Immunochemical Methods for Environmental ... - ACS Publications

Chapter 11 Immunochemical

Technology

in Environmental

Analysis Addressing Critical Problems

Downloaded by UNIV OF LIVERPOOL on December 9, 2015 | http://pubs.acs.org Publication Date: November 11, 1989 | doi: 10.1021/bk-1989-0442.ch011

Bruce D. Hammock, Shirley J. Gee, Robert O. Harrison, Freia Jung, Marvin H. Goodrow, Qing Xiao Li, Anne D. Lucas, András Székács, and K. M. S. Sundaram Department of Entomology and Department of Environmental Toxicology, University of California, Davis, CA 95616 Immunochemical technology is at a critical stage in its development for use in environmental analysis. Primary problems and issues regarding assay development and applications such as outlining common misconceptions, choice of format and choice of monoclonal or polyclonal antibodies are discussed. More far reaching concerns for the acceptance of the technology such as the roles of government and industry in assay development and standardization are also discussed. A committee to coordinate the development of immunoassay in the environmental field is proposed and its functions outlined. How some of these problems are currently being addressed are illustrated by work presented at this symposium. Work from our laboratory illustrates our approach to dealing with real world samples, more difficult target compounds and complex matrices and applying immunoassays to samples other than environmental samples. The next few years will be critical in the development of immunochemical technology for use in environmental analysis. In this light this manuscript has three objectives. The first is to address how the critical problems facing the technology can be approached. The second is to introduce aspects of this symposium by pointing out how various laboratories are approaching these problems. Finally, this manuscript will review briefly some of the topics being addressed by this laboratory. Evolution of Problems Facing Immunoassay Changes in the Last Ten Years. Based on both the Miami American Chemical Society (ACS) meeting 11 years ago and the ACS meeting 10 years ago, this symposium certainly has historical significance to

0097-6156/90/0442-0112S08.00/0 © 1990 American Chemical Society In Immunochemical Methods for Environmental Analysis; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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H A M M O C K ET A L

Problems in Immunochemical Technology 113

our l a b o r a t o r y . A t t h e Miami ACS m e e t i n g i n 1978 o n l y two p a p e r s were p r e s e n t e d on t h e immunoassay o f p e s t i c i d e s . One was on o u r immunoassay f o r the o p t i c a l isomers of the p y r e t h r o i d Sb i o a l l e t h r i n and t h e o t h e r was an immunoassay o f p a r a t h i o n . The l a t e r s t u d y was from t h e l a b o r a t o r y o f t h e l a t e C. D. E r c e g o v i c h who was one o f t h e e a r l y l e a d e r s i n t h i s f i e l d . The n e x t y e a r a t t h e 1979 ACS m e e t i n g i n Washington D. C. D r s . Harvey and Zweig r e q u e s t e d t h a t t h e l a b o r a t o r y a d d r e s s t h e p o t e n t i a l o f immunoassay f o r p e s t i c i d e r e s i d u e a n a l y s i s i n a symposium on Recent Advances in Pesticide Analytical Methodology ( 1 ) . As one might a n t i c i p a t e , t h i s t a l k drew a g r e a t d e a l o f c r i t i c i s m b a s e d on many m i s c o n c e p t i o n s r e g a r d i n g immunoassay. Some t h i n g s c e r t a i n l y have changed i n t h e t e n y e a r s l e a d i n g t o t h i s 1989 ACS m e e t i n g . S i m p l y t h e i n c r e a s e from one p a p e r t o 27 p a p e r s on t h e immunochemical a n a l y s i s o f p e s t i c i d e s and o t h e r e n v i r o n m e n t a l c h e m i c a l s i l l u s t r a t e s a major change i n t h e i n t e r e s t o f p e s t i c i d e c h e m i s t s i n immunoassay. T h i s change has n o t been due to some magical improvement in immunochemical t e c h n o l o g y . I n f a c t , immunochemical t e c h n o l o g y as i t a p p l i e s t o t h e a n a l y s i s o f s m a l l m o l e c u l e s i n e n v i r o n m e n t a l samples has n o t changed g r e a t l y i n t h e l a s t 10 y e a r s , w h i l e g r e a t changes have b e e n made i n c h r o m a t o g r a p h i c and s p e c t r a l d e t e c t i o n systems. The change h a s b e e n i n an i n c r e a s e d awareness o f t h e c a p a b i l i t i e s o f immunoassays i n t h e e n v i r o n m e n t a l f i e l d . M i s c o n c e p t i o n s . Then v s Now. At f i r s t glance t h i s increase i n i n t e r e s t i n immunoassay might i n d i c a t e t h a t p e s t i c i d e immunoassay has matured and l e f t i t s d o u b t e r s and problems b e h i n d . Such i s n o t the c a s e . A l t h o u g h t h e r e i s wide i n t e r e s t i n t h e t e c h n o l o g y and most a g r i c u l t u r a l c h e m i c a l companies have i n - h o u s e e x p e r t i s e i n immunochemical technology, immunoassays have n o t b e e n u s e d t o r e g i s t e r a s i n g l e p e s t i c i d e n o r i s an A s s o c i a t i o n o f O f f i c i a l Analytical Chemistry (AOAC) validation of immunoassays f o r p e s t i c i d e s a common phenomenon. I n f a c t , t h e t e c h n o l o g y seems t o have t r a d e d one s e t o f problems f o r a n o t h e r . A c c e p t a n c e o f t h e t e c h n o l o g y was s t i f l e d f o r y e a r s because many s c i e n t i s t s c o n c l u d e d t h a t immunochemistry h a d no p l a c e i n e n v i r o n m e n t a l c h e m i s t r y b a s e d on l i t t l e a p p r e c i a t i o n o f i t s power. We now f i n d t h e major p r o b l e m f a c i n g t h e t e c h n o l o g y i s t h a t i t i s b e i n g over s o l d , i n some c a s e s as a panacea, by p e o p l e who do n o t u n d e r s t a n d t h e l i m i t a t i o n s o f the technology. A major theme o f t h i s book c o u l d be t h e same one we a d v o c a t e d t e n y e a r s ago. T h a t i s t h a t immunochemistry r e p r e s e n t s a v e r y p o w e r f u l a n a l y t i c a l t o o l w h i c h i s a p p l i c a b l e t o many b u t c e r t a i n l y n o t a l l problems i n e n v i r o n m e n t a l c h e m i s t r y . Thus, i t complements b u t does n o t r e p l a c e o t h e r a n a l y t i c a l methods. The t e c h n o l o g y i s so p o w e r f u l and v e r s a t i l e t h a t i t s h o u l d be i n t h e r e p e r t o i r e o f e v e r y a n a l y t i c a l c h e m i s t . Y e t t h e r e must be an u n d e r s t a n d i n g t h a t t h e t e c h n o l o g y i s v e r y u s e f u l f o r some compounds and some problems, b u t t h a t i t i s no panacea. Ten y e a r s ago t h e c h a l l e n g e was t o encourage u n d e r s t a n d i n g o f t h e tremendous power o f t h e t e c h n o l o g y , w h i l e t o d a y we must p r e a c h t h e l i m i t a t i o n s .

In Immunochemical Methods for Environmental Analysis; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS

The major problem f a c i n g the t e c h n o l o g y t e n y e a r s ago was a l a c k o f u n d e r s t a n d i n g o f immunochemical a n a l y s i s by e n v i r o n m e n t a l c h e m i s t s . A t the time the m i s c o n c e p t i o n s seemed c h a l l e n g i n g t o overcome, b u t i n many ways t h e y a r e l e s s s e r i o u s t h a n the problems t h a t t h e t e c h n o l o g y f a c e s t o d a y . One s i g n i f i c a n t p r o b l e m was the j a r g o n t h a t s u r r o u n d e d immunochemical t e c h n o l o g y . A major r o l e o f the scientists involved in advocating immunoassay in the e n v i r o n m e n t a l f i e l d o v e r the l a s t decade has n o t b e e n t o p i o n e e r new immunochemical t e c h n o l o g y b u t r a t h e r t o t r a n s l a t e the j a r g o n used in clinical immunochemistry to the jargon used in e n v i r o n m e n t a l c h e m i s t r y . As p o i n t e d o u t by Ken H u n t e r f o r m e r l y o f Westinghouse B i o a n a l y t i c a l , our j o b became d r a m a t i c a l l y e a s i e r w i t h t h e advent o f the microcomputer which generated a s t a n d a r d curve f o r a n a l y t e v s r e s p o n s e . Such s t a n d a r d c u r v e s appear more f a m i l i a r t o a n a l y t i c a l c h e m i s t s , r e a s s u r i n g them t h a t t h e y a r e d e a l i n g w i t h real analytical c h e m i s t r y and not some q u a l i t a t i v e biological phenomenon. A n o t h e r o f the major m i s c o n c e p t i o n s t e n y e a r s ago was that immunoassays were b i o a s s a y s , and some even thought t h a t a r a b b i t d i e d each time an a n a l y s i s was r u n . We had a generation of a n a l y t i c a l c h e m i s t s who had f o u g h t t o have c h r o m a t o g r a p h i c methods a c c e p t e d o v e r b i o a s s a y s f o r r e s i d u e a n a l y s i s , and immunochemistry seemed t o o f f e r a g r e a t l e a p backwards. The realization that immunoassays are physical assays which simply use biological reagents i s now wide spread. The fact that the cyclodiene a n t i b o d i e s p u b l i s h e d by Langone and Van V u n a k i s i n 1975 (2) a r e s t i l l i n use, i s an e x c e l l e n t i l l u s t r a t i o n t h a t i f p r o p e r l y h a n d l e d immunochemical r e a g e n t s a r e v e r y s t a b l e . A new m i s c o n c e p t i o n now exists i n some q u a r t e r s i n t h i s decade o f b i o t e c h n o l o g y . Ten y e a r s ago the b i o l o g i c a l s o u r c e o f a n t i b o d i e s t a i n t e d immunoassays as a p o o r l y r e p r o d u c i b l e b l a c k a r t p r a c t i c e d by b i o l o g i s t s and n o t by r e a l c h e m i s t s . Now i n some quarters the b i o l o g i c a l source o f a n t i b o d i e s seems t o impart m a g i c a l q u a l i t i e s t o immunoassays. Some p e o p l e i n d i c a t e t h a t t h e s e a s s a y s c a n d e t e c t b i o l o g i c a l e f f e c t s , b u t l i k e any p h y s i c a l a s s a y , immunoassays d e t e c t m o l e c u l e s w h i c h may o r may n o t be a s s o c i a t e d with biological activity. Certainly the specificity of an immunoassay can correlate with that of a receptor molecule. However, such c o r r e l a t i o n s a r e i n c i d e n t a l . There i s an e f f o r t t o apply immunoassays to a l l compounds and problems with no a p p r e c i a t i o n f o r the t e c h n o l o g y ' s l i m i t a t i o n s o r the s t r e n g t h s o f competing t e c h n o l o g i e s . A major c h a l l e n g e f a c i n g a l l competent a n a l y t i c a l c h e m i s t s i s t o make s u r e t h a t the t e c h n o l o g y i s a d v o c a t e d b a s e d on i t s r e a l s t r e n g t h s . I f the t e c h n o l o g y i s o v e r s o l d b a s e d e i t h e r on i g n o r a n c e o r on a d e s i r e t o advance a p r o d u c t f o r p r o f i t o r one's c a r e e r , t h e r e i s c e r t a i n t o be a b a c k l a s h when immunochemistry f a i l s t o provide magical r e s u l t s . There also is the indication that immunoassays allow untrained a n a l y s t s to run highly sensitive assays. Although immunoassays may be v e r y f o r g i v i n g and easy t o p e r f o r m , the q u a l i t y o f the d a t a g e n e r a t e d f o r any p h y s i c a l a s s a y w i l l depend upon the



