Immunoassays for Crop Management Systems and Agricultural

Chapter 19 Immunoassays for C r o p Management Systems and Agricultural Chemistry Richard K. Lankow, G. David Grothaus, and Sally A. Miller Downloaded by NORTH CAROLINA STATE UNIV on December 31, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch019

Agri-Diagnostics Associates, 2611 Branch Pike, Cinnaminson, NJ 08077 Immunoassays are analytical procedures based upon the specific binding of animal-derived antibodies to a target molecule. When coupled with a visualization method, immunoassays provide simple, specific and sensitive detection and quantification of a very broad range of analytes. Many medical diagnostic procedures and products, including many home diagnostics, are based upon antibody reagents. Agricultural analyses are a natural extension of this technology. Targets include plant pathogens, pests, pesticides, crop components, nutrient markers, and genetic markers. Immunoassays will be applied as informational tools at many levels of crop production to guide management of crop inputs. Rapid analysis of pesticide residues, early detection of plant diseases, and quantification of pest levels will provide information useful in determining crop rotation patterns, variety selection, pesticide selection, pesticide application timing, harvest dates, post harvest handling and many other management aspects. O f t h e many d i s c i p l i n e s w i t h i n " b i o t e c h n o l o g y " , t h e f i e l d o f immunochemistry has had t h e e a r l i e s t commercial impact p r i m a r i l y t h r o u g h t h e d e v e l o p m e n t o f d i a g n o s t i c p r o d u c t s f o r t h e human h e a l t h c a r e i n d u s t r y . H i g h l y e f f e c t i v e immunoassays have become powerful t o o l s i n t h e a n a l y s i s o f such d i v e r s e m a t e r i a l s as t h e r a p e u t i c drugs, drugs o f abuse, m i c r o o r g a n i s m s , tumor markers, and h o r m o n e s . S u c h a s s a y s a r e r o u t i n e l y e m p l o y e d i n c l i n i c a l l a b o r a t o r i e s a n d , i n r e c e n t y e a r s , have been moved i n t o d o c t o r s o f f i c e s a n d t h e home. T h e s i m p l i f i c a t i o n o f t e s t f o r m a t s , a v a i l a b i l i t y o f e l e c t r o n i c i n s t r u m e n t a t i o n , and t h e development o f monoclonal a n t i b o d y techniques have s t i m u l a t e d r a p i d growth i n t h e m e d i c a l d i a g n o s t i c s i n d u s t r y o v e r t h e p a s t 10 y e a r s . Immunoassays a r e based on t h e a b i l i t y o f b i o l o g i c a l molecules, antibodies, t o bind very s p e c i f i c a l l y with other m o l e c u l e s . When t h e b i n d i n g i s m e a s u r e d o r v i s u a l i z e d i n some w a y , i t becomes t h e b a s i s f o r a s i m p l e , s p e c i f i c and s e n s i t i v e assay. A n t i b o d i e s d i s p l a y remarkable s p e c i f i c i t y and bind o n l y t o a 1

0097-6156/87/0334-0228$07.25/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Downloaded by NORTH CAROLINA STATE UNIV on December 31, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch019

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narrow range o f t a r g e t m o l e c u l a r s t r u c t u r e s and c o n f i g u r a t i o n s . V e r t e b r a t e immune s y s t e m s a r e e q u a l l y r e m a r k a b l e i n t h a t t h e y c a n produce s p e c i f i c a n t i b o d i e s w h i c h r e c o g n i z e and b i n d t o a v i r t u a l l y u n l i m i t e d range o f molecular s t r u c t u r e s . T h e s e t w o c h a r a c t e r i s t i c s o f immune s y s t e m s - ( 1 ) t h e a b i l i t y t o p r o d u c e a n t i b o d i e s t o an a l m o s t u n l i m i t e d r a n g e o f t a r g e t s and (2) t h e a b i l i t y o f a n t i b o d i e s t o r e c o g n i z e and b i n d t i g h t l y t o a s p e c i f i c molecular c o n f i g u r a t i o n - permit the development of unique a n a l y t i c a l procedures. Immunoassays d i f f e r from o t h e r a n a l y t i c a l methods i n t h a t s p e c i f i c i t y i s c o n f e r r e d by t h e reagents employed r a t h e r t h a n b y an a p p a r a t u s o f some t y p e . S e n s i t i v i t y i s c o n f e r r e d by t h e s t r e n g t h o f t h e a n t i b o d y : a n t i g e n i n t e r a c t i o n and b y t h e v i s u a l i z a t i o n method employed t o d e t e c t t h e b i n d i n g . A g r i c u l t u r a l r e s e a r c h and d e v e l o p m e n t and c r o p management r e q u i r e v a s t amounts o f a n a l y t i c a l d a t a t h a t p r o v i d e i n f o r m a t i o n upon w h i c h k e y d e c i s i o n s a r e b a s e d . T o x i c i t y v a l u e s , c h e m i c a l r e s i d u e l e v e l s , n u t r i e n t l e v e l s , and i n s e c t counts a r e representative of the diverse array of analytical information that i s d i r e c t l y u t i l i z e d by modern a g r i c u l t u r e . D e s p i t e t h e i r wide use i n m e d i c i n e , immunoassays have been u t i l i z e d i n crop a g r i c u l t u r e p r i m a r i l y as r e s e a r c h t o o l s i n a l i m i t e d number o f l a b o r a t o r i e s . R e l a t i v e l y f e w c o m m e r c i a l p r o d u c t s b a s e d on i m m u n o r e a g e n t s a r e intended f o r detection of a g r i c u l t u r a l chemicals or other targets o f a g r i c u l t u r a l i n t e r e s t . H o w e v e r , m a n y o f t h e same f a c t o r s t h a t f a v o r e d t h e d e v e l o p m e n t and w i d e s p r e a d use o f immunoassays i n m e d i c i n e o v e r t h e p a s t 2 0 y e a r s a r e now a t p l a y i n t h e a g r i c u l t u r a l arena. S u c h a s s a y s c a n be e x p e c t e d t o p l a y an i n c r e a s i n g r o l e i n a l l a s p e c t s o f c r o p management o v e r t h e n e x t 10 y e a r s . The a p p l i c a b i l i t y o f immunoreagents t o t h e r a p i d and c o s t - e f f e c t i v e d e t e c t i o n of a broad range o f t a r g e t s i s very a p p e a l i n g f o r u s e i n many c r o p management s y s t e m s . T h i s r e v i e w b r i e f l y e x a m i n e s t h e b a s i s o f i m m u n o a s s a y a s an a n a l y t i c a l method. I t o u t l i n e s c u r r e n t and f u t u r e a p p l i c a t i o n s o f these methods t o d e t e c t i n g m a t e r i a l s o f d i r e c t importance t o a g r i c u l t u r a l c h e m i s t r y and c r o p management. The

Technology

The t e c h n o l o g y w h i c h h a s e n a b l e d s c i e n t i s t s t o d e v e l o p i m m u n o a s s a y d e t e c t i o n s y s t e m s i s b a s e d on u n i q u e p r o p e r t i e s o f t h e n a t u r a l l y o c c u r r i n g defense mechanism o f higher v e r t e b r a t e animals, t h e immune r e s p o n s e . T h e immune r e s p o n s e c o n s i s t s o f a s e r i e s o f events which enable t h e body t o r e c o g n i z e f o r e i g n organisms o r s u b s t a n c e s and t o respond a g a i n s t t h e s e agents i n a p r o t e c t i v e m a n n e r . T h e p o r t i o n o f t h e immune r e s p o n s e u t i l i z e d i n i m m u n o a s s a y technology i s that involving production of proteins c a l l e d a n t i b o d i e s . A n t i b o d i e s a r e p r o d u c e d a s p a r t o f t h e h u m o r a l immune r e s p o n s e when f o r e i g n m a t e r i a l s a r e i n t r o d u c e d i n t o t h e b o d y o f a n animal. A n t i b o d i e s then bind very s p e c i f i c a l l y t o t h e f o r e i g n s u b s t a n c e , d e s t r o y o r n e u t r a l i z e i t , and h e l p c l e a r i t from t h e system. Antibodies recognize s p e c i f i c portions of a f o r e i g n substance, t h e b i n d i n g p a r t n e r a l l o w i n g immunoassays t o d i s t i n g u i s h between t h a t b i n d i n g p a r t n e r and s i m i l a r substances and t o d e t e c t s m a l l a m o u n t s o f t h e t a r g e t s u b s t a n c e . A n t i b o d i e s a r e p r o d u c e d f o r u s e i n an i m m u n o a s s a y b y e x p o s i n g



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an a n i m a l o r s p e c i a l i z e d c e l l s f r o m a n a n i m a l t o a t a r g e t s u b s t a n c e . F o r e x a m p l e , a l a b o r a t o r y a n i m a l s u c h a s a r a b b i t may be i m m u n i z e d w i t h a p r e p a r a t i o n o f t h e t a r g e t s u b s t a n c e t o s t i m u l a t e 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 . Larger animals such a s g o a t s , sheep o r h o r s e s a r e commonly used t o produce l a r g e r b a t c h e s of a n t i b o d i e s . For t h e p r o d u c t i o n o f monoclonal a n t i b o d i e s , mice are immunized and a n t i b o d y p r o d u c i n g c e l l s from t h e mice a r e u t i l i z e d i n the c r e a t i o n o f the antibody producing "hybridoma" c e l l s . More r e c e n t l y , c e l l s w i t h t h e c a p a c i t y t o produce a n t i b o d y have been i s o l a t e d f r o m t h e animal and t h e i r growth e s t a b l i s h e d i n v i t r o before they are exposed t o t h e t a r g e t substance. T h i s p r o c e d u r e has been p a r t i c u l a r l y i m p o r t a n t ( f o r o b v i o u s r e a s o n s ) i n t h e p r o d u c t i o n o f human m o n o c l o n a l a n t i b o d i e s (1). The a n t i b o d i e s a r e t h e n i n c o r p o r a t e d i n t o an i m m u n o a s s a y w h i c h c a n be u s e d t o determine whether t h a t t a r g e t substance i s present i n a t e s t sample. B a s i c P r i n c i p l e s o f V e r t e b r a t e Humoral Immunity. P h y s i c a l b a r r i e r s s u c h as t h e s k i n and mucous membranes p r o v i d e an a n i m a l ' s f i r s t line of defense against organisms or other substances i n the e n v i r o n m e n t . When t h e p h y s i c a l b a r r i e r i s b r o k e n , t h e immune response i s l a r g e l y r e s p o n s i b l e f o r e l i m i n a t i n g or i n a c t i v a t i n g f o r e i g n s u b s t a n c e s o r o r g a n i s m s . T h e immune " s u r v e i l l a n c e " s y s t e m recognizes foreign substances that are introduced into the animal a s " n o n - s e l f " . T h i s r e c o g n i t i o n a c t i v a t e s t h e immune r e s p o n s e . T h e immune s y s t e m i s c o m p r i s e d o f a n e t w o r k o f s p e c i a l i z e d c e l l s a n d t i s s u e s w h i c h , when a c t i v a t e d , p r o d u c e v a r i o u s m o l e c u l e s . T h e s e m o l e c u l e s , which i n c l u d e a n t i b o d i e s , p r o t e c t t h e body from harm caused by f o r e i g n agents by n e u t r a l i z i n g o r d e s t r o y i n g them. T h e immune s y s t e m n o r m a l l y r e s p o n d s o n l y t o f o r e i g n o r n o n - s e l f s u b s t a n c e s . When t h e immune s y s t e m f a i l s t o r e c o g n i z e a s u b s t a n c e as f o r e i g n o r responds s l o w l y t o t h e s u b s t a n c e , t h e r e s u l t may b e l i f e - t h r e a t e n i n g i l l n e s s o r i n f e c t i o n . S o m e t i m e s t h e immune s y s t e m l o s e s i t s a b i l i t y t o d i s t i n g u i s h b e t w e e n s e l f a n d n o n - s e l f , and a r e s p o n s e i s d i r e c t e d a g a i n s t " s e l f " t i s s u e s . D i s e a s e s r e s u l t i n g f r o m t h i s immune m a l f u n c t i o n a r e c o l l e c t i v e l y r e f e r r e d t o as autoimmune ( a g a i n s t " s e l f " ) d i s e a s e s . Examples o f autoimmune d i s e a s e i n c l u d e r h e u m a t o i d a r t h r i t i s and s y s t e m i c l u p u s e r y t h e m a t o s i s . T h e immune s u r v e i l l a n c e s y s t e m a l s o h a s t h e a b i l i t y to d e t e c t a l t e r e d c o m p o s i t i o n o f s e l f t i s s u e s so these t i s s u e s o r c e l l s c a n b e d e s t r o y e d . When a l t e r e d s e l f t i s s u e s a r e n o t d e t e c t e d o r e f f e c t i v e l y d e s t r o y e d , c a n c e r may r e s u l t . C l e a r l y , t h e immune r e s p o n s e i s an i n t r i c a t e s y s t e m r e q u i r i n g a n u m b e r o f c o m p l e x f u n c t i o n s t o be e f f e c t i v e . T h e Immune R e s p o n s e . T h e immune r e s p o n s e o f a v e r t e b r a t e a n i m a l has two major arms, t h e e e l 1-mediated r e s p o n s e s and a n t i b o d y - m e d i a t e d o r humoral r e s p o n s e s . The c e l l - m e d i a t e d r e s p o n s e , w h i c h i s i n v o l v e d i n p r o t e c t i o n a g a i n s t c a n c e r a n d some i n t r a c e l l u l a r p a t h o g e n s (2!), w i l l n o t be r e v i e w e d h e r e b e c a u s e i t i s n o t r e l e v a n t t o most immunoassay t e c h n o l o g y . The humoral response, which involves production of a n t i b o d i e s t o f o r e i g n s u b s t a n c e s , i s t h e a r m o f t h e immune s y s t e m w h i c h p r o v i d e s t h e b a s i s f o r immunoassay systems. T h e h u m o r a l immune s y s t e m c a n r e s p o n d t o f o r e i g n s u b s t a n c e s



