Biotechnology for Crop Protection - American Chemical Society

Chapter

24

Immunochemical Methods

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of Pesticide Residue Analysis Robert O. Harrison, Shirley J. Gee, and Bruce D. Hammock Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616 Immunochemical methods are rapidly gaining acceptance as analytical techniques for pesticide residue analysis. Unlike most quantitative methods for measuring pesticides, they are simple, rapid, precise, cost effective, and adaptable to laboratory or f i e l d situations. The technique centers around the development of an antibody for the pesticide or environmental contaminant of interest. The work hinges on the synthesis of a hapten which contains the functional groups necessary for recognition by the antibody. Once this aspect is complete, immunochemical detection methods may take many forms. The enzyme-linked immunosorbent assay (ELISA) is one form that has been found useful i n residue applications. This technique will be illustrated by examples from this laboratory, particularly molinate, a thiocarbamate herbicide used in rice culture. Immunoassay development will be traced from hapten synthesis to validation and f i e l d testing of the final assay. Emphasis will be placed on the justification of and the resources required for the successful incorporation of immunochemical technology into an existing analytical laboratory. Special attention will be given to aspects of immunochemical and related technology not covered i n other recent reviews. Present use of immunoassay for pesticide analysis will be described and future potential applications and problems will be discussed. Some basic immunology and definition of a few common terms w i l l allow understanding of the central concepts of immunoassay. Antibodies are serum proteins which bind to specific molecules, called antigens, due to a complementarity of chemical structure between antibody and antigen. Immunization with an antigen preferentially induces the production of antibodies specific for c

0097-6156/88/0379-0316$06.00/0 1988 American Chemical Society

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


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that antigen. W h i l e most a n t i g e n s a r e n a t u r a l l y o c c u r r i n g p r o t e i n s o r complex c a r b o h y d r a t e s , the e x t r a o r d i n a r y g e n e t i c f l e x i b i l i t y o f the immune system a l l o w s f o r the p r o d u c t i o n o f a n t i b o d i e s a g a i n s t v i r t u a l l y any c h e m i c a l s t r u c t u r e , i n c l u d i n g s y n t h e t i c a n t i g e n s . A n t i g e n s below a p o o r l y d e f i n e d s i z e t h r e s h o l d o f a p p r o x i m a t e l y 1000 d a l t o n s w i l l n o t s t i m u l a t e the 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 . However, s m a l l m o l e c u l e s , c a l l e d h a p t e n s , c a n e l i c i t s p e c i f i c a n t i b o d i e s when a t t a c h e d t o l a r g e r c a r r i e r m o l e c u l e s such as proteins. The phenomenon o f immune response t o h a p t e n s has been s t u d i e d f o r o v e r f i f t y y e a r s w i t h the s u c c e s s f u l p r o d u c t i o n o f a n t i b o d i e s t o an a s t o u n d i n g range o f n a t u r a l and s y n t h e t i c c h e m i c a l structures. Thus the p o t e n t i a l c l e a r l y e x i s t s f o r the development o f immunoassays f o r n e a r l y any compound. An o c c a s i o n a l m i s c o n c e p t i o n i s t h a t immunoassays a r e b i o a s s a y s because o f t h e i r use o f b i o l o g i c a l l y d e r i v e d a n t i b o d i e s . It is i m p o r t a n t t o r e a l i z e t h a t a l t h o u g h immunoassays a r e dependent upon a n t i b o d i e s , t h e s e a n t i b o d i e s obey the Law o f Mass A c t i o n j u s t as r e a g e n t s i n any o t h e r l i g a n d b i n d i n g a s s a y and a r e t h e r e f o r e w e l l s u i t e d to q u a n t i t a t i v e a n a l y s i s . T h e o r e t i c a l understanding of q u a n t i t a t i v e immunoassays has e v o l v e d s i g n i f i c a n t l y i n the l a s t t h r e e decades t h r o u g h e x t e n s i v e c l i n i c a l and r e s e a r c h use (26)• i n d i c a t i o n o f the massive commercial s u p p o r t f o r immunochemical t e c h n i q u e s i s the degree o f s o p h i s t i c a t i o n o f d a t a a n a l y s i s a v a i l a b l e i n numerous commercial software packages w h i c h e x p l o i t t h e s e developments (HP-Genenchem, M o l e c u l a r D e v i c e s , D y n a t e c h , and many o t h e r s ) . T h i s s u p p o r t i l l u s t r a t e s the m a t u r i t y o f immunochemical t e c h n i q u e s and t e c h n o l o g y , much o f w h i c h c a n be d i r e c t l y a p p r o p r i a t e d f o r r e s i d u e a n a l y s i s by immunoassay. The development o f immunochemical methods f o r a n a l y s i s o f h a p t e n s such as drugs and s t e r o i d s ( 4 , 1 1 ) o c c u r r e d d u r i n g the dominance o f the p e s t i c i d e market by h i g h l y n o n p o l a r o r g a n o c h l o r i n e s , w h i c h a r e e a s i l y a n a l y z e d by gas chromatography w i t h i o n s e l e c t i v e d e t e c t o r s due t o t h e i r l i p o p h i l i c i t y and halogenation. There i s a l o n g h i s t o r y o f e x t e n s i v e c l i n i c a l and r e s e a r c h use o f immunoassays f o r a n a l y s i s o f drugs and hormones, i n c l u d i n g s t e r o i d s , p e p t i d e s , and o t h e r s ( 4 , 1 1 , 2 4 , 2 5 ) . The reasons f o r the l a c k o f use 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 seem t o be p r i m a r i l y h i s t o r i c a l . Present t e c h n o l o g y makes i t f e a s i b l e t o a n a l y z e f o r n e a r l y a l l p e s t i c i d e s i n c u r r e n t use by immunochemical methods. A

n

Reasons f o r A p p l y i n g Immunochemical Technology t o P e s t i c i d e s S i n c e the use 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 was f i r s t r e v i e w e d by E r c e g o v i c h i n 1971 (9) s e v e r a l h e l p f u l r e v i e w s o f t h i s a p p l i c a t i o n have been p u b l i s h e d (11,14,23,33). D e s p i t e the p o t e n t i a l demonstrated d u r i n g t h i s p e r i o d , few r e s e a r c h e r s and r e g u l a t o r s a p p l y immunochemical t e c h n o l o g y t o t h e i r own problems 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 . Immunochemical t e c h n o l o g y f o r r e s i d u e a n a l y s i s o f f e r s the e x i s t i n g a n a l y t i c a l l a b o r a t o r y many advantages. S e n s i t i v i t y and s p e c i f i c i t y a r e g e n e r a l l y comparable t o e x i s t i n g t e c h n i q u e s w i t h l a r g e improvements i n speed and c o s t . The g e n e r a l a p p l i c a b i l i t y o f immunochemical t e c h n o l o g y i s a l s o a v a l u a b l e a s s e t . Immunoassays c a n be modular; b y s u b s t i t u t i n g two r e a g e n t s , a n a l y s i s f o r a



