Biotechnology for Crop Protection - ACS Publications - American

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Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch022

Applications of Immunochemistry in Crop Protection and Biotechnology Bruce D. Hammock Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616 It is very appropriate that a symposium on immunochemical applications be included i n a text on Biotechnology i n Crop Protection for a number of reasons. Immunoassays are physical assays yet clearly represent a biotechnology themselves which will play an increasing role i n many aspects of crop protection. In addition, immunochemical methods are central to the discovery and analysis of other biotechnology products (1). Rather than repeat other texts in describing how immunochemical applications can be carried out, this symposium was designed to provide a sampling of the great range of immunochemical applications i n crop protection today and in the foreseeable future. These applications range from the very practical work of plant diagnostics which is well accepted in many countries, to analysis of classical and genetically engineered pest control agents which has aroused wide interest i n industrial and regulatory circles, to the use of immunochemistry in the fundamental research efforts which will lead to new generations of crop protection agents. In many ways it is a pity that immunochemical assays were not extensively u t i l i z e d i n the crop protection f i e l d over a decade before. New assay formats have made assays a little easier to use, but the same techniques available i n 1970 still provide excellent assays today. However, the final article of this section introduces the concept that there will be a revolution i n immunochemical technology as proteins such as antibodies are coupled with solid state electronic and optical devices. In 1980 an article appeared on the Potential of Immunochemical Technology for Pesticide Residue Analysis (2). As evidenced by the articles in this section, that potential is beginning to be realized. However, of greater importance, we can envision far more applications in the future. 0097-6156/88/0379-0298$06.00/0 1988 American Chemical Society c

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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M i s c o n c e p t i o n s . J a r g o n , and D e f i n i t i o n s A common m i s c o n c e p t i o n i s t h a t one must be a n i m m u n o l o g i s t t o u t i l i z e immunochemical t e c h n o l o g y . T h i s i s no more t r u e t h a n t h e a s s u m p t i o n t h a t one must be a p h y s i c i s t t o use NMR. L i k e any o t h e r b i o t e c h n o l o g y , however, immunochemical methods u s u a l l y a r e s u r r o u n d e d by l a y e r upon l a y e r o f j a r g o n . This verbal i n s u l a t i o n , i n p a r t , i s a n a t u r a l e v o l u t i o n i n any f i e l d . However, t h e r e a l s o i s a c o m m e r c i a l r e a s o n f o r t h e c o n f u s i n g j a r g o n i n t h e immunoassay field. A s i n g l e a n t i b o d y p o o l c a n be u s e d i n a number o f d i f f e r e n t a s s a y f o r m a t s , each w i t h i t s own r e l a t i v e advantages w i t h r e g a r d t o s e n s i t i v i t y , speed, ease o f use and o t h e r p a r a m e t e r s . Usually, development o f an a n t i b o d y p o o l o c c u r s w i t h the use o f w e l l e s t a b l i s h e d t e c h n o l o g y so t h a t one does n o t have p a t e n t p r o t e c t i o n on t h e a n t i b o d y . However, i t i s p o s s i b l e t o p a t e n t some a s s a y formats. Thus t h e r e i s a r e w a r d i n t h e c o m m e r c i a l s e c t o r t o make o n e ' s assay format (whether p a t e n t e d o r n o t ) appear as u n i q u e as possible. C e r t a i n assay formats o f f e r c l e a r advantages f o r a p a r t i c u l a r a p p l i c a t i o n , however a l l immunoassays a r e b a s e d on t h e r e v e r s i b l e b u t v e r y s p e c i f i c i n t e r a c t i o n o f a p r o t e i n a n t i b o d y w i t h an antigen. T h i s i n t e r a c t i o n c a n be d e s c r i b e d by t h e l a w o f mass action. To d e a l w i t h t h e c h a p t e r s i n t h i s s e c t i o n , one o n l y needs the d e f i n i t i o n o f t h r e e s p e c i a l i z e d t e r m s . F i r s t , antibodies are a group o f serum p r o t e i n s t h a t r e a c t s p e c i f i c a l l y w i t h an a n t i g e n . An a n t i g e n i s a l a r g e m o l e c u l e ( u s u a l l y a p r o t e i n ) w h i c h r e a c t s w i t h a n a n t i b o d y and a l s o i s c a p a b l e o f i n d u c i n g a n t i b o d y p r o d u c t i o n when i n j e c t e d . F i n a l l y , haptens are molecules which can r e a c t w i t h a n t i b o d i e s , but which are too s m a l l to e l i c i t antibody production unless coupled to a p r o t e i n c a r r i e r . To use immunoassays one n o r m a l l y employs a r e p o r t e r s u b s t