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i n t e g r i t y o f t h e samples and t h e s k i l l o f t h e a n a l y s t . As t h e a s s a y s u s e d become more d i f f i c u l t and t h e l i m i t o f d e t e c t i o n lower, the s k i l l o f t h e a n a l y s t must be g r e a t e r as w i t h any a n a l y t i c a l system. In some ways a more d i f f i c u l t p r o b l e m has been t h a t t h e r e l u c t a n c e o f r e s i d u e c h e m i s t s t o embrace immunoassay has l e d t o the development o f immunoassays f o r e n v i r o n m e n t a l c h e m i c a l s i n metabolism, biotechnology, or c l i n i c a l l a b o r a t o r i e s . Among this group o f s c i e n t i s t s t h e r e sometimes i s a r e v e r s e a r r o g a n c e towards the r e s i d u e c h e m i s t who f a i l e d t o adopt t h i s t e c h n o l o g y . However, the a s s a y s d e v e l o p e d o u t s i d e o f an a n a l y t i c a l l a b o r a t o r y o f t e n u s e such simplistic hapten design that key r e c o g n i t i o n s i t e s are masked. A l s o , t h e r e i s a v a s t d i f f e r e n c e i n m a t r i x e f f e c t s between clinical and e n v i r o n m e n t a l samples. The use o f enzyme linked immunosorbant a s s a y s (ELISA's) o r o t h e r immunoassays i n a r e a l a n a l y t i c a l program w i l l n o r m a l l y r e v e a l new m a t r i x problems t o t a l l y unfamiliar to the c l i n i c a l chemist. The e x p e r i e n c e i n this l a b o r a t o r y i s t h a t a good a n a l y t i c a l c h e m i s t c a n be t r a i n e d t o p e r f o r m ELISA i n a m a t t e r o f days. However, t h e c o n v e r s i o n o f an immunochemist i n t o an e n v i r o n m e n t a l c h e m i s t r e p r e s e n t s a major change i n c a r e e r and p h i l o s o p h y . The c o l l e c t i o n , h a n d l i n g , and p r o c e s s i n g o f samples as w e l l as t h e d e s i g n o f a n a l y t i c a l s t u d i e s and t h e h a n d l i n g o f d a t a a r e e v e r y b i t as s o p h i s t i c a t e d as t h e p r e p a r a t i o n o f m o n o c l o n a l a n t i b o d i e s . I t i s c r i t i c a l t h a t when t h e d a t a from immunoassays a r e t o be u s e d f o r i m p o r t a n t d e c i s i o n s , t h a t w e l l d e s i g n e d a s s a y s a r e p e r f o r m e d by t r a i n e d a n a l y t i c a l c h e m i s t s Q). When i s Immunochemistry Most A p p l i c a b l e ? As i n d i c a t e d above i t i s v e r y i m p o r t a n t t h a t as a d v o c a t e s o f t h i s t e c h n o l o g y , we p o i n t o u t when i t i s b e s t a p p l i e d and a l s o when i t s h o u l d n o t be a p p l i e d . T h i s t o p i c has b e e n c o v e r e d i n a v a r i e t y o f p r e v i o u s r e v i e w s ( 1 . 4 - 8 ) , however we have f o u n d two f i g u r e s w h i c h convey s e v e r a l c o n c e p t s about a p p l i c a b i l i t y v e r y w e l l . For i n s t a n c e i n F i g u r e 1 we r e p r e s e n t a l l o f t h e compounds f o r w h i c h t h e a n a l y s t may need methods. F o r some compounds such as the v o l a t i l e o r g a n i c s i n water, gas chromatography systems o f f e r g r e a t advantages. A t t h e o t h e r extreme t h e r e a r e compounds such as paraquat, the s u l f o n y l u r e a s or benzoylphenylureas which lend themselves w o n d e r f u l l y t o immunoassay development. T h e r e i s an i m p o r t a n t s e t o f compounds w h i c h c a n be a n a l y z e d r e a d i l y by s e v e r a l d i f f e r e n t methods. The t h i o c a r b a m a t e s o r t r i a z i n e s a r e compounds h a n d l e d i n t h i s l a b o r a t o r y w h i c h f a l l i n t o such a s i t u a t i o n . Here the d e c i s i o n on t h e t e c h n o l o g y would depend upon t h e r e s o u r c e s o f the laboratory i n q u e s t i o n and t h e p r o b l e m a t hand. I f the compounds were p a r t o f a m u l t i a n a l y t e p r o b l e m o r i f o n l y a few samples needed t o be a s s a y e d , c h r o m a t o g r a p h i c systems o f f e r an advantage. I n c a s e s where f i e l d a s s a y s a r e needed o r where a l a r g e sample l o a d i s l i k e l y , immunoassay c l e a r l y i s t h e method o f c h o i c e . from

The most common q u e s t i o n from b i o t e c h n o l o g y companies and t h e a g r i c u l t u r a l c h e m i c a l i n d u s t r y c o n c e r n s w h i c h compounds



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Figure 1. Applicability of immunochemistry to analytical p r o b l e m s . The background indicates a l l compounds f o r which analysis i s needed w h i l e t h e r e s p e c t i v e c i r c l e s i n d i c a t e the s u b s e t o f compounds f o r w h i c h gas chromatography (GC), h i g h p e r f o r m a n c e l i q u i d chromatography (HPLC), and immunoassay (IA) a r e most a p p l i c a b l e . F o r t h o s e compounds w h i c h c a n be r e a d i l y a n a l y z e d by a v a r i e t y o f methods, the d e c i s i o n o f w h i c h a s s a y t o use s h o u l d be made b a s e d on the a n a l y t i c a l q u e s t i o n s t o be answered.



11. HAMMOCK ET AL.


a r e a p p r o p r i a t e t a r g e t s f o r immunoassay development. P a r t o f t h i s answer of course i n v o l v e s market a n a l y s i s , which i s not an a p p r o p r i a t e t o p i c h e r e . I t i s c l e a r t h a t t h e r e w i l l be a r e a s o n a b l e market f o r immunodiagnostics i n the e n v i r o n m e n t a l f i e l d , b u t l a r g e o b v i o u s markets do n o t now e x i s t . W i t h the management o f the a g r i c u l t u r a l c h e m i c a l i n d u s t r y i n t h e p a s t the e r r o r has been n o t t o ask what a r e the p r o p e r t a r g e t s . R a t h e r immunochemical t e c h n o l o g y i s i g n o r e d u n t i l o t h e r a n a l y t i c a l methods have f a i l e d , the c h e m i s t r y o f the compounds has become cold, and there is tremendous pressure from marketing and r e g i s t r a t i o n groups f o r immediate a n a l y t i c a l methods f o r a v e r y difficult compound. Only then i s the task of developing an immunoassay g i v e n t o a new employee w i t h few r e s o u r c e s . T h i s h a r d l y represents the optimum way f o r a company t o d e v e l o p in-house e x p e r t i s e i n immunoassay. Immunoassays a r e v e r y v e r s a t i l e , and i f one c o u l d s e l e c t b u t a s i n g l e method, i t c o u l d be the method o f c h o i c e . F o r t u n a t e l y we have a v a r i e t y o f t e c h n i q u e s a v a i l a b l e and a good a n a l y s t s h o u l d know when t o a p p l y them. T a b l e I p r o v i d e s some g e n e r a l r u l e s f o r d e t e r m i n i n g how d i f f i c u l t an immunoassay w i l l be. The terms u s e d a r e r e l a t i v e and p o s s i b l y o t h e r d i m e n s i o n s t o the t a b l e c o u l d be the laboratory's experience with immunoassay and the problems f a c e d . T h i s t a b l e does n o t i n d i c a t e t h a t good a s s a y s c a n n o t be d e v e l o p e d f o r h a r d compounds; i t j u s t i n d i c a t e s t h a t the expense, s k i l l and time r e q u i r e d may be g r e a t e r f o r t h o s e compounds. F o r instance we have developed successful immunoassays for some lipophilic, small, unstable, volatile compounds. However, such compounds would be a p o o r c h o i c e t o use f o r one's f i r s t venture i n t o immunoassay development.

Table

I . P r o p e r t i e s o f Compounds w h i c h L e a d t o D i f f i c u l t i e s Immunoassay Development

in

PROPERTIES EASY

HYDROPHILIC LARGE STABLE NONVOLATILE FOREIGN

Gaining

F u l l Use

of

HARD

LIPOPHILIC SMALL UNSTABLE VOLATILE NATURAL

Immunochemistry

F o r o v e r a decade the d a t a have been i n the l i t e r a t u r e t o s u p p o r t the contention that for appropriate molecules and problems immunochemical methods a r e f a r s u p e r i o r t o competing t e c h n o l o g i e s .


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Y e t , t h e methods s t i l l a r e n o t i n r o u t i n e use. A d v o c a t e s o f the t e c h n o l o g y have e n t e r t a i n e d t h e m s e l v e s by making one immunoassay a f t e r a n o t h e r . A l t h o u g h t h i s a c t i v i t y i s i m p o r t a n t , we now f a c e the more d i f f i c u l t c h a l l e n g e o f v a l i d a t i n g t h e s e a s s a y s , e n s u r i n g t h a t t h e y a r e i n the p r o p e r hands, and t h a t t h e y a r e u s e d e f f e c t i v e l y . T h i s c h a l l e n g e c a n be b r o k e n down i n t o a number o f s m a l l e r problems which do not differ greatly from problems faced with c h r o m a t o g r a p h i c systems. However, some o f the p r o b l e m s w i l l still be d i f f i c u l t t o a d d r e s s . F o r t u n a t e l y o t h e r problems w h i c h i n i t i a l l y w i l l seem d i f f i c u l t w i l l t u r n o u t t o be no p r o b l e m a t a l l . S h o u l d Immunoassays be Q u a l i t a t i v e o r Q u a n t i t a t i v e ? T h i s i s an e x c e l l e n t example o f a n o n q u e s t i o n w h i c h sometimes i s d i s c u s s e d s e r i o u s l y . As d i s c u s s e d below, the answer i s t h a t once one has an a n t i b o d y and t r a c e r the a s s a y c a n be p u t i n t o e i t h e r a q u a n t i t a t i v e or q u a l i t a t i v e f o r m a t d e p e n d i n g upon the q u e s t i o n t o be a d d r e s s e d . Q u a l i t a t i v e f o r m a t s w i l l be v e r y i m p o r t a n t i n t h e e n v i r o n m e n t a l a r e n a as f a s t f i e l d t e s t s . However, i t i s our o p i n i o n t h a t a t l e a s t u n t i l the t e c h n o l o g y i s w e l l e s t a b l i s h e d t h a t q u a l i t a t i v e t e s t s i n t h e e n v i r o n m e n t a l f i e l d s h o u l d be b a s e d on r e a g e n t s w h i c h have b e e n examined i n a q u a n t i t a t i v e f o r m a t . U s e r s o f q u a l i t a t i v e k i t s w h i c h have no q u a n t i t a t i v e d a t a s u p p o r t i n g them c o u l d be v e r y embarrassed i f they t r y to over i n t e r p r e t t h e i r data. What Format S h o u l d Be Used? A g r e a t s t r e n g t h o f immunoassay i s that the same r e a g e n t s c a n be u s e d i n many f o r m a t s . We have employed V o i l e r ' s ELISA f o r m a t ( £ ) , b u t even t h i s format has numerous v a r i a t i o n s . The f o r m a t g i v e s adequate s e n s i t i v i t y f o r most e n v i r o n m e n t a l q u e s t i o n s , does n o t r e q u i r e r a d i o a c t i v e compounds, c a n be o p t i m i z e d f o r speed, c o s t , s e n s i t i v i t y o r o t h e r f a c t o r s , and maybe most i m p o r t a n t , i t has a p l e a s i n g and n o n i n t i m i d a t i n g name. In a d d i t i o n we have a d v o c a t e d t h i s f o r m a t s i n c e t h e u n d e r s t a n d i n g t h a t the same a n t i b o d y c a n be u s e d i n numerous f o r m a t s i s n o t w i d e s p r e a d . We f e e l t h a t c u r r e n t l y i t i s i m p o r t a n t t o n o t f r i g h t e n new u s e r s and r e g u l a t o r y a g e n c i e s w i t h f o r m a t s f o r w h i c h t h e y have no name r e c o g n i t i o n . However, i n the l o n g term, ELISA i s an ephemeral f o r m a t . Even when s t r e a m l i n e d and automated, i t has t o o many s t e p s . C e r t a i n l y we s h o u l d r e a l i z e t h a t i t w i l l be r e p l a c e d by o t h e r systems, t h e most e x c i t i n g o f w h i c h w i l l be b i o s e n s o r s . A l s o , o t h e r f o r m a t s o f f e r a p r o p r i e t a r y edge i n the market p l a c e w h i c h w i l l be v e r y i m p o r t a n t in t h e m a t u r a t i o n o f immunoassay systems i n t h e environmental f i e l d . F i n a l l y , d i f f e r e n t formats w i l l l e n d themselves to d i f f e r e n t e n v i r o n m e n t a l p r o b l e m s . We s h o u l d c o n t i n u a l l y emphasize t h a t the same r e a g e n t s c a n be u s e d i n many f o r m a t s . P o s s i b l y i n s m a l l letters we also should caution that certain antibody c h a r a c t e r i s t i c s may be more i m p o r t a n t i n one f o r m a t t h a n a n o t h e r , t h a t some f o r m a t s a r e more r e s i s t a n t t o m a t r i x e f f e c t s , and t h a t r e l a t i v e c r o s s r e a c t i v i t i e s o f compounds c a n change as one changes the s u b t l e p r i n c i p l e s upon w h i c h an immunoassay works. F o r t h i s r e a s o n a c l e a r c h o i c e o f f o r m a t s s h o u l d be made b e f o r e i n i t i a t i n g validation studies.