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d i f f e r e n t a n a l y t e can be performed u s i n g the same i n s t r u m e n t a t i o n . M u l t i a n a l y t e p r o c e d u r e s c a n be d e v e l o p e d t h r o u g h p a r a l l e l processing. The f a c t o r s l i m i t i n g the p r e s e n t use o f immunochemical t e c h n o l o g y f o r r e s i d u e a n a l y s i s are p r i m a r i l y l a c k o f a c c e p t a n c e and poor a n t i b o d y a v a i l a b i l i t y r a t h e r t h a n the d i f f i c u l t y o f d e v e l o p i n g a n t i b o d i e s t o t a r g e t compounds. T h i s s i t u a t i o n appears t o be c h a n g i n g as the advantages o f immunoassay f o r e n v i r o n m e n t a l a n a l y s i s a r e r e c o g n i z e d by a n a l y t i c a l c h e m i s t s . One r e a s o n f o r t h i s r e c o g n i t i o n i s t h a t immunoassay i s the b e s t c h o i c e among p r e s e n t t e c h n o l o g i e s f o r the a n a l y s i s o f the l a r g e number o f samples needed f o r thorough e n v i r o n m e n t a l a n a l y s i s . I t c a n be shown by s a m p l i n g t h e o r y t h a t a l a r g e number o f samples must be a n a l y z e d t o o b t a i n h i g h c o n f i d e n c e e s t i m a t e s o f low c o n t a m i n a t i o n r a t e s ( F i g u r e 1 ) . W i t h immunochemical t e c h n o l o g y a v a i l a b l e , the a n a l y t i c a l c h e m i s t no l o n g e r need be dismayed by the p r o s p e c t o f a n a l y z i n g such l a r g e sample l o a d s . The t e c h n o l o g y f o r a u t o m a t i o n o f some types o f immunoassays i s a d v a n c i n g r a p i d l y and sample p r o c e s s i n g r a t e s a r e i n c r e a s i n g d r a m a t i c a l l y . The a p p l i c a t i o n o f r o b o t i c systems t o the r e s i d u e l a b o r a t o r y has been d i s c u s s e d (22) and an automated ELISA system u s i n g c o m m e r c i a l l y a v a i l a b l e r o b o t s and automated e x t r a c t i o n systems has been d e s c r i b e d w h i c h c a n a n a l y z e 10,000 o r more seed and p l a n t samples p e r day f o r v i r a l and b a c t e r i a l d i s e a s e s ( 3 4 ) . A p p l i c a t i o n s o f immunoassay t o p e s t i c i d e c h e m i s t r y have been d e s c r i b e d w h i c h address some d i f f i c u l t problems i n a n a l y s i s by c l a s s i c a l methods. These i n c l u d e s t e r e o s p e c i f i c a n a l y s i s o f o p t i c a l l y a c t i v e compounds such as p y r e t h r o i d s (38) , a n a l y s i s o f p r o t e i n t o x i n s from B a c i l l u s t h u r i n g i e n s i s ( 5 , 3 7 ) , and compounds d i f f i c u l t t o a n a l y z e by e x i s t i n g methods, such as d i f l u b e n z u r o n (35) and m a l e i c h y d r a z i d e (15; a l s o H a r r i s o n , R . O . ; B r i m f i e l d , A . A . ; H u n t e r , K . W . , J r . ; N e l s o n , J . O . J . A g r i c . Food Chem. s u b m i t t e d ) . An example o f the e x c e l l e n t s p e c i f i c i t y p o s s i b l e i s seen i n a s s a y s f o r p a r a t h i o n (10) and i t s a c t i v e form p a r a o x o n ( 3 ) . Some immunoassays c a n be used d i r e c t l y f o r a n a l y s i s w i t h o u t e x t e n s i v e sample e x t r a c t i o n o r c l e a n u p , d r a m a t i c a l l y r e d u c i n g the work needed i n t y p i c a l r e s i d u e a n a l y s i s . An example o f t h i s i s g i v e n i n F i g u r e s 2 and 3, comparing the d i r e c t ELISA a n a l y s i s o f m o l i n a t e i n r i c e paddy w a t e r t o the e x t r a c t i o n r e q u i r e d b e f o r e GC a n a l y s i s . W h i l e the use o f immunoassay f o r r e s i d u e a n a l y s i s s h o u l d c o n t i n u e t o expand, i t i s n o t the answer t o a l l problems i n environmental a n a l y s i s . Immunochemical t e c h n o l o g y s h o u l d s e r v e b e s t as a complement t o e x i s t i n g methods r a t h e r t h a n a replacement f o r them. I t i s e s p e c i a l l y i m p o r t a n t t o r e c o g n i z e the p o t e n t i a l o f immunoassay f o r impact i n e n v i r o n m e n t a l s c r e e n i n g t h r o u g h the developments w h i c h w i l l be d i s c u s s e d l a t e r i n t h i s c h a p t e r . It is i n t h i s a r e a t h a t we e x p e c t immunochemical t e c h n o l o g y t o make i t s g r e a t e s t c o n t r i b u t i o n to environmental a n a l y s i s . Resources R e q u i r e d f o r Immunoassay

Development

Perhaps the most i m p o r t a n t d i s t i n c t i o n t o be drawn i n t h i s c h a p t e r i s t h a t between the development o f immunochemical r e s i d u e methods and t h e i r i m p l e m e n t a t i o n f o r r o u t i n e u s e . The l a b o r and r e s o u r c e s r e q u i r e d f o r d e v e l o p i n g a s p e c i f i c a s s a y are s i g n i f i c a n t , as w i l l be shown i n t h i s s e c t i o n . However, the r o u t i n e use o f v a l i d a t e d a s s a y s



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F i g u r e 1. The shaded a r e a r e p r e s e n t s t h e 95% c o n f i d e n c e i n t e r v a l f o r t r u e c o n t a m i n a t i o n r a t e , assuming random d i s t r i b u t i o n o f c o n t a m i n a t e d samples and g i v e n a c o n s t a n t 0% d e t e c t e d c o n t a m i n a t i o n r a t e (adapted from r e f . 1 ) . The upper boundary o f the c u r v e r e p r e s e n t s t h e upper 95% c o n f i d e n c e l i m i t f o r t r u e c o n t a m i n a t i o n r a t e . Thus, i f t e n samples were a n a l y z e d w i t h no c o n t a m i n a t i o n d e t e c t e d , t h e t r u e c o n t a m i n a t i o n r a t e 95% c o n f i d e n c e i n t e r v a l w o u l d be between 0% and 30.8%.


