Pesticide Analytical Methodology - ACS Publications - American

18 Potential of Immunochemical Technology for Pesticide Analysis

Downloaded by UNIV OF ROCHESTER on April 10, 2017 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch018

BRUCE D. HAMMOCK Division of Toxicology and Physiology, Department of Entomology, University of California, Riverside, CA 92521 RALPH O. MUMMA Pesticide Research Laboratory, Department of Entomology, Pennsylvania State University, University Park, PA 16802 In the fields of clinical chemistry and endocrinology, immunochemistry is often the analytical method of choice. Immunochemical methods of analysis offer many advantages including sensitivity, specificity, speed of analysis, ease of automation, cost effectiveness, and general applicability. The importance of immunochemical assays was recognized by Rosalyn Yalow sharing the Nobel Prize in Physiology and Medicine based, in part, on her pioneering work in immunoassay development (1, 2). Surprisingly, immunochemistry has found l i t t l e or no practical application for the analysis of pesticides or other environmental contaminants (3). This fact is surprising because the chemical classes currently assayed by immunochemical techniques (2,4) are not fundamentally distinct from many classes of fungicides, herbicides, insecticides, nematocides, or plant growth regulators. Possibly the tremendous success of gas liquid chromatography (GLC) and ion selective detectors in the analysis of the chlorinated hydrocarbon insecticides fostered a generation of pesticide analytical chemists who were experts in and disciples of GLC. The phenomenal success of immunochemistry, s p e c i f i c a l l y radioimmunoassay (RIA), was p o s s i b l y analogous i n f o s t e r i n g a generation of c l i n i c a l chemists who look f i r s t to immunochemistry f o r the a n a l y s i s of hormones and pharmaceuticals, even i n cases when RIA i s not n e c e s s a r i l y the technique of c h o i c e . B i o l o g i c a l techniques, i n c o n t r a s t to p h y s i c a l or chemical techniques f o r r e s i d u e a n a l y s i s , have been c r i t i c i z e d by a n a l y t i c a l p e s t i c i d e chemists. I t i s a common misconception that immunochemical methods can be classed as b i o l o g i c a l techniques of r e s i d u e a n a l y s i s . Although a l i v i n g organism o r , at l e a s t , a c e l l l i n e i s r e q u i r e d f o r antibody production, immunoassays u s i n g these a n t i b o d i e s a r e based on p h y s i c a l and chemical p r o p e r t i e s , and immunoassays can be explained i n terms of the law of mass a c t i o n . A tremendous immunochemical technology has developed e s p e c i a l l y i n c l i n i c a l chemistry, and i t i s time that t h i s technology was e x p l o i t e d to s o l v e new and p r e s s i n g problems i n environmental chemistry.

0-8412-0581-7/80/47-136-321$08.00/0 © 1980 American Chemical Society

Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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In t h i s chapter the p o t e n t i a l of s e v e r a l immunochemical techniques f o r r e s i d u e a n a l y s i s w i l l be explored. Sufficient background methodology w i l l be presented to a l l o w the reader to evaluate the advantages and disadvantages of immunochemical techniques and t h e i r p o t e n t i a l a p p l i c a t i o n to r e s i d u e problems. A chapter of t h i s l e n g t h provides, at best, s u p e r f i c i a l treatment of immunochemical methodology and theory, but t h i s overview, i n c o n j u n c t i o n with the included r e f e r e n c e s , should o f f e r the reader ready access to the s p e c i f i c immunochemical l i t e r a t u r e . Hopef u l l y , t h i s a r t i c l e w i l l a s s i s t pesticide residue laboratories in a p p l y i n g e x i s t i n g immunochemical technology to s p e c i f i c problems i n p e s t i c i d e a n a l y t i c a l chemistry. The most common, but by no means the only or even the most promising, immunochemical assay f o r small molecules i s r a d i o immunoassay (RIA). As an overview, an immunoassay i n v o l v e s c h e m i c a l l y a t t a c h i n g the small molecule of i n t e r e s t (or a d e r i v a t i v e of i t ) to a c a r r i e r p r o t e i n and r a i s i n g s p e c i f i c antibody t i t e r s to i t i n the serum of an animal. Very d i l u t e antibody s o l u t i o n s a r e then used to bind the small molecule which has been r a d i o l a b e l e d . The competition of v a r y i n g known concentrations of unlabeled m a t e r i a l i s measured and the r e s u l t i n g standard curve used to determine unknown concentrations (Table I ) . The steps l e a d i n g to the development of an RIA are o u t l i n e d below followed by a d e s c r i p t i o n of other immunochemical procedures and an a n a l y s i s of the a t t r i b u t e s and l i m i t a t i o n s of immunoassay. Table I Steps i n the Development of an

RIA

Synthesize hapten

Prepare r a d i o l i g a n d

Couple hapten

Choose method f o r bound/free s e p a r a t i o n

P u r i f y antigen

Optimize assay c o n d i t i o n s

C h a r a c t e r i z e antigen

Develop standard

Immunize animal

C h a r a c t e r i z e assay

Titer

Determine assay

antibody

curve

reliability

C h a r a c t e r i z e antibody

Methodology of Antibody

Formation

Hapten Synthesis. Antibody t i t e r s are r a i s e d i n an e x p e r i mental animal i n response to an antigen or immunogen. In g e n e r a l , an e f f e c t i v e antigen must be r a t h e r l a r g e and f o r e i g n to the



