Bioregulators for Pest Control - American Chemical Society

22 Applications of Immunoassay to Paraquat and Other Pesticides J . M. VAN EMON, J. N. S E I B E R , and B. D .

HAMMOCK

Downloaded by UNIV OF ARIZONA on September 9, 2015 | http://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch022

Department of Environmental Toxicology, University of California, Davis, CA 95616

The enzyme-linked immunosorbent assay (ELISA) is a rapid immunochemical procedure which can be used for trace analysis. We have applied the procedure to paraquat and other compounds difficult to analyze by the more classical methods. The immunoassay for paraquat shows the practicality of the method for fortified and actual residue samples, and is being compared with a gas chromatography procedure. Our work with the ELISA illustrates that the immunochemical technology can be used to solve problems encountered in pesticide residue analysis. I t h a s been s t a t e d t h a t p r o g r e s s i n p e s t i c i d e a n a l y s i s w i l l no l o n g e r be made i n s e a r c h o f t h e p r o v e r b i a l z e r o r e s i d u e l e v e l o f d e t e c t a b i l i t y , but r a t h e r w i l l l i e i n d e v i s i n g methods o f g r e a t e r s e l e c t i v i t y f o r t h e p o s i t i v e i d e n t i f i c a t i o n o f nanogram q u a n t i t i e s o f p e s t i c i d e r e s i d u e s (J_). We c o u l d add t o t h i s statement t h e need f o r a s s a y s o f g r e a t l y reduced c o s t and i n c r e a s e d speed. Many r e s i d u e p r o c e d u r e s a r e t o o e x p e n s i v e t o be used r o u t i n e l y i n r e g u l a t o r y p r o c e d u r e s o r , perhaps o f g r e a t e r importance, t o be employed e f f e c t i v e l y i n o p t i m i z i n g p e s t i c i d e usage and m o n i t o r i n g worker h e a l t h and s a f e t y . Immunochemical methods o f a n a l y s i s o f f e r many advantages, i n c l u d i n g s e n s i t i v i t y , s p e c i f i c i t y , and speed o f a n a l y s i s (2). Compounds which a r e most d i f f i c u l t t o a n a l y z e by c l a s s i c a l p r o c e d u r e s due t o h i g h p o l a r i t y o r low v o l a t i l i t y a r e f r e q u e n t l y amenable t o a n a l y s i s by immunochemistry. We a r e now i n v e s t i g a t i n g enzyme-linked immunosorbent a s s a y s (ELISAs) r a t h e r t h a n radioimmunoassays (RIAs) f o r p e s t i c i d e r e s i d u e work. ELISAs a r e q u i c k e r , cheaper, and s a f e r than RIAs a s r a d i o a c t i v i t y i s n o t used. However, a s a l l immunoassays f u n c t i o n on t h e p r i n c i p l e o f mass a c t i o n , t h e same immunochemical t o o l s c a n be used t o d e v i s e a number o f d i f f e r e n t a s s a y p r o c e d u r e s . G e n e r a l ELISA Methodology Immunoassays a r e p h y s i c a l r a t h e r than b i o l o g i c a l a s s a y s ; they p o s s e s s t h e s p e c i f i c i t y and s e n s i t i v i t y o f b i o a s s a y s w i t h t h e speed