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r e q u i r e s l i t t l e e x p e r t i s e , equipment, o r s u p p l i e s n o t a l r e a d y found i n most a n a l y t i c a l l a b s . A summary o f the r e s o u r c e s needed f o r immunoassay development i s g i v e n i n T a b l e 1. TABLE 1 Resources needed f o r immunoassay development O r g a n i c c h e m i s t r y : h a p t e n s y n t h e s i s and c o n j u g a t i o n t o carriers Biology: antibody production Immunochemistry: immunoassay d e s i g n and o p t i m i z a t i o n A n a l y t i c a l and r e s i d u e c h e m i s t r y : sample p r e p a r a t i o n , d a t a a n a l y s i s , a s s a y v a l i d a t i o n and a p p l i c a t i o n T h i s t a b l e i l l u s t r a t e s one o f the major impediments t o the r a p i d a s s i m i l a t i o n o f immunochemical t e c h n o l o g y i n t o p e s t i c i d e r e s i d u e analysis labs. Because o f the amount and v a r i e t y o f work i n v o l v e d , new method development c o s t s may be h i g h when compared t o r o u t i n e c h r o m a t o g r a p h i c methods. However, the low c o s t p e r r u n a l l o w s f o r r a p i d r e c o v e r y o f the i n i t i a l i n v e s t m e n t w i t h s u f f i c i e n t l y h i g h sample l o a d s . F o r example, the c o s t o f r e a g e n t s and s u p p l i e s f o r an ELISA f o r d i f l u b e n z u r o n was e s t i m a t e d t o be $ 0 . 2 0 / s a m p l e as compared w i t h $4 f o r HPLC o r $11 f o r GC ( 3 5 ) . I n a d d i t i o n t o the l o w e r r e a g e n t and s u p p l y c o s t s , the major economic advantage o f immunoassay i s the d r a m a t i c d e c r e a s e i n l a b o r c o s t s . Steps i n the Development o f P e s t i c i d e Immunoassays The s t e p s i n v o l v e d i n the development o f p e s t i c i d e immunoassays have been d e s c r i b e d p r e v i o u s l y (14) and numerous r e p o r t s o f p e s t i c i d e immunoassay development now i l l u s t r a t e t h i s p r o c e s s (2,10,12,15,19,32,35,36,38). These s t e p s w i l l be r e i t e r a t e d h e r e t o r e i n f o r c e s a l i e n t p o i n t s and t o emphasize the d i s t i n c t i o n between the development and i m p l e m e n t a t i o n o f immunoassays. Hapten s y n t h e s i s . The g e n e r a l s y n t h e t i c approach chosen w i l l be d i c t a t e d by the d e s i r e d a s s a y s p e c i f i c i t y . Compound o r c l a s s s p e c i f i c a n t i b o d i e s can be produced depending on w h i c h p a r t o f the molecule i s used f o r c o n j u g a t i o n . Antibody s p e c i f i c i t y i s g e n e r a l l y h i g h e s t f o r the p a r t o f the m o l e c u l e f u r t h e s t from the c a r r i e r . The importance o f c a r e f u l l y p l a n n e d and r a t i o n a l h a p t e n s y n t h e s i s cannot be o v e r e m p h a s i z e d . The chosen s y n t h e t i c r o u t e s h o u l d p r e s e r v e as much s t r u c t u r e as p o s s i b l e o f the m o l e c u l e t o be a n a l y z e d and must p r o v i d e a 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 t o a c a r r i e r m o l e c u l e (usually protein). The optimum h a p t e n s t r u c t u r e f o r a n t i b o d y p r o d u c t i o n may however l a c k an a p p a r e n t l y e s s e n t i a l m o i e t y and t h i s i s a t r i c k y a r e a f o r the non-immunochemist t o p r e d i c t . F o r example, p r e s e r v a t i o n o f the n i t r o group o f p a r a t h i o n by c o n j u g a t i o n t h r o u g h the n o r m a l l y u n s u b s t i t u t e d a r o m a t i c r i n g p o s i t i o n s l e d t o the p r o d u c t i o n o f a n t i s e r a which d i d not recognize free p a r a t h i o n (31), w h i l e p a r a t h i o n s p e c i f i c a n t i s e r a were p r o d u c e d a g a i n s t c o n j u g a t e s o f a m i n o p a r a t h i o n ( 1 0 ) . C o n j u g a t i o n p o s i t i o n was a l s o i m p o r t a n t i n the p r o d u c t i o n o f a n t i b o d i e s t o m a l e i c h y d r a z i d e ( 1 5 ; a l s o H a r r i s o n , R . O . ; B r i m f i e l d , A . A . ; H u n t e r , K . W . , J r . ; N e l s o n , J . O . J . A g r i c . Food Chem. s u b m i t t e d ) . Immunization w i t h N-conjugates o f m a l e i c h y d r a z i d e l e d t o m a l e i c h y d r a z i d e s p e c i f i c and h a p t e n s p e c i f i c a n t i b o d i e s , w h i l e i m m u n i z a t i o n w i t h O-conjugates o f m a l e i c h y d r a z i d e