a n c e o r label. T h i s l a b e l c a n be one o f many m a t e r i a l s i n c l u d i n g a r a d i o c h e m i c a l , a heavy m e t a l , o r commonly, an enzyme. A w i d e l y u s e d format i s t h e enzyme l i n k e d immunosorbent a s s a y o r E L I S A . T h i s t e c h n o l o g y i s d i s c u s s e d i n the c h a p t e r s by Cheung, H a r r i s o n and V a n V u u r d e . There a r e numerous v a r i a t i o n s on a theme as t h i s a s s a y i s a d j u s t e d t o f i t a p a r t i c u l a r a n a l y t i c a l need. However, as an example o f t h e c o m p l e x i t y o f t e r m i n o l o g y , one c a n examine the a p p l i c a t i o n o f t h e ELISA system i n s l i g h t l y d i f f e r e n t f o r m a t s . F o r i n s t a n c e when t h e ELISA i s c a r r i e d o u t on t h e s u r f a c e o f a n i t r o c e l l u l o s e membrane, i t has the c l e v e r name o f d o t b l o t s i n c e the end r e s u l t s appear as t i n y d o t s on a p i e c e o f p a p e r . If a n t i g e n s a r e s e p a r a t e d by e l e c t r o p h o r e s i s b e f o r e t h e d o t b l o t i s r u n , one has a Western b l o t . I f the a n t i g e n i s s t a i n e d in situ one m i g h t term i t enzyme a m p l i f i e d i m m u n o h i s t o c h e m i s t r y even though t h e b i o c h e m i c a l s t e p s a r e t h e same i n each c a s e . I n the commercial s e c t o r t h i s same t e c h n o l o g y has dozens o f names. Thus, when one u n d e r s t a n d s the p r i n c i p l e o f one immunoassay most o t h e r s a r e seen as a s i m p l e v a r i a t i o n on a theme. Use o f Immunoassay i n P l a n t D i a g n o s t i c s Immunoassays have been used i n r e s e a r c h i n t h e p l a n t d i a g n o s t i c f i e l d f o r o v e r 25 y e a r s Q ) . K i t s f o r t h e d e t e c t i o n o f a v a r i e t y o f p l a n t d i s e a s e s now a r e a p p e a r i n g on t h e market, b u t r e g u l a t o r y


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a g e n c i e s i n t h i s c o u n t r y have n o t p l a c e d an emphasis on the use o f immunoassays f o r p l a n t d i a g n o s t i c s o r q u a r a n t i n e . As i n d i c a t e d i n the a r t i c l e by Van Vuurde and c o - w o r k e r s , immunochemical d i a g n o s t i c s a r e a major component o f the p l a n t p r o t e c t i o n e f f o r t i n the N e t h e r l a n d s and i n many o t h e r European c o u n t r i e s . Considering the l a b o r i n t e n s i v e a l t e r n a t i v e s o f t e n i n wide use i n t h i s c o u n t r y , we c e r t a i n l y c o u l d b e n e f i t from the e x p e r i e n c e o f our European c o l l e a g u e s w i t h i m m u n o d i a g n o s t i c s . The major impact o f immunodiagnosties f o r p l a n t pathogens i n Europe a l s o i n d i c a t e s t h a t the t e c h n o l o g y i s o f s u f f i c i e n t m a t u r i t y t o a p p l y t o d i a g n o s i s o f p e s t i c i d e s and t o o t h e r problems i n the c r o p p r o t e c t i o n a r e a . A v a r i e t y o f immunochemical d i a g n o s t i c s a r e u s e d i n E u r o p e , b u t ELISA based assays are o f i n c r e a s i n g importance. C o n s i d e r i n g t h a t over 1 0 E L I S A ' s were u s e d i n the N e t h e r l a n d s f o r r e g u l a t o r y d e t e c t i o n o f p l a n t v i r u s e s a l o n e i n 1986, t h i s format seems s u f f i c i e n t l y mature f o r i t t o be u s e d i n o t h e r c r o p p r o t e c t i o n a r e a s . Van Vuurde et. al. a l s o b r i n g up a *low t e c h n o l o g y ' approach t o b a c t e r i a l i d e n t i f i c a t i o n where a n t i b o d i e s a r e u s e d t o e n r i c h a b a c t e r i a l p o p u l a t i o n b e f o r e p l a t i n g . T h i s i n n o v a t i v e approach o f i m m u n o i s o l a t i o n i l l u s t r a t e s a n o t h e r power o f a n t i b o d i e s i n e n r i c h i n g o r p u r i f y i n g a m o l e c u l e o r even an o r g a n i s m by a f f i n i t y chromatography ( 4 ) . L i b r a r i e s o f a n t i b o d i e s a r e e x c e l l e n t t o o l s f o r the i d e n t i f i c a t i o n o f any o r g a n i s m whether i t i s a r e s i s t a n t i n s e c t , a c r o p p e s t , o r even the meal o f a p r e d a t o r u s e d i n b i o l o g i c a l control. Immunodiagnostic systems a r e p e r f e c t f o r i n t e g r a t i o n i n t o e x p e r t systems f o r d i a g n o s t i c a p p l i c a t i o n s . Immunodiagnostics and e x p e r t systems w i l l g r e a t l y improve the q u a l i t y o f r o u t i n e d i a g n o s t i c work and w i l l prove a v a l u a b l e time s a v e r even t o the expert. However, immunodiagnostic systems need t o be checked by e x p e r t s f a m i l i a r w i t h b o t h the pathogen and h o s t . Poorly defined d i a g n o s t i c k i t s , e s p e c i a l l y i f n o t u s e d by t r a i n e d i n d i v i d u a l s , c o u l d p r o v e m i s l e a d i n g as d i s c u s s e d b e l o w .