11. HAMMOCK ET AL



C l e a r l y , ELISA i s the p r i n c i p l e f o r m a t u s e d f o r i n t r o d u c i n g immunoassay i n t o the e n v i r o n m e n t a l f i e l d . We c e r t a i n l y hope t h a t i n the n e a r f u t u r e t h a t a l l a s s a y s w i l l be c h a r a c t e r i z e d i n t h i s format to a v o i d c o n f u s i o n . S h o u l d the Development o f A n t i b o d i e s and A n t i g e n s be i n the Open Literature? T h i s q u e s t i o n c e r t a i n l y i s open t o d e b a t e . On the n e g a t i v e s i d e some companies t r y i n g t o d e v e l o p a p r o p r i e t a r y n i c h e i n the e n v i r o n m e n t a l market may f e e l t h a t t h e y n e e d p r o t e c t i o n and n o t d i v u l g e t h e i r c o u p l i n g s t r a t e g i e s and o t h e r t e c h n i q u e s i n a s s a y development. I t i s v e r y d i f f i c u l t t o o b t a i n p a t e n t s i n the a r e a o f h a p t e n c h e m i s t r y s i n c e r e g a r d l e s s o f how s o p h i s t i c a t e d we f e e l our i n d i v i d u a l work i s , the c o u p l i n g p r o c e d u r e s a r e r a t h e r o b v i o u s . T h i s i s n o t t o say t h a t a g r e a t d e a l o f s k i l l and even a r t a r e n o t i n v o l v e d i n h a p t e n d e s i g n and c o u p l i n g , b u t t h a t the t e c h n o l o g i e s u s u a l l y appear o b v i o u s i n the eyes o f p a t e n t a u t h o r i t i e s . P o s s i b l y we s u f f e r from an academic b i a s , b u t t h i s l a b o r a t o r y strongly advocates that a general outline of immunoassay development (including the origin and characteristics of the a n t i b o d y as w e l l as the p o s i t i o n and c h e m i s t r y o f h a p t e n c o u p l i n g t o the a n t i g e n , and t r a c e r o r c o a t i n g a n t i g e n ) s h o u l d be a v a i l a b l e to the user. The presence of such i n f o r m a t i o n i n the open literature would n o t j e o p a r d i z e a company's a r t since myopic details are not needed for an analyst to predict the c h a r a c t e r i s t i c s o f the a s s a y . By k e e p i n g h a p t e n d e s i g n s e c r e t , companies can c o n f u s e many u s e r s , b u t their true competitors usually can discern the general methods used from the c h a r a c t e r i s t i c s o f the a n t i b o d y . We s t r o n g l y s u g g e s t t h a t t h e time and p u b l i c money w h i c h must be i n v e s t e d f o r v a l i d a t i o n s t u d i e s o n l y be i n v e s t e d f o r w e l l c h a r a c t e r i z e d a s s a y s . To do o t h e r w i s e would be l i k e d e v e l o p i n g a c h r o m a t o g r a p h i c a s s a y w i t h o u t t e l l i n g the u s e r what t y p e o f d e t e c t o r was b e i n g u s e d on t h e gas chromatograph. Who S h o u l d Develop Immunoassays? The answer t o t h i s q u e s t i o n i s s i m p l e - e v e r y o n e . The more complex q u e s t i o n i s once t h e s e a s s a y s a r e d e v e l o p e d how do we g e t them i n the hands o f u s e r s ? C e r t a i n l y the agricultural chemical industry s h o u l d be involved i n the development o f a s s a y s f o r t h e i r p r o d u c t s . Even i f the a s s a y s a r e n e v e r u s e d f o r r e g i s t r a t i o n , the a s s a y s w i l l save companies money by b e i n g u s e d i n - h o u s e as r e s e a r c h t o o l s . I n most companies t h e r e i s s u c h a b a c k l o g o f r e s i d u e samples t o r u n t h a t i n - h o u s e a s s a y s t o test formulation, plant distribution, p r o c e s s c o n t r o l and many o t h e r problems r e c e i v e low p r i o r i t y . Immunoassays c a n have a major impact on t h e s e problems. T h e r e a l s o i s a n o t h e r answer t o the q u e s t i o n i f a g r i c u l t u r a l c h e m i c a l companies s h o u l d d e v e l o p a s s a y s f o r t h e i r own compounds. T h a t answer i s t h a t i f t h e y do n o t , someone e l s e w i l l . I f t h e a s s a y i s d e v e l o p e d i n - h o u s e , one has c o n t r o l o f the c h a r a c t e r i s t i c s and d i s t r i b u t i o n o f the a s s a y and h o p e f u l l y the a s s a y development i s done c o r r e c t l y . I f the a s s a y i s n o t done i n - h o u s e t h e company w i l l have no c o n t r o l o v e r q u a l i t y , s e n s i t i v i t y , o r o t h e r a s p e c t s .



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Ultimately i t may be c o s t effective f o r a company t o s u b c o n t r a c t a s s a y development t o a c l i n i c a l d i v i s i o n i n - h o u s e o r t o a t h i r d p a r t y . However, i f t h i s i s done b e f o r e t h e p a r e n t company has t h e i n - h o u s e e x p e r t i s e t o m o n i t o r a s s a y development, t h i s c a n be a v e r y dangerous and e x p e n s i v e p r o c e s s . O f t e n t h e e x p e r t i s e on t h e c h e m i s t r y o f t h e compound c l a s s i s n o t t r a n s f e r r e d and i n f e r i o r a s s a y s a r e d e v e l o p e d a t g r e a t expense. I f i t i s n e c e s s a r y t o d e v e l o p e a r l y a s s a y s o u t s i d e t h e company, i t i s i m p o r t a n t t h a t t h e a s s a y development i s approached as a c o l l a b o r a t i v e p r o j e c t w i t h a group p o s s e s s i n g an e s t a b l i s h e d r e c o r d i n t h e development o f a s s a y s for environmental samples. Involvement of scientists from t h e clinical field c a n be v e r y u s e f u l s i n c e t h e y have decades o f a c c u m u l a t e d knowledge on a s s a y f o r m a t t i n g and development. However, the involvement of scientists w i t h an a p p r e c i a t i o n o f m a t r i x effects, metabolism, and the r e g u l a t o r y questions posed is critical. Certainly universities and government a g e n c i e s s h o u l d be i n v o l v e d i n a s s a y development. I f an i n d u s t r i a l c o l l a b o r a t i o n c a n be e s t a b l i s h e d , one g a i n s tremendous advantages with regard to chemical libraries and e x p e r t i s e . Universities and government a g e n c i e s have done an e x c e l l e n t j o b o f p i o n e e r i n g t h e development o f t h e ELISA t e c h n o l o g y i n t h e e n v i r o n m e n t a l f i e l d , b u t t h e y have two major l i m i t a t i o n s . The f i r s t c o u l d be a t t r i b u t e d t o a v a r i c e , administrative incompetence in the institutions, or petty j e a l o u s i e s among t h e i n v e s t i g a t o r s o r a g e n c i e s . T h e r e c e r t a i n l y i s l i t t l e e a s y money t o be made from immunoassays f o r e n v i r o n m e n t a l compounds i n t h e n e a r f u t u r e . U n i v e r s i t i e s and government a g e n c i e s need t o have a p o l i c y o f p r o v i d i n g t h e a s s a y s a t no o r low c o s t f o r r e s e a r c h o r r e g u l a t o r y use and some f a i r and s y s t e m a t i c method o f g e t t i n g t h e r e a g e n t s t o t h i r d p a r t y v e n d o r s . As an example, i t has taken over a dozen years of pressure from this and o t h e r l a b o r a t o r i e s b e f o r e t h e U n i v e r s i t y o f C a l i f o r n i a has begun t h e development o f s t r e a m l i n e d l i c e n s i n g p r o c e d u r e s f o r immunochemical reagents. Hopefully this problem i s being solved i n other institutions. The o t h e r p r o b l e m w i t h u n i v e r s i t y and government l a b o r a t o r i e s i s t h a t they l a c k e x p e r t i s e i n the v a r i e t y o f s o p h i s t i c a t e d formats w h i c h w i l l be v e r y u s e f u l i n t h e e n v i r o n m e n t a l a r e a and t h e methods f o r s t a b i l i z i n g and d i s t r i b u t i n g r e a g e n t s . Not o n l y s h o u l d t h e s e agencies p r o v i d e the reagents i n a s t a n d a r d format t o i n t e r e s t e d s c i e n t i s t s , b u t by p r o v i d i n g them a t a r e a s o n a b l e c o s t t o t h e t h i r d p a r t y vendors the environmental f i e l d w i l l g a i n t h e i r e x p e r t i s e i n stabilization, packaging, f o r m a t t i n g and m a r k e t i n g the assays. Which company o f f e r s t h e b e s t system t h e n c a n be d e t e r m i n e d i n t h e market p l a c e . C l e a r l y b i o t e c h n o l o g y companies ( t h i r d p a r t y v e n d o r s ) s h o u l d be developing k i t s and i n some c a s e s the assays themselves. Hopefully they can get access t o the reagents available from government and academic l a b o r a t o r i e s i n a d d i t i o n t o t h e a s s a y s d e v e l o p e d i n - h o u s e . As d i s c u s s e d above, we s t r o n g l y f e e l t h a t i t w i l l be a good p o l i c y f o r t h e s e companies t o quote t h e s o u r c e o f


11.

H A M M O C K ET AL.



a n t i b o d i e s used i n t h e i r k i t s r e a g e n t s were d e v e l o p e d .

or to provide

an o v e r v i e w o f how t h e

S h o u l d M o n o c l o n a l o r P o l y c l o n a l A n t i b o d i e s be A d v o c a t e d ? This s u b j e c t w i l l be t r e a t e d e l s e w h e r e i n t h i s book (10-11) and h a s been discussed i n numerous previous reviews. To most people with e x p e r i e n c e i n t h e f i e l d , t h i s i s a n o t h e r n o n i s s u e where t h e answer w i l l be b a s e d upon t h e p r o b l e m a t hand and t h e r e s o u r c e s a v a i l a b l e . The answer t o t h e q u e s t i o n s h o u l d n o t be b a s e d on t h e i d e a t h a t m o n o c l o n a l a n t i b o d i e s come from h i g h t e c h n o l o g y and p o l y c l o n a l from low technology. The sophistication and skill in antibody development c a n be j u s t as g r e a t w i t h e i t h e r t e c h n o l o g y . The c r i t e r i a f o r a p p r o v a l o f a p a r t i c u l a r a s s a y s h o u l d be b a s e d on r i g o r o u s performance s p e c i f i c a t i o n s o f the f i n a l product (whether from a c o m m e r c i a l o r academic s o u r c e ) , r a t h e r t h a n t h e d e s i g n o f the t e s t , an a p p r o a c h s i m i l a r t o t h a t u s e d i n t h e m a n u f a c t u r e o f c h r o m a t o g r a p h i c columns. T h i s way o f a d d r e s s i n g t e s t p e r f o r m a n c e renders most of the questions of antibody s e l e c t i o n or s t a n d a r d i z a t i o n moot. Another misconception i s that monoclonals provide an u n l i m i t e d a n t i b o d y s u p p l y from immortal c e l l s . Hybridoma l i n e s a r e immortal only so l o n g as they are maintained with constant s e l e c t i o n b y a s k i l l e d t e c h n i c i a n o r f r o z e n i n a s i t u a t i o n where t h e y c a n be a r c h i v e d , m a i n t a i n e d , and t h e n thawed by a s k i l l e d i n d i v i d u a l . I n p r a c t i c e t h e AOAC sees no d i f f e r e n c e between t h e v a l i d a t i o n o f a p o o l o f mono- o r p o l y c l o n a l a n t i b o d i e s u s e d f o r immunoassay. A l s o r e p u t a b l e immunochemical companies t r e a t monoand p o l y c l o n a l a n t i b o d i e s t h e same. A s u f f i c i e n t p o o l o f m o n o c l o n a l o r p o l y c l o n a l a n t i b o d y i s p r o d u c e d and s t o r e d such t h a t t h e company w i l l n o t have t o thaw t h e hybridoma c e l l s o r reimmunize a n i m a l s i n the f o r e s e e a b l e f u t u r e . A serious error involves t h e attempt t o use expensive hybridoma s c r e e n i n g t o overcome p o o r h a p t e n d e s i g n and h a n d l e recognition. I f one i s g o i n g t o t h e expense o f monoclonal production, c e r t a i n l y a s i m i l a r investment i n hapten d e s i g n to r e d u c e h a n d l e r e c o g n i t i o n i s w a r r a n t e d . I t i s p o o r economy t o u s e thousands o f d o l l a r s o f hybridoma t e c h n o l o g y t o make up f o r t h e l a c k o f a few h u n d r e d d o l l a r s o f h a p t e n d e s i g n and s y n t h e s i s . B o t h mono- and p o l y c l o n a l a n t i b o d i e s have a major r o l e and, we w i l l see t h e r o l e o f m o n o c l o n a l a n t i b o d i e s e x p a n d i n g . F o r most problems, p o l y c l o n a l s e r a w i l l p r o v i d e adequate s e n s i t i v i t y and s p e c i f i c i t y f a s t e r and a t a f r a c t i o n o f t h e c o s t o f m o n o c l o n a l a n t i b o d i e s . The i d e a t h a t any m o n o c l o n a l a n t i b o d y will provide greater sensitivity and s p e c i f i c i t y than a p o l y c l o n a l i s not c o r r e c t . I f one i s t o i n v e s t i n m o n o c l o n a l t e c h n o l o g y i t s h o u l d be used t o develop a l a r g e l i b r a r y t o the hapten o f choice. This l i b r a r y c a n t h e n be s c r e e n e d t o o b t a i n t r u l y s u p e r i o r a n t i b o d i e s f o r d e f i n e d a p p l i c a t i o n s . F o r i n s t a n c e one c a n s c r e e n t h e l i b r a r y f o r a n t i b o d i e s o f h i g h s p e c i f i c i t y o r a n t i b o d i e s w h i c h may be c l a s s s p e c i f i c . One a l s o c o u l d s c r e e n t h e l i b r a r y f o r a n t i b o d i e s w h i c h w i l l g i v e h i g h s e n s i t i v i t y o r even i n some c a s e s l o w e r s e n s i t i v i t y . Once a n t i b o d i e s a r e found which g i v e optimum s p e c i f i c i t y and