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such as v i r a l , b a c t e r i a l and f u n g a l p a t h o g e n s as w e l l as c h e m i c a l s or p a r t i c u l a t e s which gain access t o t h e body. Any substance which t r i g g e r s t h e p r o d u c t i o n o f an a n t i b o d y a n d b i n d s t o t h a t a n t i b o d y i s c a l l e d an a n t i g e n . C u r r e n t t h e o r y p o s t u l a t e s t h a t a n t i b o d i e s c a n be f o r m e d a g a i n s t a n t i g e n s c o n t a i n e d i n v i r t u r a l l y a l l materials. In t h e r e t i c u l o e n d o t h e l i a l system o f a n i m a l s , v a r i o u s c e l l p o p u l a t i o n s c o o p e r a t e t o p r o d u c e t h e h u m o r a l immune r e s p o n s e . These c e l l s c i r c u l a t e t h r o u g h o u t t h e body i n t h e b l o o d and lymph systems. They a r e found i n e s p e c i a l l y high c o n c e n t r a t i o n s i n t h e s p l e e n and t h e lymph nodes. F o r e i g n a n t i g e n s a r e i n i t i a l l y r e c o g n i z e d by scavenger s u r v e i l l a n c e c e l l s c a l l e d macrophages. Some s u b s t a n c e s a r e e n g u l f e d a n d d e s t r o y e d b y m a c r o p h a g e s . Macrophages process t h e degraded antigens then "present" these a n t i g e n s t o o t h e r c e l l s c a l l e d Τ and Β l y m p h o c y t e s . The Τ l y m p h o c y t e s h a v e a n t i g e n - b i n d i n g r e c e p t o r s . When s p e c i f i c a n t i g e n s bind these r e c e p t o r s , the T - c e l l s produce s i g n a l molecules which h e l p a c t i v a t e o r b o o s t t h e a c t i v i t y o f both macrophages and B - c e l l s . Β lymphocytes a l s o bind the s p e c i f i c antigens presented by m a c r o p h a g e s . B - c e l l r e s p o n s e s a r e e n h a n c e d by s i g n a l m o l e c u l e s from macrophages and Τ c e l l s . As a r e s u l t o f a n t i g e n r e c o g n i t i o n and m o l e c u l a r s i g n a l i n g , B - c e l l s a r e i n d u c e d t o d i f f e r e n t i a t e i n t o plasma c e l l s . The plasma c e l l s then produce a n t i b o d i e s t h a t b i n d to t h e a n t i g e n which t r i g g e r e d t h i s s e r i e s o f e v e n t s . Each plasma c e l l produces a n t i b o d i e s o f one s p e c i f i c i t y . That i s , they b i n d t o a small but s p e c i f i c region of the inducing antigen called the antigenic determinant. D i f f e r e n t plasma c e l l s produce a n t i b o d i e s s p e c i f i c f o r d i f f e r e n t portions of the foreign substance. This r e s u l t s i n t h e o v e r a l l production o f a n t i b o d i e s which bind t o d i f f e r e n t s i t e s on a g i v e n s u b s t a n c e . T h i s i s r e f e r r e d t o as a polyclonal antibody response. A n t i b o d i e s produced d u r i n g t h e humoral r e s p o n s e a r e s e c r e t e d by plasma c e l l s and a r e f o u n d i n high concentrations i n the blood stream. The h u m o r a l r e s p o n s e i s c o m p l e x , and s e v e r a l d a y s a r e r e q u i r e d before a n t i b o d i e s a r e produced a g a i n s t a f o r e i g n s u b s t a n c e o r a n t i g e n t h a t e n t e r s t h e b o d y . When a n i m a l s a r e purposely exposed t o a substance t o s t i m u l a t e antibody production, s e v e r a l w e e k s o f r e p e a t e d e x p o s u r e t o t h e a n t i g e n may b e r e q u i r e d before high l e v e l s o f s p e c i f i c antibody a r e present i n t h e serum. Immunoglobulins - S t r u c t u r e and F u n c t i o n . A n t i b o d i e s a r e g l o b u l a r p r o t e i n s p r o d u c e d d u r i n g t h e immune r e s p o n s e a n d c o n s e q u e n t l y , t h e y a r e a l s o c a l l e d i m m u n o g l o b u l i n s ( I g ) . P o r t e r a n d E d e l m a n won t h e Nobel p r i z e i n P h y s i o l o g y and M e d i c i n e i n 1972 f o r t h e i r f u n d a m e n t a l r e s e a r c h on t h e s t r u c t u r e a n d c h e m i s t r y o f I g (3). The c l a s s e s o f I g p r o d u c e d b y h u m a n s i n c l u d e I g A, G, Ε , M a n d D. A n t i b o d i e s o f d i f f e r e n t c l a s s e s r a n g e f r o m 150 t o 900 K i l o d a l t o n s (Kd) i n m o l e c u l a r w e i g h t and m e d i a t e d i f f e r e n t i m m u n o l o g i c a l f u n c t i o n s . Some o t h e r v e r t e b r a t e a n i m a l s p r o d u c e f e w e r c l a s s e s , but most p r o d u c e IgG and IgM. These two a n t i b o d y c l a s s e s a r e f o u n d i n s e r u m a n d may b e r e a d i l y u s e d i n i m m u n o a s s a y s . I g A i s a l s o u s e d i n some c a s e s . An I g G m o l e c u l e c o n s i s t s o f t w o h e a v y c h a i n s a n d t w o l i g h t p o l y p e p t i d e c h a i n s connected through d i s u l f i d e bonds ( F i g u r e 1 ) . IgG a n t i b o d i e s have a m o l e c u l a r w e i g h t o f 150 Kd and a c c o u n t f o r a p p r o x i m a t e l y 8 0 % o f t h e t o t a l i m m u n o g l o b u l i n i n human s e r u m .



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F i g u r e 1. I g G a n t i b o d y m o l e c u l e c o n s i s t s o f f o u r c h a i n s , t w o h e a v y and two l i g h t , h e l d t o g e t h e r by d i s u l f i d e b o n d s . Antibody b i n d s t o a n t i g e n s as a r e s u l t o f t h e m o l e c u l a r c o n f i g u r a t i o n of the v a r i a b l e region.



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The F a b p o r t i o n o f t h e a n t i b o d y ( s e e F i g u r e 1) c o n t a i n s t h e variable region that i s responsible f o r the s p e c i f i c i t y of the molecule. V a r i a t i o n s i n t h e polypeptide sequence of t h i s region a r e c o m p l i m e n t a r y t o t h e a n t i g e n i c déterminent t h u s p r o v i d i n g t h e b a s i s f o r a n t i g e n : a n t i b o d y b i n d i n g . An I g G m o l e c u l e h a s t w o antigen binding sites. The F c r e g i o n o f an a n t i b o d y m o l e c u l e ( s e e F i g u r e 1) i s c o n s t a n t w i t h i n a p a r t i c u l a r a n t i b o d y c l a s s . The F c r e g i o n m e d i a t e s secondary immunological f u n c t i o n s such as complement f i x a t i o n (4). L a b e l s o r " t a g s " w h i c h a r e u s e d f o r v i s u a l i z a t i o n o f immunoassays a r e g e n e r a l l y a t t a c h e d t o t h e Fc r e g i o n so t h e antibody r e t a i n s t h e antigen-binding c a p a c i t y of t h e Fab r e g i o n . The p o l y c l o n a l n a t u r e o f t h e h u m o r a l r e s p o n s e r e s u l t s i n t h e production of antibodies with d i f f e r e n t binding s p e c i f i c i t i e s . In a d d i t i o n , antibodies vary i n binding a f f i n i t y , or strength of antibody-antigen binding. P o l y c l o n a l a n t i s e r a e x h i b i t a range of s p e c i f i c i t y and a f f i n i t y w i t h i n a g i v e n pool o f serum. Immunoassays. Immunoassays a r e t e s t s i n which a n t i b o d i e s a r e used as a n a l y t i c a l c h e m i s t r y r e a g e n t s . T h e immune r e s p o n s e o f an a n i m a l can be m a n i p u l a t e d t o p r o d u c e a n t i b o d i e s t h a t r e a c t w i t h a p a r t i c u l a r t a r g e t a n t i g e n . Serum from t h e blood o f t h e a n i m a l , w h i c h c o n t a i n s t h e a n t i b o d i e s , c a n t h e n be c o l l e c t e d a t t h e a p p r o p r i a t e t i m e . F i n a l l y , t h e a n t i b o d i e s a r e p u r i f i e d and i n c o r p o r a t e d i n t o a d e t e c t i o n s y s t e m . The k e y t o s u c c e s s f u l d e v e l o p m e n t o f an i m m u n o a s s a y l i e s i n p r o d u c i n g t e s t s t h a t p r o v i d e accurate information; that i s to say, t e s t s with high l e v e l s of s p e c i f i c i t y , s e n s i t i v i t y and r e l i a b i l i t y . D i a g n o s t i c s e n s i t i v i t y means t h a t a t e s t i s p o s i t i v e w h e n e v e r t h e t a r g e t a n t i g e n i s p r e s e n t , even a t low l e v e l s . E q u a l l y important i s s p e c i f i c i t y , w h e r e t h e t e s t i s n e g a t i v e when t h e t a r g e t a n t i g e n i s n o t p r e s e n t . The c o m b i n a t i o n o f s p e c i f i c i t y a n d s e n s i t i v i t y a r e u s e d t o determine the p r e d i c t i v e value of a p o s i t i v e or negative t e s t , or the percent r e l i a b i l i t y o f the t e s t . R e l i a b i l i t y o f an i m m u n o a s s a y i s e s s e n t i a l t o i t s s u c c e s s i n t h e m a r k e t . T h e c o n s u m e r may t h e n b e n e f i t f r o m t h e i r u s e b y b e i n g a b l e t o take t i m e l y a c t i o n . The r a p i d advances i n b i o t e c h n o l o g y and i m m u n o l o g y o v e r t h e p a s t d e c a d e h a v e made i t p o s s i b l e t o improve both t h e s p e c i f i c i t y and s e n s i t i v i t y o f immunoassays. To a l a r g e e x t e n t , t h e a n t i b o d y i s r e s p o n s i b l e f o r t h e s p e c i f i c i t y and s e n s i t i v i t y o f t h e immunoassay. Antibodies s u i t a b l e f o r u s e i n some i m m u n o a s s a y s may b e p r o d u c e d b y immunizing a l a b o r a t o r y animal with a r e l a t i v e l y complex p r e p a r a t i o n c o n t a i n i n g t h e a n t i g e n t o be a s s a y e d . Such antigens may b e c o m p o n e n t p a r t s o f m i c r o o r g a n i s m s s u c h a s b a c t e r i a , viruses, fungi, protozoa, or helminths. A n t i g e n s may a l s o b e chemicals, plants, or plant products. Additional materials called a d j u v a n t s a r e commonly i n j e c t e d i n c o n j u n c t i o n w i t h t h e a n t i g e n - c o n t a i n i n g p r e p a r a t i o n t o enhance t h e production o f antibodies. Some m o l e c u l e s a r e t o o s m a l l t o e l i c i t an e f f e c t i v e h u m o r a l response. T h e s e m o l e c u l e s , d e f i n e d as h a p t e n s , must be c o u p l e d t o a l a r g e r p r o t e i n before being introduced t o t h e body, i n order t o e l i c i t an a n t i b o d y r e s p o n s e t o t h e h a p t e n . The l a r g e r m o l e c u l e ,