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l e d to only hapten s p e c i f i c a n t i b o d i e s . There i s e x p e r i m e n t a l e v i d e n c e t h a t haptens a r e more immunogenic when s e p a r a t e d from t h e c a r r i e r b y a s p a c e r arm. P r e s e n t a t i o n o f t h e h a p t e n t o r e c e p t o r s on c e l l s o f t h e immune system i s l e s s l i k e l y t o be s t e r i c a l l y h i n d e r e d i f t h e h a p t e n i s on a s p a c e r arm s e v e r a l atoms l o n g . The p r o d u c t i o n o f haptens f o r c o n j u g a t i o n i s t h e l a s t p o i n t i n the a s s a y development p r o c e s s where r i g o r o u s s t r u c t u r a l a n a l y s i s i s possible. Because the a s s a y s p e c i f i c i t y i s d e t e r m i n e d b y t h e s t r u c t u r e ( s ) o f the c o n j u g a t e d h a p t e n s , i t i s c r i t i c a l l y i m p o r t a n t f o r t h e s e compounds t o be pure and s t r u c t u r a l l y w e l l c h a r a c t e r i z e d before conjugation renders t h i s impossible. Conjugation to c a r r i e r s . A wide range o f p r o v e n methods a r e a v a i l a b l e f o r c o n j u g a t i o n o f haptens t o t h e i r c a r r i e r s , most o f them u s i n g common c o m m e r c i a l l y a v a i l a b l e r e a g e n t s . These have been summarized p r e v i o u s l y (11,14) and many examples o f t h e i r use i n p e s t i c i d e immunoassay development e x i s t ( 3 . 1 0 . 1 2 . 1 5 . 1 9 . 3 2 . 3 5 . 3 6 . 3 8 ) . V e r i f i c a t i o n o f conjugation. As n o t e d above, s t r u c t u r a l a n a l y s i s i s d i f f i c u l t or impossible after conjugation, but i t i s p o s s i b l e to v e r i f y c o n j u g a t i o n even though the s t r u c t u r e o f t h e c o n j u g a t e d m o l e c u l e s i s unknown. I f t h e h a p t e n has an a p p r o p r i a t e UV a b s o r p t i o n spectrum, d i f f e r e n c e s p e c t r a between c o n j u g a t e s and o r i g i n a l c a r r i e r s c a n be u s e d f o r b o t h q u a l i t a t i v e d e m o n s t r a t i o n o f c o n j u g a t i o n and e s t i m a t i o n o f the m o l a r r a t i o o f h a p t e n t o c a r r i e r , o f t e n c a l l e d the hapten d e n s i t y . T h i s method has been a p p l i e d t o b o t h UV a b s o r b i n g p a r e n t compounds (15) and haptens c o n t a i n i n g a UV a b s o r b i n g s p a c e r group (3.) . A l t e r n a t i v e methods i n c l u d e q u a n t i t a t i o n o f f r e e p r i m a r y amino groups and c o n j u g a t i o n s u s i n g r a d i o l a b e l l e d haptens. A n t i b o d y p r o d u c t i o n . A n t i b o d i e s used f o r immunoassay c a n be e i t h e r p o l y c l o n a l , c o n t a i n e d i n serum from r a b b i t s o r o t h e r a n i m a l s , o r m o n o c l o n a l , produced by mouse lymphocytes i m m o r t a l i z e d f o r i n v i t r o c u l t u r e ( c a l l e d hybridomas) . The advantages o f each have o n l y r e c e n t l y begun t o be examined s y s t e m a t i c a l l y i n t h e c o n t e x t o f q u a n t i t a t i v e h a p t e n a n a l y s i s ( 2 ) . B o t h m o n o c l o n a l ( 3 , 1 5 ) and p o l y c l o n a l ( 5 . 1 0 . 1 2 . 1 9 . 3 2 . 3 5 - 3 8 ) a n t i b o d i e s have p r o v e n e f f e c t i v e f o r p e s t i c i d e a n a l y s i s , a l t h o u g h most r e p o r t e d immunoassays have used r a b b i t s e r a . P r o d u c t i o n o f monoclonal a n t i b o d i e s i s l a b o r i n t e n s i v e and may n o t w a r r a n t t h e e x t r a e f f o r t f o r t h e p l a n n e d application. F o r s t a n d a r d i z e d methods w h i c h w i l l be w i d e l y u s e d , m o n o c l o n a l a n t i b o d i e s may be s u p e r i o r t o p o l y c l o n a l s e r a because o f t h e i r b i o c h e m i c a l u n i f o r m i t y and d e f i n e d a f f i n i t y and s p e c i f i c i t y . S t a b l e hybridoma c e l l l i n e s p r o v i d e a c o n t i n u o u s s o u r c e o f t h e i r m o n o c l o n a l a n t i b o d i e s as l o n g as they c a n be m a i n t a i n e d i n c u l t u r e . M o n o c l o n a l a n t i b o d i e s w i l l a l s o be u s e f u l i n t h e d i s c r i m i n a t i o n o f r e l a t e d m u l t i v a l e n t a n t i g e n s such as t h o s e produced b y r e l a t e d s t r a i n s such as B a c i l l u s t h u r i n g i e n s i s i s r a e l e n s i s and B a c i l l u s t h u r i n g i e n s i s k u r s t a k i o r g e n e t i c a l l y e n g i n e e r e d o r g a n i s m s . The s p e c i f i c i t y o f monoclonal a n t i b o d i e s f o r s i n g l e p r o t e i n determinants w i l l l i k e l y a l s o prove u s e f u l f o r d i s t i n g u i s h i n g a c t i v e and i n a c t i v e forms o f p r o t e i n t o x i n s , such as from B a c i l l u s t h u r i n g i e n s i s .