0097-6156/ 85/ 0276-0307S06.00/ 0 © 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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and p r e c i s i o n o f p h y s i c a l a s s a y s . S p e c i f i c antibodies are raised i n an e x p e r i m e n t a l a n i m a l i n r e s p o n s e t o a l a r g e f o r e i g n m o l e c u l e ( a n t i g e n ) . Most p e s t i c i d e m o l e c u l e s a r e n o t l a r g e enough t o s t i m u l a t e the immune system, and must be c o n j u g a t e d t o a l a r g e m o l e c u l e such as a p r o t e i n . A n t i b o d i e s a g a i n s t t h e p e s t i c i d e a r e then obtained u s i n g t h i s p e s t i c i d e - p r o t e i n conjugate. A small m o l e c u l e which becomes immunogenic a f t e r attachment t o t h e l a r g e c a r r i e r m o l e c u l e i s termed a h a p t e n . The p e s t i c i d e may a l r e a d y have a f u n c t i o n a l i t y , such as -OH, -SH, -C00H, o r -NHg, u s e f u l f o r c o n j u g a t i o n t o the c a r r i e r , but f r e q u e n t l y a d e r i v a t i v e o f t h e p e s t i c i d e p o s s e s s i n g such f u n c t i o n a l i t y must f i r s t be s y n t h e s i z e d b e f o r e c o n j u g a t i o n can o c c u r . I n e i t h e r c a s e a n t i b o d i e s can be o b t a i n e d which a r e d i r e c t e d a g a i n s t t h e o r i g i n a l p e s t i c i d e . Serum a n t i b o d i e s a r e heterogeneous m o l e c u l e s o f v a r y i n g a n t i g e n s p e c i f i c i t y and a f f i n i t y . Within t h i s p o l y c l o n a l p o p u l a t i o n , there are probably a n t i b o d i e s present which r e c o g n i z e t h e c a r r i e r p r o t e i n , but t h e c o n t r i b u t i o n o f t h e s e a n t i b o d i e s t o a s s a y b i n d i n g can be e l i m i n a t e d by u s i n g a d i f f e r e n t c a r r i e r ( £ ) . M o n o c l o n a l a n t i b o d y t e c h n o l o g y c o u l d be used t o s e l e c t the c l o n e with the h i g h e s t r e c o g n i t i o n t o hapten. Although p o l y c l o n a l a n t i b o d i e s a r e adequate f o r most ELISAs, m o n o c l o n a l a n t i b o d i e s c o u l d be d e v e l o p e d a g a i n s t p e s t i c i d e h a p t e n s y i e l d i n g an a n a l y t i c a l r e a g e n t t h a t i s p h y s i c a l l y , c h e m i c a l l y , and i m m u n o l o g i c a l l y homogeneous. As m o n o c l o n a l a n t i b o d i e s would be i n a v i r t u a l l y u n l i m i t e d s u p p l y , ELISAs c o u l d be e a s i l y s t a n d a r d i z e d as s e v e r a l l a b o r a t o r i e s would be u s i n g t h e same a n t i b o d y c l o n e . A l t h o u g h a v e r y s m a l l amount o f a n t i b o d y i s needed p e r ELISA, having a l a r g e supply o f monoclonals could a l l e v i a t e f e a r s o f e v e n t u a l l y e x h a u s t i n g one's s u p p l y . Hammock and Mumma (£) d i s c u s s some o f t h e advantages o f hybridoma t e c h n o l o g y . Yet, i t i s i m p o r t a n t t o c o n s i d e r t h a t , i n some c a s e s , p o l y c l o n a l s w i l l be s u p e r i o r t o m o n o c l o n a l a n t i b o d i e s , u n l e s s one i s d e v e l o p i n g a s o p h i s t i c a t e d system f o r a v o i d i n g t h e s e p a r a t i o n s t e p i n p e s t i c i d e Immunoassay, i t w i l l be a r a r e s i t u a t i o n where p r o d u c 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 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 can be j u s t i f i e d on a p u r e l y s c i e n t i f i c b a s i s . However, as immunoassay o f p e s t i c i d e s moves i n t o t h e p r i v a t e s e c t o r , t h e r e w i l l be c o m p e l l i n g a d m i n i s t r a t i v e and l e g a l p r e s s u r e s t o employ m o n o c l o n a l a n t i b o d i e s . F o r t h e s e r e a s o n s t h e y may dominate t h e f i e l d i n a few y e a r s . The ELISA i s based on t h e f a c t t h a t a n t i g e n o r a n t i b o d y can be attached t o a solid-phase support while r e t a i n i n g immunological a c t i v i t y , and t h a t e i t h e r a n t i g e n o r a n t i b o d y can be l i n k e d t o an enzyme w i t h t h e complex r e t a i n i n g both immunological and enzymatic activity. A v a r i e t y o f enzymes, i n c l u d i n g a l k a l i n e phosphatase, h o r s e r a d i s h p e r o x i d a s e , and g l u c o s e o x i d a s e have been l i n k e d t o a n t i b o d i e s and a n t i g e n s . T h i s method has been u s e d s u c c e s s f u l l y f o r d e t e c t i o n o f e i t h e r a n t i g e n o r a n t i b o d y (3-4) and i t has been u s e d by us f o r t h e d e t e c t i o n o f n u c l e o t i d e s , i n s e c t i c i d e s , s u r f a c t a n t s and a v a r i e t y o f o t h e r compounds. To p e r f o r m a m i c r o p l a t e ELISA f o r the measurement o f a n t i g e n , p l a t e s s e n s i t i z e d with the s p e c i f i c antigen are incubated with a m i x t u r e o f r e f e r e n c e a n t i b o d y and the t e s t sample. I f a n t i g e n i s p r e s e n t i n t h e t e s t s o l u t i o n , i t combines w i t h t h e r e f e r e n c e a n t i b o d y which cannot then r e a c t w i t h the s e n s i t i z e d p l a t e . The amount o f a n t i b o d y a t t a c h e d t o t h e s o l i d phase i s t h e n i n d i c a t e d by f