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Use o f Immunoassay i n P e s t i c i d e R e s i d u e A n a l y s i s The p o t e n t i a l o f immunochemical t e c h n o l o g y i n p e s t i c i d e r e s i d u e a n a l y s i s has been c l e a r f o r many y e a r s ( 2 ) , b u t i t s a c t u a l a p p l i c a t i o n i s j u s t b e i n g r e a l i z e d . The number o f l a b o r a t o r i e s r e p o r t i n g on the t e c h n o l o g y a t the r e c e n t IUPAC Congress o f P e s t i c i d e C h e m i s t r y a t t e s t s t o the f a c t t h a t w i d e s p r e a d use o f the t e c h n o l o g y soon w i l l be a r e a l i t y ( 5 , 6 , 7 , 8 ) . Of g r e a t e r importance a r e the l a r g e number o f a g r i c u l t u r a l c h e m i c a l companies t h a t have major i n house e f f o r t s . As w i t h HPLC t e c h n o l o g y o n l y a f t e r r e g i s t r a t i o n s a r e g r a n t e d b a s e d on immunoassay w i l l a c c e p t a n c e o f the t e c h n o l o g y become w i d e s p r e a d . Of e q u a l importance a r e the i n house e f f o r t s i n r e g u l a t o r y a g e n c i e s i n b o t h the c l a s s i c a l c h e m i c a l and b i o t e c h n o l o g y f i e l d . As r e g u l a t o r y a g e n c i e s d e v e l o p the i n house e x p e r t i s e t o e v a l u a t e the t e c h n o l o g y , i n d u s t r y w i l l f e e l more c o m f o r t a b l e i n a d v a n c i n g i t . The m a n u s c r i p t by H a r r i s o n et. al. p r o v i d e s some examples o f immunochemical a p p l i c a t i o n s t o c l a s s i c a l r e s i d u e a n a l y s i s . It summarizes some o f the advantages and l i m i t a t i o n s o f the t e c h n o l o g y as i t a p p l i e s t o the f i e l d and p r o v i d e s an o u t l i n e f o r development o f the t e c h n o l o g y i n house.