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animal to be immunized; ( p r o t e i n s of greater than 10,000 mw are common a n t i g e n s ) . By comparison, most p e s t i c i d e s are r a t h e r small molecules, and t h e r e f o r e they must f i r s t be conjugated to a p r o t e i n or other l a r g e a n t i g e n i c molecule before they can be used as a n t i g e n s . Such small molecules which become immunogenic a f t e r attachment to a l a r g e c a r r i e r molecule are c a l l e d haptens. I f the p e s t i c i d e has a r e a c t i v e f u n c t i o n a l i t y s u i t a b l e f o r conjugat i o n i t may i t s e l f be the hapten. Otherwise, a d e r i v a t i v e of the p e s t i c i d e must be synthesized s u i t a b l e f o r attachment to the c a r r i e r . C o o r d i n a t i o n between a hapten and c a r r i e r p r o t e i n may be s u f f i c i e n t f o r r a i s i n g antibody t i t e r s , but c o v a l e n t l i n k a g e s are more r e l i a b l e and d e f i n i t i v e ( 4 ) . For many p e s t i c i d e s , p o t e n t i a l l y u s e f u l haptens have a l r e a d y been described as metab o l i t e standards or environmental degradation products. The c h o i c e of the hapten and the c o n j u g a t i o n procedure used may profoundly a f f e c t the u l t i m a t e s e n s i t i v i t y and s p e c i f i c i t y of the immunochemical assay. G e n e r a l l y , antibody s p e c i f i c i t y i s highest f o r the part of the molecule d i s t a l or f u r t h e s t from the c a r r i e r p r o t e i n . T h i s knowledge has f r e q u e n t l y been u t i l i z e d to develop immunoassays which w i l l , on one hand, d e t e c t general c l a s s e s of compounds which have common f u n c t i o n a l i t i e s and to develop other assays which are h i g h l y s p e c i f i c . In an h y p o t h e t i c a l system ( F i g . 1), three s i m i l a r molecules are represented. I f molecule I i s used as a hapten and i t i s conjugated to the prot e i n through f u n c t i o n a l i t y b, the r e s u l t i n g antibody p o p u l a t i o n i s l i k e l y to c r o s s - r e a c t with the c l o s e l y r e l a t e d molecules I I and I I I . Such an antibody p o p u l a t i o n might f i n d u t i l i t y i n developing an assay to the c l a s s of compounds represented by molecules I , I I and I I I . A l t e r n a t i v e l y , i f molecule I i s c o n j u gated through f u n c t i o n a l i t y a, the r e s u l t i n g antibody p o p u l a t i o n i s l i k e l y to d i s t i n g u i s h among the three molecules and be u s e f u l f o r a s p e c i f i c assay of molecule I with minimal i n t e r f e r e n c e from r e l a t e d compounds I I and I I I . The importance of the s i t e of c o n j u g a t i o n of a hapten to a p r o t e i n has been demonstrated many times with s t e r o i d s and pharmaceuticals such as the b a r b i t u r a t e s ( 5 ) , and i t was r e c e n t l y demonstrated with the i n s e c t i c i d e S - b i o a l l e t h r i n ( I R ^ R j A ' S ^ a l l e t h r i n ) ( F i g . 2) ( J D , 7 ) . The S - b i o a l l e t h r i n was conjugated to a c a r r i e r p r o t e i n v i a an hydroxyl f u n c t i o n a l i t y of the propene side c h a i n of the r e t h r e l o n e moiety ( F i g . 2A). RIA based on the r e s u l t i n g antibody p o p u l a t i o n i n d i c a t e d a high degree of s p e c i f i c i t y f o r the a b s o l u t e configuration* of the chrysanthemate moiety d i s t a l from the point of conjugation and much lower s p e c i f i c i t y f o r the more proximal c h i r a l center i n the a l l e t h r e l o n e moiety. In a d d i t i o n , the antibody could not d i s t i n g u i s h S - b i o a l l e t h r i n from p y r e t h r i n I probably because p y r e t h r i n I has an i d e n t i c a l c o n f i g u r a t i o n and d i f f e r s from a l l e t h r i n only i n the propene s i d e c h a i n (6,7). I f i t were important to r a i s e an antibody t i t e r capable of d i s t i n g u i s h i n g between a l l e t h r i n and the p y r e t h r i n s I, the hapten could have been conjugated through i t s carbo-


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Figure 1. Illustration of importance of hapten selection on immunoassay spe cificity. A hapten molecule (I) coupled to a protein through functionality "a" would be ex pected to raise an antibody titer useful for an assay of molecule I but not II or III. A hapten molecule (I) coupled to a protein through functionality "b" would be likely to raise an antibody titer useful for the assay of the class of molecules represented by I, II, or III.

Ο S-BIOALLETHRIN

Figure 2. The structure of S-bioallethrin (7R, 3R, 4'S allethrin) and possible hap tens for the formation of antigens for allethrin. The hemisuccinate of an alcohol derivative of allethrin's propene side chain (A) illus trates the use of a spacer arm between the carrier protein and the molecule of interest. Antibodies to this antigen demonstrated the greatest specificity for the chrysanthemate end of the molecule. The allethrin CMO derivative (B) was prepared at the V ketone. Haptens attached through a gem dimethyl group (C) or the isobutenyl group (D) would be expected to lead to antibodies with a greater specificity for the allethrelone end of the molecule. Ρ indicates protein.