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k n o w n as a c a r r i e r , h e l p s p r o d u c e t h e n e c e s s a r y r e c o g n i t i o n s i g n a l s f o r a c t i v a t i o n o f t h e immune r e s p o n s e . T h i s t y p e o f s y s t e m i s employed t o p r o d u c e a n t i b o d i e s w h i c h can then b i n d t o c e r t a i n c h e m i c a l s and t o s m a l l p o l y p e p t i d e s (!5, 6 ) . O p t i m a l i m m u n i z a t i o n s c h e d u l e s ( n u m b e r o f i n j e c t i o n s and t i m e between i n j e c t i o n s ) v a r y w i d e l y d e p e n d i n g upon t h e c h a r a c t e r i s t i c s of the p a r t i c u l a r a n t i g e n . Serum, which c o n t a i n s the a n t i b o d y , i s c o l l e c t e d f r o m t h e a n i m a l and u s e d i n t h e i m m u n o a s s a y . Antibodies p r o d u c e d b y i m m u n i z i n g an a n i m a l and c o l l e c t i n g t h e a n t i b o d i e s d i r e c t l y f r o m t h e s e r u m o f t h e a n i m a l a r e r e f e r r e d t o as p o l y c l o n a l a n t i b o d i e s . P o l y c l o n a l antibodies e x h i b i t a range of s p e c i f i c i t i e s and a f f i n i t i e s w i t h i n a g i v e n p o o l r e f l e c t i n g t h e c o m p l e x i t y o f t h e p r e p a r a t i o n w h i c h was i n j e c t e d i n t o t h e a n i m a l . O f t e n , a m o r e u s e f u l p o l y c l o n a l s e r u m c a n be p r o d u c e d i f t h e m o s t d e s i r a b l e a n t i g e n i c c o m p o n e n t ( s ) c a n be i d e n t i f i e d a n d p u r i f i e d before the p r e p a r a t i o n i s i n j e c t e d i n t o the animal. For example, when a w h o l e o r g a n i s m i s i n j e c t e d , a n t i b o d i e s w i t h h u n d r e d s o f s p e c i f i c i t i e s a r e p r o d u c e d . A l t h o u g h some o f t h e a n t i b o d i e s a r e s p e c i f i c t o t h e i n j e c t e d o r g a n i s m , many o f t h e a n t i b o d i e s a r e l i k e l y t o a l s o r e a c t w i t h r e l a t e d o r g a n i s m s and some a n t i b o d i e s may e v e n r e a c t w i t h u n r e l a t e d o r g a n i s m s . T h i s problem can s o m e t i m e s be e l i m i n a t e d b y i m m u n i z i n g o n l y w i t h an a n t i g e n t h a t i s unique to the t a r g e t organism. O b v i o u s l y , t h i s a p p r o a c h may r e q u i r e a c o n s i d e r a b l e a m o u n t o f a n a l y t i c a l and p r e p a r a t i v e w o r k before the immunizations are i n i t i a t e d . Commercial q u a n t i t i e s of p o l y c l o n a l a n t i b o d i e s can be o b t a i n e d s i m p l y by immunizing l a r g e numbers o f a n i m a l s . Even under t h e b e s t o f c i r c u m s t a n c e s , h o w e v e r , l a c k o f r e p r o d u c i b i l i t y may be encountered because of c o n s i d e r a b l e d i f f e r e n c e s i n the response of i n d i v i d u a l a n i m a l s t o an i d e n t i c a l a n t i g e n p r e p a r a t i o n . Some o f t h i s d i f f i c u l t y may be o v e r c o m e b y u s i n g l a r g e a n i m a l s s u c h a s s h e e p , g o a t s , h o r s e s o r by p o o l i n g s e r a t o p r o d u c e l a r g e b a t c h e s or l o t s . M o n o c l o n a l A n t i b o d i e s . One o f t h e m o s t u s e f u l , r e c e n t s c i e n t i f i c a d v a n c e s t o be a p p l i e d t o i m m u n o a s s a y t e c h n o l o g y h a s b e e n t h e d e v e l o p m e n t o f m o n o c l o n a l a n t i b o d i e s (7). T h i s t e c h n o l o g y was f i r s t r e p o r t e d i n 1975 b y K o h l e r and M i l s t e i n ( 8 ) , who won t h e N o b e l P r i z e f o r t h e i r w o r k . A l t h o u g h much o f t h e c u r r e n t a p p l i c a t i o n o f m o n o c l o n a l a n t i b o d y t e c h n o l o g y i s r e l a t e d t o human m e d i c a l r e s e a r c h and d i a g n o s t i c s , v e t e r i n a r y and a g r i c u l t u r a l a p p l i c a t i o n s are e x p a n d i n g r a p i d l y (£). Monoclonal a n t i b o d i e s are produced i n a s e r i e s of steps b e g i n n i n g w i t h t h e i m m u n i z a t i o n o f a m o u s e and r e m o v a l o f i t s s p l e e n a f t e r an a p p r o p r i a t e p e r i o d o f t i m e . Antibody-producing c e l l s a r e i s o l a t e d f r o m t h e s p l e e n and f u s e d w i t h " i m m o r t a l " myeloma c e l l s from t i s s u e c u l t u r e through the use o f p o l y e t h y l e n e g l y c o l . C e l l s r e s u l t i n g f r o m t h e f u s i o n o f a B - c e l l and a m y e l o m a c e l l are c a l l e d hybridomas. Hybridomas d e r i v e the a b i l i t y to p r o d u c e a s i n g l e t y p e o f a n t i b o d y f r o m t h e B - c e l l p a r t n e r and t h e a b i l i t y t o s u r v i v e and p r o l i f e r a t e o u t s i d e t h e b o d y o f t h e a n i m a l f o r an e x t e n d e d p e r i o d o f t i m e f r o m t h e m y e l o m a c e l l p a r t n e r . Through a s e r i e s of manipulations i n t i s s u e c u l t u r e , i n d i v i d u a l h y b r i d o m a s a r e i s o l a t e d and a l l o w e d t o d i v i d e and p r o d u c e antibody. A n t i b o d y i n t h e c e l l c u l t u r e medium i s then t e s t e d f o r



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d e s i r a b l e a n t i g e n - b i n d i n g c h a r a c t e r i s t i c s . S i n c e each hybridoma c e l l l i n e produces o n l y one t y p e o f monoclonal a n t i b o d y , s e l e c t i o n o f an a p p r o p r i a t e h y b r i d o m a l i n e m a k e s i t p o s s i b l e t o a v o i d m a n y of t h e p o t e n t i a l problems encountered w i t h a n t i b o d i e s i n p o l y c l o n a l s e r a . H y b r i d o m a s may b e f r o z e n i n l i q u i d n i t r o g e n , s t o r e d and r e c o v e r e d a t any t i m e t o c o n t i n u e r e p r o d u c i b l e production of antibody. Monoclonal antibody p r o d u c t i o n i s "scaled-up" e i t h e r by growing l a r g e numbers o f c e l l s i n t i s s u e c u l t u r e o r by i n j e c t i n g t h e c e l l s back i n t o t h e p e r i t o n e a l c a v i t y o f a mouse t o produce antibody-rich a s c i t e s f l u i d . Tissue c u l t u r e scale-up procedures u t i l i z e a variety of bioreactors, hollow-fiber f i l t e r s , r e - c i r c u l a t i n g m e d i a and c e l l e n c a p s u l a t i o n t e c h n o l o g i e s d e s i g n e d t o promote r a p i d growth o f t h e c e l l s and i n c r e a s e t h e q u a n t i t y and p u r i t y o f antibody produced. A s c i t e s p r o d u c t i o n takes advantage o f the f a c t t h a t t h e best environment f o r growth o f t h e hybridoma c e l l s and p r o d u c t i o n o f a n t i b o d y i s p r o b a b l y i n t h e animal h o s t species from which the c e l l s o r i g i n a t e d . A s c i t e s f l u i d c o n t a i n s a much h i g h e r c o n c e n t r a t i o n o f a n t i b o d i e s t h a n t i s s u e c u l t u r e s u p e r n a t a n t s . However, t h e q u a n t i t y and c o m p l e x i t y o f contaminating materials i s also greater i n ascites f l u i d . A n t i b o d i e s m u s t be p u r i f i e d f r o m t h e t i s s u e c u l t u r e s u p e r n a t a n t , a s c i t e s f l u i d , o r serum b e f o r e t h e y c a n be " t a g g e d " o r o t h e r w i s e e f f e c t i v e l y u t i l i z e d i n an i m m u n o a s s a y . Purification o f a n t i b o d i e s c a n be a c c o m p l i s h e d t o v a r y i n g d e g r e e s e m p l o y i n g a number o f p h y s i c a l , b i o c h e m i c a l and i m m u n o l o g i c a l t e c h n i q u e s ( 1 0 ) . Visualization/Detection of Antibody/Antigen Interactions. For p r a c t i c a l a p p l i c a t i o n o f the unique p r o p e r t i e s o f a n t i b o d i e s i n a d i a g n o s t i c a s s a y , t h e u s e r must be a b l e t o d e t e r m i n e w h e t h e r t h e a n t i g e n / a n t i b o d y r e a c t i o n has i n f a c t o c c u r r e d . A v a r i e t y o f v i s u a l i z a t i o n and d e t e c t i o n methods have been d e s i g n e d f o r t h i s purpose. P r e c i p i t a t i o n / A g g l u t i n a t i o n . One o f t h e o l d e s t t e c h n i q u e s f o r v i s u a l i z a t i o n of antigen-antibody r e a c t i o n s i s through the observation of a precipitation or agglutination reaction. A l l a n t i b o d i e s a n d some a n t i g e n s a r e m u l t i v a l e n t ( h a v e m o r e t h a n o n e b i n d i n g s i t e ) . A l a t t i c e o r m a t r i x i s f o r m e d when t h e a n t i b o d i e s r e a c t w i t h t h e a n t i g e n s . I n some c a s e s , t h e l a t t i c e i s l a r g e enough t h a t i t i s v i s i b l e t o t h e naked e y e . The p r e c i p i t a t i o n r e a c t i o n c a n be c a r r i e d o u t i n s o l u t i o n ( t u r b i d i t y t e s t o r r i n g p r e c i p i t a t i o n ) or i n a g e l matrix (Ouchterlony double d i f f u s i o n or r a d i a l i m m u n o d i f f u s i o n ) . P r e c i p i t a t i o n r e a c t i o n s i n g e l s c a n be e n h a n c e d by s t a i n i n g . R e a c t a n t s c a n a l s o be a t t a c h e d t o p a r t i c l e s s u c h a s r e d b l o o d c e l l s o r l a t e x b e a d s t o make t h e r e a c t i o n e a s i e r to v i s u a l i z e . P r e c i p i t a t i o n / a g g l u t i n a t i o n assays are extremely s i m p l e t o p e r f o r m b u t t h e r e s u l t s c a n be d i f f i c u l t t o i n t e r p r e t a t low c o n c e n t r a t i o n s o f t h e t a r g e t r e a c t a n t . C o m p l e m e n t F i x a t i o n . C o m p l e m e n t i s a c o m p o n e n t o f t h e immune s y s t e m c a p a b l e o f b i n d i n g t o an a n t i b o d y a f t e r t h e a n t i b o d y h a s r e a c t e d w i t h an a n t i g e n . I f t h a t a n t i g e n i s p a r t o f a w h o l e c e l l , complement mediates a s e r i e s o f events which r e s u l t s i n t h e l y s i s of the c e l l ( 1 1 ) .



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B I O T E C H N O L O G Y IN A G R I C U L T U R A L C H E M I S T R Y