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o f Immunoassay i n D e t e c t i o n o f G e n e t i c a l l y E n g i n e e r e d M a t e r i a l s

Most a t t e m p t s t o d e v e l o p g e n e t i c a l l y e n g i n e e r e d p r o d u c t s have c o n c e n t r a t e d on t h e p r o d u c t i o n o f s i n g l e gene p r o d u c t s w h i c h b y n e c e s s i t y a r e p e p t i d e s and p r o t e i n s . I f t h e s e p r o d u c t s a r e t o be u s e d i n c r o p p r o t e c t i o n one needs a n a l y t i c a l methods t o answer r e g u l a t o r y q u e s t i o n s as w e l l as f o r q u a l i t y c o n t r o l . There i s some q u e s t i o n whether one s h o u l d m o n i t o r t h e gene, t h e message o r t h e translated product. The answer i s s i m p l e , a t l e a s t i n e a r l y s t a g e s o f t h e t e c h n o l o g y , i n t h a t one s h o u l d have t e c h n i q u e s a v a i l a b l e f o r a l l three. F o r t u n a t e l y , t h e probes f o r t h e a n a l y t i c a l methods needed p r o b a b l y were a l r e a d y d e v e l o p e d d u r i n g t h e r e s e a r c h l e a d i n g to the product. A v e r y common way t o i s o l a t e a message i s t o use an a n t i b o d y t o t h e d e s i r e d p r o t e i n p r o d u c t t o s c r e e n an e x p r e s s i o n library. T h i s screening procedure i s another a d a p t a t i o n o f the b a s i c ELISA f o r m a t . For i n s t a n c e , i n our l a b o r a t o r y the i s o l a t i o n o f t h e message f o r i n s e c t j u v e n i l e hormone e s t e r a s e by H a n z l i k and c o - w o r k e r s was a c c o m p l i s h e d by immunochemical s c r e e n i n g o f an e x p r e s s i o n l i b r a r y ( 9 ) . The same a n t i b o d y u s e d t o i s o l a t e t h e message c a n t h e n be used t o m o n i t o r t h e p r o t e i n p r o d u c e d i n an e x p r e s s i o n system as i t i s d e v e l o p e d f o r p e s t c o n t r o l . T h i s p r i n c i p l e i s amply i l l u s t r a t e d i n numerous c h a p t e r s o f t h i s volume. A p o t e n t i a l problem i s t h a t , i n an i n d u s t r i a l s e t t i n g , t h e probes f o r t h e a n a l y t i c a l method n o r m a l l y w o u l d be d e v e l o p e d i n a molecular b i o l o g y laboratory. The d e v e l o p e r s o f t h e t e c h n o l o g y p r o b a b l y w i l l l a c k t h e a n a l y t i c a l s k i l l s , time and i n t e r e s t needed t o reduce immunochemical and h y b r i d i z a t i o n t e c h n i q u e s t o r e l i a b l e a n a l y t i c a l methods t o be u s e d w i t h a v a r i e t y o f m a t r i c e s . Unless t h e r e i s a l e v e l o f a p p r e c i a t i o n among a n a l y t i c a l c h e m i s t s on how t o use t h e s e t e c h n o l o g i e s , t h e y a r e l i k e l y t o be l o s t o r t h e a n a l y t i c a l d u t i e s thrown on m o l e c u l a r b i o l o g i s t s r a t h e r t h a n p l a c e d i n an a n a l y t i c a l group where t h e y b e l o n g . The m a n u s c r i p t b y Cheung e t . a l . a d d r e s s e s t h e p r o b l e m o f immunochemical d e t e c t i o n o f b i o l o g i c a l s u s i n g t h e Bacillus thuringiensis s p e c i e s as an example. The e x c i t i n g developments r e p o r t e d i n t h i s t e x t r e g a r d i n g t h e p r o d u c t i o n o f g e n e t i c a l l y e n g i n e e r e d p l a n t s as w e l l as m i c r o b i a l i n s e c t i c i d e s i n d i c a t e t h a t t h e r e w i l l be an immediate need f o r immunochemical d e t e c t i o n o f g e n e t i c a l l y e n g i n e e r e d c r o p p r o t e c t i o n agents. Use

o f Immunoassay i n Fundamental R e s e a r c h and P r o d u c t D i s c o v e r y

As has been d i s c u s s e d by many w o r k e r s , we have r e a c h e d a p o i n t o f d i m i n i s h i n g r e t u r n s w i t h r e g a r d t o random d i s c o v e r y o f c r o p p r o t e c t i o n agents by c l a s s i c a l s c r e e n i n g methods ( 1 0 - 1 3 ) . Immunoassays p r o b a b l y w i l l be i n t r o d u c e d i n t o t h e i n d u s t r i a l s e t t i n g f o r s h o r t term g o a l s i n b i o t e c h n o l o g y and a n a l y t i c a l c h e m i s t r y . However, i t i s l i k e l y t h a t t h e major impact o f t h e t e c h n o l o g y w i l l be i n fundamental r e s e a r c h on c r o p p r o t e c t i o n . A number o f s i m p l i s t i c a p p l i c a t i o n s c o u l d r e s u l t from a s s a y s t o a group o f c a n d i d a t e p e s t i c i d e s e a r l y i n development. Such a s s a y s w o u l d be v e r y c o s t e f f e c t i v e i n m o n i t o r i n g p e n e t r a t i o n and translocation. I f a m o l e c u l e i s l a r g e , t h e r e i s hope t h a t immunochemical t e c h n i q u e s c o u l d even be u s e d t o l o c a l i z e t h e compound i n t h e t a r g e t s p e c i e s and p o s s i b l y t o p u r i f y t h e t a r g e t .