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methoxyoxime (CM0) d e r i v a t i v e at C - l of the a l l e t h r e l o n e moiety ( F i g . 2B) , or b e t t e r through a hydroxyl s u b s t i t u e n t on a gem dimethyl group or a f u n c t i o n a l i t y on the i s o b u t e n y l s i d e c h a i n of the chrysanthemic acid moiety ( F i g . 2C,2D). S e v e r a l s t u d i e s have emphasized that i t i s o f t e n important f o r maximal s p e c i f i c i t y to have the hapten separated from the c a r r i e r p r o t e i n by a spacer arm. A hemisuccinate moiety was used f o r t h i s purpose i n the case of S _ - b i o a l l e t h r i n . S e v e r a l of the conjugation procedures discussed i n the f o l l o w i n g paragraphs i n s e r t a spacer arm between the hapten and the c a r r i e r p r o t e i n due to the nature of the conjugation reagent i n v o l v e d , while i n some cases a more d e l i b e r a t e attempt to i n s e r t a spacer may be made (8_,j0 . Hapten Coupling. The f u n c t i o n a l i t i e s on a p r o t e i n u s u a l l y used f o r coupling haptens i n c l u d e NH, SH, OH, and C00H. Numerous coupling techniques have been u t i l i z e d and are described i n d e t a i l i n the pharmacology and endocrinology l i t e r a t u r e . Coup l i n g techniques f o r a f f i n i t y chromatography are a l s o o f t e n a p p l i c a b l e to h a p t e n - c a r r i e r c o u p l i n g (10). An overview of the most widely used c o u p l i n g techniques i s presented below. T h i s over view i s not exhaustive; r a t h e r , i t i s intended to i l l u s t r a t e some of the many s y n t h e t i c routes open to the p e s t i c i d e a n a l y t i c a l chemist. When p o s s i b l e , examples have been drawn from the areas of entomology or p e s t i c i d e or environmental chemistry. Langone and Van Vunakis (11) used an N-hydroxysuccinimide (NHS) (12) a c t i v e ester of a carboxyl s u b s t i t u t e d analog s i m i l a r to a l d r i n and d i e l d r i n formed by dehydration with Ν,Ν -dicyclohexylcarbod i i m i d e (DCC) to conjugate with human serum albumin ( F i g . 3, Rn 1 ) . S i m i l a r a c t i v e ester methods have been used to conjugate to p r o t e i n s c a r b o x y l i c a c i d d e r i v a t i v e s of a l l e t h r i n ( 6 ) , d i flubenzuron (13), j u v e n i l e hormone (14,15), ecdysone (15), polypodine β-oxime (16), and numerous compounds of m e d i c i n a l i n t e r e s t . The a c t i v e e s t e r can be p u r i f i e d (12,17,18), and i t i s f a i r l y s t a b l e under a c i d i c c o n d i t i o n s . A l t e r n a t i v e l y , water-soluble carbodiimides such as 1 - c y c l o hexyl-3-(2-morpholinyl-4-ethyl) carbodiimide methyl £-toluene s u l fonate (CMC) or l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide* HC1 (EDC) are a v a i l a b l e which allow a d i r e c t coupling of an amine and c a r b o x y l i c a c i d without the i s o l a t i o n of an a c t i v e e s t e r ( F i g . 3, Rn 2) (19,20,21). Such procedures may be very u s e f u l with r a t h e r water-soluble or u n s t a b l e haptens, and the r e s u l t i n g c r o s s - l i n k i n g of the p r o t e i n may a c t u a l l y i n c r e a s e i t s a n t i g e n i c i t y (4) although s o l u b i l i t y i s commonly reduced. In d e s i g n ing subsequent assays one should remember that water-soluble carbodiimides and some other coupling agents may r e a c t d i r e c t l y with a p r o t e i n and subsequent a n t i b o d i e s may be d i r e c t e d , i n p a r t , against the r e s u l t i n g guanidino of a c y l urea d e r i v a t i v e s ( F i g . 3, Rn 3 ) . When u s i n g immunodiffusion (discussed l a t e r ) f o r e s t i m a t i o n of antibody t i t e r s , t h i s l a b o r a t o r y has used haptens coupled to d i f f e r e n t p r o t e i n s by c h e m i c a l l y d i s t i n c t 1



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ACTIVE ESTER

R'NH R'l

Ο

®

I " -°-

T£S*

(CH ) I COOH 2

A N A L Y T I C A L

H NP^ 2

C · NR"

c

3

"DIFLUBENZURON BUTYRATE"

DIIMIDE

Ο

©

R'NH

P C - 0 - C =N

DIIMIDE

©

RCOOH

+ CICOCH CH(CH )

ISOBUTYL

2 - ABZI

2

3

0

2

t ^"»

2

3

2

"

2

Ν

Ρ

»

RCNP

CHLOROCARBONATE

THIOPHOSGENE

6-AMIN0BENZ0PYRENE

RCOCOCH CH(CH )

PHOSGENE

ISOTHIOCYANATE

ISOCYANATE

Figure 3. Some methods of hapten-protein coupling. Except for phosphorous in parathion, Ρ indicates protein. See text for a description of reagents.