A complement f i x a t i o n assay uses red blood c e l l s and a n t i b o d y to t h e r e d b l o o d c e l l s as an i n d i c a t o r system ( s e e F i g . 2 ) . An antibody produced a g a i n s t the t a r g e t antigen i s reacted w i t h t h e unknown t e s t s a m p l e . I f t h e t a r g e t a n t i g e n i s n o t p r e s e n t i n t h e t e s t s a m p l e , no a n t i g e n - . a n t i b o d y c o m p l e x i s formed b y the t e s t system and complement i s n o t " f i x e d " by the t e s t system. The c o m p l e m e n t s u b s e q u e n t l y l y s e s t h e r e d b l o o d c e l l s when t h e i n d i c a t o r system i s added. I f t a r g e t antigen i s present i n t h e t e s t sample, the a n t i g e n : a n t i b o d y complex i nthe t e s t system t i e s up t h e c o m p l e m e n t , a n d t h e r e d b l o o d c e l l s a r e n o t l y s e d w h e n t h e i n d i c a t o r system i s added. The degree o f l y s i s o f the i n d i c a t o r system i s determined by observing the supernatant o f the r e a c t i o n mixture. Hemoglobin from the red blood c e l l s c o l o r st h e supernatant red i f the red blood c e l l s are l y s e d . Enzyme Immunoassay. Enzyme immunoassay o r E I A i s p r o b a b l y t h e i m m u n o a s s a y t e c h n o l o g y m o s t w i d e l y a p p l i e d t o d a y (12!). I n a n E I A , e i t h e r the a n t i b o d y o r t h e a n t i g e n i s tagged w i t h an enzyme. A f t e r the assay i s performed, the presence o r absence o f t h e e n z y m e - l a b e l e d component i s d e t e c t e d b y the a d d i t i o n o f an a p p r o p r i a t e s u b s t r a t e . EIA's are q u i t e s e n s i t i v e , reagents a r e r e l a t i v e l y inexpensive and w i d e l y a v a i l a b l e , and the reagents a r e r e l a t i v e l y s t a b l e . Q u a n t i t a t i v e r e s u l t s c a n be o b t a i n e d u s i n g i n s t r u m e n t a t i o n and E I A i s e a s i l y automated. Immunoassays which r e q u i r e t h a t unbound r e a c t a n t s be s e p a r a t e d from the bound r e a c t a n t s ( u s u a l l y b y w a s h i n g ) b e f o r e t h e s i g n a l i s generated o r detected are r e f e r r e d t o as heterogeneous i m m u n o a s s a y s . One p o p u l a r c o n f i g u r a t i o n o f a h e t e r o g e n e o u s E I A i s i l l u s t r a t e d i n F i g u r e 3. T h i s f o r m a t i s n o t f r e q u e n t l y used f o r e v a l u a t i n g unknown s a m p l e s b e c a u s e o f t h e p o s s i b i l i t y o f i n t e r f e r e n c e from i r r e l e v a n t molecules i nthe sample p r e p a r a t i o n . The f o r m a t i s w i d e l y u s e d f o r s c r e e n i n g o f m o n o c l o n a l a n t i b o d i e s and e v a l u a t i n g p o l y c l o n a l a n t i b o d i e s i n s i t u a t i o n s w h e r e a r e l a t i v e l y pure p r e p a r a t i o n o f the t a r g e t antigen i s a v a i l a b l e . The t a r g e t a n t i g e n i s f i r s t a t t a c h e d t o t h e s o l i d s u p p o r t s u c h a s a 9 6 - w e l l m i c r o t i t e r p l a t e a n d e x c e s s a n t i g e n i s washed away. T h e t e s t antibody i s then incubated i n the antigen-coated w e l l . A f t e r an i n c u b a t i o n p e r i o d , t h e u n r e a c t e d a n t i b o d y i s w a s h e d a w a y . A n e n z y m e - t a g g e d a n t i b o d y made t o r e a c t w i t h t h e immunoglobulin species o f the t e s t antibody i s the next reagent placed i n t h e w e l l . A f i n a l wash s t e p i s performed and c o l o r l e s s s u b s t r a t e i s added. Generation o f c o l o r by the s u b s t r a t e (mediated by the enzyme) i s i n d i c a t i v e o f t h e p r e s e n c e o f an a n t i b o d y which r e a c t e d with the t a r g e t antigen. F o r e v a l u a t i o n o f t h e amount o f a t a r g e t a n t i g e n p r e s e n t i n an u n k n o w n s a m p l e , a p u r e a n t i g e n p r e p a r a t i o n i s b o u n d t o t h e p l a t e . T h e t e s t sample i s c o m b i n e d w i t h m e a s u r e d amount o f a n t i b o d y w h i c h i s known t o r e a c t w i t h t h e t a r g e t a n t i g e n . T h e sample antibody mixture i s then incubated i n the antigen coated w e l l and the q u a n t i t y o f a n t i b o d y bound t o the w e l l i s subsequently determined as p r e v i o u s l y d e s c r i b e d . This format i s g e n e r a l l y r e f e r r e d t o as a b i n d i n g - i n h i b i t i o n assay because i f antigen i s present i nthe t e s t sample, the antibody i s depleted by t h e t e s t sample and i s n o t bound t o the a n t i g e n on the p l a t e . I n t h i s case, the generation o f c o l o r i nthe w e l l s would i n d i c a t e that the t a r g e t antigen i s not present i nthe t e s t sample.



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i g u r e 2. C o m p l e m e n t f i x a t i o n i m m u n o a s s a y . R e d c o l o r r e l e a s e d d u e to the l y s i s of red blood c e l l s i n d i c a t e s a negative assay, i . e . , the absence of the t a r g e t antigen.



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F i g u r e 3. E n z y m e l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) r e p r e s e n t s a heterogeneous assay i n which unreacted m a t e r i a l s a r e washed away between s t e p s . A n t i b o d y - a n t i g e n b i n d i n g i s v i s u a l i z e d by t h e g e n e r a t i o n o f a c o l o r e d p r o d u c t .



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Another u s e f u l heterogeneous EIA i s the double a n t i b o d y or " s a n d w i c h " i m m u n o a s s a y . I n a s a n d w i c h i m m u n o a s s a y , an u n t a g g e d a n t i b o d y or c a p t u r e a n t i b o d y i s the f i r s t r e a g e n t bound t o the s o l i d phase. The t e s t s a m p l e and t h e n t h e e n z y m e l a b e l e d a n t i b o d y a r e a d d e d s e q u e n t i a l l y . The u n b o u n d c o m p o n e n t s a r e r e m o v e d b y washing a f t e r each i n c u b a t i o n . Enzyme-labeled a n t i b o d y i s d e t e c t e d by a d d i t i o n o f a s u b s t r a t e as a b o v e . The c a p t u r e a n t i b o d y i s p a r t i c u l a r l y u s e f u l when t h e t e s t s a m p l e c o n s i s t s o f a c o m p l e x m i x t u r e o f m a t e r i a l s . The c a p t u r e a n t i b o d y " f i s h e s - o u t " t h e t a r g e t a n t i g e n and o t h e r c o m p o n e n t s o f t h e t e s t s a m p l e ( w h i c h c o u l d i n t e r f e r e w i t h subsequent s t e p s i n the immunoassay) are more e f f e c t i v e l y washed away. S e n s i t i v i t i e s o f m a n y E I A s c a n be i n c r e a s e d t h r o u g h t h e u s e o f a v i d i n - b i o t i n a m p l i f i c a t i o n s y s t e m s (1.3)· A v i d i n i s a 68 k d g l y c o p r o t e i n w h i c h c a n be p u r i f i e d f r o m e g g w h i t e s o r f r o m some s p e c i e s of b a c t e r i a . B i o t i n i s a small molecular weight v i t a m i n . A v i d i n b i n d s b i o t i n i n an e s s e n t i a l l y i r r e v e r s i b l e r e a c t i o n . The a f f i n i t y o f t h i s r e a c t i o n i s o v e r one m i l l i o n t i m e s h i g h e r t h a n t h e a f f i n i t y o f a n t i b o d i e s f o r a n t i g e n s . I n o n e e x a m p l e o f an a m p l i f i e d EIA, a n t i b o d y prepared a g a i n s t the t a r g e t a n t i g e n i s t a g g e d w i t h s e v e r a l m o l e c u l e s o f b i o t i n . The e n z y m e i s b o u n d t o a v i d i n . The h i g h a f f i n i t y r e a c t i o n b e t w e e n a v i d i n a n d b i o t i n and t h e f a c t t h a t each a v i d i n m o l e c u l e has f o u r b i n d i n g s i t e s f o r b i o t i n a m p l i f i e s t h e number o f enzyme m o l e c u l e s p r e s e n t f o r each b o u n d a n t i b o d y m o l e c u l e . The l a r g e r n u m b e r o f e n z y m e m o l e c u l e s p r e s e n t g e n e r a t e s a s t r o n g e r s i g n a l when s u b s t r a t e i s a d d e d . An i m m u n o a s s a y t h a t i s e x t r e m e l y u s e f u l i n t h e d e t e c t i o n o f s m a l l m o l e c u l a r w e i g h t compounds ( i . e . , p e s t i c i d e s , drugs) i s the homogeneous enzyme-immunoassay. In a homogeneous immunoassay t h e r e i s no n e e d t o s e p a r a t e t h e b o u n d r e a c t a n t s f r o m t h e f r e e r e a c t a n t s b e f o r e the s i g n a l i s measured. In o r d e r t o d e v e l o p a homogeneous i m m u n o a s s a y , t h e a n t i g e n t o be d e t e c t e d m u s t be o b t a i n e d i n p u r e f o r m . Enzyme i s c o v a l e n t l y bound t o t h e a n t i g e n i n such a p o s i t i o n t h a t t h e enzyme a c t i v i t y i s s t e r i c a l l y a l t e r e d i f t h e a p p r o p r i a t e a n t i b o d y b i n d s t o t h e t a g g e d a n t i g e n ( s e e F i g u r e 4 ) . To p e r f o r m t h e a s s a y , t h e e n z y m e - t a g g e d a n t i g e n and t h e n t h e a n t i b o d y a r e a d d e d t o t h e t e s t s a m p l e . The f i n a l r e s u l t i s t h e n o b t a i n e d b y t h e a d d i t i o n of s u b s t r a t e which can r e a c t o n l y w i t h enzyme i n i t s u n i n h i b i t e d form. A v a r i e t y of other u s e f u l c o n f i g u r a t i o n s f o r E I A s h a v e b e e n d e m o n s t r a t e d d e p e n d i n g on t h e p a r t i c u l a r application (14). Other D e t e c t i o n Systems f o r Heterogenous Immunoassays. Tags o t h e r than enzymes which have been used s u c c e s s f u l l y i n heterogeneous i m m u n o a s s a y s y s t e m s i n c l u d e r a d i o i s o t o p e s , f l u o r e s c e n t d y e s , and c o l l o i d a l g o l d . S i m i l a r assay procedures are performed using these t a g s , although d i f f e r e n t d e t e c t i o n methods are employed to determine the r e s u l t of the assay. I n an i m m u n o f l o r e s c e n c e a s s a y , f l u o r e s c e n t d y e s s u c h as f l u o r e s c e i n a n d r h o d a m i n e c a n be v i s u a l i z e d on a m i c r o s c o p e s l i d e t h r o u g h the use of u l t r a v i o l e t l i g h t or d e t e c t e d w i t h i n s t r u m e n t s which measure t h e i r e m i s s i o n s . F l u o r e s c e n t immunoassays are w i d e l y u s e d i n c l i n i c a l l a b o r a t o r i e s . T h e y a r e q u i t e s e n s i t i v e b u t may be t i m e - c o n s u m i n g and r e a g e n t s may be u n s t a b l e and r e q u i r e s p e c i a l handling.



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F i g u r e 4. H o m o g e n e o u s i m m u n o a s s a y i n w h i c h r e s u l t s a r e o b t a i n e d without washing procedures. The a s s a y d e p i c t e d i s c o m p e t i t i v e - unlabeled antigen i n sample competes f o r a n t i b o d y b i n d i n g s i t e w i t h enzyme l a b e l e d a n t i g e n .



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R a d i o i s o t o p e s have been w i d e l y u s e d an i n d i c a t o r s i n a s s a y s t h a t a r e a p p r o p r i a t e l y r e f e r r e d t o as r a d i o i m m u n o a s s a y s ( R I A ) . Y a l o w won t h e N o b e l P r i z e f o r w o r k l e a d i n g t o t h e d e v e l o p m e n t o f RIA ( 1 5 h RIA's t y p i c a l l y have a h i g h d e g r e e o f s e n s i t i v i t y and r e p r o d u c i b i l i t y . A d d i t i o n a l l y , automated i n s t r u m e n t s have been d e s i g n e d t h a t make i t p o s s i b l e t o p r o c e s s a l a r g e v o l u m e o f samples. RIAs a r e also very popular i n c l i n i c a l m i c r o b i o l o g y l a b o r a t o r i e s . Drawbacks f o r RIA's i n c l u d e t h e need f o r e x p e n s i v e e q u i p m e n t t o r e a d t h e r a d i o a c t i v e s i g n a l , s h o r t h a l f l i f e o f some l a b e l s , p o t e n t i a l h e a l t h h a z a r d s t o p e r s o n n e l , and w a s t e d i s p o s a l problems. C o l l o i d a l g o l d l a b e l e d a n t i b o d i e s a r e u s e f u l when t h e i m m u n o a s s a y i s p e r f o r m e d u s i n g an a f f i n i t y m e m b r a n e o r p a p e r ( s u c h as n i t r o c e l l u l o s e ) a s t h e s o l i d s u p p o r t m a t r i x . T h e c o l o r g e n e r a t e d f r o m a c o l l o i d a l g o l d t a g g e d r e a c t a n t c a n be v i s u a l i z e d d i r e c t l y . The s e n s i t i v i t y o f t h e i m m u n o a s s a y c a n be i n c r e a s e d b y an a d d i t i o n a l s t e p d u r i n g w h i c h t h e g o l d i s r e d u c e d a n d g e n e r a t e s additional color (16). Assay Formats. Recent m o d i f i c a t i o n s i n t h e a c t u a l formats used f o r t h e e x e c u t i o n o f i m m u n o a s s a y s h a v e g e n e r a l l y b e e n d e s i g n e d t o make the t e s t s f a s t e r and more c o n v e n i e n t t o p e r f o r m and i n t e r p r e t . Most commercial d i a g n o s t i c a p p l i c a t i o n s a r e changing from l a b o r a t o r y assays which r e q u i r e t r a i n e d t e c h n i c a l p e r s o n n e l and e x p e n s i v e i n s t r u m e n t a t i o n t o a s s a y s t h a t c a n be p e r f o r m e d i n t h e d o c t o r s o f f i c e , i n p r i v a t e homes o r i n t h e f i e l d . O r i g i n a l l y , most immunoassays were performed i n t e s t t u b e s , on m i c r o s c o p e s l i d e s o r i n 9 6 w e l l m i c r o - t i t e r p l a t e s . T h i s " s o l i d phase" component o f t h e immunoassay has been r e p l a c e d by a v a r i e t y o f p a r a p h e r n a l i a d e s i g n e d t o make t h e t e s t e a s i e r t o p e r f o r m a n d i n t e r p r e t a n d t o make t h e a s s a y c o m p a t i b l e w i t h t h e m a n y d i f f e r e n t types o f samples c u r r e n t l y being analyzed. The b a s i c t e c h n o l o g i e s u s e d t o d e t e c t a n t i g e n i n t h e s a m p l e are unchanged or o n l y s l i g h t l y m o d i f i e d from t h e p r e v i o u s l y d e s c r i b e d t e c h n i q u e s i n m o s t o f t h e new i m m u n o a s s a y " k i t " f o r m a t s . A l a r g e v a r i e t y o f e x e c u t i o n s have been developed t o f i l l t h e i n c r e a s i n g and d i v e r s e demand f o r i m m u n o d i a g n o s t i c a s s a y s . The k e y c o m p o n e n t s o f some o f t h e m o r e p o p u l a r c o m m e r c i a l i m m u n o d i a g n o s t i c k i t s are described below. I m m u n o a s s a y s c a n b e p e r f o r m e d on t h e s u r f a c e o f b e a d s o r o t h e r p a r t i c l e s . T h e c o m p o s i t i o n a n d s i z e o f t h e b e a d s may v a r y d e p e n d i n g on t h e a p p l i c a t i o n . S u r f a c e a r e a a v a i l a b l e f o r t h e r e a c t i o n s i s g r e a t l y i n c r e a s e d by u s i n g beads i n s t e a d o f a f l a t s u r f a c e as t h e s o l i d p h a s e . A d d i t i o n a l l y , beads c a n be e a s i l y t r a n s f e r r e d from one r e a g e n t t o another and w a s h i n g s t e p s a r e r a p i d and c o n v e n i e n t ( e . g . beads c o n t a i n i n g m e t a l c a n be transferred magnetically). One o f t h e s i m p l e s t t o u s e e x e c u t i o n s o f t h e i m m u n o a s s a y u s e s a d i p s t i c k as t h e s o l i d p h a s e . The d i p s t i c k i s moved from one r e a g e n t t o t h e next and t h e a s s a y r e s u l t i s then d e t e r m i n e d by e x a m i n i n g t h e d i p s t i c k . The c o l o r r e a c t i o n i n many E I A - d i p s t i c k a s s a y s i s p r o d u c e d when a n e n z y m e l a b e l c o n v e r t s t h e s u b s t r a t e i n t o an i n s o l u b l e c o l o r e d p r o d u c t t h a t b i n d s t o t h e d i p s t i c k . S e m i - q u a n t i t a t i v e r e s u l t s c a n be o b t a i n e d b y c o m p a r i n g t h e c o l o r