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Antibody c h a r a c t e r i z a t i o n . A u s e f u l d e s c r i p t i o n o f some i m p o r t a n t c o n s i d e r a t i o n s i n a n t i b o d y c h a r a c t e r i z a t i o n has been g i v e n by Hammock and Mumma ( 1 4 ) . S p e c i f i c i t y i s g e n e r a l l y t e s t e d u s i n g some form o f c o m p e t i t i v e b i n d i n g , such as a c o m p e t i t i v e ELISA. An e s t i m a t i o n o f the average a f f i n i t y c o n s t a n t may be u s e f u l i n the c o m p a r i s o n o f d i f f e r e n t a n t i b o d i e s and i n l a t e r o p t i m i z a t i o n o f the a s s a y . T h i s v a l u e can be c o n v e n i e n t l y e s t i m a t e d from a n t i s e r u m d i l u t i o n c u r v e s (27) f o r s e l e c t i o n o f the b e s t a n t i b o d i e s i n a screening process. T h i s r e q u i r e s the c h o i c e o f an a s s a y format (see l a t e r s e c t i o n on a s s a y f o r m a t s ) , such as ELISA, p r e f e r a b l y u s i n g a d i f f e r e n t c a r r i e r to e l i m i n a t e c a r r i e r c r o s s r e a c t i v i t y . It is i m p o r t a n t t h a t s p e c i f i c i t y and s e n s i t i v i t y be e v a l u a t e d a t t h i s p o i n t and be deemed a c c e p t a b l e b e f o r e i n v e s t i n g f u r t h e r r e s o u r c e s i n a s s a y s based on i n a d e q u a t e a n t i b o d i e s . A s s a y o p t i m i z a t i o n . An o p t i m i z a t i o n s t e p n o t always t a k e n , b u t n o n e t h e l e s s i m p o r t a n t t o the s u c c e s s o f any immunochemical method o f a n a l y s i s i s the s e l e c t i o n o f m a t e r i a l s , such as t e s t tube o r p l a s t i c p l a t e s , w h i c h maximize a s s a y performance. An example o f t h i s i s the s e l e c t i o n of 96-well m i c r o t i t e r p l a t e s for enzyme-linked immunosorbent a s s a y (ELISA) w h i c h g i v e maximum p r o t e i n b i n d i n g c a p a c i t y and minimum i n t e r w e l l v a r i a b i l i t y ( 2 8 , 3 0 ; a l s o H a r r i s o n , R . O . ; N e l s o n , J . O . J . Immunoassay, s u b m i t t e d ) . The type o f m i c r o t i t e r p l a t e may be the most i m p o r t a n t s i n g l e d e t e r m i n a n t o f ELISA performance and t h i s s e l e c t i o n s h o u l d n o t be made c a r e l e s s l y . S i g n i f i c a n t e r r o r may a l s o o c c u r i n the r e a d i n g o f a s s a y s performed i n 9 6 - w e l l m i c r o t i t e r p l a t e s , due t o a l i g n m e n t e r r o r s o f a u t o m a t i c p l a t e r e a d e r s u n d e t e c t e d i n normal use ( H a r r i s o n , R . O . ; N e l s o n , J . O . J . Immunoassay, s u b m i t t e d ; H a r r i s o n , R . O . ; Hammock, B . D . J . A s s o c . O f f . A n a l . Chem.. s u b m i t t e d ) , and a p l a t e r e a d e r t e s t s h o u l d a l l o w further reduction of error. These s t e p s s h o u l d be t a k e n b e f o r e a major i n v e s t m e n t i n t i m e , e f f o r t , o r money i s made i n an a s s a y system w h i c h may l a t e r be found t o be l e s s t h a n a c c e p t a b l e . Once d e v e l o p e d , any a s s a y system s h o u l d be o p t i m i z e d t o p r o v i d e the b e s t p o s s i b l e s e n s i t i v i t y . One i m p o r t a n t v a r i a b l e a f f e c t i n g ELISA performance i s the s e l e c t i o n o f c o a t i n g a n t i g e n s . Wie and Hammock (36) showed t h a t p r u d e n t c h o i c e o f c o a t i n g a n t i g e n s c a n s i g n i f i c a n t l y improve s e n s i t i v i t y . A u s e f u l e m p i r i c a l approach t o o p t i m i z a t i o n i s d e s c r i b e d by Hunter and Bosworth ( 1 7 ) , c o n s i s t i n g m a i n l y o f i d e n t i f y i n g r e a g e n t c o n c e n t r a t i o n s w h i c h p r o v i d e the h i g h e s t s i g n a l t o n o i s e r a t i o below b i n d i n g s a t u r a t i o n . A s s a y a p p l i c a t i o n . A t t h i s p o i n t major d i f f e r e n c e s appear between the h i s t o r i c a l use o f c l i n i c a l immunoassays and the p o t e n t i a l a p p l i c a t i o n s o f e n v i r o n m e n t a l and p e s t i c i d e immunoassays. Most c l i n i c a l a s s a y s have been a p p l i e d t o s i m p l e o r w e l l d e f i n e d and c o n s i s t e n t m a t r i c e s such as u r i n e o r serum. I n c o n t r a s t , most m a t r i c e s l i k e l y t o be a n a l y z e d f o r p e s t i c i d e s are more complex, l e s s w e l l d e f i n e d , and more v a r i a b l e . The p o t e n t i a l f o r s e r i o u s problems w i t h m a t r i x e f f e c t s i n the e n v i r o n m e n t a l f i e l d i s f a r g r e a t e r t h a n most c l i n i c a l immunoassays have e n c o u n t e r e d . The a p p l i c a t i o n o f immunoassays t o e n v i r o n m e n t a l a n a l y s i s r e q u i r e s s a m p l i n g s t r a t e g i e s , c l e a n u p p r o c e d u r e s , and d a t a h a n d l i n g f u n d a m e n t a l l y s i m i l a r t o those p r e s e n t l y i n use i n any good a n a l y t i c a l l a b . The c r i t i c a l f a c t o r i n the s u c c e s s o f immunochemical t e c h n o l o g y w i l l l i k e l y be competence