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r-A

/N = C = O

t

/

DIISOCYANATE

(9)

(C H 0) P 2

5

2

TYROSINE AMINO PARATHION

DIAZOTIZED PARATHION

Ο ^ - | - S C C H Ρ - NH

2

SCCH Η I PNC-CH CH 3

3

0

Ο

H NOH 2

2

P N C - CH C H II Ο COOH 2

COOH

THIOLATED PROTEIN

H NP^ 2

@

ROH *

^

PHOSGENE

°H ROCNP

CHLOROCARBONATE

Figure 3. Continued



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procedures. For instance, d i f l u b e n z u r o n d e r i v a t i v e s were coupled using both the p u r i f i e d NHS a c t i v e ester and v i a a water-soluble carbodiimide ( F i g . 3, Rn 1,2) (13) to s e v e r a l d i f f e r e n t p r o t e i n s . S e v e r a l other routes r e s u l t i n g i n conjugation of a carboxyl i c acid group to an amine i n c l u d e r e a c t i o n of the acid with an a l k y l c h l o r o c a r b o n a t e (chloroformate) ( F i g . 3, Rn 4 ) . The e t h y l and i s o b u t y l c h l o r o c a r b o n a t e s are commonly used; f o r i n s t a n c e , deReggi et a l . (22) used ethylchlorocarbonate to make a conjugate of a s u c c i n y l a t e d ecdysterone d e r i v a t i v e while V a l l e j o and Ercegovich (23) used sec-butylchlorocarbonate f o r the conjugation of a s u c c i n y l a t e d s o l a n i d i n e . P i o n e e r i n g work on immunochemical assays f o r p e s t i c i d e s involved the s y n t h e s i s of haptens f o r DDT and malathion. Haas and Guardia (24) used the acid c h l o r i d e s of malathion h a l f e s t e r and DDA ( 2 , 2 - b i s - [ p - c h l o r o p h e n y l ] a c e t i c a c i d ) f o r conjugation while Centeno et a l . (25) used the anhydrides of DDA and malat h i o n d i a c i d (0,0-dimethyl S - [ l , 2 - b i s - c a r b o x y e t h y l ] p h o s p h o r o d i t h i o a t e ) . In r e t r o s p e c t , more s p e c i f i c a n t i b o d i e s of a higher t i t e r may have been obtained had a spacer arm been used. The above methods were u t i l i z e d to conjugate a carboxyl group on a hapten to an amino r e s i d u e on a p r o t e i n . Obviously, the above r e a c t i o n s could be, and have been, u t i l i z e d to c o n j u gate an amino r e s i d u e on a hapten to the carboxyl residues on p r o t e i n s . However, there are a d d i t i o n a l methods which have proven u s e f u l f o r conjugating amine c o n t a i n i n g haptens to proteins . For i n s t a n c e , Lukens et a l . (26) attached 2-aminobenzimidazole (2-ABZI - a degradation product of the carbamate f u n g i c i d e Benomyl) to ovalbumin by r e a c t i n g the amine of 2-ABZI with t h i o phosgene to produce the i s o t h i o c y a n a t e followed by a d d i t i o n of ovalbumin ( F i g . 3, Rn 5). Benzo[a]pyrene was conjugated to bovine serum albumin (BSA) by forming the isocyanate a t C-6 by r e a c t i o n of phosgene with the corresponding amine (27) ( F i g . 3, Rn 6). S i m i l a r approaches could be a p p l i e d to the development of an immunoassay f o r tetrachlorodibenzo-£-dioxin using the r e c e n t l y synthesized l-amino-2,3,7,8-tetrachlorodibenzo-jD-dioxin as a hapten (28). The d i f f e r e n t i a l r e a c t i v i t y of the s t e r i c a l l y hindered and unhindered isocyanate groups of t o l y l e n e - 2 , 4 - d i i s o c y a n a t e f a c i l i t a t e s the stepwise conjugation of hapten (R) and p r o t e i n (P) amino groups ( F i g . 3, Rn 7 ) . £,_p/-Difluoro-m,m -dinitrobenzene (DFDNB) r e a c t s with numerous f u n c t i o n a l i t i e s i n c l u d i n g primary and secondary amines, imidazoles, and phenols to y i e l d mixtures of conjugated m a t e r i a l s ( F i g . 3, Rn 8 ) . T h i s r e a c t i o n i s appare n t l y harder to c o n t r o l than the d i i s o c y a n a t e r e a c t i o n s , but i t i s much more v e r s a t i l e . Aromatic amines may be converted to t h e i r diazonium s a l t s with n i t r o u s a c i d . The hapten may then be bound v i a azo l i n k a g e s to the t y r o s i n e (shown), h i s t i d i n e , l y s i n e , and p o s s i b l y a r g i n i n e and tryptophane r e s i d u e s of the c a r r i e r p r o t e i n by mixing the T