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an enzyme l a b e l l e d anti-immunoglobulin conjugate and enzyme substrate. There i s a proportional r e l a t i o n s h i p between the amount of i n h i b i t i o n of substrate converted to products i n the t e s t sample and to the amount of antigen i n the test system. We routinely run samples i n p l a s t i c plates containing 50 wells with which a l l of the procedures can be executed very r a p i d l y . The end point of the assay i s the bright yellow color of p-nitrophenol, an end product from the a l k a l i n e phosphatase-mediated hydrolysis of p-nitrophenyl phosphate; t h i s product can be v i s u a l l y estimated f o r semi-quantitative answers or r a p i d l y and p r e c i s e l y measured i n an inexpensive colorimeter f o r quantitation. Immunoassays can be designed to analyze parent compounds and metabolites separately or as a group. Ercegovich (5.) obtained antibodies against parathlon which also detected i t s metabolite p-nitrophenol. Other workers were successful i n r a i s i n g antibodies to DDT and malathion metabolites (6-7). Although the s p e c i f i c i t y of immunoassays are usually very high there i s no guarantee against cross r e a c t i v i t y . Just as good chromatographic techniques require controls, so do immunoassays. Fortunately immunoassays are quickly and e a s i l y performed so that the necessary controls can be run to check f o r interferences. The s e n s i t i v i t y and s e l e c t i v i t y of immunoassays can also greatly reduce the cost of analysis by minimizing the amount of sample preparation. Examples of Pesticide Immunoassays We w i l l f i r s t review several of the immunoassays developed i n t h i s laboratory as they i l l u s t r a t e some of the advantages of immunoassay i n pesticide residue analysis. Then we w i l l move on to a more detailed discussion of our recent a n a l y t i c a l work with the herbicide paraquat. Diflubenzuron. The benzoylphenyl urea insect growth regulators, f o r example, pose a formidable residue analysis problem. The compounds are nonvolatile and thus must be derivatized f o r GC analysis by a rather arduous chemical procedure. The immunoassay developed i n t h i s laboratory i s much more s e n s i t i v e and reproducible at a f r a c t i o n of the cost and can be used to analyze the more d i f f i c u l t matrices such as milk. For instance, a s e n s i t i v i t y of 1 ppb i s routinely obtained when milk i s added d i r e c t l y to the assay (£.)· A s e r i e s of p a r t i t i o n steps can also be added to further clean diflubenzuron milk extracts y i e l d i n g a s e n s i t i v i t y i n the low ppt range (£.). However t h i s increase i n s e n s i t i v i t y may not be needed since methods i n current use provide a detection l i m i t of only 10-50 ppb. An impressive aspect of Immunoassays i s t h e i r s p e c i f i c i t y . One immunoassay f o r diflubenzuron can d i s t i n g u i s h i t from the very c l o s e l y r e l a t e d BAY SIR 8514 as well as a v a r i e t y of other c l o s e l y related materials (£)· High resolution HPLC columns can resolve these compounds but the analysis i s slow and expensive. The ELISA can d i s t i n g u i s h these materials when applied d i r e c t l y , or l e s s s p e c i f i c assays can be used as a highly selective detector when used as an adjunct to HPLC.