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Longer term work w i l l i n v o l v e the use o f immunochemical probes t o e x p l o r e the c o m p a r a t i v e b i o c h e m i s t r y o f a v a r i e t y o f t a r g e t and nontarget species. E x c e l l e n t examples o f t h i s work a r e seen i n the i n s e c t m o l e c u l a r b i o l o g y s e c t i o n o f t h i s book. A c l e a r example has been the use o f a n t i b o d i e s r a i s e d a g a i n s t mammalian neurohormones as a l e a d i n the i s o l a t i o n o f i n v e r t e b r a t e hormones. These m a t e r i a l s a r e p o t e n t i a l t a r g e t s t o use as i n s e c t c o n t r o l agents when p r o d u c e d i n i n a p p r o p r i a t e l e v e l s i n i n s e c t s f o l l o w i n g i n f e c t i o n w i t h an a p p r o p r i a t e e x p r e s s i o n v e c t o r . The p e p t i d e s t r u c t u r e s a l s o may p r o v i d e l e a d s f o r the development o f i n h i b i t o r s o f p r o c e s s i n g enzymes. I n d u s t r i a l r e s e a r c h on i n s e c t neurohormones i s t o t a l l y j u s t i f i e d even i f one n e v e r d e v e l o p s an i n s e c t c o n t r o l agent based on them. J u s t as probes d e r i v e d from the s t r u c t u r e s o f mammalian neurohormones have p r o v e n u s e f u l i n work on i n s e c t n e u r o e n d o c r i n o l o g y ( 1 4 ) , i n v e r t e b r a t e neurohormones a r e l i k e l y to p r o v i d e immunochemical and o l i g o n u c l e i c a c i d probes f o r use i n i n v e s t i g a t i n g p e p t i d e n e u r o t r a n s m i t t e r s i n the human c e n t r a l nervous system l e a d i n g t o new g e n e r a t i o n s o f p h a r m a c e u t i c a l s . A n t i b o d i e s to candidate h e r b i c i d e s can a i d i n t r a n s l o c a t i o n s t u d i e s , and f o r l a r g e m o l e c u l e s can even a s s i s t i n l o c a l i z i n g b i n d i n g to r e c e p t o r s . A y e r s and coworkers i n t h i s s e c t i o n p r o v i d e an e x c e l l e n t i l l u s t r a t i o n o f the a p p l i c a t i o n o f b o t h c l a s s i c a l and i n n o v a t i v e immunochemical t e c h n o l o g y i n a p p r o a c h i n g problems w i t h plant disease resistance. A l t h o u g h t h i s work i s fundamental, t h e r e i s a c l e a r p a t h towards e x p l o i t a t i o n o f such r e s e a r c h i n c r o p protection. T h i s s t u d y c l e a r l y i l l u s t r a t e s how i m p o r t a n t immunochemical t e c h n o l o g y i s t o the modern b i o c h e m i s t , and analogous examples appear i n each s e c t i o n o f t h i s t e x t . P o t e n t i a l o f New A s s a y Formats S e v e r a l o f the m a n u s c r i p t s i n t h i s s e c t i o n demonstrate t h a t even mature immunoassay formats i n common use o v e r a decade ago s t i l l provide very valuable a n a l y t i c a l data. However, i t i s c l e a r t h a t competing a n a l y t i c a l t e c h n o l o g i e s , e s p e c i a l l y i n the mass s p e c t r a l a r e a , have advanced d r a m a t i c a l l y . Thus, some o f the g r e a t advantages o v e r c h r o m a t o g r a p h i c a s s a y s o f f e r e d by immunoassay a r e n o t as d r a m a t i c when compared t o modern p h y s i c a l methods as t h e y were p r e v i o u s l y . I n the n e x t decade we w i l l see a n t i b o d i e s and o t h e r b i o l o g i c a l m o l e c u l e s combined w i t h s o l i d s t a t e e l e c t r o n i c and o p t i c a l d e v i c e s i n many ways t o y i e l d h y b r i d b i o l o g i c a l - p h y s i c a l sensors. These so c a l l e d b i o s e n s o r s a r e l i k e l y t o have a d r a m a t i c impact on a n a l y t i c a l t e c h n o l o g y . There can be v e r y s i m p l e ways t o combine immunochemistry w i t h c l a s s i c a l r e s i d u e methodology. P o s s i b l y the s i m p l e s t example would be t o a n a l y z e f r a c t i o n s from an HPLC r u n by immunoassay. However, d i r e c t c o m b i n a t i o n s o f immunochemical and m i c r o e l e c t r o n i c systems o f f e r the p o s s i b l e advantages o f r e a l time a n a l y s i s , g r e a t e r l i n e a r i t y , and i n c r e a s e d s e n s i t i v i t y due t o s m a l l e r sample s i z e s . The a r t i c l e by Stanbro et. al. e x p l a i n s the o p e r a t i o n o f one such evolving biosensor technology. T h i s a r t i c l e i s e x c i t i n g i n i t s own r i g h t and even more e x c i t i n g i n p r o p h e s i z i n g t h i n g s t o come.