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i n t e n s i t y caused by t h e t e s t sample t o c o l o r i n t e n s i t y caused by known s t a n d a r d s . Q u a n t i t a t i v e r e s u l t s c a n be o b t a i n e d t h r o u g h t h e u s e o f an i n e x p e n s i v e f i e l d a d a p t a b l e r e f l e c t o m e t e r w h i c h m e a s u r e s t h e c o l o r i n t e n s i t y on t h e d i p s t i c k . Dry c h e m i s t r y t e c h n o l o g y e l i m i n a t e s t h e need f o r r e a g e n t s i n l i q u i d s o l u t i o n s . Some r e a g e n t s a r e i m m o b i l i z e d i n l a y e r s o f porous m a t e r i a l and d r i e d . The immunoassay i s performed by a l l o w i n g t h e sample s o l u t i o n t o migrate through t h e reagent l a y e r s where t h e r e a c t i o n s take p l a c e i n t h e order i n which t h e reagents have been l a y e r e d . M i g r a t i o n o f t h e sample s o l u t i o n g e n e r a l l y t a k e s p l a c e b y c a p i l l a r y a c t i o n . I n some l a y e r e d a s s a y s , f o r c e i s a p p l i e d i n o r d e r t o c a u s e a s p e c i f i c l a y e r o f r e a g e n t t o come i n t o c o n t a c t w i t h t h e sample. I t i s possible to obtain s e m i - q u a n t i t a t i v e and q u a n t i t a t i v e r e s u l t s by u s i n g methods s i m i l a r t o those described f o rt h e d i p s t i c k assay. The A p p l i c a t i o n s Commercial immunoassays a r e used i n g r e a t q u a n t i t i e s i n m e d i c a l c h e m i s t r y l a b o r a t o r i e s t o p r o v i d e a wide range o f i n f o r m a t i o n used by t h e p h y s i c i a n i n m a n a g i n g p a t i e n t c a r e . D i a g n o s t i c t e s t s f o r i n f e c t i o u s d i s e a s e s , t e s t s f o r i n f e c t i o u s agents i n blood s u p p l i e s , assays f o r markers o f s p e c i f i c d i s e a s e s , and procedures f o r d e t e c t i n g drugs and o t h e r s m a l l m o l e c u l e s a r e r o u t i n e l y a v a i l a b l e . Immunoassay p r o c e d u r e s f o r t h e d o c t o r ' s o f f i c e a r e b e c o m i n g common a n d a r e a l l o w i n g r o u t i n e a n a l y s e s t o b e p e r f o r m e d o n - s i t e by the primary care p h y s i c i a n . Testing k i t s targeted f o r u s e b y c o n s u m e r s a t home a r e a l s o b e i n g m a r k e t e d a t a n i n c r e a s i n g r a t e . M o s t n o t a b l e a r e home p r e g n a n c y t e s t k i t s a n d k i t s f o r d e t e c t i n g S t r e p t o c o c c u s i n t h r o a t swabs. A g r i c u l t u r e has a n a l y t i c a l t a r g e t s analogous t o those d e s c r i b e d i n t h e m e d i c a l a r e a s . C o n s e q u e n t l y , t h e u t i l i t y o f a new a n a l y t i c a l procedure such as immunoassay i s r e a d i l y apparent. However, s e v e r a l f a c t o r s have slowed t h e development o f a n t i b o d y - b a s e d assays f o r a g r i c u l t u r a l a p p l i c a t i o n s . The p r i n c i p l e f a c t o r i s t h e n a t u r e o f t h e m a t r i x t o be a n a l y z e d . I n m e d i c a l immunoassays t h e m a t r i x i s u s u a l l y blood serum o r u r i n e from a s i n g l e host s p e c i e s - humans. A g r i c u l t u r a l c h e m i s t s and b i o l o g i s t s deal with a wide v a r i e t y o f matrices ranging from r e l a t i v e l y s i m p l e ground water t o complex s o i l s and macerated crop samples. Crop v a r i e t i e s , c r o p s p e c i e s , and w i d e l y v a r i a b l e e n v i r o n m e n t a l c o n d i t i o n s add l a y e r s o f c o m p l e x i t y t h a t a r e n o t r o u t i n e l y encountered i n the c l i n i c a l laboratory. D e s p i t e t h e c o m p l e x i t y o f a g r i c u l t u r a l systems, immunoassays are being i n c r e a s i n g l y e x p l o i t e d because o f t h e i r a t t r a c t i v e c h a r a c t e r i s t i c s o f speed, r e l a t i v e low c o s t , and s i m p l i c i t y . T h e s e c t i o n s below o u t l i n e a p p l i c a t i o n s o f these t o o l sf o r e x p e r i m e n t a l use and f o r d i r e c t a p p l i c a t i o n t o c r o p p r o d u c t i o n . D e t e c t i o n and D i a g n o s i s o f Crop Diseases. Diagnosis o f i n f e c t i o u s d i s e a s e s o f crops i s one o f t h e near term uses o f immunoassays i n a g r i c u l t u r e (21)· E f f e c t i v e management o f p l a n t d i s e a s e s r e q u i r e s t h a t crop managers have a c c e s s t o t i m e l y and a c c u r a t e i n f o r m a t i o n on t h r e a t s t o t h e i r c r o p . E x i s t i n g p l a n t d i s e a s e d i a g n o s t i c procedures require t r a i n e d personnel, well-equipped l a b o r a t o r i e s ,



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immunoassays for


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and a g o o d d e a l o f t i m e . D i a g n o s i s may r e q u i r e a f e w d a y s t o s e v e r a l m o n t h s , d e p e n d i n g on t h e n a t u r e o f t h e p a t h o g e n t o be detected. I m m u n o a s s a y s o f f e r s i m p l e , r a p i d , and s e n s i t i v e d e t e c t i o n o f p l a n t p a t h o g e n s and p e s t s i n c r o p s , s o i l s , and o t h e r m a t r i c e s . P l a n t v i r o l o g i s t s p i o n e e r e d t h e use o f a n t i b o d y - b a s e d a s s a y s f o r p l a n t v i r a l d i s e a s e d i a g n o s i s (Γ7). D i a g n o s i s o f t h e s e p l a n t v i r u s d i s e a s e s i s p a r t i c u l a r l y d i f f i c u l t b e c a u s e t h e v i r u s e s c a n n o t be c u l t u r e d , and o t h e r d i a g n o s t i c t e s t s s u c h as l o c a l l e s i o n a s s a y s and e l e c t r o n m i c r o s c o p y a r e t i m e c o n s u m i n g , l a b o r i n t e n s i v e a n d / o r r e q u i r e expensive equipment. Since plant v i r u s e s are d i s t i n c t e n t i t i e s w i t h a f a i r l y w e l l d e f i n e d s t r u c t u r e t h e y c o u l d be p u r i f i e d and u s e d as a n t i g e n s t o i n d u c e f a i r l y s p e c i f i c p o l y c l o n a l a n t i b o d i e s . V i r o l o g i s t s c o u l d use t h e s e a n t i b o d i e s t o d e t e c t viruses in plant extracts for rapid diagnosis. The c o m p l e x n a t u r e o f f u n g i , b a c t e r i a , and n e m a t o d e s made i t d i f f i c u l t t o d e v e l o p s p e c i f i c a n t i b o d i e s t h a t c o u l d be u s e d f o r d i a g n o s t i c p u r p o s e s . The a d v e n t o f m o n o c l o n a l a n t i b o d y technology stimulated i n t e r e s t i n developing d i a g n o s t i c reagents f o r these c o m p l e x p l a n t p a t h o g e n s . The a b i l i t y t o s c r e e n a b a t t e r y o f monoclonal a n t i b o d i e s to i d e n t i f y those which recognize the t a r g e t p a t h o g e n i s an a p p e a l i n g f e a t u r e o f t h i s t e c h n o l o g y s i n c e t h e b i o c h e m i c a l c o m p o s i t i o n o f m o s t p l a n t p a t h o g e n s and p e s t s i s n o t w e l l u n d e r s t o o d . S u b s e q u e n t l y , t h e s p e c i f i c a n t i b o d i e s c a n be u s e d t o c h a r a c t e r i z e d i f f e r e n c e s among p a t h o g e n s a t t h e g e n u s , s p e c i e s , and e v e n r a c e l e v e l s . W h i l e t h e b u l k o f w o r k i n t h i s a r e a r e p o r t e d t o d a t e has i n v o l v e d p l a n t v i r u s e s , m o n o c l o n a l a n t i b o d i e s have been p r o d u c e d a g a i n s t p l a n t p a t h o g e n i c b a c t e r i a , s p i r o p l a s m a s , m y c l o p l a s m a - l i k e o r g a n i s m s , and f u n g i ( 1 7 , 1£, 2 0 ) . The b e n e f i c i a l c h a r a c t e r i s t i c s of monoclonal a n t i b o d i e s are e s p e c i a l l y a p p l i c a b l e t o p l a n t d i s e a s e d i a g n o s t i c s due t o t h e c o m p l e x i t y o f a n t i g e n p r e p a r a t i o n s , d i f f i c u l t y i n o b t a i n i n g o b l i g a t e p a r a s i t e s , and t h e l a c k o f b a s i c i n f o r m a t i o n on t h e b i o c h e m i c a l s t r u c t u r e o f p l a n t pathogens. D i a g n o s t i c p r o d u c t s b a s e d on a n t i b o d i e s w i l l p r o v i d e v a l u a b l e i n f o r m a t i o n t o g u i d e many c r o p d i s e a s e management d e c i s i o n s ( 1 8 , 1 9 ) . Some o f t h e m o r e i m p o r t a n t u s e s o f i m m u n o a s s a y s i n d e t e c t i n g p l a n t p a t h o g e n s and p e s t s i n c l u d e : Rapid diagnosis of disease to confirm a v i s u a l d i a g n o s i s . T h i s w i l l be m o s t u s e f u l w h e n s y m p t o m s a r e s p a r s e , u n d e v e l o p e d , o r a r e p r e s e n t on r o o t s . B e c a u s e o f the s p e c i f i c i t y of the antibody reagents used, primary p a t h o g e n s c a n be d e t e c t e d e v e n i f s e c o n d a r y o r g a n i s m s have invaded the t i s s u e . M u l t i p l e i n f e c t i o n s i n which m o r e t h a n one p a t h o g e n i s p r e s e n t c a n a l s o be d e t e c t e d . Presymptomatic d e t e c t i o n of i n f e c t i o n i n which a c t i v e i n f e c t i o n s a r e d e t e c t e d b e f o r e s e c o n d a r y s p r e a d and s e r i o u s economic l o s s are s u s t a i n e d . D e t e c t i o n and q u a n t i f i c a t i o n o f p a t h o g e n s w i l l a l l o w d e t e r m i n a t i o n o f p e s t and p a t h o g e n l e v e l s i n s o i l , p l a n t i n g m e d i a , w a t e r and p r o p a g a t i n g m a t e r i a l s s u c h as s e e d s and c u t t i n g s . L e v e l s c a n t h e n be r e l a t e d t o f u t u r e


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y i e l d l o s s so t h a t a p p r o p r i a t e management measures c a n be e m p l o y e d .