310


^ 0 Η 0 H

^ 0 Η 0 H -N-C-N-^^-0CF

Diflubenzuron


F

3

BAY SIR 8514

Surfactants have many i n d u s t r i a l applications and are found i n such diverse products as pesticide formulations and cosmetics. These nonionic compounds are d i f f i c u l t to extract, clean up, and analyze and, consequently, no s e n s i t i v e method existed f o r t h e i r analysis. An ELISA was developed i n t h i s laboratory which distinguishes between the nonionic surfactants T r i t o n X and T r i t o n Ν (10). These compounds, mixtures o f ethoxylates o f varying length of 4-(1,1,3f3-tetramethylbutyl)phenol and 4-nonylphenol, give numerous overlapping peaks upon chromatographic analysis and have almost i d e n t i c a l UV and IR spectra. An ELISA has been developed f o r class s e l e c t i v e detection o f the T r i t o n X s e r i e s ; the antibody detects the 4-(1,1,3 3-tetramethylbutyl)phenyl moiety and does not distinguish among molecules with ethoxylated side chains of varying lengths. f

CH

3

HC 3

CH -Ç-CH -ÇH^^-(OCH CH )^OH 3

2

2

CH

3

2

C H -^^-(0CH CH )^0H 9

I 9

2

H C ~~ Triton X

2

3

Triton Ν ,

S - B i o a l l e t h r i n . The pyrethroid S - b i o a l l e t h r i n (1R,3R,4 S) and i t s i n a c t i v e isomer (1S,3S,4 R) can be r e a d i l y distinguished by another ELISA procedure, i l l u s t r a t i n g the assay's a b i l i t y to determine c h i r a l i t y at the residue l e v e l (Figure 1). Antibodies were raised against S - b i o a l l e t h r i n using the a l l e t h r i n hemisuccinate conjugated to various proteins (11-12). f

0 S-Bioallethrin

0 1S,3S,4'R Allethrin

Pyrethroids, as well as some carbamate and organophosphate i n s e c t i c i d e s , are marketed as isomer mixtures, each isomer having a d i f f e r e n t degree of a c t i v i t y . I t can be expected that environmental degradation and metabolism w i l l occur p r e f e r e n t i a l l y with some isomers. Thus the a b i l i t y to distinguish between o p t i c a l isomers a t the residue l e v e l may be of c r i t i c a l importance i n monitoring the safety of treated substances.

Paraquat During a recent year, over 950,000 pounds o f paraquat dichloride


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22.