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P e r i l s o f a S u c c e s s f u l Technology C l i n i c a l d i a g n o s t i c s have matured t o t h e p o i n t where p e o p l e a s k how w e l l an assay performs r a t h e r t h a n w o r r y i n g about t h e c h e m i c a l b a s i s o f the assay. By c o n t r a s t i n t h e e n v i r o n m e n t a l f i e l d t h e r e i s a c o n c e n t r a t i o n on the t e c h n i q u e u s e d r a t h e r t h a n t h e r e s u l t s i t produces. The e n v i r o n m e n t a l f i e l d must mature t o t h e p o i n t where t h e r e a r e c l e a r c r i t e r i a f o r a c c e p t a b l e a s s a y performance r e g a r d l e s s o f t h e t e c h n o l o g y upon w h i c h t h a t assay i s b a s e d . Immunochemical t e c h n o l o g y was v i r t u a l l y i g n o r e d f o r many y e a r s , b u t now i s b e i n g w i d e l y advanced as a panacea. Possibly this n o t o r i e t y i s due t o p r e s s u r e s on a n a l y s t s t o a c c e p t b i o t e c h n o l o g y approaches o r p o s s i b l y i t i s o u r p e r p e t u a l d e s i r e f o r an easy solution. Recent a t t e m p t s t o l e g i s l a t e t h e type o f a s s a y s u s e d by a n a l y t i c a l c h e m i s t s i n d i c a t e t h a t o u r f i e l d may f a c e a s e v e r e problem. There i s no q u e s t i o n t h a t immunochemistry w i l l make a major c o n t r i b u t i o n t o many a s p e c t s o f e n v i r o n m e n t a l a n a l y s i s , b u t t h e r e i s a s e v e r e danger t h a t i t w i l l be v i e w e d as a p a n a c e a . Many a u t h o r s have l i s t e d the numerous advantages o f i m m u n o d i a g n o s t i c s . I t i s i m p o r t a n t t o r e a l i z e t h a t some o f t h e s e advantages may be m u t u a l l y e x c l u s i v e . Even though t h e same a n t i b o d y p o o l i s u s e d , one format may s a c r i f i c e speed f o r a c c u r a c y and a n o t h e r s a c r i f i c e some s e n s i t i v i t y f o r c o s t . I f the c o n c e p t i s advanced t h a t e v e r y a s s a y w i l l have a l l b e n e f i t s , p e o p l e w i l l be g r e a t l y d i s a p p o i n t e d w i t h the technology. As mentioned e a r l i e r , immunochemistry i s a p p l i c a b l e t o a g r e a t many s t r u c t u r e s , however, i n t h i s case as w e l l , t h e p r o m i s e o f immunoassay c a n be o v e r s o l d . The r e v e r s i b l e b i n d i n g o f a n t i b o d i e s t o a m o l e c u l e i s b a s e d on t h e summation o f a number o f weak molecular i n t e r a c t i o n s . Hydrogen bonds a r e v e r y i m p o r t a n t s i n c e t h e y p r o v i d e a g r e a t d e a l o f b i n d i n g energy as w e l l as d i r e c t i o n a l and d i s t a n c e s p e c i f i c i t y . Many o f t h e o t h e r bonds formed a r e even more dependent upon c l o s e f i t . These b i n d i n g e n e r g i e s however do i n d i c a t e t h a t there i s a lower l i m i t t o the s i z e o f the molecule to w h i c h one c a n e x p e c t t o r a i s e good a n t i b o d i e s . Although i t i s p o s s i b l e t o o b t a i n a n t i b o d i e s t o m o l e c u l e s w h i c h have o n l y t h r e e o r f o u r c a r b o n s , the l i k e l i h o o d o f g e n e r a t i n g h i g h a f f i n i t y and v e r y s p e c i f i c antibodies i s not great. S i n c e immunoassays must be r u n i n a p r e d o m i n a n t l y aqueous system, l i p o p h i c i t y o f t h e t a r g e t compound a l s o i s i m p o r t a n t . S o l u b i l i t y o f t h e t a r g e t compound i n an aqueous system seldom i s a problem s i n c e the compound c a n be p r e s e n t e d i n a w a t e r s o l u b l e c o s o l v e n t o r as a m i c e l l e . However, s e p a r a t i n g the t a r g e t compound from a l i p o p h i l i c m a t r i x c a n be a nightmare. Such p r o c e d u r