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s e n s i t i v i t y , one c a n r e s c r e e n f o r a n t i b o d i e s w h i c h a r e r e s i s t a n t t o s o l v e n t s and/or m a t r i x e f f e c t s . W i t h p r o p e r h a p t e n d e s i g n and a l a r g e l i b r a r y , one can s c r e e n f o r the a n t i b o d i e s o f the d e s i r e d characteristics. With the development of biosensors, the a v a i l a b i l i t y o f d e f i n e d monoclonal a n t i b o d i e s o f v a r y i n g a f f i n i t y and a v i d i t y w i l l be v e r y i m p o r t a n t . U n f o r t u n a t e l y many hybridoma p r o j e c t s end w i t h s c i e n t i s t s f i n d i n g the a n t i b o d y t h e y s c r e e n e d f o r b u t n o t the a n t i b o d y t h a t t h e y wanted. A l t h o u g h e x p e n s i v e i n d o l l a r s and time, the i n v e s t m e n t needed f o r s u p e r i o r m o n o c l o n a l a n t i b o d i e s i s d r o p p i n g . T h i s c o s t may seem h i g h i n i t i a l l y , b u t i t i s a s m a l l i n v e s t m e n t compared t o the major i n v e s t m e n t needed t o c h a r a c t e r i z e and v a l i d a t e an a s s a y . The c o s t i s even s m a l l compared t o a modern chromatograph and work s t a t i o n . F o r many compounds t h i s i n v e s t m e n t w i l l be v e r y c o s t e f f e c t i v e so l o n g as t h e p l a n i s t o o b t a i n a l i b r a r y o f s u p e r i o r m o n o c l o n a l a n t i b o d i e s r a t h e r t h a n any m o n o c l o n a l a n t i b o d y . Once a m o n o c l o n a l a n t i b o d y e x i s t s , the cDNAs c o d i n g f o r the r e s p e c t i v e l i g h t and heavy c h a i n s c a n be c l o n e d . These cDNAs t h e n c a n be e n g i n e e r e d t o p r o v i d e v e r y i n e x p e n s i v e a n t i b o d i e s w h i c h c a n be f u r t h e r t a i l o r e d for a p p l i c a t i o n s i n immunoaffinity chromatography o r biosensor development. A l t h o u g h t h i s added i n v e s t m e n t seems v e r y h i g h a t t h i s time, the technologies involved are advancing rapidly and r e c o m b i n a n t a n t i b o d i e s c a n be a n t i c i p a t e d t o have a f u t u r e r o l e i n the immunodiagnostic a r e a (12-14). T h e r e i s even the hope t h a t one may be able to screen for antibodies i n b a c t e r i a by using r e c o m b i n a n t DNA t e c h n o l o g y . Development o f t h e s e t e c h n o l o g i e s i s i n the f u t u r e . However, i t i s o b v i o u s t h a t the f i e l d o f a n t i b o d y p r o d u c t i o n i s i n f o r some e x c i t i n g changes. Based on t h i s p o t e n t i a l we a r e p l a c i n g a major e f f o r t i n the a r e a o f a n t i b o d y e n g i n e e r i n g . How S h o u l d Immunoassays f o r E n v i r o n m e n t a l Samples be Standardized? T h i s q u e s t i o n c a n be b r o k e n down i n t o many s u b t o p i c s r e l a t i v e t o good laboratory practice, assay criteria, specifications for immunoassay readers and many more. Obviously the need for s t a n d a r d i z a t i o n w i l l v a r y d e p e n d i n g upon the u s e s o f the assay. A l s o d i f f e r e n t r e g u l a t o r y agencies w i l l develop d i f f e r i n g c r i t e r i a . I n i t i a l l y a t a r g e t c o u l d be t o use the c r i t e r i a s e t f o r w a r d by the AOAC and d i s c u s s e d i n p a r t by H i n t o n e t a l . (16) and o t h e r s (17-20) a t t h i s m e e t i n g . As d i s c u s s e d below, i f some w o r k i n g p a p e r s appear on s t a n d a r d i z a t i o n o r a committee c o u l d be e s t a b l i s h e d t o p r o v i d e a d v i c e on s t a n d a r d i z a t i o n i t would s t r e a m l i n e a c c e p t a n c e by n o t r e q u i r i n g e a c h agency t o r e d i s c o v e r the c r i t e r i a w h i c h a r e u s e f u l f o r acceptance. I n g e n e r a l immunoassay i s n o t hardware i n t e n s i v e . However, the p o o r r e p r o d u c i b i l i t y o f b i n d i n g t o some ELISA p l a t e s , i s a recurring nightmare to a n a l y s t s . While sources of intraplate v a r i a b i l i t y o t h e r t h a n the p l a t e s t h e m s e l v e s (washing, p i p e t t i n g error, thermal gradients) may contribute significantly, major d i f f e r e n c e s i n v a r i a b i l i t y among p l a t e s have been documented (21). One o f our s t u d i e s (22) has i d e n t i f i e d i n t e r w e l l v a r i a b i l i t y t o be by f a r the l a r g e s t s o u r c e of v a r i a b i l i t y . This v a r i a b i l i t y i s analogous to chromatographic b a s e l i n e n o i s e , so i t i s a c r i t i c a l



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determinant o f assay performance i n m i c r o p l a t e systems. Also, d i m e n s i o n a l s t a n d a r d i z a t i o n i s a t p r e s e n t a d i s t a n t dream. I n our experience, at l e a s t 6 d i f f e r e n t sets of dimensional s p e c i f i c a t i o n s a r e u s e d by the few l a r g e s t m a n u f a c t u r e r s o f p l a t e s . M a n u f a c t u r e r s o f r e a d e r s who do n o t make m a t c h i n g p l a t e s must t h e n compromise t h e i r s p e c i f i c a t i o n s t o be a b l e t o r e a d a l l o f the p l a t e s on the market. As w i t h the equipment f o r c h r o m a t o g r a p h i c systems, n o t a l l r e a d e r s a r e i d e n t i c a l i n performance (23). I t i s c r i t i c a l that users look carefully at the specifications of the equipment purchased and have a r o u t i n e system of rechecking instrument performance. I t would be useful t o have a committee t o make manufacturers o f p l a t e s and r e a d e r s f u l l y aware o f the unique demands o f r i g o r o u s l y q u a n t i t a t i v e m i c r o p l a t e methods. T h i s would h o p e f u l l y l e a d t o the s e t t i n g o f d i m e n s i o n a l and q u a l i t y s t a n d a r d s f o r p l a t e s and r e a d e r s . These changes c o u l d have a d r a m a t i c e f f e c t on s p e e d i n g a c c e p t a n c e o f the t e c h n o l o g y and thus e x p a n d i n g the market f o r enzyme immunoassay (EIA) p l a t e s . What Can I n d u s t r i e s and R e g u l a t o r y A g e n c i e s Do t o Advance the Technology? A major c o n t r i b u t i o n t h a t t h e s e groups c a n make t o the advancement o f the t e c h n o l o g y i s t o d e v e l o p the i n - h o u s e e x p e r t i s e to e v a l u a t e the s t r e n g t h s and l i m i t a t i o n s o f the t e c h n o l o g y . As d i s c u s s e d above a major t h r e a t t o the t e c h n o l o g y comes when i t i s advocated f o r i n a p p r o p r i a t e a p p l i c a t i o n s . In the chemical i n d u s t r y the best way to advance the t e c h n o l o g y i s t o have an in-house s u c c e s s . T h i s c a n be a c c o m p l i s h e d by s e l e c t i n g a c h e m i c a l l y r e a s o n a b l e t a r g e t and p l a n n i n g ahead t o o b t a i n adequate c h e m i c a l s u p p o r t . As mentioned above, i t may n o t be good t o s e l e c t a new p r o d u c t where t h e r e w i l l be a g r e a t d e a l o f time p r e s s u r e on the new a s s a y . Agencies e s p e c i a l l y can p r o v i d e a l e a d e r s h i p r o l e i n s e v e r a l ways. F o r example the r o l e p l a y e d by the C a l i f o r n i a Department o f Food and A g r i c u l t u r e has been v e r y p o s i t i v e ( 1 2 ) , and h o p e f u l l y other agencies with responsibilities at the national and i n t e r n a t i o n a l l e v e l s w i l l take a c t i v e r o l e s as w e l l . An i m p o r t a n t c o n t r i b u t i o n i s t o d e v e l o p s t r a t e g i c p l a n s f o r the development o f the t e c h n o l o g y and t h e n attempt t o f u n d work w h i c h does n o t l e a d t o duplication of effort. The private sector w i l l be greatly encouraged i f agencies can provide clear procedures for the v a l i d a t i o n o f a s s a y s and c l e a r r e q u i r e m e n t s f o r the d a t a needed. The most s i g n i f i c a n t r o l e t h a t government c o u l d p l a y i s i n the a r e a o f assay s t a n d a r d i z a t i o n ; c e r t a i n l y a v e r y a c t i v e r o l e i s p o s s i b l e . A p r o c e d u r e now u s e d by the Food and Drug A d m i n i s t r a t i o n (FDA) c o u l d be immediately implemented by the Environmental P r o t e c t i o n Agency (EPA). T h i s would s i m p l y i n v o l v e t e s t i n g the c l a i m s o f a m a n u f a c t u r e r w i t h r e g a r d t o the s p e c i f i c a t i o n s o f t h e i r p a r t i c u l a r a s s a y . Any l e a d e r s h i p t h a t a g e n c i e s c a n p r o v i d e w i l l b e n e f i t the f i e l d g r e a t l y , and the c u r r e n t e f f o r t o f the Las Vegas EPA l a b o r a t o r y w i l l have a major impact i n t h i s a r e a . As d i s c u s s e d above g e t t i n g a s s a y s i n t o the hands o f u s e r s i s a major g o a l . T h i s sometimes i s seen as a major h u r d l e t h a t i s d i f f e r e n t from c l a s s i c a l c h r o m a t o g r a p h i c methods. The v i e w i s t h a t r e a g e n t s may someday v a n i s h and the a s s a y cannot be p e r f o r m e d .