311


312


( I j I ' - d i m e t h y l - ^ ' - b i p y r i d i n i u m d i c h l o r i d e ) was used i n C a l i f o r n i a a g r i c u l t u r e (11). Much o f t h i s h e r b i c i d e was used on c o t t o n , p r i m a r i l y as a h a r v e s t a i d . The v a l u e o f t h i s c o t t o n p r o d u c t i o n i s e s t i m a t e d t o exceed one b i l l i o n d o l l a r s ( H ) . Although i t i s e x t e n s i v e l y used, l i t t l e i s known r e g a r d i n g t h e l o n g term c h r o n i c e f f e c t s o f paraquat exposure. P a r a q u a t has a number o f b i o l o g i c a l e f f e c t s , but i t s main b i o c h e m i c a l a c t i o n i s a p p a r e n t l y as a p o t e n t redox u n c o u p l e r . R e g a r d l e s s o f t h e r o u t e o f a d m i n i s t r a t i o n , symptoms o f p a r a q u a t p o i s o n i n g a r e c e n t e r e d i n the l u n g s l e a d i n g t o f i b r o s i s and pneumonia (15.). Submicrogram q u a n t i t i e s o f p a r a q u a t d e p o s i t e d i n t h e l u n g can cause f i b r o t i c l e s i o n s (16) which c o u l d l e a d t o asthma and emphysema symptoms i n c h r o n i c a l l y exposed i n d i v i d u a l s (11). u n f o r t u n a t e l y , the c o n v e n t i o n a l methods o f a n a l y s i s o f p a r a q u a t a r e t o o l a b o r i o u s and/or i n s e n s i t i v e t o h a n d l e t h e l a r g e sample l o a d g e n e r a t e d by r i g o r o u s s t u d i e s needed t o e v a l u a t e human exposure. Immunochemical p r o c e d u r e s were d e v e l o p e d t o overcome t h i s difficulty. F a c t o r ! and H u n t e r (18.) r e p o r t e d a radioimmunoassay f o r p a r a q u a t , and N i e w o l a e t a l . , (H) r e p o r t e d an ELISA f o r e s t i m a t i n g p a r a q u a t i n serum. T h i s ELISA i s s i m i l a r t o o u r s but u s e s a d i f f e r e n t enzyme, i n c u b a t i o n t e m p e r a t u r e and time, and shows a h i g h degree o f c r o s s r e a c t i v i t y w i t h e t h y l p a r a q u a t . Methods s i m i l a r t o t h o s e p r e v i o u s l y r e p o r t e d were u s e d t o g e n e r a t e p a r a q u a t h a p t e n s f o r our s t u d y . N - M e t h y l - 4 - ( 4 - p y r i d y l ) p y r i d i n i u m bromide was r e a c t e d w i t h e t h y l 5 - b r o m o v a l e r a t e f o r m i n g a p a r a q u a t analogue c a p a b l e o f c o n j u g a t i n g w i t h a p r o t e i n . New Z e a l a n d w h i t e r a b b i t s were i n j e c t e d w i t h a p a r a q u a t - p r o t e i n c o n j u g a t e and a n t i b o d i e s c a p a b l e o f r e c o g n i z i n g p a r a q u a t were o b t a i n e d . The a n t i b o d i e s a r e q u i t e s e l e c t i v e showing no c r o s s r e a c t i v i t y t o compounds s t r u c t u r a l l y r e l a t e d t o paraquat (e.g. e t h y l paraquat, N j N ' - d i m e t h y l - i j j l p - b i p i p e r i d i n e ) o r compounds used i n c o n j u n c t i o n w i t h paraquat (e.g. d i q u a t ) . Using the s e l e c t i v e antibody f o r paraquat, environmental samples can be a n a l y z e d w i t h l i t t l e o r no c l e a n u p . Sample t h r o u g h p u t can be measured i n samples p e r hour r a t h e r t h a n days p e r sample. T h i s was a r e m a r k a b l e d i s c o v e r y as p a r a q u a t i s n o t o r i o u s f o r r e q u i r i n g e x t e n s i v e sample p r e p a r a t i o n . The l i m i t o f d e t e c t i o n (2 ng/ml) i n the ELISA p r o c e d u r e i s a l s o much l o w e r t h a n the c o l o r i m e t r i c p r o c e d u r e , which has a s e n s i t i v i t y o f 200 ng/ml ( 2 0 ) , and l o w e r t h a n t h e GC p r o c e d u r e ( F i g u r e 2) ( 2 1 ) . Perhaps the g r e a t e s t advantage i s t h e speed o f t h e ELISA e n a b l i n g l a r g e numbers o f samples t o be a n a l y z e d w i t h o u t r e q u i r i n g an e x t e n s i v e c l e a n u p procedure, thus r e d u c i n g a n a l y t i c a l time. G a s - l i q u i d chromatography f o l l o w i n g r e d u c t i o n o f p a r a q u a t t o t h e mono- and d i u n s a t u r a t e d d e r i v a t i v e s (£1) i s o f adequate s e n s i t i v i t y f o r most work when N - s e l e c t i v e d e t e c t o r s a r e employed. S e i b e r and Woodrow (22.) m o d i f i e d t h i s method f o r a s s a y i n g p a r a q u a t i n a i r samples. The method i s time consuming and l a b o r i n t e n s i v e , i n v o l v i n g a c i d e x t r a c t i o n and many c o n c e n t r a t i o n and evaporation steps. The maximum sample o u t p u t per a n a l y s t p e r day i s 6-8 w i t h no d u p l i c a t e s . The r e p o r t e d r e c o v e r y e f f i c i e n c y was 75$ (22.) a l t h o u g h an e f f i c i e n c y c l o s e r t o 50$ i s f r e q u e n t l y encountered i n p r a c t i c e . A m o d i f i e d a c i d e x t r a c t i o n combined w i t h a n a l y s i s by t h e ELISA p r o v i d e s r e c o v e r i e s o f 75$ ( F i g u r e 3 ) . This 9



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PARAQUAT

PARAQUAT

CATION

CATION

ng/ml

ug/mI

F i g u r e 2. S t a n d a r d c u r v e s f o r p a r a q u a t u s i n g ELISA ( m i d d l e ) , and c o l o r i m e t r y ( b o t t o m ) .

