Hapten Synthesis for Pesticide Immunoassay ... - ACS Publications

Chapter 2

Hapten Synthesis for Pesticide Immunoassay Development Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on November 22, 2014 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch002

1

Robert O. Harrison , Marvin H. Goodrow, Shirley J . Gee, and Bruce D. Hammock Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616

The production of pesticide specific antibodies requires presentation of a conjugated form of the pesticide to an animal's immune system. This in turn demands the design of an appropriate chemical structure (a hapten) which can be covalently coupled to a carrier, but which will s t i l l elicit the production of antibodies recognizing the target analyte. The immunoassay literature does not adequately address the question of what chemical structure is required for the production of specific antibodies to low molecular weight compounds, especially in the pesticide area. Hapten design historically is considered an art, with minimal or no explanation for hapten structures which failed to produce the desired antibodies. Most failed attempts remain unpublished and thus have not shed light on the structural requirements for antibody production. We present here an examination of selected examples of successful and unsuccessful hapten designs from our laboratory and from the literature. These examples are used herein to illustrate several criteria deemed critical for successful hapten synthesis strategies.

The Development And Utilization Of Pesticide-Specific Antibodies in Immunoassays The procedures for production of specific antibodies and their application in a competitive inhibition ELISA (Enzyme-Linked Immunosorbent Assay) are discussed in detail in the preceding chapter (Vanderlaan et a l . , this volume). In addition, other comprehensive overviews of the immunoassay development process in the pesticide field are available (.5,7,8,19). in general, a 1

Current address: ImmunoSystems, Inc., 4 Washington Avenue., Scarborough, ME 04074 0097-6156/91/0451-0014$06.00/0 © 1991 American Chemical Society In Immunoassays or Trace Chemical Analysis; Vanderlaan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on November 22, 2014 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch002

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s y n t h e t i c a n t i g e n (immunogen o r immunizing a n t i g e n ) i s u s e d t o immunize an a n i m a l f o r a n t i b o d y development. T h i s immunogen i s composed o f a s y n t h e s i z e d h a p t e n ( m i m i c k i n g t h e s t r u c t u r e o f t h e t a r g e t compound) which i s c o v a l e n t l y a t t a c h e d t o a c a r r i e r p r o t e i n . A n t i b o d i e s a r e p r o d u c e d a g a i n s t many s i t e s on t h e immunogen, i n c l u d i n g t h e conjugated hapten. A n t i b o d i e s t h u s made may r e c o g n i z e e i t h e r t h e c a r r i e r p r o t e i n , t h e hapten, o r a s i t e c o m b i n i n g p a r t s o f both. Because s e v e r a l q u i t e d i f f e r e n t r e s u l t s a r e p o s s i b l e from an i m m u n i z a t i o n , a n i m a l s o r c u l t u r e d c e l l s must be t e s t e d c a r e f u l l y t o assess t h e u s e f u l n e s s o f t h e a n t i b o d i e s produced. The more common u n d e s i r e d r e s u l t s i n c l u d e e x c e s s i v e r e c o g n i t i o n o f t h e s p a c e r arm, r e c o g n i t i o n o f t h e immunizing hapten w i t h o u t adequate r e c o g n i t i o n o f the t a r g e t compound, and f a i l u r e o f t h e h a p t e n t o e l i c i t a s p e c i f i c response d e s p i t e a s u c c e s s f u l immunization procedure. A n t i b o d i e s t h u s p r o d u c e d must be e v a l u a t e d f o r s p e c i f i c i t y t o a l l o w a n a l y s i s o f t h e s u c c e s s o r f a i l u r e o f t h e h a p t e n d e s i g n . The p r o b l e m o f a n t i b o d y s c r e e n i n g r e q u i r e s s p e c i a l c o n s i d e r a t i o n due t o the p o t e n t i a l f o r e r r o r and m i s i n t e r p r e t a t i o n o f d a t a . Careful design of the screening protocol i s e s p e c i a l l y c r u c i a l f o r development o f m o n o c l o n a l o r recombinant a n t i b o d i e s ; c r i t i c a l d e c i s i o n s must be made r a p i d l y t o p r e s e r v e growing c e l l s , b u t one can p o t e n t i a l l y be overwhelmed by c e l l s p r o d u c i n g u n d e s i r a b l e antibodies. A n t i b o d y s c r e e n i n g must a d d r e s s t h e f o l l o w i n g q u e s t i o n s for e i t h e r p o l y c l o n a l o r monoclonal procedures. a.

Does t h e a n t i b o d y b i n d t o t h e immunizing a n t i g e n ? ( i . e . D i d t h e a n i m a l r e s p o n d s p e c i f i c a l l y t o t h e immunogen? T h i s t e s t i s needed o n l y i n t h e r a r e r e t r o s p e c t i v e analysis of a t o t a l f a i l u r e . Our s c r e e n i n g g e n e r a l l y s t a r t s a t b; i f b i s s u c c e s s f u l , a i s moot.)

b.

Does t h e a n t i b o d y b i n d t o t h e c o n j u g a t e d h a p t e n on t h e ELISA a n t i g e n ( p l a t e c o a t i n g a n t i g e n ; s e e V a n d e r l a a n e t a l . , t h i s volume, F i g u r e 2a) , which has a d i f f e r e n t c a r r i e r p r o t e i n t h a n t h e immunogen, b u t i s o t h e r w i s e identical? ( i . e . Are a n t i b o d i e s p r e s e n t which r e c o g n i z e the h a p t e n p o r t i o n o f t h e immunogen?)

c.

Does t h e f r e e immunizing hapten i n h i b i t t h e b i n d i n g shown i n b? ( i . e . A r e a n t i b o d i e s p r e s e n t which r e c o g n i z e t h e u n c o n j u g a t e d h a p t e n s t r u c t u r e i n d e p e n d e n t o f attachment to a c a r r i e r p r o t e i n ? )

d.

Does t h e t a r g e t a n a l y t e i n h i b i t t h e b i n d i n g shown i n b? (i.e. A r e a n t i b o d i e s p r e s e n t which r e c o g n i z e t h e a n a l y t e p o r t i o n o f t h e h a p t e n independent o f t h e s p a c e r arm?)

e.