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A c t u a l l y the same s i t u a t i o n e x i s t s i n the c h r o m a t o g r a p h i c a r e a . F o r i n s t a n c e v e r y few l a b o r a t o r i e s a r e c a p a b l e o f b u i l d i n g t h e i r own gas chromatograph o r mass s p e c t r o p h o t o m e t e r . While t h e r e i s no g u a r a n t e e by i n d u s t r y t h a t s u c h equipment always w i l l be a v a i l a b l e , the market p l a c e p r o v i d e s an i n c e n t i v e f o r t h i r d p a r t y companies t o p r o v i d e such equipment. The same s i t u a t i o n now e x i s t s w i t h EIA r e a d e r s . I f r e g u l a t o r y a g e n c i e s would s u g g e s t a system where a p o o l o f a n t i b o d y and h a p t e n t r a c e r i s p r o v i d e d t o them f o r a r c h i v i n g o r to a l a r g e c h e m i c a l o r b i o c h e m i c a l s u p p l y company, t h i s fear r e g a r d i n g the a v a i l a b i l i t y o f r e a g e n t s might v a n i s h . A major d i f f e r e n c e between immunoassay development and the development o f a c h r o m a t o g r a p h i c a s s a y i s t h a t f o r t h e former a s i n g l e moderate i n v e s t m e n t i s needed t o d e v e l o p a n t i b o d i e s and t r a c e r s . A d a p t i n g the r e s u l t i n g a s s a y s t o hundreds o f l a b o r a t o r i e s t h e n i s r e l a t i v e l y cheap. However, w i t h c h r o m a t o g r a p h i c a s s a y s the d e v e l o p e r can assume a heavy i n v e s t m e n t i n equipment i n i n d i v i d u a l u s e r l a b o r a t o r i e s . A l t h o u g h i n some c a s e s the i n i t i a l c o s t o f a s s a y development may be a l i t t l e l e s s f o r a c h r o m a t o g r a p h i c system, the t o t a l c o s t t o s o c i e t y i s d r a m a t i c a l l y r e d u c e d i f immunoassays a r e d e v e l o p e d . I f government a g e n c i e s can f u n d the i n i t i a l development of a v a r i e t y o f a s s a y s o r make the development o f s u c h a s s a y s a t t r a c t i v e t o t h i r d p a r t y companies, the r a t e o f a c c e p t a n c e o f the technology w i l l increase dramatically. How Can We A v o i d " T u r f Wars" i n the Immunoassay F i e l d ? A major p r o b l e m t h a t the t e c h n o l o g y has f a c e d o v e r the l a s t decade has b e e n t h a t t h e r e were too few a s s a y s . I t has b e e n d i f f i c u l t t o j u s t i f y the amount o f time needed t o l e a r n the t e c h n o l o g y t o a n a l y t i c a l l a b o r a t o r i e s when t h e r e a r e so few a p p l i c a t i o n s . We s t i l l a r e i n a s i t u a t i o n where f a r too few a s s a y s e x i s t . C e r t a i n l y o v e r the n e x t few y e a r s a d d i t i o n a l groups e n t e r i n g the f i e l d w i l l be o f g r e a t a s s i s t a n c e , and we soon w i l l be t o the p o i n t where enough a s s a y s e x i s t f o r i t t o be a t t r a c t i v e f o r a r e s i d u e l a b o r a t o r y t o d e v o t e a component o f i t s r e s o u r c e s t o immunochemical a n a l y s i s . In s p i t e o f the numerous p r o j e c t s i n need o f scientists w o r k i n g on them, the s i t u a t i o n seems t o be e v o l v i n g where s e v e r a l l a b o r a t o r i e s a r e w o r k i n g on the same compounds. W i t h some major problems such as the d i o x i n s and d i b e n z o f u r a n s o r t r i a z i n e s t h i s c l e a r l y i s j u s t i f i e d . The v a r i e t y o f i s o m e r s and m e t a b o l i t e s w h i c h need t o be a n a l y z e d as w e l l as the p o l i t i c a l importance o f the c l a s s o f compounds r e q u i r e the i n p u t o f s e v e r a l l a b o r a t o r i e s . I n addition a common group of compounds targeted by several l a b o r a t o r i e s w i l l f a c i l i t a t e comparison o f d i f f e r i n g t e c h n o l o g i e s . W i t h o t h e r p r o j e c t s the r e s o u r c e s c o u l d be b e t t e r u t i l i z e d w i t h o u t d u p l i c a t i o n , b u t a t t h i s s t a g e o f development i n the t e c h n o l o g y , i t certainly helps the technology to have procedures repeated i n d e p e n d e n t l y i n s e v e r a l l a b o r a t o r i e s . T h i s s i t u a t i o n c l e a r l y i s no different from classical methodology where hundreds of "new" a n a l y t i c a l methods have been p u b l i s h e d f o r DDT, but i t is a s i t u a t i o n where we need t o a v o i d n o n p r o d u c t i v e d u p l i c a t i o n . Some d u p l i c a t i o n can be a v o i d e d by the a g e n c i e s t h a t f u n d the r e s e a r c h . Those o f us who r u n s o f t money l a b o r a t o r i e s o f t e n a r e i n



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t h e p o s i t i o n o f p r o v i d i n g the a s s a y s f o r w h i c h we r e c e i v e f u n d s . When d i f f e r e n t a g e n c i e s need the same a s s a y , u n l e s s t h e r e i s a major e f f o r t at coordination, s e v e r a l l a b o r a t o r i e s may receive funds to develop assays f o r the same compound. The chemical i n d u s t r y a l s o s h o u l d r e a l i z e t h a t i f a r e g u l a t o r y agency does n o t have access to their assays, that they will have to fund development o f a d u p l i c a t e a s s a y s e p a r a t e l y . When the c h e m i c a l i n d u s t r y r e a l i z e s t h a t i t i s t o t h e i r b e n e f i t t o make a s s a y s f o r t h e i r compounds a v a i l a b l e , t h e n t h e r e w i l l be few c a s e s o f academic and government l a b o r a t o r i e s d e v e l o p i n g a s s a y s w h i c h a l r e a d y e x i s t i n i n d u s t r y . Immunochemical companies who s e l l q u a l i t a t i v e k i t s o r assays where the methods u s e d f o r assay p r o d u c t i o n are not available to the p u b l i c should r e a l i z e again that regulatory a g e n c i e s o r government l a b o r a t o r i e s may have t o d e v e l o p competing assays. A b u r d e n c e r t a i n l y w i l l f a l l on academic l a b o r a t o r i e s f o r the n e x t few y e a r s t o ensure t h a t a s p i r i t o f c o l l a b o r a t i o n e x i s t s among the l a b o r a t o r i e s i n the f i e l d . The q u e s t i o n s i s n o t who d e v e l o p s the * f i r s t ' a s s a y f o r a compound o r the * b e s t ' a s s a y b u t r a t h e r t h a t the f i e l d advances and a s s a y s g e t i n t o the hands o f u s e r s . The t e c h n o l o g y i s complex enough t h a t the f i e l d w i l l b e n e f i t from d i f f e r e n t methods even on the same compound. H o p e f u l l y w i t h w i d e l y u s e d a s s a y s such as t h o s e f o r the t r i a z i n e s the l a b o r a t o r i e s i n v o l v e d w i l l exchange haptens and a n t i b o d i e s and j o i n t l y use a l i b r a r y o f r e a g e n t s t o g e n e r a t e the m u l t i l a b o r a t o r y d a t a needed f o r validation. A l l l a b o r a t o r i e s now have the o b l i g a t i o n , n o t o n l y t o d e v e l o p a s s a y s , b u t t o g e t the a s s a y s i n t o the hands o f u s e r s . R a t h e r t h a n r a c i n g t o d e v e l o p new a s s a y s , p o s s i b l y we s h o u l d j u d g e our s u c c e s s b a s e d on our a b i l i t y t o t r a n s f e r the t e c h n o l o g y s u c c e s s f u l l y t o u s e r l a b o r a t o r i e s . We r o u t i n e l y send r e a g e n t s t o o t h e r l a b o r a t o r i e s in the f i e l d . We t r y t o send t h e s e r e a g e n t s w i t h a detailed p r o t o c o l as w e l l . Not t h a t t h i s i s the b e s t way t o r u n the a s s a y , b u t i t r e p r e s e n t s a method t h a t w i l l g i v e r e l i a b l e r e s u l t s i n the hands o f b o t h e x p e r t s and n e o p h y t e s . T h e r e i s a Need f o r a Committee t o C o o r d i n a t e the Development o f Immunoassays i n the E n v i r o n m e n t a l F i e l d . A committee such as the one outlined above now exists i n Europe. In the following p a r a g r a p h s we s u g g e s t t h a t such a committee may be o f b e n e f i t i n the U n i t e d S t a t e s . However, t h e r e i s a c a u t i o n t h a t the committee comes w i t h a v a r i e t y o f problems. F o r the purpose o f t h i s p a p e r the acronym f o r t h i s Committee f o r the E v a l u a t i o n o f Immunoassay i n E n v i r o n m e n t a l C h e m i s t r y w i l l be the p a l i n d r o m e CEIEC. P o s s i b l e R o l e s o f CEIEC. The major r o l e o f CEIEC would be t o a c t as a c a u t i o u s a d v o c a t e f o r the o v e r a l l t e c h n o l o g y r a t h e r t h a n a s i n g l e a s s a y . I t a l s o c o u l d a c t as a c l e a r i n g h o u s e f o r i n f o r m a t i o n and p e o p l e d e a l i n g w i t h immunoassay and a way f o r United States r e s e a r c h e r s t o c o o r d i n a t e w i t h s c i e n t i s t s i n t e r n a t i o n a l l y . A major g o a l would be t o s e r v e as a forum f o r d i s c u s s i o n o f problems r e l e v a n t t o the e n t i r e f i e l d . Such a committee c o u l d encourage


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i n v e s t m e n t i n the f i e l d i n g e n e r a l from b o t h the p u b l i c and p r i v a t e s e c t o r . P o s s i b l y the most u r g e n t r o l e f o r such a committee would be t o f a c i l i t a t e and c o o r d i n a t e v a l i d a t i o n e f f o r t s f o r the t e c h n o l o g y i n g e n e r a l as w e l l as f o r s p e c i f i c a s s a y s . Problems w i t h CEIEC. In advocating the e s t a b l i s h m e n t o f such a committee one must c o n s i d e r that i t can have b o t h real and p e r c e i v e d p r o b l e m s . CEIEC c o u l d be s e e n as an u n f a i r a d v o c a t e o f one t e c h n o l o g y o r company o v e r a n o t h e r . T h e r e i s a danger t h a t some r e g u l a t o r y a g e n c i e s would see i t as u s u r p i n g t h e i r r o l e s . This would have t o be a v e r y c a r e f u l l y drawn l i n e . The committee c o u l d a c t f a s t e r t h a n an agency s i n c e i t s a c t i o n s would n o t be legally b i n d i n g , b u t the committee's l i f e c o u l d be v e r y s h o r t i f i t was s e e n as a t h r e a t t o e x i s t i n g a g e n c i e s . C e r t a i n l y the committee would have t o a v o i d the charge t h a t i t was raising funds or a d v a n c i n g the reputation o f one laboratory a t the expense of a n o t h e r . C l e a r l y the g o a l o f CEIEC would be t o expand r a t h e r t h a n to restrict representation which could lead to an unwieldy o r g a n i z a t i o n . I t s management would p r e s e n t a p o l i t i c a l t i g h t rope where p o l i c i e s c o n s i d e r e d good f o r the f i e l d w o u l d have t o be e n f o r c e d by mutual a c c e p t a n c e r a t h e r t h a n r e g u l a t i o n . I n t h i s l i g h t the committee would be s i m i l a r t o some o f the i n d u s t r i a l groups trying to avoid pesticide resistance problems. Finally, as a scientific community we must ask i f the benefits of such an o r g a n i z a t i o n w i l l t r u l y outweigh the added a d m i n i s t r a t i v e l o a d and even p o t e n t i a l dangers s h o u l d the committee be r u n i n a negative way. R e c e n t Work i n the

Immunoassay A r e a

The 198th ACS m e e t i n g c e r t a i n l y i s a landmark m e e t i n g i n the immunoassay f i e l d . F o r the f i r s t time a t t h i s m e e t i n g we have s e e n r e p o r t s from a v a r i e t y o f major a g r i c u l t u r a l c h e m i c a l companies about the i n - h o u s e e f f o r t s i n immunodiagnostics (11.24-29) as w e l l as c o l l a b o r a t i v e v a l i d a t i o n s t u d i e s between a b i o t e c h n o l o g y company and a u n i v e r s i t y (20) and a c o n t r a c t l a b o r a t o r y ( H ) . I n a d d i t i o n to the development o f p o l y c l o n a l based systems, there is an i n c r e a s e d i n t e r e s t i n the development o f m o n o c l o n a l a n t i b o d i e s f o r environmental chemicals (luiHi22.) · Deschamps and Hall (32) p r e s e n t e d a n i c e c o m p a r i s o n o f the r e l a t i v e a t t r i b u t e s o f mono- v s p o l y c l o n a l b a s e d systems f o r the h e r b i c i d e p i c l o r a m . It is reassuring in the Agrochemical Division to see p r e s e n t a t i o n o f r e s u l t s on v e t e r i n a r y drugs (21) and e n v i r o n m e n t a l compounds w h i c h a r e n o t p e s t i c i d e s as w e l l as t o see the e n t r a n c e o f s y n t h e t i c c h e m i s t s i n t o the a r e a ( 3 3 ) . As the t a r g e t s s e l e c t e d for immunoassay development become more difficult, chemical e x p e r t i s e i n h a p t e n d e s i g n w i l l become more c r i t i c a l . T h i s m e e t i n g was notable f o r the f i r s t r e p o r t o f the use o f computer aided design i n a n a l y s i s of hapten p r e s e n t a t i o n (10). D i f f e r e n t animals o f t e n have c o m p l e t e l y d i f f e r e n t a n t i b o d y c o m b i n i n g s i t e s t o the same a n t i g e n . Thus, one can a n t i c i p a t e an element o f a r t and l u c k i n h a p t e n d e s i g n . However, one can s t a c k the odds on the s i d e o f a



11.



