goal of immunogen design and preparation is to maximize the quantity of specific antibodies in the antisera having high affinity for the antigen (analyte). Although it can not be denied that the development of a good immunogen at the outset is the most effective way to obtain a good antibody, the design of the immunogen used to produce monoclonal antibodies may not be as critical as that required for polyclonal antibody production. An effective screening program will enable the investigator to select and expand the hybridoma cell clone(s) producing the desired antibody even if such clones are rare. In the present study, the same immunogen that yielded a polyclonal antisera with a low average affinity also yielded a monoclonal antibody of high affinity based on the I50values reported here. These immunoassays could be incorporated on a routine basis in most laboratories to serve one of two functions. The assays could be used as a rapid, inexpensive method for herbicide quantitation with little or no sample clean-up. Alternatively, they may be implemented as a preliminary screen to rank samples for follow-up determination by gas chromatography. In either function, the immunoassays represent savings in time, labor, and materials. Acknowledgments The provision of radiolabeled [^C]picloram and analytical picloram, fluroxypyr, clopyralid, and triclopyr by the Dow Chemical Company are gratefully acknowledged. Suppport for R.J.A.D. was provided by a Natural Sciences and Engineering Research Council of Canada PostGraduate Scholarship. The research was supported by grants to J.C.H. from the Natural Science and Engineering Research Council of Canada, the Ontario Ministry of the Environment, The Ontario Pesticide Advisory Committee, the Ontario Minsitry of Agriculture and Food, as well as Agriculture Canada. Literature Cited 1. 2. 3. 4. 5.

Hamaker, J . W.; Johnston, H.; Martin, R. T.; Redemann, C. T. Science 1963, 141, 363. Frank, B.; Clegg, B. S.; Ripley, B. D.; Braun, H. E. Arch. Environ. Contam. Toxicol. 1987, 16, 9-22. Baur, J . R.; Bovey, R. W.; Merkle, M. G. Weed Sci. 1972, 20, 309-313. Ragab, M. T. H. Can. J . Soil Sci. 1975, 55, 55-59. Cheung, P. Y. K.; Gee, S. J.; Hammock, B. D. In Impact of Chemistry on Biotechnology; Phillips, Μ., Shoemaker, S.


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P., Middlekauf, R. D., Ottenbrite, R. Μ., Eds.; ACS Symposium Series 362; American Chemical Society: Washington, DC, 1988; pp 217-229. 6. Ercegovich, C. D.; Vallejo, R. P.; Gettig, R. R.; Woods, L.; Bogus, E. R.; Mumma, R. O. J . Agric. F. Chem. 1981, 29, 559-563. 7. Hammock, B. D.; Gee, S. J.; Cheung, P. Y. K.; Miyamato, T.; Goodrow, M. H.; Van Emon, J . Seiber, J . N. In Pesticide Science and Biotechnology; Greenhalgh, R.; Roberts, T. R., Eds.; Blackwell Scientific Publications: Oxford, 1987, pp 309-316. 8. Hammock, B. D.; Mumma, R. O. In Recent Advances in Pesticide Analytical Methodology; Harvey, J . J r . ; Zweig, G., Eds.; ACS Symposium series 136; American Chemical Society: Washington, DC, 1980; pp 321-352. 9. Hunter, K. W., J r . ; Lenz, D. E. Life Sci. 1982, 30, 355-361. 10. Mumma, R. O.; Brady, J . F. In Pesticide Science and Biotechnology; Greenhalgh, R.; Roberts, T. R., Eds., Blackwell Scientific Publications: Oxford, 1987; pp 341-348. 11. Newsome, W. H.; Shields, J . B. J . Agric. F. Chem. 1981, 29, 220-222. 12. Zola, H. Monoclonal Antibodies: A Manual of Techniques: CRC Press: Boca Raton, 1987; p 214. 13. Vanderlaan, M.; Van Emon, J.; Stanker, L. In Pesticide Science and Biotechnology: Greenhalgh, R.; Rooberts, T. R., Eds.; Blackwell Scientific Publications: Oxford, 1987; pp 597-602. 14. Vinas, J . Pure Appl. Chem. 1985, 57, 577-582. 15. Hall, J . C.; Deschamps, R. J . Α.; Krieg, Κ. K. J . Agric. Food Chem. 1989, 37, 981-984. 16. Deschamps, R. J . Α.; Hall, J . C.; McDermott, M. R. J . Agric. Food Chem. 1989, (submitted). 17. Fleeker, J . R. J . Assoc. Off. Anal. Chem. 1987, 70, 874878. 18. Shortman, Κ. Ν., Williams, Ν., Adams, T. J . Immunologic Meth. 1972, 1, 273-279. 19. Libich, S.; To, J . C.; Frank, R.; Sirons, G. J . Am. Ind. Hyg. Assoc. J . 1984, 45, 56-62. 20. Knopp, D.; Nuhn, P.; Dobberkau, H.-J. Arch. Toxic. 1985, 58, 27-32. 21. Rinder, D. F.; Fleeker, J . R. Bull. Environ. Contam. Toxicol. 1981, 26, 375-380. 22. Singh, P.; Ram, B. P.; Sharkov, N. J . Agric. Food Chem. 1989, 37, 109-114. 23. Wie, S. I.; Hammock, B. D. J . Agric. Food Chem. 1982, 30, 949-957. 24. Jung, F.; Gee, S. J.; Harrison, R. O.; Goodrow, M. H.; Karu, A. E.; Braun, A. L . ; L i , Q. X.; Hammock, B. D. Pestic. Sci. 1989, 26, 303-317. 25. Vallejo, R. P.; Bogus, E. R.; Mumma, R. O. J . Agric. Food Chem. 1982, 30, 572-580. 26. Wie, S. I.; Hammock, B. D. J . Agric. Food Chem. 1984, 32, 1294-1301. 27. Wie, S. I.; Sylwester, A. P.; Wing, K. D.; Hammock, Β. D. J. Agric. Food Chem. 1982, 30, 943-948. RECEIVED May 8, 1990


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