Does t h e a n t i b o d y b i n d t o a c o n j u g a t e d h e t e r o l o g o u s hapten (a p l a t e c o a t i n g a n t i g e n made w i t h a h a p t e n r e l a t e d t o but n o t i d e n t i c a l t o t h e immunizing h a p t e n ) , and c a n t h i s b i n d i n g be i n h i b i t e d by t h e t a r g e t a n a l y t e ? ( i . e . Do the a n t i b o d i e s a g a i n s t t h e immunizing h a p t e n a l s o b i n d o t h e r c o n j u g a t e d haptens h a v i n g s i g n i f i c a n t d i f f e r e n c e s i n s t r u c t u r e from t h e immunizing hapten? I f the a n t i b o d i e s g i v e poor s e n s i t i v i t y i n t e s t d, c a n t h e

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

16

IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS


problem be overcome by t h e use o f h e t e r o l o g o u s a s s a y systems?) Some s t u d i e s (10,21,22.) have r e p o r t e d a n t i b o d i e s which meet t e s t s b and c above, b u t n o t t e s t d. T h i s r e s u l t i n d i c a t e s much s t r o n g e r r e c o g n i t i o n o f t h e c o n j u g a t e d o r f r e e immunizing h a p t e n t h a n t h e t a r g e t a n a l y t e . Such a n t i b o d i e s may s t i l l y i e l d a u s e f u l a s s a y , as n o t e d i n t e s t e above and t h e s e c t i o n below. Other s t u d i e s (6,17) have r e p o r t e d a n t i b o d i e s which meet t e s t s b and c above, b u t n o t t e s t s d o r e. This r e s u l t i n d i c a t e s r e c o g n i t i o n of t h e c o n j u g a t e d o r f r e e immunizing hapten w i t h o u t s i g n i f i c a n t r e c o g n i t i o n o f t h e t a r g e t a n a l y t e . In t h i s c a s e t h e a n t i b o d i e s a r e n o t u s e f u l f o r a n a l y s i s o f t h e t a r g e t compound. We s u s p e c t b o t h o f t h e s e problems may be more f r e q u e n t t h a n i s a p p a r e n t from t h e l i t e r a t u r e , e s p e c i a l l y f o r small target molecules. We a l s o s u s p e c t t h a t more c a r e f u l e x p l o r a t i o n o f h e t e r o l o g o u s a s s a y systems, f a c i l i t a t e d by more e x t e n s i v e hapten s y n t h e s i s , would i n c r e a s e t h e s e n s i t i v i t y o f many e x i s t i n g a s s a y s . In some c a s e s , as d e s c r i b e d below, such an approach has s a l v a g e d a f u n c t i o n a l a s s a y from what o t h e r w i s e would have been a t o t a l f a i l u r e . A common m i s c o n c e p t i o n i s t h a t monoclonal a n t i b o d i e s can be u s e d t o c i r c u m v e n t problems o f h a n d l e r e c o g n i t i o n . Although t h i s i s c o n c e p t u a l l y p o s s i b l e , i n p r a c t i c e most m o n o c l o n a l a n t i b o d i e s t o s m a l l m o l e c u l e s demonstrate e x t e n s i v e h a n d l e r e c o g n i t i o n . Proper and e x t e n s i v e s c r e e n i n g o f numerous c e l l l i n e s , as o u t l i n e d above, can y i e l d t r u l y s u p e r i o r a n t i b o d i e s w i t h e x c e l l e n t d e t e c t i o n o f t h e a n a l y t e ( 2 3 ) . However, i t i s u n r e a s o n a b l e t o c o n s i d e r such a major expense u n l e s s t h e r e has been a p a r a l l e l i n t e l l e c t u a l i n v e s t m e n t i n good hapten d e s i g n . E x c e l l e n t , rugged immunochemical a s s a y s r e s u l t from a combined i n v e s t m e n t i n b o t h c h e m i s t r y and immunology. Hapten D e s i g n And

Utilization

The i n i t i a l and c r i t i c a l s t e p i n t h e development o f e f f e c t i v e immunoassays f o r p e s t i c i d e s and o t h e r low m o l e c u l a r weight e n v i r o n m e n t a l c h e m i c a l s l i e s i n t h e s e l e c t i o n o f a p p r o p r i a t e haptens w h i c h w i l l e l i c i t t h e p r o d u c t i o n o f a n t i b o d i e s d e m o n s t r a t i n g maximum s p e c i f i c i t y and s e n s i t i v i t y f o r t h e t a r g e t m o l e c u l e . It i s i m p o r t a n t t o d i s t i n g u i s h between t h e problem o f a n t i b o d y p r o d u c t i o n and t h e development o f many h a p t e n - p r o t e i n c o n j u g a t e s f o r a s s a y optimization using existing antibodies. A s s a y s p e c i f i c i t y and s e n s i t i v i t y a r e d e t e r m i n e d p r i m a r i l y by t h e a n t i b o d y produced i n r e s p o n s e t o t h e immunogen; t h u s i t s d e s i g n and p r e p a r a t i o n a r e critical. However, f o r a g i v e n a n t i b o d y , o t h e r ( h e t e r o l o g o u s ) haptens used i n t h e l a t e r s t a g e s o f a s s a y d e s i g n may o f f e r c r i t i c a l improvements i n s e n s i t i v i t y (10,20,22; below and e a b o v e ) . Recent r e v i e w s on immunoassay t e c h n i q u e s (5,7,8,19) emphasize t h e i m p o r t a n c e o f a t t e n t i o n t o hapten s e l e c t i o n d u r i n g a s s a y development. The p r i n c i p l e s o f good hapten d e s i g n a r e s i m p l e and straightforward, given a basic understanding of the process of antibody production. However, t h i s i s not an e x a c t s c i e n c e . F o r i n s t a n c e , even hybridomas o r i n b r e d a n i m a l s which y i e l d a n t i b o d i e s r e c o g n i z i n g t h e same m o l e c u l e may produce a n t i b o d i e s w i t h v e r y d i f f e r e n t amino a c i d s i n t h e i r combining s i t e s . In d e s i g n i n g



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haptens f o r a n t i b o d y p r o d u c t i o n , s e v e r a l g e n e r a l g u i d e l i n e s a r e clear. I t i s p r e f e r a b l e t o a v o i d s p a c e r attachment a t o r n e a r f u n c t i o n a l groups o f t h e t a r g e t m o l e c u l e as t h i s c a n r e d u c e t h e number o f p o t e n t i a l s i t e s c o n t r i b u t i n g t o a n t i b o d y b i n d i n g ( n i t r o , amine, hydroxy, h a l i d e , e t c . ) , e i t h e r d i r e c t l y by c h e m i c a l m o d i f i c a t i o n o r i n d i r e c t l y by s t e r i c a l l y b l o c k i n g a c c e s s t o t h e s e groups. To enhance compound s p e c i f i c i t y , s y n t h e s i s s t r a t e g i e s s h o u l d maximize e x p o s u r e o f u n i q u e d e t e r m i n a n t s by attachment o f t h e s p a c e r a t a p o s i t i o n on t h e t a r g e t m o l e c u l e where members o f a c l a s s have i d e n t i c a l ( o r v e r y s i m i l a r ) s t r u c t u r a l f e a t u r e s . Spacers c o n t a i n i n g s t r o n g d e t e r m i n a n t groups, such as a r o m a t i c r i n g s , c o n j u g a t e d d o u b l e bonds, o r heteroatoms s h o u l d be a v o i d e d i f p o s s i b l e , t o minimize the p r o d u c t i o n of s p a c e r - s p e c i f i c a n t i b o d i e s . The