(top),



Incubate with Antibody Glass Fiber F i l t e r

^ELISA

60$ E x t r a c t i o n

I

Efficiency

2 steps

E x t r a c t w i t h 6N HC1 Concentrate & C e n t r i f u g e


Paraquat i n 6N HC1

Evaporate

>ELISA

75$ E x t r a c t i o n

Efficiency

5 Steps Extract with sat'd

NH HC0 4

3

Centrifuge

P a r a q u a t i n s a t ' d NH^HCO,

Evaporate D i s s o l v e i n NaOH (NaBH^) Heat (60°C) f o r 12 min Cool, extract with ethyl acetate

Reduced P a r a q u a t

(monoene & d i e n e ) i n e t h y l a c e t a t e

Gas Chromatography

50$ E x t r a c t i o n E f f i c i e n c y 11 S t e p s DB-1 30 m c a p i l l a r y NP d e t e c t i o n (HP 5710A) Sum monoene and d i e n e peaks

F i g u r e 3. Comparison o f sample p r e p a r a t i o n s t e p s f o r a n a l y s i s of p a r a q u a t on a i r f i l t e r s u s i n g ELISA and gas chromatography.



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VAN EMON ET AL.


315

i l l u s t r a t e s t h e s u c c e s s f u l a d a p t a t i o n o f an e x t r a c t i o n t e c h n i q u e i n t e n d e d f o r use w i t h GC d e t e r m i n a t i o n t o an immunochemical method o f a n a l y s i s . A l t e r n a t i v e l y , t h e a n t i b o d y i t s e l f can be used t o e x t r a c t p a r a q u a t from a g l a s s f i b e r f i l t e r , e l i m i n a t i n g the time-intensive e x t r a c t i o n procedure. Using the s p e c i f i c a n t i b o d i e s f o r e x t r a c t i o n w i t h subsequent d e t e c t i o n by ELISA, 50 samples can be a n a l y z e d I n t r i p l i c a t e i n one day w i t h a r o u t i n e e x t r a c t i o n e f f i c i e n c y o f 60$ ( F i g u r e 3). However, t h i s method u s e s a r a t h e r l a r g e amount o f a n t i b o d y making t h e m o d i f i e d a c i d e x t r a c t i o n p r o c e d u r e p r e f e r a b l e f o r a n a l y z i n g a l a r g e sample l o a d . T h i s r a p i d s a m p l e - p r o c e s s i n g c a p a b i l i t y makes i t f e a s i b l e t o measure exposure o f f i e l d w o r k e r s l i t e r a l l y on t h e same day t h e exposures occur. I n o r d e r t o t e s t t h i s c a p a b i l i t y a i r s a m p l i n g was c o n d u c t e d i n t h e San J o a q u i n v a l l e y d u r i n g a p a r a q u a t a p p l i c a t i o n on c o t t o n , and t h e samples a r e now b e i n g a n a l y z e d u s i n g both ELISA and GC methods t o compare t h e two t e c h n i q u e s i n terms o f speed, p r e c i s i o n (by c a l c u l a t i n g t h e p e r c e n t c o e f f i c i e n t o f v a r i a t i o n ) , and a c c u r a c y (by comparing r e s u l t s from f o r t i f i e d samples u s i n g both techniques). P r e l i m i n a r y r e s u l t s i n d i c a t e t h a t t h e ELISA compares w e l l w i t h GC l i t e r a t u r e v a l u e s i n t h e s e t h r e e p a r a m e t e r s and a d d i t i o n a l l y has the a n t i c i p a t e d g r e a t e r sample throughput capability. Conclusions I t i s p r o b a b l e t h a t i n c e r t a i n s i t u a t i o n s immunochemical methods w i l l provide d i s t i n c t advantages over c o n v e n t i o n a l a n a l y t i c a l methods. However, i t i s u n l i k e l y t h a t immunochemical methods w i l l c o m p l e t e l y r e p l a c e c u r r e n t , e s t a b l i s h e d a n a l y t i c a l methods o f p e s t i c i d e a n a l y s i s (JL). T h i s i s i n s p i t e o f the f a c t t h a t c h e m i c a l c l a s s e s c u r r e n t l y a s s a y e d by immunochemical t e c h n i q u e s i n c l i n i c a l a n a l y t i c a l l a b s c o n t a i n t h e same t y p e o f f u n c t i o n a l groups as many p e s t i c i d e s . ELISA c o u l d p o t e n t i a l l y be used a d v a n t a g e o u s l y i n many t y p e s o f exposure and m o n i t o r i n g s i t u a t i o n s , f o r p a r a q u a t and o t h e r p e s t i c i d e s amenable t o ELISA a n a l y s i s . An o b v i o u s use o f ELISA i s t h e d e t e c t i o n o f p e s t i c i d e r e s i d u e l e v e l s i n p l a n t and a n i m a l t i s s u e s , o r f o o d e x t r a c t s . B i o l o g i c a l specimens such as plasma and u r i n e c u r r e n t l y a n a l y z e d by RIA seem p a r t i c u l a r l y amenable t o a n a l y s i s by ELISA. P o r t a b l e f i e l d k i t s c o u l d be d e v e l o p e d t o d e t e r m i n e s a f e worker r e - e n t r y t i m e s i n t o t r e a t e d f i e l d s . E n v i r o n m e n t a l samples s u c h as s o i l , water, and a i r , can be a n a l y z e d by t h e ELISA. P e s t i c i d e c o n j u g a t e s have been p r o p o s e d f o r s k i n t e s t i n g o f i n d i v i d u a l s suspected o f s e n s i t i v i t y t o p e s t i c i d e s (£) ; the ELISA c o u l d be used t o d e t e c t s p e c i f i c a n t i b o d i e s i n t h e s e i n d i v i d u a l s and a i d i n exposure s t u d i e s . A n t i b o d i e s have been r a i s e d a g a i n s t r e p r e s e n t a t i v e compounds from t h e major c l a s s e s o f p e s t i c i d e s . A l t h o u g h t h e ELISA w i l l be u s e f u l f o r i n d i v i d u a l a n a l y s i s o f a wide v a r i e t y o f compounds, i f one needed t o a n a l y z e s e v e r a l d i f f e r e n t compounds s i m u l t a n e o u s l y i n one m a t r i x immunoassay may n o t be the method o f c h o i c e , due t o the l a r g e amount o f c o n t r o l s and s t a n d a r d s needed. However, i t c o u l d be s u c c e s s f u l l y used f o r t h e r a p i d s c r e e n i n g o f a l a r g e number o f samples f o r t h e p r e s e n c e o f s p e c i f i c t y p e s o f p e s t i c i d e s and f o r c o n f i r m a t o r y t e s t s (JL). The work r e p o r t e d h e r e w i t h p a r a q u a t ,