f a v o r a b l e a s s a y by t h e a p p l i c a t i o n o f c a r e f u l h a p t e n d e s i g n . A n i c e example o f c l e v e r h a p t e n d e s i g n was p r e s e n t e d b y M e i and Y i n (34) on c o u p l i n g t h e c a r b o x y l i c a c i d o f methoprene. I n g e n e r a l i n t h e j u v e n i l e hormone f i e l d t h e s i m p l i s t i c a p p r o a c h o f a t t a c h i n g t h e acid directly t o a l y s i n e has b e e n used. T h i s work u s e d 4hydroxybutanoic acid t o minimize handle recognition. A similar a p p r o a c h was u s e d w i t h a l a c h l o r where a s u l f u r was u s e d t o mimic a chlorine (2£). This assay illustrates both a s t r e n g t h and l i m i t a t i o n o f immunoassay. The h a p t e n d e s i g n i n d i c a t e s t h a t t h e a s s a y w i l l d e t e c t some major d e g r a d a t i o n p r o d u c t s o f a l a c h l o r i n a d d i t i o n t o t h e p a r e n t . S i n c e t h e s e workers have d e s c r i b e d how t h e i r a s s a y was made, t h e c h a r a c t e r i s t i c c a n be u s e d t o advantage in exposure s t u d i e s (22) o r t h e i n t e r f e r i n g m a t e r i a l s easily removed. The Dupont work (2A,2JL) i n a d d i t i o n t o s e v e r a l o t h e r s t u d i e s provides an excellent correlation between immunochemical and c l a s s i c a l methods. The work a l s o p r o v i d e s a u s e f u l c a u t i o n t h a t with such sensitive assays extreme c a r e i s needed i n sample handling. Excellent correlations also were obtained between c l a s s i c a l and immunochemical methods w i t h clomazone (2â). A c a u t i o n common t o b o t h a s s a y s i s t h a t n e i t h e r c o r r e l a t e s p e r f e c t l y w i t h b i o a s s a y . T h i s i s a reminder t h a t immunoassays a r e p h y s i c a l a s s a y s w i t h no m a g i c a l b i o l o g i c a l p r o p e r t i e s . An i n t e r e s t i n g v a l i d a t i o n study u s i n g t r i a z i n e a n t i b o d i e s i n d i c a t e d that h i g h pressure l i q u i d chromatography (HPLC) detected atrazine in a sample while immunoassay d i d n o t . When t h e sample was f u r t h e r a n a l y z e d by gas chromatography-mass s p e c t r o m e t r y , t h e ELISA r e s u l t s were c o n f i r m e d ( 1 1 ) . T h i s c e r t a i n l y does n o t i n d i c a t e t h a t ELISA i s s u p e r i o r t o HPLC, b u t t h a t t h e d i f f e r e n t methods complement each o t h e r and c a n be u s e d t o c r o s s check each o t h e r . As w i t h o u r work w i t h a v a r i e t y o f compounds, t h e work from Dupont on t r i a z i n e s (£8) i l l u s t r a t e s t h a t t h e same a n t i b o d y c a n g i v e a s s a y s o f v a s t l y d i f f e r e n t s p e c i f i c i t y and s e n s i t i v i t y i f i t i s used with a d i f f e r e n t c o a t i n g antigen. T h i s a g a i n i l l u s t r a t e s the importance o f a l a b o r a t o r y d e v e l o p i n g a l i b r a r y o f a n t i b o d i e s and a n t i g e n s f o r a whole c l a s s o f compounds and t h e r e s p e c t i v e metabolites, rather than a piece meal approach to assay development. S e v e r a l o f t h e p a p e r s p r e s e n t e d d e m o n s t r a t e t h a t t h e same a n t i b o d y c a n be u s e d i n a v a r i e t y o f d i f f e r e n t f o r m a t s (31-32). T h i s c h a r a c t e r i s t i c w i l l become i n c r e a s i n g l y i m p o r t a n t . Certainly t h e same a s s a y c a n be u s e d b o t h f o r a n a l y s i s o f e n v i r o n m e n t a l samples and i n t h e a n a l y s i s o f human body f l u i d s as a b i o m a r k e r approach (22,3^); i n the l a t t e r a p p l i c a t i o n immunoassay offers numerous advantages. Many o f t h e problems now f a c e d by immunoassay m e n t i o n e d above and i n other a r t i c l e s (3,4,36) c l e a r l y a r e b e i n g a d d r e s s e d by s c i e n t i s t s i n academic, governmental and i n d u s t r i a l l a b o r a t o r i e s . W i t h t h e l e v e l o f e x p e r t i s e t h a t i s now e v i d e n t i n t h e f i e l d , one c a n have c o n f i d e n c e t h a t t h e problems w i l l be s o l v e d and t h a t immunochemical a s s a y s w i l l assume t h e i r r i g h t f u l r o l e as one o f t h e t o o l s o f t h e modern e n v i r o n m e n t a l c h e m i s t .


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R e p r e s e n t a t i v e Work from T h i s

Laboratory

A s s a y development c a n be d e s c r i b e d i n a t i m e l i n e s u c h as shown i n F i g u r e 2. We w i l l i l l u s t r a t e p o i n t s on t h i s t i m e l i n e w i t h examples from our l a b o r a t o r y . An example o f an a s s a y d e v e l o p e d i n our l a b o r a t o r y w h i c h i s w e l l a l o n g the t i m e l i n e i s the one f o r the h e r b i c i d e m o l i n a t e . T h i s compound i s r e l a t i v e l y v o l a t i l e and has a r e l a t i v e l y h y d r o l y t i c a l l y unstable thiocarbamate bond. Four h a p t e n s were s y n t h e s i z e d by a t h i o l replacement r e a c t i o n with thiocarbamate s u l f o n e s which l e f t the h e x a h y d r o a z e p i n e r i n g u n m o d i f i e d . Two o f the h a p t e n s had a l k y l c h a i n s p a c e r s t e r m i n a t i n g i n a c a r b o x y l i c a c i d . The o t h e r two had ρ-aminophenyl s p a c e r s . A n t i b o d i e s a g a i n s t an a l k y l c h a i n d e r i v a t i v e conjugated to keyhole l i m p e t hemocyanin (KLH) were u s e d i n an indirect competitive ELISA format with a ρ-aminophenyl hapten c o n j u g a t e d t o c o n a l b u m i n as the c o a t i n g a n t i g e n . T h i s a s s a y had a l i m i t o f d e t e c t a b i l i t y o f about 3 ppb and an I50 o f a p p r o x i m a t e l y 100 ppb. A l a b o r a t o r y d i s s i p a t i o n s t u d y was c o n d u c t e d and samples a n a l y z e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g and ELISA. Samples were e i t h e r added d i r e c t l y t o l i q u i d s c i n t i l l a t i o n c o c k t a i l and c o u n t e d or diluted i n b u f f e r and mixed w i t h antibody f o r the ELISA determination. T h i s p i l o t study confirmed t h a t the ELISA c o u l d q u a n t i t a t i v e l y measure m o l i n a t e i n samples, w i t h the advantage o f n o t n e e d i n g e x t r a c t i o n p r i o r t o a n a l y s i s . D e t a i l s o f the h a p t e n s y n t h e s i s , a s s a y development and o p t i m i z a t i o n were r e p o r t e d by Gee et a l . (37). To f u r t h e r v a l i d a t e the a s s a y f o r use w i t h environmental samples, water samples s p i k e d w i t h m o l i n a t e were e x t r a c t e d and a n a l y z e d by ELISA and GC. R e c o v e r y comparisons were made between ELISA and GC f o r b o t h l i q u i d - l i q u i d and s o l i d phase e x t r a c t i o n methods. R e c o v e r i e s were g r e a t e r t h a n 90% f o r l e v e l s as low as l p p b f o r a l l a n a l y s i s and e x t r a c t i o n method comparisons (38)· T h i s s t u d y a l s o d e s c r i b e d the u t i l i t y and c o m p a t i b i l i t y between s o l i d phase e x t r a c t i o n and ELISA f o r m e a s u r i n g low c o n c e n t r a t i o n s o f m o l i n a t e . As much as 10% a c e t o n i t r i l e / p r o p y l e n e g l y c o l (1:1) o r 5% methanol had no e f f e c t on the m o l i n a t e a s s a y . D e t a i l s o f t h i s s t u d y were r e p o r t e d by L i e t a l . ( 3 8 ) . S u b s e q u e n t l y we have c o m p l e t e d an e x t e n s i v e v a l i d a t i o n s t u d y u s i n g f i e l d samples w h i c h c o n t a i n e d h i g h c o n c e n t r a t i o n s o f m o l i n a t e f o l l o w i n g an a e r i a l a p p l i c a t i o n . These samples were a n a l y z e d by ELISA d i r e c t l y a f t e r b u f f e r i n g and c o n f i r m e d by GC a n a l y s i s o f split samples. One of the most valuable lessons from this v a l i d a t i o n s t u d y was the importance o f the v a r i o u s q u a l i t y c o n t r o l c o n s i d e r a t i o n s ( 2 2 ) . From s i g m o i d a l s t a n d a r d c u r v e s , 20-60 p e r c e n t o f the c o n t r o l absorbance was d e t e r m i n e d e x p e r i m e n t a l l y t o be the r e g i o n o f g r e a t e s t p r e c i s i o n . Thus sample c o n c e n t r a t i o n s a r i s i n g from d a t a g e n e r a t e d o u t s i d e t h i s a r e a would be less reliable. C o n t r o l c h a r t s were c o n s t r u c t e d f o r b o t h p o s i t i v e and negative c o n t r o l samples as a means o f e v a l u a t i n g a s s a y p e r f o r m a n c e o v e r the s t u d y p e r i o d . Such c h a r t s can be u s e f u l i n d i c a t o r s o f changes i n the a s s a y t h a t may a f f e c t r e p o r t e d r e s u l t s and a r e a commonly u s e d



11.

HAMMOCK E T A L


tool in clinical c h e m i s t r y . These c o n t r o l d a t a were a l s o run t h r o u g h a n e s t e d a n a l y s i s o f v a r i a n c e . The l a r g e s t r e l a t i v e e r r o r contributions arose from well to well (or well replicate) v a r i a b i l i t y . O t h e r d e t a i l s o f t h i s s t u d y such as d a t a h a n d l i n g and o t h e r s o u r c e s o f p r o c e d u r a l e r r o r c a n be f o u n d i n H a r r i s o n e t a l . (22). We v i e w t h i s s e r i e s o f s t u d i e s as an e s s e n t i a l p r o t o t y p e f o r the e n t i r e development and v a l i d a t i o n p r o c e s s . F o r example, we have a l s o completed a s i m i l a r s t u d y f o r m o l i n a t e u s i n g an improved format t o a n a l y z e low c o n c e n t r a t i o n samples o b t a i n e d from the Sacramento R i v e r and a s s o c i a t e d d r a i n a g e c a n a l s . We a r e a l s o u s i n g t h i s p r o t o t y p e i n the development and v a l i d a t i o n o f our a s s a y s f o r t r i a z i n e s ( 3 9 ) . Our e x p e r i e n c e i n the v a l i d a t i o n o f the m o l i n a t e assay, especially our understanding of the quality control problems, has been c r u c i a l t o our successful transfer of the triazine assays to the California Department of Food and A g r i c u l t u r e (CDFA) and o t h e r l a b o r a t o r i e s f o r r o u t i n e a p p l i c a t i o n t o e n v i r o n m e n t a l samples. The same a s s a y which was t r a n s f e r r e d t o CDFA has been u s e d by our l a b o r a t o r y t o demonstrate the u s e f u l n e s s o f immunoassay f o r s c r e e n i n g water samples. I n t h i s s t u d y , 75 w e l l w a t e r samples were a n a l y z e d by GC and immunoassay f o r t r i a z i n e s . The b a c k g r o u n d l e v e l o f the immunoassay was 15 p p t , d e t e r m i n e d by r e p e a t e d s o l i d phase e x t r a c t i o n and a n a l y s i s o f r e a g e n t water b l a n k s ; the h i g h e s t l e v e l d e t e c t e d was a p p r o x i m a t e l y 0.3 ppb. The c o e f f i c i e n t o f v a r i a t i o n f o r a s i n g l e sample r u n 6 times was 10%. The v a r i a b i l i t y o f the two methods was comparable b a s e d on a n a l y s i s o f 18 p a i r e d samples; the mean c o e f f i c i e n t o f v a r i a t i o n was 11% f o r the ELISA and 13% f o r the GC. The most v a l u a b l e a t t r i b u t e o f t h i s a p p l i c a t i o n i s the low f a l s e n e g a t i v e r a t e . None o f the 40 samples h a v i n g the l o w e s t c o n c e n t r a t i o n s by ELISA were p o s i t i v e by GC. T h i s a s s a y i s now b e i n g u s e d i n a l a r g e s c a l e f i e l d t e s t by CDFA as w e l l as i n a worker exposure s t u d y . An important extension of our large validation studies involves the use of data bases from field studies in the development of improved statistical methods f o r a v a r i e t y of problems in quantitative applications of immunoassays. These problems i n c l u d e the p r e p a r a t i o n and analysis of calibration c u r v e s , t r e a t m e n t o f " o u t l i e r s " and v a l u e s below d e t e c t i o n l i m i t s , and the o p t i m i z a t i o n o f r e s o u r c e a l l o c a t i o n i n the analytical procedure. This last area is a difficult one because o f the m u l t i p l e l e v e l n e s t e d d e s i g n s f r e q u e n t l y u s e d i n l a r g e s t u d i e s such as o u r s ( 2 2 ) . We have d e v e l o p e d c o l l a b o r a t i o n s w i t h D a v i d Rocke and D a v i s Bunch ( s t a t i s t i c i a n s and n u m e r i c a l a n a l y s t s a t D a v i s ) i n order to address t h e s e problems w i t h i n the c o n t e x t o f working a s s a y s . H o p e f u l l y we a l s o can a d d r e s s the m a t h e m a t i c a l b a s i s o f u s i n g m u l t i p l e immunoassays as b i o c h e m i c a l " t a s t e r s " t o approach multianalyte situations. As mentioned above and i n v a r i o u s r e v i e w s (1.4.6-8). hapten s y n t h e s i s i s the f i r s t and p r o b a b l y one o f the most i m p o r t a n t s t e p s i n a s s a y development. The most g e n e r a l " r u l e s " i n h a p t e n d e s i g n a r e to l o c a t e the s p a c e r attachement distal to important haptenic