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i n a n a l y t i c a l and r e s i d u e c h e m i s t r y r a t h e r t h a n immunochemical expertise. There a r e s e v e r a l c o n s i d e r a t i o n s u n i q u e t o immunoassay, s u c h as t h e t o l e r a n c e o f a n t i b o d i e s f o r o r g a n i c s o l v e n t s o r v a r i a t i o n s i n pH o r i o n i c s t r e n g t h , and s t a b i l i t y o f b i o l o g i c a l reagents i n storage. Sample p r e s e n t a t i o n t o t h e r e a g e n t a n t i b o d y i s an i m p o r t a n t c o n s i d e r a t i o n . Immunoassay o f l i p o p h i l i c m a t e r i a l s i n a l i p o p h i l i c environment i s n o t l i k e l y t o suceed w i t h o u t some means o f p r e s e n t i n g the l i p o p h i l i c a n a l y t e to the h y d r o p h i l i c antibody. T h i s c a n be done i n most cases u s i n g w a t e r m i s c i b l e o r g a n i c c o s o l v e n t s o r detergents t o induce m i c e l l e f o r m a t i o n . Additionally, the e f f e c t o f t h e m a t r i x b e i n g a n a l y z e d on t h e a n t i b o d y u s e d f o r a n a l y s i s must be e v a l u a t e d . I f the m a t r i x e f f e c t s a r e m i n i m a l w i t h l i t t l e o r no sample c l e a n u p , t h e n a n a l y s i s c a n be done d i r e c t l y . I n a d d i t i o n t o the a n a l y s i s o f molinate i n r i c e f i e l d water (Figures 2 & 3 ) , immunoassays f o r s e v e r a l o t h e r compounds have been r e p o r t e d w h i c h r e q u i r e l i t t l e o r no sample c l e a n u p , i n c l u d i n g p a r a t h i o n ( 1 0 ) , c h l o r s u l f u r o n (19) , p a r a q u a t (32.) , and m a l e i c h y d r a z i d e ( 1 5 ) . The sample workup n e c e s s a r 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 w i l l v a r y w i t h each c o m b i n a t i o n o f a n a l y t e and a n t i b o d y , each o f w h i c h may have a d i f f e r e n t t o l e r a n c e f o r t h e m a t r i x and o t h e r factors. The e f f e c t s o f t h e s e f a c t o r s must be c o n s i d e r e d as w i t h the development o f any o t h e r a n a l y t i c a l t e c h n i q u e . Matrix effects f o r one ELISA system a r e summarized i n F i g u r e 4 . W h i l e t h e e f f e c t o f t h e m a t r i x on t h e a n t i b o d i e s i n F i g u r e 4 i s d i f f e r e n t f o r each a n t i b o d y - s o l v e n t - m a t r i x c o m b i n a t i o n , t h e c o m p e t i t i v e ELISA s t a n d a r d c u r v e s f o r most o f t h e s e c o m b i n a t i o n s a r e s i m i l a r when e x p r e s s e d as p e r c e n t o f t h e a p p r o p r i a t e c o n t r o l . Some systems may n o t r e q u i r e e x t e n s i v e adjustment, b u t t h i s must be t e s t e d w i t h each i n d i v i d u a l system. F o r example, o u r m o l i n a t e a s s a y performs e q u a l l y w e l l i n a v a r i e t y o f water types a t h i g h concentrations o f molinate (Figure 5). The s m a l l d i f f e r e n c e seen between t h e b u f f e r and w a t e r s t a n d a r d c u r v e s i n F i g u r e 5 was e l i m i n a t e d by t h e a d d i t i o n o f s m a l l amounts o f c o n c e n t r a t e d b u f f e r t o w a t e r samples t o e q u a l i z e them t o t h e buffer composition. A s s a y v a l i d a t i o n . The v a l i d a t i o n p r o c e d u r e c o n s i s t s p r i m a r i l y o f c o m p a r i s o n o f the immunoassay method t o an e x i s t i n g method and v e r i f i c a t i o n o f t h e s t a t i s t i c a l r e l i a b i l i t y o f t h e new method according to w e l l established p r i n c i p l e s i n a n a l y t i c a l chemistry. A g e n e r a l approach t o t h e p r o b l e m o f v a l i d a t i o n has been summarized i n a u s e f u l form b y H o r w i t z ( 1 6 ) . P a r t i c i p a t i o n o f groups such as t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists (A0AC) and t h e U . S . E n v i r o n m e n t a l P r o t e c t i o n Agency (EPA) i n t h e o f f i c i a l v a l i d a t i o n process should lead to w e l l defined v a l i d a t i o n protocols s u i t a b l e f o r g e n e r a l immunoassay u s e . These p r o t o c o l s must d e a l r e a l i s t i c a l l y w i t h m a t r i x e f f e c t s such as t h o s e shown i n F i g u r e 4 . D e m o n s t r a t i o n o f s t a n d a r d c u r v e s i n t h e m a t r i x o f i n t e r e s t ( s u c h as shown i n F i g u r e 5) i s i m p o r t a n t , b u t i s n o t s u f f i c i e n t t o p r e d i c t s u c c e s s o f t h e method. B i o t r a n s f o r m a t i o n and v a r i a b i l i t y o f m a t r i x e f f e c t s among samples must a l s o be e v a l u a t e d b y t h e a n a l y s i s o f f i e l d t r e a t e d samples.


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F i g u r e 4 . M a t r i x e f f e c t s i n ELISA o f aqueous and m e t h a n o l i c e x t r a c t s o f p o t a t o and t o b a c c o u s i n g p u r i f i e d a n t i - m a l e i c h y d r a z i d e m o n o c l o n a l a n t i b o d i e s ( m o d i f i e d from r e f . 1 5 ) .