The u s e o f t h e i n f o r m a t i o n g e n e r a t e d w i t h i m m u n o a s s a y s i s t h e c r i t i c a l factor i n determining the ultimate u t i l i t y of this t e c h n o l o g y i n a g r i c u l t u r e . C r o p m a n a g e m e n t d e c i s i o n s w h i c h may b e i n f l u e n c e d b y s p e c i f i c i n f o r m a t i o n on p e s t a n d p a t h o g e n l e v e l s include: C r o p r o t a t i o n p a t t e r n s b a s e d on t h e p r e s e n c e or absence o f t h r e a t e n i n g l e v e l s o f pathogens or pests. P l a n t i n g decisions f o r s p e c i f i c f i e l d s or groups o f f i e l d s can be made w i t h k n o w l e d g e o f p e s t p o p u l a t i o n s a n d p r o b a b l e need f o r c h e m i c a l treatments. V a r i e t y s e l e c t i o n w i l l be i n f l u e n c e d s i m i l a r l y . F o r example, a high y i e l d i n g but d i s e a s e - s u s c e p t i b l e v a r i e t y m i g h t be p l a n t e d i f immunoassays i n d i c a t e r e l a t i v e l y l o w pathogen l e v e l s i n a c e r t a i n f i e l d . Choice of chemical treatment e i t h e r at p l a n t i n g or d u r i n g t h e g r o w i n g s e a s o n c a n b e b a s e d on i n f o r m a t i o n o n p e s t d i s t r i b u t i o n , d e n s i t y , and i d e n t i t y . Such i n f o r m a t i o n c a n be used b y t h e k n o w l e d g e a b l e g r o w e r o r crop c o n s u l t a n t t o s e l e c t c h e m i c a l s , use r a t e s , and t i m i n g o f a p p l i c a t i o n w i t h i n t h e ranges approved f o r use. C e r t i f i c a t i o n o f seeds and o t h e r p r o p a g a t i n g m a t e r i a l s as being d i s e a s e - f r e e o r d i s e a s e - t e s t e d i s another short term a p p l i c a t i o n o f immunoassays. Commercial products o r s e r v i c e s employing antibody-based t e s t s are a v a i l a b l e f o r d e t e c t i n g v i r u s e s i n seed p o t a t o e s and l e t t u c e seed. These assays a r e p e r f o r m e d p r i m a r i l y i n c e n t r a l l a b o r a t o r i e s , b u t s i m p l i f i c a t i o n of assay f o r m a t s and p r o c e d u r e s w i l l p e r m i t seed c r o p s t o be m o n i t o r e d more c l o s e l y throughout the production c y c l e t o assure that e l i t e stock remains pathogen or p e s t - f r e e . D i a g n o s t i c m a t e r i a l s f o r f i e l d use a r e under development a t a number o f c o m m e r c i a l c o n c e r n s and r e s e a r c h c e n t e r s . Over t h e n e x t 3-5 y e a r s s e v e r a l i m m u n o a s s a y s i n k i t f o r m w i l l b e a v a i l a b l e t o c r o p managers t o use as t o o l s f o r managing a g r o n o m i c p r a c t i c e s . The m a n a g e r w i l l u s e i n f o r m a t i o n f r o m d i a g n o s t i c k i t s a s a p o r t i o n of t h e t o t a l i n f o r m a t i o n t h a t goes i n t o t h e complex judgements made i n s u c c e s s f u l m o d e r n a g r i c u l t u r e . D e t e c t i o n and Q u a n t i f i c a t i o n o f Crop P r o t e c t i o n Chemicals. Immunoassay procedures p r o v i d e a d d i t i o n a l a n a l y t i c a l t o o l s f o r d e t e r m i n i n g t h e presence and q u a n t i t y o f s p e c i f i c crop p r o t e c t i o n c h e m i c a l s i n v a r i o u s m a t r i c e s . T h e s p e c i f i c i t y o f an i m m u n o a s s a y i s provided by t h e antibody reagent r a t h e r than by t h ea n a l y t i c a l a p p a r a t u s . The s p e c i f i c i t y , as noted above, a l l o w s t h e a n a l y s i s o f r e l a t i v e l y crude preparations r a p i d l y i f t h e appropriate reagents ( i e . antibodies) are r e a d i l y a v a i l a b l e . Since a s p e c i f i c reagent must be p r e p a r e d f o r e a c h a n a l y t e , t h e g r e a t e s t u t i l i t y f o r t h e s e



19.

L A N K O W ET

AL.

Immunoassays for


245

a s s a y s l i e s i n a n a l y s i s o f c r o p , s o i l and e n v i r o n m e n t a l s a m p l e s f o r s p e c i f i c p e s t i c i d e s and a l i m i t e d n u m b e r o f d e g r a d a t i o n products. S u c h a s s a y s c a n g e n e r a l l y be c o n d u c t e d w i t h much c r u d e r p r e p a r a t i o n s than are p o s s i b l e w i t h c o n v e n t i o n a l methods (HPLC a n d / o r GLC) w h i c h r e s u l t s i n t i m e and c o s t s a v i n g s . One i m m u n o a s s a y m e t h o d ( 2 2 ) f o r t h e i n s e c t g r o w t h r e g u l a t o r d i f l u b e n z u r o n was l e s s t h a n 10% as c o s t l y as H P L C o r GLC, a l l o w e d 10-50 t i m e s as m a n y s a m p l e s t o be a n a l y z e d i n one d a y , and r e q u i r e d lower c a p i t a l costs (Table 1). Assay s e n s i t i v i t y i n s t a g n a n t w a t e r and m i l k was a t l e a s t e q u a l t o t h a t o b t a i n e d w i t h t h e s t a n d a r d m e t h o d s . The g r e a t e s t t i m e and c o s t s a v i n g s r e s u l t e d from the r e d u c t i o n or e l i m i n a t i o n of cleanup steps r e q u i r e d . Other authors have r e p o r t e d s i m i l a r r e s u l t s f o r a v a r i e t y of p e s t i c i d e s a n a l y z e d w i t h i m m u n o l o g i c a l a s s a y s (23!, 2 4 ) . TABLE 1 COMPARISON OF H P L C , GLC, AND IMMUNOASSAY Analyte: Diflubenzuron i n s e c t i c i d e Substrates: Stagnant Water, M i l k Reagent Cost per Sample

Sensiti vity Limit

Method

Matrix

Clean Up Steps

HPLC ELISA

Water Water

5 0

5 >50

1.6 H r 5 mi η

$4.00 0.20

lOppb l.Oppb

250ml 0.5ml

GLC ELISA

Milk Milk

19 0

0.5 >50

16 H r 7 mi η

$11.00 0.20

50ppb 40ppb

20ml 0.2ml

Assays Per man-day

Labor: Sample

Adapted from

Sample Size

(22)

I m m u n o a s s a y s f o r p e s t i c i d e a n a l y s i s c a n r e a d i l y be s t r u c t u r e d as l a b o r a t o r y a n a l y t i c a l m e t h o d s e m p l o y i n g m a n u a l , s e m i a u t o m a t e d and a u t o m a t e d f o r m a t s . The k e y s t e p i n d e v e l o p i n g t h e a n a l y t i c a l p r o c e d u r e i s g e n e r a t i n g a n t i b o d i e s w h i c h r e c o g n i z e and b i n d t o t h e p r o p e r c h e m i c a l s p e c i e s . T a r g e t m o l e c u l e s m u s t be c o v a l e n t l y l i n k e d t o c a r r i e r p r o t e i n s i n s u c h a way t h a t t h e c h a r a c t e r i s t i c p o r t i o n s of the t a r g e t are f r e e to bind w i t h the v a r i a b l e regions o f an a n t i b o d y . I t i s necessary, t h e r e f o r e , that the exact s t r u c t u r e o f t h e t a r g e t m o l e c u l e s and o f n o n - t a r g e t c h e m i c a l s be known b e f o r e a n t i b o d i e s a r e d e v e l o p e d . One o f t h e m a j o r d i f f e r e n c e s b e t w e e n i m m u n o a s s a y s and c o n v e n t i o n a l a n a l y t i c a l procedures, t h a t immunoassays f o r small molecules are not g e n e r a l l y s u i t a b l e f o r d e t e c t i n g or determining the s t r u c t u r e of u n k n o w n m a t e r i a l s . I m m u n o a s s a y s may be u s e f u l f o r i n d i c a t i n g w h e t h e r an u n k n o w n s u b s t a n c e s h a r e s some common s t r u c t u r e w i t h a k n o w n m a t e r i a l f o r w h i c h an a n t i b o d y e x i s t s , h o w e v e r . Once a n t i b o d i e s t o a s p e c i f i c c h e m i c a l have been g e n e r a t e d , t h e y m u s t be e v a l u a t e d t o d e t e r m i n e t h e r a n g e o f s t r u c t u r a l v a r i a t i o n the a n t i b o d i e s recognize. Antibodies with d i f f e r i n g l e v e l s o f s p e c i f i c i t y c a n o f t e n be g e n e r a t e d t h r o u g h c a r e f u l d e s i g n o f t h e immunogen. For e x a m p l e , c l a s s - s p e c i f i c a n t i b o d i e s c a n be d e v e l o p e d b y l e a v i n g a common m o i e t y e x p o s e d s o t h a t



246


a n t i b o d i e s t h a t r e c o g n i z e f e a t u r e s s h a r e d by a f a m i l y o f a n a l o g s c a n be s e l e c t e d . I n m a n y i n s t a n c e s c r o s s - r e a c t i v i t y i s u n d e s i r a b l e a n d a n t i b o d i e s m u s t be d e v e l o p e d w h i c h do n o t r e c o g n i z e c l o s e l y r e l a t e d m o l e c u l e s . Many e x a m p l e s e x i s t o f t h e d e v e l o p m e n t o f h i g h l y s p e c i f i c a n t i b o d i e s . Wie and Hammock (25») r e p o r t e d p o l y c l o n a l a n t i s e r a which d i f f e r e n t i a t e d the i n s e c t e d d i f l u b e n z u r o n and t h e c h e m i c a l l y r e l a t e d c o m p o u n d , BAY-SIR 8514. In extreme examples, s e v e r a l workers have r e p o r t e d a n t i b o d y r e a g e n t s w h i c h d i s t i n g u i s h e d o p t i c a l l y a c t i v e i s o m e r s and c i s - and t r a n s - forms of the t a r g e t molecule. S e n s i t i v i t y of immunoassays f o r small molecules i s g e n e r a l l y c o m p a r a b l e t o t h a t o b t a i n e d w i t h HPLC and GLC m e t h o d s . As w i t h a l l a n a l y t i c a l p r o c e d u r e s , s e v e r a l q u a l i f y i n g s t a t e m e n t s m u s t be made r e g a r d i n g a b s o l u t e s e n s i t i v i t y o f an i m m u n o a s s a y . T h e l e v e l o f a n a l y t e d e t e c t e d w i l l vary w i t h the s u b s t r a t e being examined, i n t e r f e r i n g s u b s t a n c e s , and t h e n a t u r e o f t h e a n a l y t e and r e a g e n t s t h e m s e l v e s . M o s t i m m u n o a s s a y s c a n be s u c c e s s f u l l y p e r f o r m e d o n c r u d e e x t r a c t s o f c r o p and e n v i r o n m e n t a l s a m p l e s w i t h no o r m i n i m a l c l e a n - u p s t e p s . I n c l i n i c a l u s e , h i g h l y p r e c i s e and a c c u r a t e a n a l y s e s a r e r o u t i n e l y p e r f o r m e d on p a t i e n t s a m p l e s w i t h no s a m p l e p r e p a r a t i o n o t h e r t h a n f i l t r a t i o n and d i l u t i o n . The k e y f a c t o r i n t h i s i n s t a n c e i s d e v e l o p i n g an a n t i b o d y r e a g e n t w h i c h h a s a h i g h a f f i n i t y f o r t h e a n a l y t e . The a n t i b o d y : a n t i g e n ( o r analyte) r e a c t i o n i s r e v e r s i b l e , consequently assay s e n s i t i v i t y w i l l be p r o p o r t i o n a l t o s t r e n g t h o f t h e b i n d i n g a n d t o t h e n a t u r e o f t h e s i g n a l g e n e r a t e d by t h e a n t i g e n : a n t i b o d y complex. T h e a b i l i t y t o p e r f o r m q u a n t i t a t i v e a s s a y s on c o m p l e x m i x t u r e s w i t h l i t t l e sample c l e a n - u p i s perhaps the most a t t r a c t i v e f e a t u r e of immunoassays f o r a p p l i c a t i o n t o a g r i c u l t u r a l c h e m i s t r y . A l a r g e p o r t i o n o f t h e c o s t and l a b o r i n v o l v e d 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 i s i n v e s t e d i n s a m p l e e x t r a c t i o n and c l e a n - u p s t e p s t o r e m o v e s u b s t a n c e s w h i c h may i n t e r f e r e w i t h subsequent c h e m i c a l a n a l y s i s . S i n c e most p r e p a r a t o r y s t e p s are not r e q u i r e d p r i o r t o p e r f o r m i n g an i m m u n o a s s a y , s a m p l e s c a n b e a n a l y z e d much l e s s e x p e n s i v e l y . T h i s w i l l p e r m i t t h e v a s t n u m b e r o f d a t a p o i n t s r e q u i r e d f o r p e s t i c i d e r e g i s t r a t i o n t o be g a t h e r e d i n a m o r e t i m e l y and c o s t - e f f e c t i v e m a n n e r . S t u d i e s w h i c h w e r e p r o h i b i t i v e l y e x p e n s i v e because t h e y would have r e q u i r e d l a r g e n u m b e r s o f e x p e n s i v e a s s a y s c a n be c o m p l e t e d u s i n g i m m u n o a s s a y p r o c e d u r e s . S u c h s t u d i e s may i n c l u d e a n a l y s i s o f p e s t i c i d e m o v e m e n t f r o m a p p l i c a t i o n a r e a s and t h e r a t e o f d i s s i p a t i o n o f p e s t i c i d e f r o m c r o p t i s s u e , s o i l s , and p r o c e s s e d f o o d s . A second h i g h l y a t t r a c t i v e f e a t u r e of immunoassays i s the s p e e d o f most p r o c e d u r e s and t h e number o f a s s a y s t h a t can b e c o m p l e t e d i n one day. U s i n g s e m i - a u t o m a t e d e q u i p m e n t c o u p l e d w i t h a p e r s o n a l c o m p u t e r , a s i n g l e p e r s o n c a n g e n e r a t e 1,000 t o 1 0 , 0 0 0 d a t a p o i n t s per day. S i n c e the s p e c i f i c i t y of the a n a l y s i s i s p r o v i d e d b y t h e r e a g e n t , t h e e q u i p m e n t u s e d c a n be a p p l i e d t o m a n y d i f f e r e n t assays. Again the only l i m i t a t i o n i s the supply of high quality antibody reagents. A l t h o u g h a n t i b o d y - b a s e d a s s a y s h a v e many a t t r a c t i v e f e a t u r e s , we m u s t e m p h a s i z e t h a t c l a s s i c a l a n a l y t i c a l m e t h o d s m u s t be u s e d t o c o n f i r m and v a l i d a t e i m m u n o a s s a y s w h i c h a r e u s e d f o r q u a n t i t a t i v e and/or q u a l i t a t i v e a n a l y s i s of s p e c i f i c m o l e c u l a r s t r u c t u r e s . S i n c e most i m m u n i z a t i o n p r o c e d u r e s g e n e r a t e a n t i b o d i e s