130


determinants t o maximize their exposure f o r antibody binding; s p a c e r s c o n t a i n i n g s t r o n g d e t e r m i n a n t groups s h o u l d be a v o i d e d t o m i n i m i z e the p r o d u c t i o n o f s p a c e r s p e c i f i c a n t i b o d i e s ; f u n c t i o n a l groups u s e d f o r c o u p l i n g must be c o m p a t i b l e w i t h t a r g e t m o l e c u l e f u n c t i o n a l groups t o a v o i d c r o s s - l i n k i n g o r m o d i f y i n g the t a r g e t d u r i n g c o n j u g a t i o n ; c o n s i d e r a t i o n o f h a p t e n and t a r g e t s t a b i l i t y under c o n j u g a t i o n , i m m u n i z a t i o n and a s s a y c o n d i t i o n s ; s e l e c t i o n o f h a p t e n t o improve s o l u b i l i t y o r a t l e a s t a v o i d s o l u b i l i t y problems and t o m i n i m i z e the number o f s y n t h e t i c s t e p s by u s i n g commerically a v a i l a b l e m a t e r i a l s o r , i n some c a s e s , direct c o u p l i n g to the t a r g e t . We have d e s c r i b e d and examined t h e s e b a s i c c r i t e r i a f o r h a p t e n s y n t h e s i s i n some d e t a i l u s i n g examples from our l a b o r a t o r y and the l i t e r a t u r e ( 4 0 ) . The s i g n i f i c a n c e o f t h e s e c r i t e r i a i s d e m o n s t r a t e d r o u t i n e l y i n our l a b o r a t o r y . I n some c a s e s , however, where the development o f an immunoassay may be d i f f i c u l t , the c h o i c e o f the h a p t e n t o be s y n t h e s i z e d may depend g r e a t l y on the u l t i m a t e use o f the a s s a y w i t h samples. F o r example, b e n t a z o n , a r i c e h e r b i c i d e , i s a s m a l l m o l e c u l a r w e i g h t (MW 240) m o l e c u l e w i t h an u n i q u e a c i d i c s e c o n d a r y sulfonamide (pKa 3.4). An N - d e r i v a t i z e d b e n t a z o n compound was s y n t h e s i z e d . T h i s changed the m o l e c u l e from an a c i d i c s e c o n d a r y sulfonamide to a n e u t r a l t e r t i a r y sulfonamide. Antisera raised a g a i n s t t h i s Ν-derivative c o n j u g a t e d t o KLH showed 2 - o r d e r s o f magnitude g r e a t e r b i n d i n g t o N - e t h y l b e n t a z o n t h a n t o b e n t a z o n ( F i g u r e 3 ) . An a s s a y such as t h i s c o u l d be u s e f u l i n a s s e s s i n g b e n t a z o n c o n c e n t r a t i o n s a f t e r the sample has been e t h y l a t e d . Sample d e r i v a t i z a t i o n p r i o r t o GC a n a l y s i s i s a commonly u s e d technique. W i t h compounds f o r w h i c h immunoassay development i s d i f f i c u l t , due t o the p r e s e n c e o f m u l t i p l e r e a c t i v e groups, a n t i b o d i e s a g a i n s t d e r i v a t i z e d compounds i s an a l t e r n a t i v e . Not a l l o f our a s s a y development work i s s u c c e s s f u l and we have f o u n d i t i n s t r u c t i v e t o a n a l y z e our n e g a t i v e r e s u l t s i n some d e t a i l . T h i s i s e s p e c i a l l y important f o r f a i l u r e s o f hapten design, where no u s e a b l e a n t i b o d i e s t o the t a r g e t compound a r e obtained. T h e r e a r e a number o f s t r a t e g i e s f o r a t t a c h i n g the h a p t e n t o a c a r r i e r m o l e c u l e . One i s t o a t t a c h the s p a c e r arm t o the p r o t e i n , t h e n a t t a c h the h a p t e n t o the f r e e f u n c t i o n a l group o f the s p a c e r arm ( 4 1 ) . We have f o u n d t h a t t h i s c o n j u g a t i o n s t r a t e g y f a i l e d t o p r o d u c e h i g h a f f i n i t y a n t i b o d i e s f o r b o t h a m i t r o l e ( F i g u r e 4) and b e n t a z o n , y i e l d i n g i n s t e a d a n t i b o d i e s w h i c h p r i m a r i l y r e c o g n i z e the s p a c e r . S i m i l a r d a t a have been o b t a i n e d by o t h e r s (42-43). These examples emphasize the v a l u e o f the a p p r o a c h t o a n t i b o d y s c r e e n i n g d e s c r i b e d by H a r r i s o n e£ a l . (40) i n u n d e r s t a n d i n g n e g a t i v e d a t a . I n o p t i m i z i n g an a s s a y d u r i n g development the n a t u r e o f the interaction of the analyte with the antibody is particularly important. Assays u s u a l l y are carried out under p h y s i o l o g i c a l c o n d i t i o n s and f r e q u e n t l y no e f f o r t i s made t o o p t i m i z e f o r pH, i o n i c s t r e n g t h , o r o t h e r f a c t o r s . These f a c t o r s can d i r e c t l y a f f e c t the a s s a y by m o d i f y i n g the p r e s e n t a t i o n o f the s o l u b l e a n a l y t e t o the a n t i b o d y o r c h a n g i n g the i n t e r a c t i o n o f the a n t i b o d y and the c o n j u g a t e d h a p t e n u s e d i n the a s s a y . F o r example, a s s a y s f o r some compounds show a d i s t i n c t pH dependence. I n an i n d i r e c t c o m p e t i t i v e


11.



Hapten Synthesis

-

ι Conjugation to Proteins

—

—

—

Immunization

Antibody Characterization


Assay Development

Assay Vafldatton Figure 2. Timeline i l l u s t r a t i n g the r e l a t i o n s h i p among t h e various assay development and implementation steps. It is c r i t i c a l that hapten p r e p a r a t i o n o c c u r f i r s t . However e x i s t i n g a s s a y s c a n be improved by r a t i o n a l improvements i n r e a g e n t s o r f o r m a t . Once a v a l i d a t i o n s t u d y i s u n d e r t a k e n , i t i s i m p o r t a n t t o use a c o n s t a n t f o r m a t and r e a g e n t s e t .

1000

10000

Log Inhibitor Concentration (μΜ) Figure 3 . R e l a t i v e s e n s i t i v i t y o f a r a b b i t antibody to bentazon and methylated bentazon. A rabbit antisera against an Nderivatized bentazon had b e t t e r recognition of methylated b e n t a z o n t h a n b e n t a z o n . C o a t i n g a n t i g e n s were Bz(6)-0-MPAA-BSA and Bz-succ-BSA f o r m e t h y l b e n t a z o n and b e n t a z o n respectively. These c u r v e s i n d i c a t e t h a t one may f i n d a much more s e n s i t i v e assay f o r a d e r i v a t i v e t h a n f o r t h e p a r e n t compound. As i n chromatographic analysis, i t may be advantageous to run immunoassays on d e r i v a t i v e s and an immunoassay s u c h as this c o u l d e a s i l y be u s e d t o q u a n t i t a t e d e r i v a t i z e d b e n t a z o n samples.


132

I M M U N O C H E M I C A L M E T H O D S F O R ENVIRONMENTAL ANALYSIS


1.60

200

800

3200 12800 51200

0

Antibody Dilution Figure 4. Rabbit antibody specificity for different coating antigens after the second bleeding. Binding o f the a n t i aminotriazole antibody t o t h e homologous coating antigen, a m i t r o l e - s u c c i n y l a t e d o v a l b u m i n (OVA), compared t o t h e n a t i v e p r o t e i n , OVA, and s u c c i n y l a t e d OVA as c o a t i n g a n t i g e n s . The a n t i b o d i e s show low b i n d i n g t o t h e homologous a n t i g e n b u t h i g h r e c o g n i t i o n o f t h e s u c c i n y l a t e d p r o t e i n . These d a t a show a common p r o b l e m when r a i s i n g a n t i b o d i e s t o v e r y s m a l l m o l e c u l e s . The antibodies from t h i s b l e e d have a low a f f i n i t y f o r t h e a m i n o t r i a z o l e h a p t e n w h i l e t h e h e m i s u c c i n a t e u s e d as a s p a c e r on the s u c c i n y l a t e d p r o t e i n i s a n t i g e n i c .


11.




ELISA, the s e n s i t i v i t y o f the a s s a y f o r the h e r b i c i d e g l y p h o s a t e was improved by a t l e a s t one o r d e r o f magnitude when the a s s a y was conducted at pH 5.8, rather than 7.3 (Figure 5). Glyphosate ( p h o s p h o n o m e t h y 1 g l y c i n e ) has s e v e r a l Z w i t t e r i o n i c forms, so i t i s n o t s u r p r i s i n g t h a t c a r e f u l o p t i m i z a t i o n o f pH l e d t o a d r a m a t i c a s s a y improvment as d i d a s h i f t away from phosphate b u f f e r s . T h i s work by Dr. J u n g i s a d r a m a t i c d e m o n s t r a t i o n o f how a s e r i e s of o p t i m i z a t i o n s c a n improve the s e n s i t i v i t y o f a s s a y s s e v e r a l o r d e r s o f magnatude. A l o n g the same l i n e , Sharp e t a l . (24) have r e p o r t e d t h a t 0.01M C a C l 2 g r e a t l y improves the s e n s i t i v i t y o f some, b u t n o t a l l assays f o r c h l o r s u l f u r o n . Although o p t i m i z a t i o n of s e n s i t i v i t y i s i m p o r t a n t , i t i s a l s o n e c e s s a r y t o r e c o g n i z e t h a t t h e r e may be a t r a d e o f f between the i n c r e a s e d s e n s i t i v i t y and a s s a y r u g g e d n e s s . F i g u r e 6 shows the c l a s s r e c o g n i t i o n o f one o f the t r i a z i n e antibodies produced in our laboratory. Immunoassays for the t r i a z i n e s w i l l be v e r y i n t e r e s t i n g due t o the e x i s t a n c e o f numerous s t r u c t u r a l analogs i n t h i s important c l a s s of h e r b i c i d e s . Although most d e g r a d a t i o n p r o d u c t s l a c k h e r b i c i d a l a c t i v i t y , t h e y can be important analytical targets as indicators of human or environmental exposure. The antibody in Figure 6 recognizes t r i a z i n e s h a v i n g a -CI o r - S C H 3 i n the 2 p o s i t i o n o f the r i n g , such as a t r a z i n e ( I 5 0 - 6.5 ppb), s i m a z i n e ( I C 5 0 - 54 ppb) and ametryne (I5O 130 ppb), regardless o f minor changes i n the N-alkyl s u b s t i t u t i o n p a t t e r n . The m o n o d e a l k y l a t e d o r 2-hydroxy m e t a b o l i t e s a r e a l s o r e c o g n i z e d , though t o a l e s s e r degree ( I 5 0 > 3500 p p b ) . We have o b t a i n e d s i m i l a r r e s u l t s f o r s e v e r a l o t h e r r a b b i t a n t i b o d i e s and f i v e mouse m o n o c l o n a l a n t i b o d i e s . Such a n t i b o d i e s c a n be u s e d f o r d i r e c t a n a l y s i s o f t r i a z i n e s by ELISA, s e p a r a t i o n o f r e l a t e d t r i a z i n e s p e c i e s by i m m u n o a f f i n i t y chromatography, o r r e m o v a l o f t r i a z i n e s from c o n t a m i n a t e d samples. The r e l a t i v e r e c o g n i t i o n o f the v a r i o u s t r i a z i n e s and t h e i r m e t a b o l i t e s depends on the h a p t e n u s e d t o p r o d u c e the a n t i b o d y . V a r i a b l e s we have e x p l o r e d thus f a r i n our work on the t r i a z i n e s include p o s i t i o n of conjugation, s p a c e r l e n g t h , and a l k y l group s u b s t i t u t i o n p a t t e r n (39-40). Use o f a l i b r a r y o f a n t i b o d i e s and c o a t i n g a n t i g e n s c a n r e s u l t i n e i t h e r c l a s s o r compound s p e c i f i c a s s a y s . A s e r i e s o f r e l a t e d a s s a y s can be used to screen samples for certain substitutions, aiding i d e n t i f i c a t i o n o f the immunoreacting compounds. We have a l s o a p p l i e d ELISA t o s e v e r a l b i o l o g i c a l p e s t i c i d e s i n c l u d i n g the e n d o t o x i n o f B a c i l l u s t h u r i n g i e n s i s k u r s t a k i ( B t k ) . I n t h i s a p p l i c a t i o n t o a m a c r o m o l e c u l a r a n a l y t e , we have u s e d a d o u b l e a n t i b o d y sandwich ELISA f o r Btk t o measure the amount o f ELISA r e a c t i v e m a t e r i a l i n f o r m u l a t i o n s o f the p e s t i c i d e . F i g u r e 7 shows the use o f an ELISA s t a n d a r d curve of g e l p u r i f i e d Btk e n d o t o x i n t o measure the immunoreactive m a t e r i a l i n d i l u t i o n s o f two B t k f o r m u l a t i o n s . I t has been d e m o n s t r a t e d t h a t ELISA c a n s e r v e as a q u i c k q u a l i t y c o n t r o l check f o r f o r m u l a t i o n s of B a c i l l u s thuringiensis israelensis (44). Such examples indicate that immunoassays w i l l be i n c r e a s i n g l y i m p o r t a n t as b i o l o g i c a l s and p r o d u c t s o f recombinant DNA r e s e a r c h impact our f i e l d ( 4 4 ) .