Importance

Of N e g a t i v e R e s u l t s

I t i s d i f f i c u l t t o c o m p l e t e l y u n d e r s t a n d t h e immune r e s p o n s e t o a p a r t i c u l a r hapten. Development o f s t r o n g d a t a on t h e i n a b i l i t y o f a h a p t e n t o produce s p e c i f i c a n t i b o d i e s r e q u i r e s c o n s i d e r a b l e r e s o u r c e s , i n c l u d i n g many a n i m a l s , time f o r i m m u n i z a t i o n , and e x t e n s i v e c h a r a c t e r i z a t i o n t o demonstrate l a c k o f a p p r o p r i a t e antibodies. I t i s p o s s i b l e t o reduce t h e d i f f i c u l t y o f o b t a i n i n g n e g a t i v e d a t a t h r o u g h t h e use o f mice r a t h e r t h a n r a b b i t s ( 6 ) , a r o u t e n o t u s u a l l y p u r s u e d e x c e p t when p l a n n i n g m o n o c l o n a l a n t i b o d y production. I n any c a s e , r i g o r o u s s y n t h e t i c c h e m i s t r y remains c r i t i c a l t o understanding of the f i n a l r e s u l t s , successful or not. The few a v a i l a b l e s t u d i e s which d e s c r i b e u n s u c c e s s f u l i m m u n i z a t i o n s p r o v i d e v a l u a b l e i n s i g h t (6,1/7), as do t h o s e f a i l u r e s w i t h i n o u r group. Based on t h e l i t e r a t u r e and o u r own e x p e r i e n c e , we have i d e n t i f i e d and examined s e v e r a l c r i t e r i a f o r t h e s u c c e s s f u l development o f immunoassays f o r p e s t i c i d e s . We p r e s e n t h e r e examples i l l u s t r a t i n g and c o n t r a d i c t i n g t h o s e c r i t e r i a . C r i t e r i a F o r Hapten S y n t h e s i s And D i s c u s s i o n Of Examples The s t r u c t u r e s o f t h e compounds d i s c u s s e d below a r e g i v e n i n F i g u r e s 1 and 2, w i t h r e f e r e n c e s i n d i c a t e d under t h e compound names. Sites o f c o n j u g a t i o n a r e i n d i c a t e d by arrows, w h i l e d o t t e d p o l y g o n s e n c l o s e p a r e n t m o l e c u l e s t r u c t u r e which i s n o t r e t a i n e d i n t h e immunizing h a p t e n . S p a c e r Arm L o c a t i o n ; Exposure o f D e t e r m i n a n t s . The l o c a t i o n o f t h e s p a c e r arm on t h e t a r g e t m o l e c u l e s h o u l d be d i s t a l t o i m p o r t a n t h a p t e n i c d e t e r m i n a n t s t o maximize t h e i r exposure f o r a n t i b o d y binding. T h i s p o i n t i s s u p p o r t e d by s e v e r a l examples. In the development o f m o l i n a t e haptens (3.), t h e u n i q u e hexamethyleneimine r i n g was r e t a i n e d , l e a d i n g t o a n t i b o d i e s which were s p e c i f i c f o r m o l i n a t e and r e c o g n i z e d EPTC p o o r l y . T h i s must be due t o t h e d i f f e r e n c e between t h e c y c l i z e d hexamethyleneimine r i n g o f m o l i n a t e and t h e open tf-dipropyl group o f EPTC. A s s a y s d e v e l o p e d f o r t h e s t r i a z i n e h e r b i c i d e s u s i n g a range o f immmunizing h a p t e n s (4, H a r r i s o n e t a l . J . A g r i c . Food Chem., i n p r e s s ) e x h i b i t e d s p e c i f i c i t y r e f l e c t i n g t h e s t r u c t u r e s o f t h e immunizing h a p t e n s ; c h l o r o and a l k y l a m i n o groups were each i m p o r t a n t d e t e r m i n a n t s o f specificity. In a s e r i e s o f papers d e t a i l i n g t h e s y n t h e s i s o f




Thiocarbamate Haptens (3) CHgCH CH » 2

2

NCSCH R

CHgCH

2

CH CH

2

11 NCSCHgR

NCSCH R

2

2

CHgCH CH 2

2

3

2 eptam

molinate

'

thiobencarb Haptens

Parent Compounds

1a, 2a, 3a: R = CHgCH COOH 2

1,2: R = C H

3

1b, 2b, 3 b : R « ( C H ) C O O H 2

1c, 2c, 3c: R = C H 1d,3d:R«

5

NH

2

4>NH

2

9

Triazine Haptens (4) CI

CI V ^ N R R Et IPr

2

R N ^ S r^N(CH2) ^N RISK R'

/

4a 4b

SCH R'

CI

n

5a 5b 5c 5d 5e 5f 5g 5h 5i 5i

R Et IPr Et Et Et Et Et IPr IPr IPr

n_ 2 2 1 2 3 4 5 1 2 5

CH CH 3

2

1

a

H (simazine) H (atrazine) COOH COOH COOH COOH COOH COOH COOH COOH

R R' Et H (simetryne) 6b IPr H (ametryne) 6c Et CH COOH 6d IPr CH COOH 6a

2

2

F i g u r e 1. S t r u c t u r e s o f t h i o c a r b a m a t e and t r i a z i n e haptens and p a r e n t compounds.


2.

HARRISON ETAL.


^ Bentazon (11)

19

Hapten Synthesis

rv%cHA> / -

Aminotriazole

Maleic Hydrazide (6)

3 2

H

>

N H

Paraquat (13,18)

S H

Fenpropimorph 9

(10)

Ethylenethiourea (etu)

° (jj

s

Endosulfan (1)

Parathion (2,17)

c.^

a a

Diflubenzuron (20,21,22)

F i g u r e 2. S t r u c t u r e s o f o t h e r compounds d i s c u s s e d i n t e x t . Arrows i n d i c a t e s i t e s o f c o n j u g a t i o n . Dotted polygons enclose p a r e n t m o l e c u l e s t r u c t u r e n o t r e t a i n e d i n t h e immunizing h a p t e n . The numbers under compound names r e f e r t o l i t e r a t u r e c i t e d .