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p r o t e i n and diazonium s a l t under basic c o n d i t i o n s . T h i s method was used i n the c l a s s i c immunochemical s t u d i e s by P a u l i n g et a l . (29) and Landsteiner (30) and more r e c e n t l y to coupled aminoparathion to bovine serum albumin (BSA) ( F i g . 3, Rn 9) (31) . The s u l f h y d r y l r e s i d u e i s commonly encountered i n p e s t i c i d e s , and i t can be u t i l i z e d to conjugate a hapten to a p r o t e i n v i a a d i s u l f i d e b r i d g e . Most p r o t e i n s do not have numerous f r e e s u l f h y d r y l groups, so the f r e e SH groups can be "enriched" by r e a c t i n g the p r o t e i n with N-acetyl-homocysteine t h i o l a c t o n e or more r e c e n t l y S-acetylmercaptosuccinic anhydride (SAMSA) ( F i g . 3, Rn 10) followed by a d d i t i o n of the hapten (32) . T h i o l a t e d p r o t e i n s can be used f o r r e a c t i o n with any compound capable of forming cov a l e n t bonds with s u l f u r . Glutathione or other conjugates of p e s t i c i d e metabolites could a l s o p o s s i b l y be used f o r c o u p l i n g to proteins. Other f u n c t i o n a l i t i e s on a hapten can be d i r e c t l y l i n k e d to a p r o t e i n by a v a r i e t y of methods or they can be converted to compounds c o n t a i n i n g a f r e e amine or carboxy1 group and then conjugated by the above methods. By r e a c t i n g aldehydes or ketones with carboxymethoxylamine hemihydrochloride (CMA) the r e s u l t i n g oxime with a f r e e carboxyl group can be formed as shown f o r the a l l e t h r i n CMO d e r i v a t i v e ( F i g . 2B) ( 6 ) . T h i s procedure has a l s o been used i n c o u p l i n g r e a c t i o n s l e a d i n g to a n t i b o d i e s f o r i n s e c t molting hormones (33,34,35) . The a l l e t h r i n CMO d e r i v a t i v e was found to be q u i t e unstable, and t h i s f a c t emphasizes the need f o r r i g o r o u s s t r u c t u r a l proof of hapten s t r u c t u r e . Hydroxylated p e s t i c i d e s are common metabolites and thus, a c h o i c e of hydroxylated m a t e r i a l s are o f t e n a v a i l a b l e f o r conjugat i o n . Exposure of metabolites with primary or secondary a l c o h o l s a v a i l a b l e to s u c c i n i c anhydride i n p y r i d i n e leads to a hemisucc i n a t e as shown f o r a l l e t h r i n d e r i v a t i v e s i n F i g . 2A. This method has been used to d e r i v a t i z e many compounds of b i o l o g i c a l i n t e r e s t i n c l u d i n g ecdysone and s o l a n i d i n e (15,22,23). A l t e r n a t i v e l y , hydroxyl groups can be exposed to equimolar phosgene r e s u l t i n g i n a chlorocarbonate which w i l l r e a c t with amino groups of p r o t e i n s ( F i g . 3, Rn 11) or reacted with e t h y l d i a z o a c e t a t e followed by h y d r o l y s i s to g i v e a carboxymethyl ether (36). Phenols and d i a z o t i z e d jj-aminobenzoic a c i d r e a c t to i n t r o duce a f r e e carboxyl group (32). Ethylbromoacetate was used to d e r i v a t i z e phenolic metabolites of the i n s e c t i c i d e d i f l u b e n z u r o n and model p y r e t h r o i d s under anhydrous c o n d i t i o n s . The r e s u l t i n g e t h y l e s t e r could be hydrolyzed i n d i l u t e methanolic base without h y d r o l y z i n g d i f l u b e n z u r o n . Longer spacers can be introduced by using bromopropionates and buterates, but harsher c o n d i t i o n s are required f o r these l e s s r e a c t i v e bromides. The bromoacids can be used f o r more water-soluble haptens and c h l o r o a c e t i c a c i d has been used f o r estrogen (13,37). Phenols can a l s o be coupled using other d i v a l e n t reagents such as t o l y l e n e - 2 , 4 - d i i s o c y a n a t e or cyanuric c h l o r i d e . A conjugated o l e f i n can be reacted with 3mercaptopropionate to a l s o y i e l d a f r e e c a r b o x y l i c a c i d at the


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end of a convenient spacer arm (38). Numerous other methods of conjugation a r e a v a i l a b l e and w i l l l i k e l y be obvious to the chemist f a m i l i a r with the p r o p e r t i e s of the p e s t i c i d e of i n t e r e s t . In a d d i t i o n to many methods i n the l i t e r a t u r e , numerous reviews g i v e e i t h e r d e t a i l e d c o n j u g a t i o n procedures (32) or r e f e r e n c e s to these procedures (4,10,39). As w i l l be discussed below, organic chemists may f i n d the d i f f i c u l t y of e s t a b l i s h i n g the s t r u c t u r e ( s ) of the f i n a l c o n j u gate d i s c o n c e r t i n g . I n c o n t r a s t to numerous papers i n the l i t e r a t u r e where " r e c i p e s ' f o r conjugation a r e simply followed, i t i s important to v e r i f y the s t r u c t u r e of the hapten a t each step of the s y n t h e s i s . I t may a l s o be important to adapt the c o n d i t i o n s of c o n j u g a t i o n to the s p e c i f i c r e a c t i o n i n question. The s t a b i l i t y of the hapten, the r e s i s t a n c e of the c a r r i e r p r o t e i n to dénaturâtion, and the r e l a t i v e s o l u b i l i t i e s of the hapten and the c a r r i e r p r o t e i n i n the r e a c t i o n medium should be considered.