19.

LANKOW ET

AL.

Immunoassays for


247

t o c a r r i e r p r o t e i n s and l i n k i n g m o i e t i e s as w e l l as t o t h e t a r g e t c h e m i c a l , v a r i o u s c o n v e n t i o n a l m e t h o d s i n c l u d i n g MNR, I R , and m a s s s p e c t o s c o p y m u s t be e m p l o y e d t o a s s u r e t h a t t h e a s s a y d e t e c t s t h e target molecule. Q u a n t i f i c a t i o n o f an a n a l y t e i n an a g r i c u l t u r a l s a m p l e r e l a t e s t o b o t h t h e a s s a y p r o c é d u r e and t h e s a m p l e p r e p a r a t i o n procedure. C o n v e n t i o n a l a n a l y s e s f r e q u e n t l y f o c u s on t o t a l r e s i d u e s and may e m p l o y r e l a t i v e l y h a r s h e x t r a c t i o n p r o c e d u r e s t o remove bound m a t e r i a l s from the s o i l or c r o p sample. The b i o l o g i c a l n a t u r e o f a n t i b o d i e s r e q u i r e s t h a t m o s t i m m u n o a s s a y s be r u n i n a q u e o u s s y s t e m s a t a pH n e a r n e u t r a l i t y . I f t o t a l r e s i d u e s a r e t o be m e a s u r e d and e x t r a c t i o n r e q u i r e s t h e u s e o f o r g a n i c s o l v e n t s , strong a c i d s , or s t r o n g bases the e x t r a c t e d m a t e r i a l s m u s t be d i s p e r s e d i n an a q u e o u s m e d i u m p r i o r t o u s i n g an i m m u n o a s s a y . M o d e r a t e l e v e l s o f m e t h a n o l ( < 1 0 % ) and o t h e r w a t e r m i s c i b l e s o l v e n t s do n o t i n t e r f e r e w i t h m o s t i m m u n o a s s a y s . Since immunoassays are aqueous, they are i d e a l f o r d e t e c t i n g and m e a s u r i n g b i o l o g i c a l l y a c t i v e r e s i d u e s . A q u e o u s e x t r a c t i o n o f s o i l s f o l l o w e d b y r a p i d i m m u n o a s s a y c o u l d p r o v i d e t i m e l y d a t a on p o t e n t i a l l y p h y t o t o x i c l e v e l s of h e r b i c i d e r e s i d u e s or c o u l d d e t e r m i n e w h e t h e r i n s e c t i c i d e l e v e l s had f a l l e n b e l o w t h e b i o l o g i c a l l y s i g n i f i c a n t p o i n t . D a t a o f t h i s n a t u r e c a n be u s e d b y c r o p m a n a g e r s as t h e y d e t e r m i n e c r o p r o t a t i o n p a t t e r n s , p e s t i c i d e p u r c h a s i n g and a p p l i c a t i o n p l a n s , and r e a p p l i c a t i o n t i m i n g . T h e s e a n a l y s e s c o u l d be p e r f o r m e d r e a d i l y i n s o i l n u t r i e n t l a b o r a t o r i e s , c h e m i c a l company a n a l y t i c a l d e p a r t m e n t s , or s i m p l e l a b o r a t o r i e s at co-ops or chemical d i s t r i b u t o r companies. L o c a l A n a l y s i s . One o f t h e m o s t p r o m i s i n g a p p l i c a t i o n s f o r immunoassays i n a g r i c u l t u r a l c h e m i s t r y i s l o c a l i z e d a n a l y s i s of p e s t i c i d e r e s i d u e s . By t h i s we mean q u a n t i t a t i v e d e t e c t i o n o f s p e c i f i c r e s i d u e s at s i t e s remote from h i g h l y s o p h i s t i c a t e d , e x p e n s i v e l y e q u i p p e d c e n t r a l l a b o r a t o r i e s . The a v a i l a b i l i t y o f l o c a l i z e d t e s t i n g w i l l speed data g e n e r a t i o n , encourage g r e a t e r v o l u m e s o f d a t a t o be g e n e r a t e d , and a l l o w a n a l y t i c a l i n f o r m a t i o n t o be u t i l i z e d i n m a k i n g c r o p m a n a g e m e n t d e c i s i o n s . C u r r e n t l y t h e o n l y c o m p a r a b l e p r a c t i c e i s s o i l and p l a n t n u t r i e n t a n a l y s i s i n w h i c h f e r t i l i z a t i o n p r o g r a m s a r e i n f l u e n c e d by a n a l y t i c a l r e s u l t s . T h i s t y p e o f t e s t i n g c a n be c o n d u c t e d w i t h s i m p l e a s s a y f o r m a t s w h i c h e m p l o y no i n s t r u m e n t a t i o n o r w i t h m o r e s o p h i s t i c a t e d a s s a y s w h i c h u t i l i z e i n e x p e n s i v e i n s t r u m e n t a t i o n . The k e y f e a t u r e s o f i m m u n o a s s a y s t h a t make t h i s p o s s i b l e a r e t h o s e d i s c u s s e d e a r l i e r : a s s a y s p e c i f i c i t y c o n f e r r e d b y t h e r e a g e n t and r e a c t i o n s v i s u a l i z e d without r a d i o i s o t o p e s or expensive instruments. O n c e a n a l y t i c a l c a p a b i l i t y and r a p i d t u r n a r o u n d t i m e a r e a v a i l a b l e l o c a l l y o r on t h e f a r m , m a n y c r o p m a n a g e m e n t d e c i s i o n s w i l l be g u i d e d b y t h i s t y p e o f a n a l y s i s . One e x a m p l e a v a i l a b l e commercially allows s t a t e i n s p e c t o r s to r a p i d l y check that p e s t i c i d e c o n t a i n e r s have been p r o p e r l y r i n s e d b e f o r e t h e y are d i s c a r d e d . C e r t a i n s t a t e s r e q u i r e a d e p o s i t on a l l r e s t r i c t e d p e s t i c i d e c o n t a i n e r s . Deposits are refunded o n l y i f the c o n t a i n e r being returned passes a r a p i d assay which confirms that the c o n t a i n e r h a s b e e n p r o p e r l y r i n s e d . The t e s t s c u r r e n t l y u s e d f o r t h i s o n - s i t e a n a l y s i s a r e b a s e d on i m m u n o a s s a y s as w e l l as a c o l o r i m e t r i c c h o l i n e s t e r a s e i n h i b i t i o n assay (26).

American Chemical Society! Library 1155

16th

St., N.W.

LeBaron et al.; Biotechnology in Agricultural Chemistry Washington, DXL 20038 ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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O t h e r o n - s i t e immunoassays f o r c h e m i c a l s w i l l become i n c r e a s i n g l y a v a i l a b l e o v e r t h e n e x t 5-10 y e a r s . T h e s e t e s t s w i l l impact a number o f e c o n o m i c c r o p management d e c i s i o n s i n c l u d i n g :


C r o p r o t a t i o n p a t t e r n s b a s e d on h e r b i c i d e l e v e l s f r o m p r e v i o u s years treatments. T h i s i n f o r m a t i o n would be c o n s i d e r e d a l o n g w i t h d a t a on p a t h o g e n a n d p e s t l e v e l s as d i s c u s s e d e a r l i e r . C h o i c e o f c h e m i c a l t r e a t m e n t b a s e d on e x i s t i n g c h e m i c a l r e s i d u e s , t h e p e s t t o be c o n t r o l l e d , and p o s s i b l e interactions with other chemicals. T i m i n g o f t r e a t m e n t a p p l i c a t i o n based upon t h e r a t e o f loss of a c t i v e i n g r e d i e n t from e a r l i e r treatments. H a r v e s t s c h e d u l i n g b a s e d on r e s i d u e l e v e l s i n i n d i v i d u a l f i e l d s . P r e h a r v e s t i n t e r v a l s a r e c u r r e n t l y based upon "worst case" i n f o r m a t i o n from d e t a i l e d chemical m a n u f a c t u r e r t r i a l s . I n t h e f u t u r e , h a r v e s t d a t e s may be s c h e d u l e d by d e t e r m i n i n g w h e t h e r r e s i d u e l e v e l s i n i n d i v i d u a l f i e l d s have reached l e g a l t o l e r a n c e s . Re-entry period f o l l o w i n g a p p l i c a t i o n of a r e s t r i c t e d u s e p e s t i c i d e i s an a r e a o f g r o w i n g c o n c e r n . Rapid i m m u n o a s s a y s c a n be e m p l o y e d t o d e t e r m i n e t h e d i s l o d g e a b l e r e s i d u e of s p e c i f i c compounds p r i o r t o allowing farm workers to enter a f i e l d . On-site i m m u n o a s s a y m e t h o d s w i l l p e r m i t and e n c o u r a g e m o r e a c t i v e management o f work e x p o s u r e . D e t e c t i o n and Q u a n t i f i c a t i o n o f N a t u r a l l y O c c u r r i n g Compounds. A n t i b o d i e s c a n be p r e p a r e d f o r n a t u r a l l y o c c u r r i n g c o m p o u n d s a s w e l l as f o r p e s t i c i d e s and d r u g s . T h i s o p e n s t h e way f o r d e v e l o p i n g r a p i d i m m u n o a s s a y s f o r p l a n t and m i c r o b i a l p r o d u c t s s u c h as m y c o t o x i n s , p l a n t h o r m o n e s , and h i g h v a l u e p l a n t c o m p o n e n t s s u c h as f l a v o r and f r a g r a n c e c o m p o u n d s and pharmaceutical precursors. S e v e r a l r e s e a r c h groups have r e p o r t e d a n t i b o d i e s f o r a f l a t o x i n s and o t h e r m y c o t o x i n s ( 2 7 ) . C o m m e r c i a l k i t s f o r a f l a t o x i n d e t e c t i o n i n v a r i o u s s u b s t r a t e s h a v e b e e n a n n o u n c e d . The i n t r o d u c t i o n of such k i t s w i l l permit o n - s i t e d e t e c t i o n of a f l a t o x i n s t o be c o n f i r m e d i m m e d i a t e l y r a t h e r t h a n h a v i n g t o w a i t for a n a l y t i c a l r e s u l t s from a remote l a b o r a t o r y f o l l o w i n g d e t e c t i o n of f l u o r e s c i n g m a t e r i a l s i n a commodity. Since a f l a t o x i n s and o t h e r m i c r o b i a l t o x i n s h a v e a n u m b e r o f s t r u c t u r a l v a r i a t i o n s , the a n t i b o d i e s used i n t h e i r a n a l y s i s must b e c a r e f u l l y s e l e c t e d t o a s s u r e t h a t t h e p r o p e r compounds are b e i n g d e t e c t e d and a c c u r a t e l y m e a s u r e d . P l a n t h o r m o n e a n a l y s i s t e n d s t o be a v e r y d i f f i c u l t t a s k due i n p a r t t o t h e low l e v e l s t h a t o c c u r n a t u r a l l y . Over the p a s t s e v e r a l y e a r s Wei 1 e r a n d h i s c o w o r k e r s a n d a n u m b e r o f o t h e r g r o u p s have d e v e l o p e d e x t r e m e l y s e n s i t i v e i m m u n o a s s a y s f o r many o f t h e n a t u r a l p l a n t h o r m o n e s (213). T h e s e a s s a y s h a v e g r e a t l y r e d u c e d t h e t i m e and e f f o r t r e q u i r e d t o a n a l y z e i n d o l e a c e t i c a c i d , c y t o k i n i n s , g i b b e r e l l i n s , and a b s c i s i c a c i d .