Continued on next page



IMMUNOASSAYS F O R T R A C E CHEMICAL

Permethrin (16)

Iprodione Diclofop-methyl (12) (15)

Figure 2 Continued


ANALYSIS


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21

h a p t e n s and p r o d u c t i o n o f a n t i b o d i e s f o r d i f l u b e n z u r o n (20-22), s e v e r a l haptens w h i c h were d e r i v a t i z e d d i s t a l t o t h e 2,6d i f l u o r o b e n z o y l u r e a m o i e t y y i e l d e d a n t i b o d i e s s p e c i f i c f o r t h i s end of the molecule. An a s s a y f o r f e n p r o p i m o r p h (9) p r o d u c e d h i g h l y s p e c i f i c a n t i b o d i e s because o f p r e s e r v a t i o n o f t h e u n i q u e dimethylmorpholine r i n g . C o n j u g a t i o n o f a p e r m e t h r i n h a p t e n (16) d i s t a l t o t h e 3-phenoxybenzyl group p r o d u c e d a n t i b o d i e s w h i c h c r o s s r e a c t e d w i t h s e v e r a l r e l a t e d p y r e t h r o i d s because t h e y s h a r e t h i s p o r t i o n o f t h e p e r m e t h r i n s t r u c t u r e . F o r i p r o d i o n e (12), conjugation d i s t a l t o the m-dichlorophenyl r i n g produced a n t i b o d i e s which were c l a s s s p e c i f i c , r e c o g n i z i n g t h e p a r e n t , a m e t a b o l i t e , a h y d r o l y s i s p r o d u c t , and two r e l a t e d f u n g i c i d e s . However, s e v e r a l e x c e p t i o n s t o t h i s g e n e r a l i z a t i o n have been observed. The c o n j u g a t i o n o f an aminophenyl t h i o b e n c a r b h a p t e n by d i a z o t i z a t i o n (Gee e t a l . , t h i s volume) p r o d u c e d a n t i b o d i e s which r e c o g n i z e d t h e t a r g e t compound. C o n j u g a t i o n o f m a l e i c hydrazide t h r o u g h t h e n i t r o g e n o f t h e d i s t i n c t i v e h y d r a z i d e group (6) y i e l d e d a n t i b o d i e s r e c o g n i z i n g t h e t a r g e t compound. P r e s e r v a t i o n o f t h e s e e m i n g l y i m p o r t a n t n i t r o group on p a r a t h i o n ' s p h e n y l r i n g (by c o n j u g a t i o n o r t h o o r meta t o t h e n i t r o group) l e d t o a n t i b o d i e s which d i d not r e c o g n i z e p a r a t h i o n (17). However, u s e f u l a n t i b o d i e s were p r o d u c e d i n r e s p o n s e t o d i a z o t i z e d a m i n o p a r a t h i o n (2; n i t r o group o f p a r e n t compound was r e d u c e d t o amino group f o r c o n j u g a t i o n ) , but a m i n o p a r a t h i o n was a l s o s t r o n g l y r e c o g n i z e d . S p a c e r Arm L o c a t i o n ; P r e s e r v a t i o n o f F u n c t i o n a l Groups. The l o c a t i o n o f t h e s p a c e r arm on t h e t a r g e t m o l e c u l e s h o u l d a v o i d attachment t o f u n c t i o n a l g r o u p s , i n c l u d i n g h e t e r o a t o m s , which might l e a d t o change i n p o l a r i t y o f t h e group and/or a r e d u c t i o n i n t h e number o f p o t e n t i a l s i t e s c o n t r i b u t i n g t o a n t i b o d y b i n d i n g . C o u p l i n g t h r o u g h a c a r b o n atom appears p r e f e r a b l e . S e v e r a l examples can be c i t e d t o i l l u s t r a t e t h i s p r i n c i p l e . In t h e development o f a s s a y s f o r t h e t r i a z i n ^ s (4, H a r r i s o n e t a l . J . A g r i c . Food Chem., i n p r e s s ) , a s u l f u r atom was used i n t h e s p a c e r t o mimic t h e s i z e and o t h e r p r o p e r t i e s o f t h e c h l o r o group, a l l o w i n g a n t i b o d i e s a g a i n s t t h i o e t h e r haptens t o r e t a i n good r e c o g n i t i o n o f c h l o r o compounds w h i l e h a v i n g e x c e l l e n t r e c o g n i t i o n o f S-methyl t r i a z i n e s . In a s s a y s f o r d i f l u b e n z u r o n (20-22.), attachment o f t h e s p a c e r t h r o u g h t h e r i n g NH produced s p e c i f i c a n t i b o d i e s , but t h e p a r e n t compound ( t a r g e t a n a l y t e ) c o u l d not compete t h e a n t i b o d y o f f t h e plate coating antigen. T h i s problem was overcome by c h a n g i n g t h e h a p t e n u s e d i n making t h e p l a t e c o a t i n g a n t i g e n . W i t h t h i s change, N - m e t h y l a t e d d i f l u b e n z u r o n was s t i l l r e c o g n i z e d more s t r o n g l y t h a n d i f l u b e n z u r o n , i n d i c a t i n g the c r i t i c a l importance of the d i f f e r e n c e between s e c o n d a r y and t e r t i a r y n i t r o g e n s . The i m p o r t a n c e o f t h i s d i f f e r e n c e between s e c o n d a r y and t e r t i a r y n i t r o g e n s has a l s o been o b s e r v e d f o r b e n t a z o n (11). Attachment o f t h e s p a c e r t o t h e r i n g n i t r o g e n p r o d u c e d s p e c i f i c a n t i b o d i e s , but t h e s e r e c o g n i z e d Nm e t h y l a t e d b e n t a z o n 100 t o 1000 t i m e s b e t t e r t h a n b e n t a z o n . S i m i l a r l y , i n the production of antibodies t o maleic hydrazide (6), c o n j u g a t i o n t h r o u g h one oxygen p r e v e n t e d t a u t o m e r i z a t i o n o f t h e remaining carbonyl to a phenol. T h i s hapten produced a n t i b o d i e s w h i c h r e c o g n i z e d t h e immunizing hapten, but not t h e p h e n o l i c p a r e n t compound. In two immunoassays f o r p a r a q u a t (.13, 13.) r r e t e n t i o n o f