316


allethrin, diflubenzuron, Triton X and Triton Ν provides evidence of the ELISA's ability to distinguish closely related compounds. The ELISA promises to be a good supplement to current methods of residue analysis.


Literature Cited 1. Zweig, G. In "Essays in Toxicology"; Blood F.R. Ed.; Academic Press: New York, 1970; Chap. 3. 2. Hammock, B. D.; Mumma R. O. In "Recent Advances in Pesticide Analytical Methodology"; Harvey J.; Zweig, G., Eds.; ACS SYMPOSIUM SERIES No. 136, American Chemical Society: Washington, D.C., 1980; Chap. 18. 3. Voller, Α.; Bidwell, D.E. ; Bartlett, A.BullWorldHealth Org. 1976, 53, 55-65. 4. Van Weeman, B.K.; Schuurs, A.H.W.M. FEBS Letters 1971, 15, 232-6. 5. Ercegovich, C.D. In "Pesticide Identification at the Residue Level"; Gould, R.F., Ed.; ADVANCES IN CHEMISTRY SERIES No. 104, American Chemical Society: Washington, D.C., 1971; Chap. 11. 6. Centeno, E.R.; Johnson, W.J.; Shehon, A.H. Int. Arch. AllergyAppl.Imm.1970, 37, 1-13. 7. Haas, G.J.; Guardia, E.J.Proc.Soc.Expt.Biol.Med. 1968, 129, 546-51. 8. Wie, S.I.; Hammock, B.D. J. Agric. Food Chem. 1984, Accepted. 9. Wie, S.I.; Hammock, B.D. J, Agric. Food Chem. 1982. 30, 949-57. 10. Wie, S.I.; Hammock, B.D. Anal. Biochem. 1982, 125, 168-176. 11. Wing, K.D.; Hammock, B.D.; Wuster, D.A. J. Agric. Food Chem. 1978, 26, 1328-33. 12. Wing, K.D.; Hammock, B.D. Experientia.1979,35, 1619-20. 13. "Pesticide Use Report by Commodity." California Department of Food and Agriculture, 1983. 14. "California Agriculture." California Department of Food and Agriculture, 1982. 15. Smith, P.; Heath, D. "Paraquat" CRC Crit Revs. Toxicol. 4, 411-45. 16. Zavala, D.C.; Rhodes, M.L. Chest 1978, 74, 418-20. 17. Maddy, K.T. "Human Health Problems with the Herbicide Paraquat in California 1965 through 1974." California Department of Food and Agriculture, 1975. 18. Fatori, D.; Hunter, W.M.ClinicChimicaActa1980,100, 81-90. 19. Niewola, Ζ.; Walsh, S.T.; Davies, G.E.Int.J.Immunopharmac. 1983, 5, 211-18. 20. Lott, P.F.; Lott, J.W. J. Chrom. Sci. 1978, 16, 390-5. 21. van Dijk, Α.; Ebberink, R.; deGroot, G.; Maes, R.A.A.; Douze, J.M.C.; van Heyst, A.N.P. J. Analy. Tox. 1977, 1, 151-54. 22. Seiber, J.N.; Woodrow, J.E. Arch. Environm. Contam. 1981, 10, 133-49. RECEIVED November 8, 1984