19.

L A N K O W ET

AL.


249

A n a l y s i s o f Crop Components. The a b i l i t y o f a n t i b o d i e s t o b i n d t o a v a r i e t y o f m o l e c u l e s l e a d s t o many a d d i t i o n a l a p p l i c a t i o n s i n c r o p a g r i c u l t u r e . Once t h e n a t u r e o f a t a r g e t m o l e c u l e i s known, a n t i b o d i e s c a n be p r e p a r e d a n d a n a p p r o p r i a t e a s s a y procedure d e v i s e d . F o r example, a n t i b o d i e s produced t o soybean seed p r o t e i n s are c u r r e n t l y b e i n g used t o d e t e c t soybean e x t e n d e r s i n meat p r o d u c t s . S i m i l a r a s s a y s c o u l d be u s e d d i r e c t l y on s o y b e a n s e e d t o s e l e c t breeding m a t e r i a l s which are q u a n t i t a t i v e l y or q u a l i t a t i v e l y d i f f e r e n t f r o m e x i s t i n g l i n e s . D u r i n g p r o d u c t i o n an a s s a y c o u l d be u s e d t o m o n i t o r s e e d m a t u r a t i o n t o g u i d e h a r v e s t s c h e d u l i n g . S i m i l a r l y , processors c o u l d employ r a p i d assays f o r key p l a n t components r e l a t i n g t o q u a l i t y o r i n t e n d e d end use o f the commodity. Isoenzyme a n a l y s i s and e l e c t r o p h o r e t i c s e p a r a t i o n have been employed t o d i f f e r e n t i a t e p l a n t s p e c i e s and v a r i e t i e s b u t a r e slow and r e l a t i v e l y c u m b e r s o m e p r o c e d u r e s . The b a c k g r o u n d i n f o r m a t i o n d e v e l o p e d has begun t o i d e n t i f y s p e c i f i c m a r k e r s c o r r e l a t e d w i t h d e s i r e d agronomic c h a r a c t e r i s t i c s . U s i n g a n t i b o d i e s and i m m u n o a s s a y s i t i s now p o s s i b l e u s i n g t h e s e r a p i d t e s t s t o i d e n t i f y p l a n t s c a r r y i n g s p e c i f i c genes. A p p l i c a t i o n s o f t h i s a s p e c t o f a g r i c u l t u r a l i m m u n o a s s a y a p p l y t o t h e p l a n t b r e e d e r who may now s c r e e n l a r g e p o p u l a t i o n s o f s e e d l i n g s f o r t h o s e i n d i v i d u a l s which express the s p e c i f i c marker. Further a p p l i c a t i o n s i n c l u d e m o n i t o r i n g seed p r o d u c t i o n f i e l d s f o r g e n e t i c p u r i t y , seed c e r t i f i c a t i o n , and v a r i e t y i d e n t i f i c a t i o n a t g r a i n e l e v a t o r s and p r o c e s s i n g p l a n t s . As g e n e t i c t r a i t s a r e e n g i n e e r e d i n t o c r o p s i m m u n o a s s a y s c a n be u s e d t o t r a c k a n d c e r t i f y t h e new v a r i e t y . T r a i t s such as s p e c i f i c h e r b i c i d e r e s i s t a n c e a r e t a r g e t s f o r r a p i d a s s a y s s i n c e g r o w e r s may n o t w i s h t o r i s k u s i n g t h e h e r b i c i d e u n t i l i t i s known t h a t t h e c r o p i s i n d e e d e x p r e s s i n g herbicide resistance prior to applying the p o t e n t i a l l y phytotoxic material. As b a s i c i n f o r m a t i o n i n p l a n t s c i e n c e i n c r e a s e s a d d i t i o n a l m a r k e r s w i l l b e i d e n t i f i e d f o r w h i c h i m m u n o a s s a y s c a n be developed. Rapid determination of the n u t r i e n t status of the growing crop w i l l a l l o w crop managers t o s e l e c t f e r t i l i z a t i o n t h a t p r o d u c e s m a x i m u m r e t u r n b a s e d on t h e s p e c i f i c n e e d s o f t h e c r o p a s i t grows i n a s p e c i f i c l o c a t i o n ( 2 9 h S i m i l a r l y , d e t e c t i o n o f w a t e r s t r e s s m a r k e r s w i l l p e r m i t i r r i g a t i o n s c h e d u l i n g b a s e d on c r o p w a t e r s t a t u s r a t h e r t h a n on s o i l m o i s t u r e l e v e l s o r o t h e r environmental measures. Conclusions Immunoassays a r e powerful a n a l y t i c a l t o o l s t h a t p r o v i d e s e n s i t i v i t y and s p e c i f i c i t y c o m p a r a b l e t o t r a d i t i o n a l c h e m i c a l a n a l y s e s . The a d v a n t a g e s o f immunoassays l i e i n t h e i r s p e e d , s i m p l i c i t y , and r e l a t i v e l y l o w c o s t . T h e s e f a c t o r s have f o s t e r e d t h e r a p i d g r o w t h o f t h e m e d i c a l d i a g n o s t i c i n d u s t r y a n d h a v e made p o s s i b l e an a g r i c u l t u r a l d i a g n o s t i c s i n d u s t r y . T h r o u g h o u t t h e c r o p p r o d u c t i o n c y c l e , m a n a g e r s m u s t make a s e r i e s o f e c o n o m i c d e c i s i o n s a b o u t t h e i r c r o p s ( F i g u r e 5 ) . As t h e season progresses, each d e c i s i o n r e p r e s e n t s a g r e a t e r expense l e v e l and, c o n s e q u e n t l y , a g r e a t e r p o t e n t i a l l o s s . Immunoassay can



250


KEY CROP INPUTS-USA

Seed $4 Billion Pesticides $5 Billion Disease Losses $7 Billion Post Harvest Loss $8 Billion Fertilizers $9 Billion Irrigation $5 Billion

Breeder ;: Biochemical, Resistance ^Markers: Proprietary

k G rower k

Variety Selection 1 Selection

CROP OUTPUT—USA

j Treatment

$80 Billion

I Insecticide / Nematicide I Fungicide ^Seedling Disease r Fungicide ^Insecticide Treatment t Supplement ^Irrigation Timing, Quantity ^Harvest Date - Quality j Protein, Oil

t_ Processor k Post Harvest Quality t Harvest Components ^Processor-Components

F i g u r e 5. C r o p p r o d u c t d e c i s i o n s i n f l u e n c e t h e u t i l i z a t i o n o f v a r i a b l e c r o p i n p u t s . Crop management d e c i s i o n s o c c u r through a l l stages o f the production c y c l e . Antibody based assays can p r o v i d e a c c u r a t e , t i m e l y i n f o r m a t i o n t o support such d e c i s i o n s .



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be a p p l i e d t h r o u g h o u t t h e c r o p p r o d u c t i o n c y c l e t o p r o v i d e s p e c i f i c i n f o r m a t i o n . The crop manager uses e x p e r i e n c e , o u t s i d e i n f o r m a t i o n s o u r c e s , and c a r e f u l e v a l u a t i o n o f t h e crop t o develop o p t i o n s f o r managing resources and r e t u r n s . T h e l e v e l o f f a r m i n p u t s i n t h e U.S. i s v e r y l a r g e ( F i g u r e 5). A s p r e s s u r e on p r o d u c e r s i n c r e a s e s , t h e i n e f f i c i e n t w i l l n o t be s u c c e s s f u l . T h e 5 0 , 0 0 0 g r o w e r s who a r e p r o j e c t e d t o b e t h e source o f 7 5 % o f A m e r i c a ' s food and f i b e r by t h e Year 2000 w i l l adopt p r o c e d u r e s and products t o m a x i m i z e t h e i r e f f i c i e n c y . Immunoassays w i l l c o n t r i b u t e t o t h a t e f f i c i e n c y by p r o v i d i n g c o s t - e f f e c t i v e i n f o r m a t i o n a t t h e f a r m , l o c a l , r e g i o n a l , and national level.

Literature Cited 1. Strike, L. E., Devens, Β. H. and Lundak, R.L. J. Immunol., 1984, 132, 1798-1803. 2. Barrett, J. H. "Textbook of Immunology"; C.V. Mosby Company: St. Louis, 1983; Chapter 12, 242-254. 3. Porter, R. R. Science 1973, 180, 713. 4. Ishizaka, T . , Ishizaka, K . , Borsos, T. and Rapp, H. J. J. Immunol., 1966, 97, 716. 5. Wie, S. I., Sylwester, A. P., Wing, K. D. and Hammock, B. D. J. Agric. Food Chem. 1982, 30, 943-948. 6. Kabat, E . A. "Structural Concepts in Immunology and Immunochemistry"; Holt, Rinehart, and Winston. 1976. 7. Scott, M. G. Trends in Biotechnology, 1985, Vol. 3, No. 7, 170-175. 8. Kohler, G. and Milstein, C. Nature (London) 1975, 256, 495. 9. Stern, N. J. and Gamble, H. R. "Hybridoma Technology in Agriculture and Veterinary Research". Rowman and Allanheld. Totowa, New Jersey. 1984. 10. Weir, D. M. "Handbook of Experimental Immunology"; Volume I; Immunochemistry. Blackwell Scientific Publications: Oxford, 1978. 11. Kolb, W. P. and Muller-Eberhard, H. J. J. Immunol. 1974, 113, 4789. 12. Langone, J. J. and Vanvunakis, H. "Methods in Enzymology, Immunochemical Techniques, Part E " . Academic Press, New York. 1983. 13. Hsu, S. M. Raine, L. and Fanger, H. J . Histochem. Cytochem., 1981, Vol. 29, No. 4, 577-580. 14. Ngo, T. T.; Lenhoff, H. M. "Enzyme-Mediated Immunoassay" Plenum Press: New York, 1985. 15. Yalow, R. S. Pharmacol. Reviews, 1973, 25, 161-178. 16. Moeremans, M . , Daneels, G . , Van Dijck, Α . , Langanger, G. and De Mey, J. J. Immunol. Methods, 1984, 74, 353. 17. Halk, E . L. and DeBoer, S. H. Ann. Rev. Phytopathol. 1985; 23, 321-350. 18. Marsden, J. F . and Miller, S. A. BioExpo '85, 1985. 19. M i l l e r , S. Α., Grothaus, G. D., Petersen, F . P., and Papa, S. L. 1986. Detection of Pythium blight in turfgrass using a monoclonal antibody-based diagnostic test (abstr.). Phytopathology. (in press)


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Hardham, A. R., Suzaki, E. and Perkin, J. L . 1986. Can. J. Bot. 64:311-321. Lankow, R. K . , Woodhead, S. H . , Patterson, R. J., Massey, R., and Schochetman, G. Plant Disease 1984, 68, 1100-1101. Wie, S. I . , and Hammock, B. D. J. Agric. Food Chem. 1982, 30, 949-957. Ercegovich, C. D . , Vallejo, R. P . , Gettig, R. R . , Woods, L., Bogus, E. R., Mumma, R. O. J. Agric. Food Chem. 1981, 29, 559-563. Newsome, W. Α., Shields, J . B. J . Agric. Food Chem., 1981, 29, 220-222. Wie, S. I. and Hammock, B. D. J. Agric. Food Chem., 1984, 32, 1294, 1301. Ferguson, B. BioExpo '86, 1986. Pestka, J . J., Li, Y., Harden, W. O., Chu, F. S. J. Assoc. of Anal. Chem., 1981, 64, 294-301. Weiler, E. W. Physiol. Plant., 1982, 54, 230-234. Notton, Β. Α . , Fido, R. J., and Galfre, G. Planta, 1985, 105, 114-119.

RECEIVED

December 10, 1986


Immunoassays for Crop Management Systems and Agricultural

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