22


t h e p o s i t i v e c h a r g e on t h e n i t r o g e n used f o r c o n j u g a t i o n l e d t o a n t i b o d i e s w h i c h bound p o o r l y t o s i n g l y charged monoquat. The f o l l o w i n g e x c e p t i o n s t o t h e above p r i n c i p l e must be n o t e d . I n an a s s a y f o r t h i o b e n c a r b (Gee e t a l . , t h i s volume), removal o f t h e c h l o r o group and c o n j u g a t i o n t h r o u g h t h a t p o s i t i o n d i d n o t prevent t h e production o f s p e c i f i c a n t i b o d i e s . For maleic hydrazide (6), c o n v e r s i o n o f t h e secondary n i t r o g e n t o t e r t i a r y by c o n j u g a t i o n through t h e n i t r o g e n d i d not prevent t h e p r o d u c t i o n o f a n t i b o d i e s r e c o g n i z i n g t h e p a r e n t compound. I n one p a r a t h i o n a s s a y (17, p r e s e r v a t i o n o f t h e n i t r o group on t h e p h e n y l r i n g (by c o n j u g a t i o n o r t h o o r meta t o t h e n i t r o group) l e d t o a n t i b o d i e s which r e c o g n i z e d t h e immunizing h a p t e n s , b u t n o t p a r a t h i o n . S e l e c t i o n o f S p a c e r Arm. The s p a c e r arm l e n g t h and s t r u c t u r e s h o u l d be chosen c a r e f u l l y t o r e d u c e s p a c e r r e c o g n i t i o n , w h i l e r e t a i n i n g s p e c i f i c i t y f o r the t a r g e t molecule. F u n c t i o n a l groups i n t h e s p a c e r arm s h o u l d be a v o i d e d , i f p o s s i b l e , t o m i n i m i z e " s p a c e r " recognition. A l k y l s p a c e r s appear p r e f e r a b l e ; heteroatoms appear undesirable. T h i s i s borne o u t by s e v e r a l examples. In t h e assays f o r b o t h m o l i n a t e (3) and EPTC (Gee e t a l . , t h i s volume), a n t i b o d i e s a g a i n s t a r a l k y l haptens produced h i g h t i t e r a n t i b o d i e s which c o u l d not be used because s p a c e r r e c o g n i t i o n was t o o s t r o n g . F o r t h i o b e n c a r b (Gee e t a l . , t h i s volume) and p a r a t h i o n ( 2 ) , t h e a r o m a t i c r i n g i n t h e s p a c e r was i m p o r t a n t t o a n t i b o d y b i n d i n g , b u t t h i s was d e s i r a b l e because i t c o n s t i t u t e d p a r t o f t h e t a r g e t a n a l y t e as w e l l . A m i n o t r i a z o l e haptens (10) c o n j u g a t e d u s i n g t h e h e t e r o b i f u n c t i o n a l r e a g e n t MBS ( m a l e i m i d o b e n z o i c a c i d Nh y d r o x y s u c c i n i m i d e ) p r o d u c e d a n t i b o d i e s which bound t h e homologous hapten-protein conjugate, but d i d not recognize t h e t a r g e t a n a l y t e . T h i s r e s u l t i n d i c a t e s r e c o g n i t i o n o f t h e b u l k y and d i s t i n c t i v e MBS s p a c e r group. The l e n g t h o f t h e s p a c e r arm may a l s o be an i m p o r t a n t factor. S e v e r a l examples a r e g i v e n below, p r i m a r i l y f o r t h e u s e o f a l t e r n a t i v e haptens i n ELISA w i t h a n t i b o d i e s a g a i n s t o t h e r h a p t e n s . I n one a s s a y f o r t h e s - t r i a z i n e s (4, H a r r i s o n e t a l . J . A g r i c . Food Chem., i n p r e s s ) , t h e ELISA u s e o f a h a p t e n w i t h r e d u c e d l e n g t h spacer, ( ( C H ) l e s s t h a n l o n g a l k y l s p a c e r o f immunizing hapten) i n c r e a s e d a s s a y s e n s i t i v i t y 100 f o l d o v e r t h e homologous system. I n our a s s a y s f o r m o l i n a t e ( 3 ) , t h i o b e n c a r b (Gee e t a l . , t h i s volume), and EPTC (Gee e t a l . , t h i s volume), a n t i b o d i e s a g a i n s t s h o r t c h a i n a l k y l a c i d haptens p r o d u c e d no i n h i b i t i o n i n homologous a s s a y systems, b u t gave a c c e p t a b l e s e n s i t i v i t i e s when t h e s p a c e r l e n g t h o f t h e ELISA a n t i g e n was i n c r e a s e d by ( C H j ^ S i m i l a r r e s u l t s were o b t a i n e d f o r d i f l u b e n z u r o n (20-22), where'the u s e o f s i t e and s p a c e r h e t e r o l o g o u s haptens improved t h e a s s a y s e n s i t i v i t y i n most c a s e s . 2

5

F o r many h a p t e n s , more complex s p a c e r s c o n t a i n i n g a r o m a t i c r i n g s o r m u l t i p l e heteroatoms do n o t p r e v e n t t h e p r o d u c t i o n o f t h e d e s i r e d a n t i b o d i e s . A b e n t a z o n hapten (11) c o n t a i n i n g a b e n z y l group i n t h e s p a c e r p r o d u c e d a n t i b o d i e s r e c o g n i z i n g t h e t a r g e t compound. S i m i l a r l y , a n t i b o d i e s r a i s e d a g a i n s t d i a z o t i z e d aminoparathion recognized parathion (2). T h i s i s not s u r p r i s i n g , s i n c e t h e p h e n y l r i n g o f t h e d i a z o t i z e d hapten i s s h a r e d by t h e t a r g e t p a r a t h i o n . We have a l s o o b s e r v e d t h e u s e f u l n e s s o f m u l t i p l e s p a c e r t y p e s f o r t h e development o f h e t e r o l o g o u s a s s a y s f o r improving s e n s i t i v i t y . I n t h e case o f t h e t h i o c a r b a m a t e s , t h i s i s t h e o n l y s i m p l e r o u t e f o r making h e t e r o l o g o u s a s s a y s . In t h e assays


2.

HARRISON ET AL.