1

Antigen P u r i f i c a t i o n and C h a r a c t e r i z a t i o n . The a n t i g e n ( h a p t e n - c a r r i e r p r o t e i n conjugate) i s u s u a l l y separated from low molecular weight by-products based on i t s l a r g e s i z e . Dialysis i s an obvious method of s e p a r a t i o n , but some l i p o p h i l i c molecules pass through a d i a l y s i s membrane with great d i f f i c u l t y . G e l f i l t r a t i o n provides another convenient method of s e p a r a t i o n . I f the p r o t e i n i s not too badly denatured, repeated p r e c i p i t a t i o n with an organic solvent such as ethanol i s a r a t h e r c e r t a i n way of removing l i p o p h i l i c i m p u r i t i e s . The most q u a n t i t a t i v e methods of determining the moles of hapten bound per mole of c a r r i e r p r o t e i n i n c l u d e the use of r a d i o l a b e l e d haptens or the monitoring of a change i n the absorbance of the h a p t e n - c a r r i e r conjugate i n a s p e c t r a l r e g i o n where the p r o t e i n i t s e l f does not s t r o n g l y absorb ( 4 ) . For the parat h i o n conjugate a phosphorus determination proved to be a u s e f u l method (31). These methods a r e o f t e n not a p p r o p r i a t e , so a l t e r nate methods such as the monitoring of the p r o t e i n s ' r e a c t i v e groups (such as f r e e amine) before and a f t e r conjugation must be employed (4,6,32,40,41). C a r e f u l c o n t r o l s a r e necessary with these procedures because s e l f conjugation or d e n a t u r a t i o n of the p r o t e i n may decrease or even i n c r e a s e the apparent f u n c t i o n a l i ties available for binding. There i s no consensus on the optimum number of hapten molec u l e s per c a r r i e r , but a t l e a s t two molecules a r e required f o r subsequent immunoprecipitin t e s t s . Many e a r l y s t u d i e s used very high l o a d i n g and u s e f u l antibody t i t e r s continue to be r a i s e d using h e a v i l y loaded antigens. Some workers f e e l that antibody t i t e r s with a higher average s p e c i f i c i t y f o r the hapten can be r a i s e d u s i n g low l o a d i n g (4,42). Numerous p r o t e i n s have been s u c c e s s f u l l y used as c a r r i e r s . Bovine and human serum albumin are very commonly used because they have numerous f r e e amine groups and a r e remarkably s o l u b l e when c r o s s - l i n k e d or even when h e a v i l y loaded with haptens. Many workers have found that mollusk


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hemocyanin i s phenomenally immunogenic. Although hemocyanin has been s u c c e s s f u l l y used as a c a r r i e r f o r p e s t i c i d e haptens, one o f t e n encounters s o l u b i l i t y problems. Many other commercially a v a i l a b l e and e x o t i c p r o t e i n s have a l s o been used as c a r r i e r s . One can be r e l a t i v e l y c e r t a i n that a p r o t e i n w i l l be immunogenic i f i t has a molecular weight >10,000 and i f i t i s ajmnunochemic a l l y f o r e i g n to the animal r e c e i v i n g the a n t i g e n . One should a l s o consider the u l t i m a t e use of the antibody when choosing the c a r r i e r p r o t e i n . For i n s t a n c e , human serum albumin would be a poor c a r r i e r f o r a hapten i f the r e s u l t i n g antibody were to be used to monitor human blood samples by ijinnunod i f f u s i o n . Choice of Animal f o r Antibody P r o d u c t i o n . Numerous v e r t e brates have been used as the source of a n t i b o d i e s . As techniques become more s e n s i t i v e , l e s s antibody i s needed. Guinea p i g s and r a b b i t s are thus commonly used. Even mice are used, e s p e c i a l l y s i n c e the major c e l l l i n e s now a v a i l a b l e f o r c l o n i n g a n t i b o d i e s are derived from mice (43) . I f l a r g e r q u a n t i t i e s of a n t i b o d i e s are needed, one can move to e i t h e r l a r g e r numbers of small mammals or to goats, sheep and l a r g e r mammals. The use of a v i a n species i s not common when haptens are used, but they may y i e l d high, broad spectrum antibody t i t e r s against mammalian p r o t e i n s . The nature of the a n t i b o d i e s obtained w i l l vary somewhat with the species used. For i n s t a n c e , goats are known to o f t e n produce a n t i b o d i e s with very high a f f i n i t y f o r haptens while guinea p i g s o f t e n y i e l d a h i g h t i t e r of complement. Immunization Procedures. The antigen i s u s u a l l y i n j e c t e d i n t o the r e c i p i e n t animal i n Freund's complete adjuvant. T h i s w a t e r - i n - o i l emulsion provides a slow r e l e a s e f o r m u l a t i o n f o r the antigen, p r o t e c t s the a n t i g e n , and with dead Mycobacteria, i t s t i m u l a t e s the immune system. Subsequent booster i n j e c t i o n s are u s u a l l y given i n adjuvant without the Mycobacteria i n order to avoid severe a l l e r g i c response. The r e s u l t i n g antibody t i t e r i n the serum i s monitored and when i t has reached an acceptably high l e v e l , blood i s withdrawn and the serum i s o l a t e d f o r use i n assay development. Many of the numerous immunization p r o t o c o l s are referenced i n Parker (4) w h i l e W i l l i a m s and Chase (32) g i v e d e t a i l e d i n s t r u c t i o n s on the handling of animals. Although the assays using a n t i b o d i e s have reached a h i g h s t a t e of s o p h i s t i c a t i o n , a d e f i n i t i v e work on immunization procedures i s s t i l l l a c k i n g . I t i s not g e n e r a l l y p o s s i b l e to reproduce the exact t i t e r and s p e c i f i c i t y of a n t i b o d i e s even i n apparently i d e n t i c a l animals. T h i s l a c k of r e p r o d u c i b i l i t y i n the r a i s i n g of antibody t i t e r s may have led to the r e l u c t a n c e on the part of p e s t i c i d e a n a l y t i c a l chemists to embrace immunochemical techniques. However, the animal i s only the t o o l used to o b t a i n the antibody, and once the antibody i s i n hand, the assays a r e p h y s i c a l i n c o n t r a s t to b i o l o g i c a l assays. Most radioimmunoassays use serum d i l u t i o n s of 1:5,000 to 1:100,000 so