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Assay

formats

Haptens and a n t i - h a p t e n a n t i b o d i e s a r e r e a g e n t s w h i c h c a n be u s e d i n a wide v a r i e t y o f a s s a y f o r m a t s . One fundamental d i v i s i o n among immunoassay formats i s b a s e d on the type o f l a b e l o r t r a c e r w h i c h i s u l t i m a t e l y detected. Two o f the most common t y p e s o f l a b e l a r e r a d i o i s o t o p e s and enzymes, u s e d r e s p e c t i v e l y i n radioimmunoassay (RIA) and enzyme immunoassay ( E I A ) . RIA i s the o l d e r o f the two and remains v e r y i m p o r t a n t and w i d e l y u s e d i n c l i n i c a l and r e s e a r c h situations. However, the use o f r a d i o i s o t o p e s c a r r i e s w i t h i t p o s s i b l e h e a l t h h a z a r d s , the need f o r s p e c i a l h a n d l i n g p r e c a u t i o n s , and mandatory r e g u l a t o r y o v e r s i g h t . Because EIA a v o i d s t h e s e p r o b l e m s , i t i s g a i n i n g p o p u l a r i t y r a p i d l y among u s e r s o f immuno as s a y s . The format p r e s e n t l y f a v o r e d f o r p e s t i c i d e immunoassay i s the c o m p e t i t i v e ELISA o r e n z y m e - l i n k e d immunosorbent a s s a y . This i s a heterogeneous a s s a y , w h i c h i s based upon c o m p e t i t i o n between an unknown and v a r i a b l e amount o f s o l u b l e a n a l y t e ( t h e sample) and a s m a l l f i x e d amount o f the a n a l y t e i m m o b i l i z e d on the s o l i d phase, f o r b i n d i n g a s m a l l amount o f s o l u b l e a n t i b o d y . The c o n c e n t r a t i o n o f a n a l y t e i n the sample i s i n d i r e c t l y measured by the q u a n t i t a t i o n o f bound a n t i b o d y a f t e r i t i s s e p a r a t e d from the f r e e a n t i b o d y . In c o n t r a s t , homogeneous a s s a y s r e q u i r e no s e p a r a t i o n s t e p and so can be performed i n a s i n g l e s t e p i n one t u b e . An example o f t h i s as a p p l i e d t o p e s t i c i d e s i s the Emit a n a l y s i s o f d i e l d r i n and 2 , 4 - D (12). B o t h heterogeneous and homogeneous a s s a y systems have been u s e d e x t e n s i v e l y i n the c l i n i c a l f i e l d ( 2 4 , 2 5 ) . Useful comprehensive r e v i e w s o f the v a r i o u s a s s a y formats have been p u b l i s h e d ( 2 1 , 2 4 ) . The c h o i c e between t h e s e two major c l a s s e s o f a s s a y format i s i m p o r t a n t f o r s e v e r a l r e a s o n s . Homogeneous formats a r e g e n e r a l l y l e s s s e n s i t i v e , b u t have fewer s t e p s and a r e e a s i e r , thus l e n d i n g themselves t o f i e l d u s e . Heterogeneous a s s a y s a r e becoming more v e r s a t i l e w i t h the development o f many d i f f e r e n t c h o i c e s o f equipment. There a r e many d i f f e r e n t format and equipment p o s s i b i l i t i e s f o r ELISA a l o n e and s e l e c t i o n from among t h e s e depends on the needs and budget o f the u s e r . T e s t s c a n be performed i n s i n g l e tubes o r i n 96 w e l l m i c r o t i t e r p l a t e s u s i n g a v a r i e t y o f manual o r s e m i a u t o m a t i c s i n g l e - t i p o r m u l t i c h a n n e l p i p e t t o r s . Manual o r a u t o m a t i c m i c r o t i t e r p l a t e r e a d e r s may be u s e d o r f u l l y automated systems c a n be s e t up w h i c h c a n p r o c e s s thousands o f samples p e r day. A v a r i e t y o f equipment i s a v a i l a b l e f o r any d e s i r e d degree o f a u t o m a t i o n . Assay implementation The l i s t above i s n o t i n t e n d e d t o be comprehensive, b u t i t w i l l s e r v e as a g e n e r a l g u i d e t o immunoassay development. We must emphasize a g a i n the d i f f e r e n c e between development and i m p l e m e n t a t i o n . A s s a y s w h i c h work i n the l a b o r a t o r y g e n e r a l l y r e q u i r e m o d i f i c a t i o n b e f o r e t h e y can be u s e d t o a n a l y z e f i e l d samples. I m p l e m e n t a t i o n o f a s s a y s t h a t have been f u l l y v a l i d a t e d f o r f i e l d samples may r e q u i r e l i t t l e a d d i t i o n a l commitment by the u s e r , o t h e r t h a n a n a l y s t t r a i n i n g . F o r the n e a r f u t u r e , t h e r e may be c o n s i d e r a b l e p r e s s u r e t o t r a n s f e r immunoassay methods t o the a n a l y t i c a l l a b as soon as p o s s i b l e a f t e r development, and the


24.

HARRISON E T A L .


327

a n a l y t i c a l c h e m i s t may need t o do a d d i t i o n a l work t o adapt and f u l l y v a l i d a t e many a s s a y s . T h i s w i l l demand t h a t the a n a l y t i c a l c h e m i s t d e v e l o p some immunochemical e x p e r t i s e . Many o f the i m p l e m e n t a t i o n problems l i k e l y t o be e n c o u n t e r e d w i l l b e s t be s o l v e d w i t h t r a d i t i o n a l a n a l y t i c a l c h e m i s t r y e x p e r t i s e tempered by an u n d e r s t a n d i n g o f p r a c t i c a l immunochemistry.


P r e s e n t Use The use 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 has now advanced beyond the p o t e n t i a l s t a g e . Some groups a r e now u s i n g ELISA f o r r e a l r e s i d u e d a t a . Dupont has s u b m i t t e d c y a n a z i n e ( B l a d e x ) ELISA d a t a t o EPA f o r p r o d u c t r e g i s t r a t i o n (Sharp, J . , Dupont, p e r s o n a l communication, 1987) and 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 i s i n i t i a t i n g a program o f m o n i t o r i n g r i c e paddy w a t e r f o r m o l i n a t e and t h i o b e n c a r b by ELISA. Some c o m m e r c i a l k i t s a r e a v a i l a b l e now, i n c l u d i n g ELISA t e s t s f o r a t r a z i n e , s i m a z i n e , and p r o p a z i n e , and f o r c a r b o f u r a n , s o l d by ImmunoSysterns, I n c . f o r $15 p e r t e s t i n a package o f t e n t e s t s . These k i t s o f f e r c o s t and s e n s i t i v i t y c o m p e t i t i v e w i t h o t h e r methods and c a n be u s e d q u a n t i t a t i v e l y i n the f i e l d w i t h a p o r t a b l e spectrophotometer. Other companies have immunoassay k i t s i n v a r i o u s s t a g e s o f development ( 2 0 , 2 3 ) . S e v e r a l r e c e n t e v e n t s i n d i c a t e the i n t e n s e i n t e r e s t i n immunoassay f o r a n a l y s i s o f p e s t i c i d e s and o t h e r e n v i r o n m e n t a l contaminants. S p e c i a l symposia on immunochemical t e c h n o l o g y have been o r a r e b e i n g p r e s e n t e d by the A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists a t t h e i r 1986, 1987, and 1988 a n n u a l meetings and by the I n t e r n a t i o n a l U n i o n o f Pure and A p p l i e d C h e m i s t r y a t the q u a d r e n n i a l P e s t i c i d e C h e m i s t r y Congress i n Ottawa i n 1986 ( 1 3 , 2 3 ) . S e v e r a l government a g e n c i e s have r e c e n t l y s o l i c i t e d p r o p o s a l s f o r development o f immunoassay methods f o r p r o d u c t o r e n v i r o n m e n t a l a n a l y s i s , i n c l u d i n g the U . S . Food and Drug A d m i n i s t r a t i o n ( 8 ) , the Food S a f e t y and I n s p e c t i o n S e r v i c e o f the U . S . Department o f A g r i c u l t u r e ( 7 ) , and the U . S . Army ( 6 ) . The U . S . E n v i r o n m e n t a l P r o t e c t i o n Agency i s p e r f o r m i n g a v a l i d a t i o n s t u d y o f an immunoassay method f o r a n a l y s i s o f p e n t a c h l o r o p h e n o l i n w a t e r , d e v e l o p e d by Westinghouse B i o a n a l y t i c Systems o f R o c k v i l l e , MD ( 1 8 ) . F u t u r e use One e s t i m a t e o f the s i z e o f the market f o r n o n - t r a d i t i o n a l immunoassays i s $24 m i l l i o n f o r p l a n t d i a g n o s t i c s , $180 m i l l i o n f o r hazardous c h e m i c a l s , and $126 m i l l i o n f o r f o o d t e s t i n g by the y e a r 2000, t o t a l i n g $330 m i l l i o n ( 2 0 ) . F o r c e r t a i n p e s t i c i d e s and hazardous c h e m i c a l s , the market f o r a n a l y s i s may be l a r g e r t h a n the market f o r u s e . A s s a y s f o r new compounds w i l l c o n t i n u e t o be developed, e s p e c i a l l y i n c l u d i n g p e s t i c i d e s which are d i f f i c u l t to a n a l y z e b y t r a d i t i o n a l methods. Other e n v i r o n m e n t a l contaminants and b i o t e c h n o l o g y p r o d u c t s , i n c l u d i n g g e n e t i c a l l y e n g i n e e r e d p e s t i c i d e s , w i l l a l s o be e x c e l l e n t c a n d i d a t e s f o r immunoassay. We e x p e c t t h a t even i f l i m i t e d t o o n l y the p r e s e n t l e v e l o f t e c h n o l o g y , immunoassays i n some form w i l l soon have a l a r g e impact on the a n a l y s i s o f p e s t i c i d e s , hazardous c h e m i c a l s , i n d u s t r i a l b y - p r o d u c t s , and n a t u r a l t o x i n s i n p r o d u c t s and the e n v i r o n m e n t . Novel assay