134

IMMUNOCHEMICAL

METHODS FOR ENVIRONMENTAL

ANALYSIS


Ο

I

Log (Molar Concentration) F i g u r e 5. I n h i b i t i o n o f a n t i - g l y p h o s a t e a n t i b o d y by glyphosate. Inhibition curves for polyclonal anti-glyphosate antibodies r a i s e d i n r a b b i t s were c o n d u c t e d i n 50 mM TRIS b u f f e r a t pH 5.8 and pH 7.3. The c u r v e s show an increased affinity between antibody and g l y p h o s a t e a t the lower pH b u f f e r , illustrating that f o r some compounds, optimization f o r pH is critical. C a r e f u l o p t i m i z a t i o n of assay c o n d i t i o n s i s e s p e c i a l l y important as the m o l e c u l e becomes s m a l l e r , f o r z w i t t e r i o n i c m a t e r i a l s , and f o r a n a l y t e s where h y d r o g e n b o n d i n g may p l a y a major r o l e i n antibody binding.



11.



0 0.0016 0.005 0.016 0.05 0.16 0.5 1.6 Inhibitor Concentration (uM)

5.0

16

50

F i g u r e 6. C o m p e t i t i v e i n h i b i t i o n ELISA r e s u l t s . R e c o g n i t i o n o f a t r a z i n e and s e v e r a l r e l a t e d compounds, i n c l u d i n g m e t a b o l i t e s , by a r a b b i t a n t i s e r u m r a i s e d a g a i n s t a c o n j u g a t e o f an a t r a z i n e h a p t e n and a c a r r i e r p r o t e i n . T h i s a n t i b o d y r e c o g n i z e s t r i a z i n e s h a v i n g e i t h e r a -CI o r -SCH^ i n the 2 p o s i t i o n o f t h e r i n g , such as a t r a z i n e and ametryne, r e g a r d l e s s o f minor changes i n the Na l k y l s u b s t i t u t i o n p a t t e r n . By c a r e f u l d e s i g n o f the immunizing and coating antigen, one can v a r y the p r o p e r t i e s of the r e s u l t i n g a s s a y t o d e t e c t a s i n g l e compound o r a p r e d i c t a b l e s e t o f r e l a t e d compounds.


136



0.6

0 Li 0

ι 1.0

ι 2.0

• ι » ι ι ι 1 1 3.9 7Λ 15.6 31.3 62.5 125 250 500 ng/mL gel purified endotoxin

i—l 1000

F i g u r e 7. S t a n d a r d c u r v e o f g e l p u r i f i e d 60 k d p r o t e i n e n d o t o x i n o f B t k was g e n e r a t e d u s i n g a double a n t i b o d y sandwich ELISA. The arrows i n d i c a t e d i l u t i o n s o f two B t k f o r m u l a t i o n s ; a b s o r b a n c e v a l u e s were u s e d t o d e t e r m i n e the e n d o t o x i n c o n c e n t r a t i o n s o f the f o r m u l a t i o n s , b a s e d on the s t a n d a r d c u r v e . The f o r m u l a t i o n d i l u t i o n s gave c u r v e s t h a t were v i r t u a l l y s u p e r i m p o s a b l e on t h e s t a n d a r d c u r v e . Such s i m i l a r i t y i n shape and s l o p e i n d i c a t e t h a t the a n t i b o d y i s l i k e l y b i n d i n g t o a s p e c i f i c d e t e r m i n a n t common t o t h e p u r i f i e d B t k and the B t k i n the f o r m u l a t i o n . I n g e n e r a l immunoassays f o r b i o p o l y m e r s a r e much e a s i e r t o d e v e l o p than assays f o r small molecules. However, o n l y r e c e n t l y has an i n t e r e s t i n t r a c e a n a l y s i s o f such m a t e r i a l s begun t o d e v e l o p i n the e n v i r o n m e n t a l f i e l d . Thus, sample c l e a n u p and h a n d l i n g i s n o t as s o p h i s t i c a t e d as w i t h s m a l l m o l e c u l e s .


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Problems in Immunochemical Technology

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Summary Immunoassays are now being seen as useful supplements to classical chromatographic analytical systems. In the near future we will see also an integration between immunochemical and classical procedures as immunochemical methods are used to prioritize or clean up samples before chromatography or as a post-column detection system. If "Green" initiatives in several countries pass we will see two striking trends. The first will be a shift of agricultural production to other areas and the second will be an acceleration in the development of biological methods of pest control. Immunochemistry offers tremendous advantages for inspection of the large increase in imported food and may be the only viable analytical method for many biological pesticides. Acknowledgments This work was supported in part by NIEHS Superfund PHS ES04699, EPA Cooperative Agreement No. CR-814709-01-0, and a grant from the California Department of Food and Agriculture. B.D.H. is a Burroughs Wellcome Scholar in Toxicology. K.M.S.S. was on leave from Forestry Canada, Forest Pest Management Institute, Sault Ste. Marie, Ontario Canada. Notice: Although the research described in this article has been supported by the United States Environmental Protection Agency (through assistance agreement CR-814709), it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Literature Cited 1. 2. 3.

4.

5.

6.

Hammock, B.D.; Mumma, R.O. In Pesticide Analytical Methodology; Zweig, G., Ed.; ACS Symposium Series No. 136; American Chemical Society: Washington, DC, 1980; pp 321-352. Langone, J . J . ; Van Vunakis, H. Res. Commun. Chem. Pathol. Pharmacol. 1975, 10, 163-171. Hammock, B.D. In Biotechnology for Crop Protection: Hedin, P.Α.; Menn J.J.; and Hollingworth, R.M., Eds.; ACS Symposium Series 379: American Chemical Society: Washington, DC, 1988; p 298. Van Emon, J.M.; Seiber, J.N.; Hammock, B.D. In Bioregulators for Pest Control; Hedin, P., Ed.; ACS Symposium Series No. 276; American Chemical Society: Washington, DC, 1985; pp 307316. Hammock, B.D.; Gee, S.J.; Cheung, P.Y.K.; Miyamoto, T.; Goodrow, M.H.; Van Emon, J.; Seiber, J.N. In Pesticide Science and Biotechnology; Greenhalgh, R. and Roberts, T.R., Eds.; Blackwell: Oxford, 1986; p 309. Harrison, R. O.; Gee, S.J.; Hammock, B.D. In Biotechnology for Crop Protection; Hedin, P.A.; Menn J . J . ; and Hollingworth,


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IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS R.M., Eds.; ACS Symposium Series 379: American Chemical Society: Washington, DC, 1988; p316. Jung, F.; Gee, S.J.; Harrison, R.O.; Goodrow, M.H.; Karu, A.E. Braun, A.L.; L i , Q.X.; and Hammock, B.D. Pestic. Sci. 1989, 26, 303-317. Van Emon, J.M.; Seiber, J.N.; Hammock, B.D. In Analytical Methods for Pesticides and Plant Growth Regulators; Sherma, ED.; Vol. XVII; Academic Press: New York, 1989; pp 217263. Voiler, Α.; Bartlett, Α.; Bidwell, D.E. J. Clin. Pathol. 1978, 31, 507-520. Karu, A.E.; Schmidt, D.J. Clarkson, C.E.; Carlson, R.E.; and Van Emon, J.M. 198th ACS National Meeting. Division of Agrochemicals, Miami Beach, Florida, 1989, paper 9. Schlaeppi, I.-M.; Fory, W.; Ramsteiner, K. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 25. Bird, R. E.; Hardman, K. D.; Jacobson, J. W.; Johnson, S; Kaufman, Β. M.; Lee, S.-M.; Lee, T.; Pope, S. H.; Riordan, G. S.; Whitlow, M. Science 1988, 242, 423-6. Rodwel, J . D. Nature 1989, 342, 99-100. Hiatt, Α.; Cafferkey R.; Bowdish, K. Nature 1989, 342, 76-78. Huse, W.D.; Sastry, L.; Iverson, S.A.; Kang, A.S.; Alting-Mees, M.; Burton, D.R.; Benkovic, S.J.; Lerner, R.A. Science 1989, 246, 1275-1281. Hinton, D. M.; Albert, R. H.; Horwitz, W. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 56. Stoddard, P. J . 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 20. Berkowitz, D. B. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 19. Van Emon, J. M. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 18. Pohland, A. E. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 17. Shekarchi, I.C.; Sever, J . L . ; Lee, Y . J . ; Castellano, G.; Madden, D.L. J . Clin. Microbiol., 1984, 19, 89-96. Harrison, R.O; Braun, A.L., Gee, S.J.; O'Brien, D.J.; Hammock, B.D. Food Agric. Immunol. 1989, 1, 37-51. Harrison, R. O.; Hammock, B. D. J. Assoc. Off. Anal Chem., 1988, 71, 981-7. Sharp, J.; Lueng, F.S.; O'Brien, D.P.; Carski, T.H. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 7. Feng, P.C.C.; Wratten, S.J.; Sharp, C.R.; Horton, S.R.; and Logusch, E.W. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 8. Dargar, R.V.; Tymonko, J.M. Van Der Werf, P. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 10.


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Wong, R.B. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 26. 28. Sharp, J.K.; Marxmiller, R.L.; Robotti, K.; Ehrmann, P.; Sereno, R. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 29. 29. Sharp, C.R.; Feng, P.C.C.; Horton, S.R.; Logusch, E.W. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 30. 30. Bushway, R.J.; Ferguson, B.S. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 4. 31. Reck, B.; Frevert, J.; Knoell, H.E. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 31. 32. Deschamps, R.J.Α.; Hall, J.C. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 27. 33. Eck, D.L.; Kurth, M.J.; Macmillan, C.B. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 11. 34. Mei, J.V.; Yin, C.-M. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 28. 35. Lindley, K.; White, R.J. Van Emon, J. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 32. 36. Seiber, J.N.; Li, Q.X.; Van Emon J. 198th ACS National Meeting, Division of Agrochemicals, Miami Beach, Florida, 1989, paper 16. 37. Gee, S.J.; Miyamoto, T.; Goodrow, M.H.; Buster, D.; Hammock, B.D. J. Agric. Food Chem. 1988, 36, 863-870. 38. L i , Q.X.; Gee, S.J., McChesney, M.M.; Hammock, B.D.; Seiber, J.N. Anal. Chem. 1989, 61, 819-823. 39. Goodrow, M.H.; Harrison, R.O.; Hammock, B.D. 1990, J . Agri. Food Chem. in press. 40. Harrison, R.O.; Goodrow, M.H.; Gee, S.J.; Hammock, B.D. To be published in an ACS Symposium Series volume based on the symposium Immunoassays for Monitoring Human Exposure to Toxic Chemicals in Food and the Environment: Vanderlaan, M., Ed.; American Chemical Society: Washington, DC. 41. Hung, D.T.; Benner, S.A.; Williams, C.M. J. Biol. Chem. 1980, 255, 6047-6048. 42. Harrison, R.O. Ph.D. Thesis, University of Maryland, College Park, 1987. 43. Jung, F.; Szekacs, Α.; Hammock, B.D. To be published in an ACS Symposium Series volume based on the symposium Immunoassays for Monitoring Human Exposure to Toxic Chemicals in Food and the Environment; Vanderlaan, Μ., Ed.; American Chemical Society: Washington, DC. 44. Cheung, P.Y.K.; Hammock, B.D. In Biotechnology for Crop Protection; Hedin, P.A.; Menn J.J.; and Hollingworth, R.M., Eds.; ACS Symposium Series 379: American Chemical Society: Washington, DC, 1988; 359-372. Received July 31, 1990


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