e s c a n be p a r t i c u l a r l y i n t i m i d a t i n g t o an inimunochemist u n f a m i l i a r w i t h h a n d l i n g o f l i p o p h i l i c m a t e r i a l s . If one must p e r f o r m many c l e a n up s t e p s p r i o r t o t h e a s s a y t o p a r t i a l l y p u r i f y a l i p o p h i l i c compound, t h e n t h e advantages o f immunoassay o v e r c h r o m a t o g r a p h i c methods a r e l o s t . Thus, h i g h l y l i p o p h i l i c m o l e c u l e s may n o t be optimum t a r g e t s f o r immunoassays. S i m i l a r l y one s h o u l d a v o i d h i g h l y s y m m e t r i c a l , w a t e r u n s t a b l e o r v o l a t i l e compounds. I t i s n o t i m p o s s i b l e t o d e v e l o p a s u c c e s s f u l immunoassay t o m o l e c u l e s t h a t do n o t seem t o l e n d themselves t o immunoassay. However, as t h e number o f c o n t r a i n d i c a t i o n s i n c r e a s e , development


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o f the a s s a y w i l l become i n c r e a s i n g l y d i f f i c u l t . One w o u l d n o t want t o t r y a s s a y development f o r a s m a l l , l i p o p h i l i c , v o l a t i l e , s y m m e t r i c a l m o l e c u l e as an i n t r o d u c t i o n t o the t e c h n o l o g y . The f i e l d faces a problem i n t h a t there i s r e g u l a t o r y pressure to apply s i m p l i s t i c immunochemistry t o m o l e c u l e s t h a t a r e b e t t e r a n a l y z e d by other technologies. I n many c a s e s we w i l l be a b l e t o d e v e l o p assays f o r these m a t e r i a l s . However, the g r e a t r e l a t i v e advantages o f immunochemical approaches w i l l be l o s t . The development o f the t e c h n o l o g y i s a t a c r i t i c a l s t a g e i n t h a t i t s h o u l d be advanced r a p i d l y , b u t i f i t i s o v e r s o l d the t e c h n o l o g y c o u l d be seen as f a i l i n g i n k e y a p p l i c a t i o n s . P o s s i b l y the b e s t s t r a t e g y i s t o t r e a t immunochemistry as s i m p l y a n o t h e r a n a l y t i c a l method w h i c h must be c a r e f u l l y v a l i d a t e d b e f o r e u s e . Commercial E x p l o i t a t i o n I f immunochemical t e c h n o l o g y i n c r o p p r o t e c t i o n i s t o r e a c h i t s f u l l p o t e n t i a l , i t must be advanced i n the c o m m e r c i a l as w e l l as other sectors (15). The g r e a t e s t c o m m e r c i a l b e n e f i t w i l l be i n money saved by l a r g e c h e m i c a l and b i o t e c h n o l o g y companies, money made b y p r o d u c t s w h i c h r e a c h the market f a s t e r thanks t o immunochemical s u p p o r t , and new p r o d u c t s g e n e r a t e d i n p a r t w i t h immunochemical methods i n c o r p o r a t e d i n t o r e s e a r c h programs. C o n s i d e r i n g o n l y i m m u n o d i a g n o s t i c s , i t w i l l be i m p o r t a n t f o r companies t o e n t e r the f i e l d where p r o f i t s w i l l be t i e d t o s a l e s o f immunochemical t e c h n o l o g i e s . The overhead i n v o l v e d w i t h l a r g e companies p r o b a b l y w i l l be too g r e a t f o r them t o make a r e a l i s t i c p r o f i t from a g r i c u l t u r a l immunodiagnostics i n the s h o r t term and s m a l l companies o r s u b s i d i a r i e s w i l l p i o n e e r the f i e l d . F o r such b i o t e c h n o l o g y companies t h e r e a r e s e v e r a l c l e a r m a r k e t s . Probably the b e s t market i s i n p r o v i d i n g a t e c h n o l o g i c a l s e r v i c e t o major companies who want i n house methods c a p a b l e o f m o n i t o r i n g p r o d u c t q u a l i t y , w o r k e r e x p o s u r e , waste d i s p o s a l , and o t h e r h o u s e k e e p i n g chores. There i s an e x i s t i n g s m a l l market i n end u s e r a s s a y s f o r many compounds, b u t o n l y t h o s e where