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that a s i n g l e r a b b i t w i l l y i e l d enough antibody f o r a staggering number of assays. I f p r o p e r l y handled and f r o z e n , a n t i b o d i e s may be stored f o r long p e r i o d s . U l t i m a t e l y , the serum from a s i n g l e animal w i l l be exhausted. Although i t may be d i f f i c u l t to o b t a i n another batch of serum of phenomenally high t i t e r , a f f i n i t y and s p e c i f i c i t y , numerous s t u d i e s have shown that f o r most molecules one has a very h i g h p r o b a b i l i t y of o b t a i n i n g u s e f u l sera f o l l o w ing the i n j e c t i o n of a l i m i t e d number of animals (44) by standard procedures. For i n s t a n c e , out of 8 r a b b i t s i n j e c t e d with s e v e r a l d i f l u b e n z u r o n antigens, antibody t i t e r s were detected i n a l l r a b b i t s a g a i n s t the c a r r i e r p r o t e i n and i n 7 a g a i n s t the hapten (13). Monoclonal antibody technology (see below) promises to improve the c o n s i s t e n t a v a i l a b i l i t y a n t i b o d i e s as reagents (45). The antibody t i t e r i s monitored i n the serum by any of the numerous a n a l y t i c a l procedures discussed below. For i n s t a n c e , a n t i b o d i e s were detected to an a l l e t h r i n - h e m o c y a n i n conjugate by immunodiffusion s t u d i e s u s i n g among other molecules, an a l l e t h rin-BSA conjugate (6) and p a s s i v e hemagglutination was s i m i l a r l y used f o l l o w i n g immunization with a parathion-BSA conjugate (31). In a d d i t i o n to immunodiffusion, a radioimmunoassay was developed using a low s p e c i f i c a c t i v i t y C d i f lubenzuron l a b e l f o r the monitoring of d i f l u b e n z u r o n antibody t i t e r s (13). In order to determine antibody t i t e r , the serum i s g e n e r a l l y d i l u t e d u n t i l a serum c o n c e n t r a t i o n i s reached which w i l l bind 50% of a constant amount of hapten (44). 1I+

Antibody C h a r a c t e r i z a t i o n . In attempting to c h a r a c t e r i z e the a n t i b o d i e s i n a serum sample i t should be kept i n mind that one i s d e a l i n g with a heterogeneous p o p u l a t i o n of antibody molec u l e s of v a r y i n g s p e c i f i c i t y and a f f i n i t y . There are undoubtedly a n t i b o d i e s present which recognize the c a r r i e r p r o t e i n , but c o n t r i b u t i o n of these a n t i b o d i e s to assay binding can be e l i m i n ated by u s i n g a d i f f e r e n t c a r r i e r or a tagged hapten. Even when only the hapten i s recognized i n the assay, one i s d e a l i n g with a heterogeneous antibody p o p u l a t i o n i n a serum sample. An estimat i o n of the average a f f i n i t y constant (K ) i s o f t e n u s e f u l i n the o p t i m i z a t i o n of competitive binding assays (46), and one estimat i o n of s e n s i t i v i t y i s taken as one t e n t h of the r e c i p r o c a l of the average b i n d i n g a f f i n i t y (44) . Estimates of the average K are obtained by p l o t t i n g a f u n c t i o n of the hapten which i s a n t i body bound vs a f u n c t i o n of the c o n c e n t r a t i o n of the hapten. Such p l o t s i n c l u d e M i c h a e l i s Menten curves, Scatchard p l o t s , and S i p s p l o t s . The l a t e r two p l o t s w i l l a l s o g i v e an estimate of the heterogeneity of the antibody p o p u l a t i o n (4,44,46). The s p e c i f i c i t y of an antibody t i t e r r e f e r s to the degree of c r o s s - r e a c t i v i t y one sees with the antiserum used. By u s i n g d i f f e r e n t tagged haptens one can vary the s p e c i f i c i t y of the r e s u l t i n g assay; however, t h e r e i s an i n t r i n s i c s p e c i f i c i t y of the antiserum which i s d i f f i c u l t (although p o s s i b l e ) to improve. The s p e c i f i c i t y of an antiserum i s u s u a l l y e s t a b l i s h e d by compea