A the

Direct C o r r e l a t i o n Between B i o a s s a y and ELISA for Bacillus thuringiensis var. israelensis δ-endotoxin.

P e t e r Y. K. Cheung and Bruce D. Hammock


Department of Entomology University of California Davis, California 95616

A partially purified Bacillus thuringiensis var. israelensis (Bti) δ-endotoxin was used to immunize rabbits. The antisera obtained have an improved spe cificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units ± 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy.

RECEIVED December 17, 1984




E u p l e c t r u s plathypenae Parasitization of Trichoplusia ni. on Weight G a i n , E c d y s t e r o i d Titer and M o l t i n g .


Thomas A. Coudron and Thomas

J.

Kelly

Effect

1/

U. S. Department o f Agriculture Biological C o n t r o l of I n s e c t s Research L a b o r a t o r y , P. O. Box A Columbia, MO 65205

E u p l e c t r u s spp. (Hymenoptera: E u l o p h i d a e ) a r e g r e g a r i o u s e c t o p a r a s i t e s of s e v e r a l l e p i d o p t e r a n s p e c i e s that are pests to agricultural crops. Parasitization of Trichoplusia ni l a r v a e by E u p l e c t r u s p l a t h y p e n a e results in an inhibition of the larval larval molting p r o c e s s in t h e h o s t . After parasitization the host t e m p o r a r i l y r e f r a i n s from e a t i n g and l a g s b e h i n d t h e s y n c h r o n i z e d n o n - p a r a s i t i z e d l a r v a e in weight g a i n . However t h e parasitized l a r v a e does resume e a t i n g and c o n t i n u e s t o i n c r e a s e its body w e i g h t p r i o r t o t h e development o f t h e p a r a s i t e l a r v a e . There is no s i g n o f new cuticle formation in parasitized larvae. A p r o l o n g e d a s s o c i a t i o n between t h e parasite and h o s t is not n e c e s s a r y t o elicit an effect on t h e h o s t . Larvae w h i c h a r e i m m e d i a t e l y s e p a r a t e d from the parasitic egg will fail t o molt i n t o the next instar. The inhibi tion of h o s t molt may be r e l a t e d t o t h e absence o f peak in t h e e c d y s t e r o i d titer i n t h e hemolymph which o c c u r s 20 h r s b e f o r e e c d y s i s in n o n - p a r a s i t i z e d T. ni. T h i s u n i q u e t y p e o f h o s t development c o n t r o l appears t o be distinct from the p a r a l y z i n g effect o f many Hymenoptera venoms and the a l g o g e n i c effects o f some Hemiptera salivary toxins.

RECEIVED February 11, 1985

* Current

address:

I n s e c t R e p r o d u c t i o n L a b o r a t o r y , BARC-E, B l d g . 3 0 6 , B e l t s v i l l e , MD 20705


Bioregulators for Pest Control - American Chemical Society

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