many u s e r s w i l l be i n v o l v e d o f f e r an immediate p r o f i t . Examples o f t h i s w o u l d be i n c a s e s where a farmer w o u l d need t o know h e r b i c i d e l e v e l s b e f o r e p l a n t b a c k . When a s s a y s a r e approved f o r r o u t i n e use i n p l a n t c e r t i f i c a t i o n and r e s i d u e a n a l y s i s , l a r g e markets f o r many a s s a y s suddenly w i l l appear. S i n c e assay v a l i d a t i o n i s b e i n g p u r s u e d on s e v e r a l f r o n t s , i t i s l i k e l y t h a t numerous markets w i l l d e v e l o p i n the n e a r f u t u r e . E s p e c i a l l y a t an e a r l y s t a g e i n the acceptance o f the t e c h n o l o g y , i t i s c r i t i c a l t h a t w e l l c h a r a c t e r i z e d a s s a y s be developed f o r c l e a r l y defined goals. Immunochemical t e c h n o l o g y i s c e r t a i n t o r e p r e s e n t a major market i n the p l a n t p r o t e c t i o n a r e a . However, the speed w i t h w h i c h the market d e v e l o p s depends t o a g r e a t e x t e n t on the q u a l i t y o f the a s s a y s used t o p i o n e e r the technology. Acknowledgments T h i s work was s u p p o r t e d i n p a r t by NIEHS Superfund G r a n t PHS ES04699-01, EPA C o o p e r a t i v e Agreement C R - 8 1 4 7 0 9 - 0 1 - 0 , and a g r a n t from the 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 S c h o l a r i n T o x i c o l o g y .


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Literature Cited 1. Cheung, P. Y. K; Gee, S. J.; Hammock, B. D. In The Impact of Chemistry on Biotechnology: Multidisciplinary Discussions: P h i l l i p s , M. P.; Shoemaker, S. P., Eds.; V o l . 362; American Chemical Society: Washington, DC, 1988; p 217-229. 2. Hammock, B. D.; Mumma, R. O. In Recent Advances i n Pesticide Analytical Methodology: Harvey, J., J r . ; Zweig, G., Eds.; ACS Publications: Washington, DC, 1980; p 321-352. 3. Clark, M. F. Ann. Rev. Plant Pathol. 1981, 19, 83-106. 4. Van Vuurde, J. W. L. Bulletin OEPP/EPPO 1987, 17, 139-148. 5. Hammock, B. D.; Gee, S. J.; Cheung, P. Y. K . ; Miyamoto, 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; p 309-316. 6. Mumma, R. O.; Brady, J. F. In Pesticide Science and Biotechnology: Greenhalgh, R.; Roberts, T. R., Eds.; Blackwell Scientific Publications: Oxford, 1987; p 341-8. 7. Newsome, W. H. In Pesticide Science and Biotechnology: Greenhalgh, R.; Roberts, T. R., Eds.; Blackwell Scientific Publications: Oxford, 1987; p 349-352. 8. Vanderlaan, M.; Van Emon, J.; Watkins, B . ; Stanker, L. In Pesticide Science and Biotechnology: Greenhalgh, R.; Roberts, T. R., Eds.; Blackwell Scientific Publications: Oxford, 1987; p 597-602. 9. Hammock, B. D.; Harshman, L. G.; Philpott, M. L.; Székács, A . ; Ottea, J . A . ; Newitt, R. A . ; Wroblewski, V. J.; Halarnkar, P. P. In USDA Beltsville Symposia Proceedings in Agricultural Research. 1988, in press. 10. Metcalf, R. L. Ann. Rev. Entomol. 1980, 25, 219-256. 11. Hollingworth, R. M. In Pesticides: Minimizing the Risks: Ragsdale, N. N . ; Kuhr, R. J., Eds.; Vol. 336; American Chemical Society: Washington, DC, 1987; p 54-76. 12. Hammock, B. D.; Soderlund, D. M. In Pesticide Resistance: Strategies and Tactics for Management: Glass, E . , Ed.; National Academy Press: Washington DC, 1986; p 111-129. 13. Hammock, B. D.; Abdel-Aal, Y. A. I.; Ashour, M . ; Buhler, A . ; Hanzlik, T. N. ; Newitt, R.; Sparks, T. C. In Human Welfare and Pest Control Chemicals-Approaches to Safe and Effective Control of Medical and Agricultural Pests: Sasa, M.; Matsunaka, S.; Yamamoto, I.; Ohsawa, K . , Eds.; Pesticide Science Society of Japan: Tokyo, Japan, 1986; p 53-72. 14. Scharrer, B. Ann. Rev. Entomol. 1987, 32, 1-16. 15. Vanderlaan, M.; Watkins, B. E . ; Stanker, L. Environ. S c i . Technol. 1988, 22, 247-254. RECEIVED May 4, 1988


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