Hapten Synthesis

23


f o r b o t h m o l i n a t e (3) and t h i o b e n c a r b (Gee e t a l . , t h i s volume), a n t i b o d i e s a g a i n s t a l k y l a c i d haptens p r o d u c e d t h e b e s t a s s a y s when ELISA c o n j u g a t e s ( p l a t e c o a t i n g a n t i g e n s ) u s e d s p a c e r h e t e r o l o g o u s aminophenyl h a p t e n s . S e l e c t i o n o f Coupling Chemistry. In p r a c t i c e , t h e s e l e c t i o n o f coupling chemistry i s c l o s e l y l i n k e d t o the points discussed i n the p r e v i o u s s e c t i o n . The c h o i c e o f c h e m i c a l r e a c t i o n f o r t h e c o n j u g a t i o n o f h a p t e n t o p r o t e i n must t a k e i n t o c o n s i d e r a t i o n t h e r e a c t i v i t y o f t h e hapten m o l e c u l e ' s o t h e r f u n c t i o n a l g r o u p s . Most o f t h e compounds d e s c r i b e d i n t h i s r e v i e w used a l k y l COOH o r a r y l NH groups f o r c o n j u g a t i o n . T h e d i f f e r e n c e s i n s t a b i l i t y among a z o , amide, e s t e r , and d i s u l f i d e l i n k a g e s i l l u s t r a t e t h e r a n g e o f p o s s i b i l i t i e s a v a i l a b l e w i t h d i f f e r e n t c o n j u g a t i o n r e a c t i o n s . The e a s i e s t (and most common) a p p r o a c h i s t o s e l e c t t h e s i m p l e s t and most s t a b l e c o n j u g a t i o n c h e m i s t r y which i s a p p r o p r i a t e f o r t h e most e a s i l y produced hapten(s). T h i s i s n o t always t h e b e s t s t r a t e g y . The s t a b i l i t y ( c h e m i c a l and b i o l o g i c a l ) o f t h e h a p t e n m o l e c u l e d u r i n g c o n j u g a t i o n and subsequent use must a l l o w f o r t h e in vivo p r o d u c t i o n o f a n t i b o d i e s and in vitro p r o d u c t i o n and use o f h a p t e n p r o t e i n conjugates. F u n c t i o n a l group p r o t e c t i o n s t r a t e g i e s may be required during conjugation. F o r example, i n a t t e m p t i n g t o p r o d u c e s t a b l e a m i n o t r i a z o l e c o n j u g a t e s (10), chromophore g r o u p s were used t o p r o t e c t t h e r e a c t i v e p r i m a r y amine, p r o v i d i n g c o l o r i m e t r i c m o n i t o r i n g o f c o n j u g a t i o n ; t h e s e groups were t h e n removed a f t e r conjugation. In t h e p r e p a r a t i o n o f bentazon conjugates (11). d i m e r i z a t i o n o f t h e a c t i v a t e d hapten o c c u r r e d d u r i n g t h e c o n j u g a t i o n p r o c e d u r e , due t o r e a c t i v i t y o f t h e u n p r o t e c t e d s e c o n d a r y n i t r o g e n . One p o s s i b l e s o l u t i o n would be t o use a s p a c e r which c a n c y c l i z e w i t h t h e secondary n i t r o g e n , t e m p o r a r i l y p r o t e c t i n g i t while p r o v i d i n g a n o t h e r f u n c t i o n a l group f o r c o n j u g a t i o n ; a f t e r c o n j u g a t i o n , r i n g o p e n i n g would r e s t o r e t h e s e c o n d a r y n i t r o g e n , l e a v i n g an a l k y l s p a c e r . Our work on e t h y l e n e t h i o u r e a (ETU) i s an example where normal hapten c o n j u g a t i o n p r o t o c o l s d i d n o t p r o d u c e v e r i f i a b l e conjugates. Because t h e u n i q u e p o r t i o n o f t h i s m o l e c u l e i s a l s o h i g h l y r e a c t i v e , s p e c i a l p r e c a u t i o n s were r e q u i r e d f o r v e r i f i c a t i o n of conjugation. 2

S o l u b i l i t y o f Hapten and C o n j u g a t e . The s o l u b i l i t y o f h a p t e n and c o n j u g a t e must a l s o be c o n s i d e r e d , due t o t h e n o n p o l a r i t y o f most pesticides. U n u s u a l measures may be r e q u i r e d t o overcome problems of hydrophobicity. For endosulfan (1), periodate conjugation (aqueous N a I 0 ) o f t h e d i o l was d i f f i c u l t due t o p o o r water s o l u b i l i t y o f t h e hapten. However, t h e N - h y d r o x y s u c c i n i m i d e a c t i v e e s t e r method c o u l d be p e r f o r m e d u s i n g o v e r 50% d i m e t h y l f o r m a m i d e as a cosolvent. I n d i f l u b e n z u r o n a n t i g e n p r e p a r a t i o n (20-22), t h e u s e o f d i a z o t i z a t i o n r e s u l t e d i n a r e a c t i v e i n t e r m e d i a t e which i s c h a r g e d and t h u s v e r y s o l u b l e i n aqueous systems. In a d d i t i o n , d i f f e r e n c e s e x i s t among t h e p o s s i b l e c a r r i e r p r o t e i n s . I n a s s a y s f o r t h e s - t r i a z i n e s (4, H a r r i s o n e t a l . J . A g r i c . Food Chem., i n p r e s s ) , t h e s o l u b i l i t y o f a l l haptens was improved by u s i n g 20% d i m e t h y l f o r m a m i d e as a c o s o l v e n t i n aqueous c o n j u g a t i o n r e a c t i o n s . The s o l u b i l i t y o f a l l b o v i n e serum a l b u m i n (BSA) c o n j u g a t e s was b e t t e r t h a n k e y h o l e l i m p e t hemocyanin (KLH) c o n j u g a t e s , due t o s o l u b i l i t y d i f f e r e n c e s i n t r i n s i c t o t h e two p r o t e i n s . Thus KLH 4