328


formats w i l l be developed which offer greater opportunity for f i e l d testing, including rapid or instantaneous procedures, such as described by Stanbro et a l . (29)• Such new technology has great potential for development of multianalyte methods by merely combining multiple antibodies and haptens on a single probe. The use of monoclonal antibodies for these and other immunoassay applications w i l l increase because of specificity, regulatory, patent, or economic considerations. Increasing automation of the entire immunoassay process w i l l occur even i n the absence of radically new technology, due to the increasing availability and sophistication of laboratory robots and dedicated automatic immunoassay aids. Acceptance of immunochemical technology for pesticide residue analysis w i l l become more widespread as more commercial immunoassay products reach the market and more programs u t i l i z i n g immunochemical technology begin producing tangible results. Increasing realization of the need for high sample loads for thorough environmental analysis w i l l increase the demand for low cost methods of analysis. Official validation of immunoassay methods w i l l accelerate as analytical chemists become familiar with immunoassay and gain experience with these methods in the validation process. This new experience w i l l undoubtedly be accompanied by better control of assay sources of error and better instrumentation specifically designed for quantitative immunoassay. Greater scientific understanding of underlying principles w i l l be gained as a direct result of the need to deal with matrix effects i n the analysis of complex samples and this understanding w i l l facilitate the application of immunochemical technology to new analytical problems. A l l immunoassays, no matter how novel, w i l l continue to depend on the production of specific antibodies as decribed above. This process i n turn depends upon rational antigen synthesis, from hapten design and synthesis through conjugation, to novel methods of antibody production, as outlined i n this chapter. Regardless of the exact configuration of the final immunoassay, the principles outlined above w i l l continue to be important for the forseeable future. Acknowledgments This work was supported i n part by NIEHS Superfund Grant PHS ES04699-01, EPA Cooperative Agreement CR-814709-01-0, and a grant from the California Department of Food and Agriculture. BDH is a Burroughs Wellcome Scholar i n Toxicology. Literature Cited 1. 2. 3. 4. 5.

Beyer, W.H., Ed. CRC Handbook of Probability and Statistics CRC Press: Cleveland, 1968; pp 219-237. Bjercke, R.J.; Cook, G.; Langone, J.J. J . Immunol. Meth. 1987, 96, 239-246. Brimfield, A.A.; Lenz, D.E.; Graham, C.; Hunter, K.W.,Jr. J. Agric. Food Chem., 1985, 33, 1237-1242. Butler, V.P.,Jr.; Beiser, S.M. Adv. Immunol. 1973, 17, 255-310. Cheung, P.Y.K.; Hammock, B.D. Curr. Microbiol. 1985, 12, 121126.


24. HARRISON ETAL.

6. 7. 8. 9.


10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31.


329

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32. Van Emon, J.; Hammock, B.D.; Seiber, J.N. Anal.Chem. 1986, 58, 1866-1873. 33. Van Emon, J.M.; Seiber, J.N.; Hammock, B.D. In Bioregulators for Pest Control: Hedin, P., Ed.; ACS Symposium Series No. 276; American Chemical Society: Washington, DC, 1985; pp 307-316. 34. van Vuurde, J.W.L.; Maat, D.Z.; Franken, A.A.J.M., Chapter 26, In Biotechnology for Crop Protection; Hedin, P.A., Menn, J.J., Hollingworth, R.M., Eds.; ACS Symposium Series No. 379; American Chemical Society: Washington, DC, 1988. 35. Wie, S.I.; Hammock, B.D. J. Agric. Food Chem. 1982, 30, 949-957. 36. Wie, S.I.; Hammock, B.D. J. Agric. Food Chem. 1984, 32, 12941301. 37. Wie, S.I.; Hammock, B.D.; Gill, S.S.; Grate, E.; Andrews, R.E.; Faust, R.M.; Bulla, L.A.; Schaefer, C.H. J. Appl. Bacteriol. 1984, 57, 447-454. 38. Wing, K.D.; Hammock, B.D.; Wustner, D.A. J. Agric. Food Chem. 1978, 26, 1328-1333. RECEIVED April 1, 1988


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