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c o n j u g a t e s were u s e d f o r immunization (emulsification for injection m i t i g a t e s s o l u b i l i t y problems) and BSA c o n j u g a t e s were used f o r ELISA. Ease of S y n t h e s i s . The ease o f h a p t e n s y n t h e s i s i s a l s o i m p o r t a n t , e s p e c i a l l y i n m i n i m i z i n g t h e number o f s y n t h e t i c s t e p s r e q u i r e d . F o r most o f t h e a s s a y s c o v e r e d h e r e , g e n e r a l l y two o r t h r e e s t e p s were r e q u i r e d from c o m m e r c i a l l y a v a i l a b l e s t a r t i n g m a t e r i a l s . I t i s a l s o c o n v e n i e n t t o use r e a d i l y a v a i l a b l e s t a r t i n g m a t e r i a l s i f possible. F o r examples, t h e d i r e c t c o n j u g a t i o n o f r e a c t i v e d i c h l o r o t r i a z i n e s y i e l d e d ELISA a n t i g e n s u s e f u l f o r a n a l y s i s o f t h e s - t r i a z i n e s (4, H a r r i s o n e t a l . J . A g r i c . Food Chem., i n p r e s s ) . D i r e c t c o n j u g a t i o n o f a c i d m e t a b o l i t e s produced s p e c i f i c a n t i b o d i e s a g a i n s t b o t h f e n p r o p i m o r p h (9) and d i c l o f o p - m e t h y l ( 1 5 ) . We must emphasize however t h a t s y n t h e t i c e x p e d i e n c y a t t h e expense o f t h e o t h e r c o n s i d e r a t i o n s we have d i s c u s s e d above i s l i k e l y t o c r e a t e more problems t h a n i t s o l v e s . F u r t h e r D i s c u s s i o n o f T h i o c a r b a m a t e s and T r i a z i n e s The h a p t e n s shown i n F i g u r e 1 were s y n t h e s i z e d f o r t h e t h i o c a r b a m a t e s m o l i n a t e , EPTC, and t h i o b e n c a r b . Two haptens f o r t h i o b e n c a r b (3a-3b) c o n t a i n e d S - a l k y l s p a c e r s ( i n p l a c e o f t h e p c h l o r o b e n z y l group) w i t h a t e r m i n a l c a r b o x y l i c a c i d group f o r c o n j u g a t i o n , w h i l e two haptens c o n t a i n e d a r a l k y l s p a c e r s (3c-3d) and were c o n j u g a t e d v i a a d i a z o n i u m s a l t . The t h i o b e n c a r b haptens w i t h a l k y l s p a c e r s e l i c i t e d adequate a n t i b o d i e s , but t h e a r a l k y l haptens produced a n t i b o d i e s with s u p e r i o r s p e c i f i c i t y f o r thiobencarb, s i n c e h a p t e n 3d i s n e a r l y i d e n t i c a l t o t h e t a r g e t compound. T h i s a n t i b o d y c o u l d be used i n b o t h h e t e r o l o g o u s and homologous a s s a y s f o r t h e parent thiobencarb with acceptable s e n s i t i v i t y . Sometimes o p t i o n s f o r s p a c e r arm l o c a t i o n a r e l i m i t e d , as i s t h e c a s e f o r EPTC ( F i g u r e 1 ) . Many t h i o c a r b a m a t e s c o n t a i n t h e Se t h y l m o i e t y ; t h u s o n l y t h e d i p r o p y l a m i n o group i s u n i q u e t o EPTC. Hence t h e s p a c e r attachment f o r a l l our EPTC haptens (2a-2c) was a t t h e s u l f u r , d i s t a l t o t h e d i p r o p y l a m i n o group. These haptens e l i c i t e d good t i t e r a n t i b o d i e s , but t h e b e s t EPTC a s s a y was 100 f o l d l e s s s e n s i t i v e than the best assays f o r molinate or thiobencarb. It a p p e a r s t h a t t h i s d e f i c i e n c y i s due t o t h e EPTC s t r u c t u r e i t s e l f . In l i g h t of t h i s r e s u l t , i t i s i n t e r e s t i n g t o note the s t r u c t u r a l s i m i l a r i t y o f m o l i n a t e and EPTC; symmetric o p e n i n g o f t h e m o l i n a t e h e x a m e t h y l e n e i m i n e r i n g y i e l d s EPTC. We s u s p e c t t h a t t h e c r i t i c a l d i f f e r e n c e between t h e s e two compounds i s t h e added r o t a t i o n a l freedom o f t h e two n - p r o p y l groups o f EPTC. W h i l e t h e r e l a t i v e a s s a y s e n s i t i v i t i e s suggest t h a t t h e a l i c y c l i c h e x a m e t h y l e n e i m i n e group i s s u p e r i o r t o t h e a l i p h a t i c d i p r o p y l a m i n o group f o r t h e p r o d u c t i o n o f a n t i b o d i e s , t o our knowledge no s y s t e m a t i c s t u d y o f t h e advantages o f r i n g systems e x i s t s . I n t h e t r i a z i n e s e r i e s ( F i g u r e 1 ) , t h e b e s t immunizing h a p t e n f o r making t r i a z i n e - c l a s s s p e c i f i c a n t i b o d i e s was compound 6d. This h a p t e n has t h e s p a c e r a t t a c h e d a t t h e 2 - p o s i t i o n and c o n t a i n s 4e t h y l a m i n o and 6 - i s o p r o p y l a m i n o g r o u p s . Hapten 6c, t h e 6-ethylamino a n a l o g , was s l i g h t l y l e s s e f f e c t i v e , but t h i s may be r e l a t e d t o t h e d e c r e a s e d s o l u b i l i t y and accompanying d i f f e r e n c e s i n c o n j u g a t i o n and in vivo a n t i g e n p r e s e n t a t i o n . I t i s not c l e a r a t p r e s e n t which i s



2.

HARRISON ET AL.

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t h e b e s t immunizing hapten f o r p r o d u c i n g s i m a z i n e s p e c i f i c antibodies. The most s e n s i t i v e m o n o c l o n a l a n t i b o d y (1-2 ppb f o r a t r a z i n e ) was made i n r e s p o n s e t o compound 6d. The immunizing hapten p r o d u c i n g t h e b e s t s p e c i f i c i t y f o r a t r a z i n e was 5 j ( c o n t a i n i n g 2 - c h l o r o and 4 - i s o p r o p y l a m i n o groups, w i t h t h e s p a c e r arm a t t h e 6 - p o s i t i o n ) . O n l y 8% c r o s s r e a c t i v i t y w i t h s i m a z i n e was n o t e d f o r t h e b e s t r a b b i t a n t i b o d y made a g a i n s t t h i s hapten, a s u r p r i s i n g r e s u l t c o n s i d e r i n g t h a t t h e s t r u c t u r e s d i f f e r o n l y by one m e t h y l e n e group. A n t i b o d i e s a g a i n s t compound 5g ( c o n t a i n i n g 2c h l o r o and 4 - e t h y l a m i n o groups, w i t h t h e s p a c e r arm a t t h e 6p o s i t i o n ) were a l s o v e r y s p e c i f i c f o r a t r a z i n e , as e x p e c t e d . However, t h e i r c r o s s - r e a c t i v i t y f o r s i m a z i n e was a p p r o x i m a t e l y 100%, but w i t h r e d u c e d s e n s i t i v i t y compared t o t h e a n t i b o d i e s a g a i n s t compound 5 j . The r e c o g n i t i o n o f v a r i a b l e s p a c e r arm l e n g t h t r i a z i n e haptens was e v a l u a t e d u s i n g c o m p e t i t i v e ELISA. U s i n g a n t i b o d i e s made a g a i n s t haptens 5 j and 6d, a c l e a r d e c r e a s e i n r e c o g n i t i o n was o b s e r v e d as t h e s p a c e r arm was s h o r t e n e d . Based on t h e s e r e s u l t s , t h e n = 1, 2, and 3 d e r i v a t i v e s o f s i m a z i n e (5c-5e) and t h e n = 1 and 2 d e r i v a t i v e s o f a t r a z i n e ( 5 h - 5 i ) were chosen f o r c o n j u g a t i o n t o a l k a l i n e phosphatase and BSA. The u l t i m a t e g o a l o f t h i s approach i s t o produce more s e n s i t i v e h e t e r o l o g o u s a s s a y s by e x p l o i t i n g t h e r e d u c e d a f f i n i t y o f t h e a n t i b o d i e s f o r t h e c o n j u g a t e d haptens, w h i l e r e t a i n i n g s p e c i f i c i t y f o r t h e t r i a z i n e c l a s s (4, H a r r i s o n e t a l . J . A g r i c . Food Chem., i n p r e s s ) . Conclusions 1.

The importance o f t h e above c r i t e r i a , e s p e c i a l l y s p a c e r r e c o g n i t i o n and p r e s e r v a t i o n o f p a r e n t m o l e c u l e f u n c t i o n a l groups, appears t o i n c r e a s e as t h e s i z e o f t h e t a r g e t m o l e c u l e d e c r e a s e s and as t h e number o f c l e a r l y r e c o g n i z a b l e f u n c t i o n a l i t i e s decreases (6,10). 2. Some m o l e c u l e s may be i n a d e q u a t e f o r t h e p r o d u c t i o n o f s p e c i f i c a n t i b o d i e s due t o s i z e , r e a c t i v i t y , o r s t r u c t u r e . L i m i t a t i o n s due t o s i z e may be m a n i f e s t e d o n l y as d e c r e a s i n g a s s a y s e n s i t i v i t y with decreasing molecular s i z e . 3. Sometimes i t i s i m p o s s i b l e o r v e r y d i f f i c u l t t o a v o i d s a c r i f i c i n g a u s e f u l d e t e r m i n a n t group i n hapten s y n t h e s i s ; e x p l o r a t i o n o f m u l t i p l e haptens i s c r u c i a l i n such c a s e s ( 6 ) . 4. M u l t i p l e haptens s h o u l d be p r e p a r e d f o r each t a r g e t compound, i n c l u d i n g d i f f e r e n t c o n j u g a t i o n p o s i t i o n s and s p a c e r l e n g t h s and structures, i f possible. W h i l e i t may be u n n e c e s s a r y t o u s e a l l o f t h e s y n t h e s i z e d haptens f o r a n t i b o d y p r o d u c t i o n , h e t e r o l o g o u s systems s h o u l d be c a r e f u l l y e x p l o r e d t o o p t i m i z e a s s a y s e n s i t i v i t y w h i l e r e t a i n i n g s p e c i f i c i t y and ruggedness. 5. The s t r a t e g y o f s y n t h e s i z i n g a l i b r a r y o f p o t e n t i a l haptens d u r i n g t h e e a r l y phase o f a g r i c u l t u r a l c h e m i c a l p r o d u c t development would be a v a l u a b l e c o r p o r a t e p o l i c y b e c a u s e i t would f a c i l i t a t e t h e l a t e r development o f immunoassays. An added b e n e f i t i s t h a t t h e s e d e r i v a t i v e s would o f f e r new compounds f o r s c r e e n i n g as p o t e n t i a l p e s t i c i d e s o r s e r v e as m e t a b o l i t e standards. 6. The a b i l i t y t o g e n e r a t e c l a s s o r compound s p e c i f i c a n t i b o d i e s depends g r e a t l y on t h e class/compound s t r u c t u r e and t h e number o f




26

c l o s e l y r e l a t e d compounds which might be e n c o u n t e r e d i n r o u t i n e analysis. The u s e o f m u l t i p l e haptens e m p l o y i n g d i f f e r e n t c o n j u g a t i o n p o s i t i o n s and s u b s t i t u t i o n p a t t e r n s a l l o w s e x p l o r a t i o n o f class/compound s p e c i f i c i t y . 7. When d e s i g n i n g a s s a y s f o r l a r g e r m o l e c u l e s , immunizing haptens c o n t a i n i n g l e s s t h a n t h e complete p a r e n t s t r u c t u r e may y i e l d a n t i b o d i e s which a d e q u a t e l y r e c o g n i z e t h e t a r g e t m o l e c u l e (12, 16). Such haptens a r e more l i k e l y t o p r o d u c e c l a s s s p e c i f i c a n t i b o d i e s due t o n o n - r e c o g n i t i o n o f t h e o m i t t e d s t r u c t u r e . 8. Study o f t h e l i t e r a t u r e on low m o l e c u l a r w e i g h t d r u g s (14 f o r r e v i e w ) may o f f e r f u r t h e r i n s i g h t ( v a l p r o i c a c i d , p h e n o b a r b i t a l , caffeine, nicotine, etc.). 9. U n d e r s t a n d i n g o f h a p t e n - a n t i b o d y i n t e r a c t i o n would b e n e f i t from p h y s i c a l - c h e m i c a l s t u d y o f b i n d i n g ( x - r a y , NMR, e t c . ) , w i t h t h e r e a l i z a t i o n t h a t f o r most m o l e c u l e s t h e r e w i l l be many p o s s i b l e antibody combining s i t e s t r u c t u r e s . Acknowledgments T h i s work was s u p p o r t e d i n p a r t by NIEHS S u p e r f u n d g r a n t PHS ES04699, EPA g r a n t CR-814709-02-0, and a g r a n t from t h e C a l i f o r n i a Department o f Food and A g r i c u l t u r e . BDH i s a Burroughs-Wellcome Scholar i n Toxicology.

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12. Newsome, W.H. In Pesticide Science and Biotechnology, Proceedings of the Sixth International Congress of Pesticide Chemistry; Greenhalgh, R., Roberts, T.R., Eds.; Blackwell: Oxford, 1987; pp 349-352. 13. Niewola, Z.; Hayward, C.; Symington, B.A.; Robson, R.T. Clin. Chim. Acta 1985, 148, 149-156. 14. Oellerich, M. J . Clin. Chem. Clin. Biochem. 1980, 18, 197-208. 15. Schwalbe, M.; Dorn, E . ; Beyermann, K. J . Agric. Food Chem. 1984, 32, 734-741. 16. Stanker, L.H.; Bigbee, C.; Van Emon, J.; Watkins, B.; Jensen, R. H.; Morris, C.; Vanderlaan, M. J . Agric. Food Chem. 1989, 37, 834-839. 17. Vallejo, R.P; Bogus, E.R.; Mumma, R.O. J . Agric. Food Chem. 1982, 30, 572-580. 18. Van Emon, J.; Hammock, B.D.; Seiber, J.N. Anal. Chem. 1986, 58, 1866-1873. 19. Van Emon, J.; Seiber, J.N.; Hammock, B.D. In Analytical Methods for Pesticides and Plant Growth Regulators: Advanced Analytical Technigues, Vol. XVII; Sherma, J., Ed.; Academic Press: New York, 1989, pp 217-263. 20. Wie, S.I.; Sylwester, A.P.; Wing, K.D.; Hammock, B.D. J . Agric. Food Chem. 1982, 30, 943-948. 21. Wie, S.I.; Hammock, B.D. J . Agric. Food Chem. 1982, 30, 949957. 22. Wie, S.I.; Hammock, B.D. J . Agric. Food Chem. 1984, 32, 12941301. 23. Weiler, E. W. In Chemistry of Plant Protection; Bowers, W. S., Ebing, W., Martin, D., Wegler, R., and Yamamoto, I., Eds.; Springer-Verlag: New York, 1990, pp. 145-220. RECEIVED August 30, 1990


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