a



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t i t i v e binding s t u d i e s . S p e c i f i c i t y i s o f t e n expressed as the c o n c e n t r a t i o n of a substance needed to d i s p l a c e 50% of an a n t i body bound hapten. The s p e c i f i c i t y of an antiserum may be very high, r e q u i r i n g many-fold higher concentrations of very c l o s e l y r e l a t e d molecules to d i s p l a c e the r a d i o l i g a n d . Such s p e c i f i c i t y i s the b a s i s of the major advantages of immunochemical assays over many c l a s s i c a l procedures; however, i t may be m i s l e a d i n g . Although an antibody may e f f e c t i v e l y d i s c r i m i n a t e among s e v e r a l very c l o s e l y r e l a t e d molecules, i t may bind q u i t e t i g h t l y to an unknown molecule i n an e x t r a c t . A l s o , even a 1000X s e l e c t i v i t y may be overcome i f very high l e v e l s of even p o o r l y r e a c t i v e contaminants are present. Such problems are most common when l i p o p h i l i c haptens are used. In c l a s s i c a l GLC assays one i s u s u a l l y l o o k i n g at a weak e l e c t r i c a l response i n d i c a t i n g the presence o f , f o r instance, a mass fragment or e l e c t r o n c a p t u r i n g m a t e r i a l . Such observations are only i n d i c a t i v e of the presence of a p e s t i c i d e i f c a r e f u l c o n t r o l runs have been performed, and s i m i l a r c o n t r o l s are a l s o necessary i n assays i n which a n t i b o d i e s are used. Competitive B i n d i n g . Competitive binding provides the p r i n c i p l e upon which most immunochemical assays are based. Enough antibody i s added to a small, constant amount of r a d i o l a b e l e d antigen to bind 35-50% of i t (the same p r i n c i p l e s apply r e g a r d l e s s of the tag used to i d e n t i f y the a n t i g e n ) . As i n c r e a s i n g amounts of unlabeled antigen are added, one decreases the amount of bound r a d i o l a b e l e d antigen which i s then separated by one of a v a r i e t y of techniques from the f r e e r a d i o l a b e l e d a n t i g e n ( F i g . 4). By monitoring the percentage bound and/or f r e e r a d i o l a b e l e d antigen as the c o n c e n t r a t i o n of unlabeled antigen i s i n c r e a s e d , one can e s t a b l i s h a standard curve. T h i s standard curve can then be used to determine the c o n c e n t r a t i o n of an unknown ( F i g . 4 ) . Bound/Free Separations. U s u a l l y the most time-consuming part of a radioimmunoassay i n v o l v e s the s e p a r a t i o n of the bound and f r e e r a d i o l a b e l e d a n t i g e n . There are s e v e r a l promising new techniques which avoid t h i s separation step, but the most s e n s i t i v e assays s t i l l employ s e p a r a t i o n . E q u i l i b r i u m d i a l y s i s i s an e s t h e t i c a l l y p l e a s i n g method of s e p a r a t i o n , but i t does not lend i t s e l f to the processing of a l a r g e number of samples. Gel permeation chromatography works on the same p r i n c i p l e because the l a r g e antibody bound antigen e l u t e s ahead of the smaller f r e e a n t i g e n . Gel permeation i s u s u a l l y too slow f o r r o u t i n e immunoassay procedure, although i t forms the b a s i s f o r s e v e r a l very rapid automated procedures. N i t r o c e l l u l o s e membranes w i l l a l l o w small antigens to pass through while r e t a i n i n g a n t i b o d i e s and they form the b a s i s of s e v e r a l rapid a n a l y t i c a l procedures. The use of dextran-coated c h a r c o a l to p r e c i p i t a t e unbound antigen i s commonly used, and i t should be g e n e r a l l y a p p l i c a b l e to p e s t i c i d e s (_6,7) . The dextran c o a t i n g on the c h a r c o a l and/or the


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Figure 4.

Illustration of the principle of competitive binding.

An increasing amount of unlabeled antigen displaces a constant amount of labeled antigen from a constant amount of antibody. Separation of antibody bound and free material results in a standard curve that can be used to determine the amount of unlabeled antigen in unknown samples.



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presence of n o n s p e c i f i c sera g r e a t l y reduce the p r e c i p i t a t i o n of a n t i b o d i e s and may i n c r e a s e the ease with which a c h a r c o a l suspension i s handled. F l o r i s i l may sometimes be s u b s t i t u t e d f o r c h a r c o a l . Although techniques which bind the antigen are g e n e r a l l y a p p l i c a b l e to l i p o p h i l i c molecules, they w i l l s h i f t the bound/free e q u i l i b r i u m with time. Thus, assays are o f t e n more time-dependent with antigen binding r a t h e r than antibody binding techniques. The b i n d i n g of a charged antibody to i o n exchange r e s i n s or hydroxyapatite i s a l s o commonly used f o r s e p a r a t i o n . The a n t i body can be p r e c i p i t a t e d using polyethylene g l y c o l or ammonium s u l f a t e and/or a second antibody such as goat a n t i - r a b b i t l e a v i n g unbound antigen i n s o l u t i o n . More r e c e n t l y a s u r f a c e p r o t e i n , p r o t e i n A, on the s u r f a c e of some Staphylococcus aureus c e l l s has been found to s p e c i f i c a l l y bind and p r e c i p i t a t e many a n t i b o d i e s . S o l i d phase systems i n which the antibody i s coated on tubes or attached to polyacrylamide or dextran p a r t i c l e s lend themselves to very r a p i d a n a l y s i s . Larger amounts of antibody are g e n e r a l l y required f o r s o l i d phase assays and some a d d i t i o n a l e f f o r t i n assay o p t i m i z a t i o n i s o f t e n needed. There are s e v e r a l l i n e s of r e s e a r c h which may lead to very s e n s i t i v e , r a p i d immunochemical assays which do not r e q u i r e separa t i o n of f r e e and bound a n t i g e n . The ELISA procedure discussed below does r e q u i r e s e p a r a t i o n , but d i r e c t i n h i b i t i o n of a haptens u b s t i t u t e d enzyme by a n t i g e n binding (EMIT procedure) may a l l e v i a t e a s e p a r a t i o n requirement. A s e n s i t i v e method f o r the a n a l y s i s of 2-ABZI has been demonstrated based on f l u o r e s c e n c e p o l a r i z a t i o n (26). Binding can a l s o be measured without separat i o n by a t t a c h i n g e l e c t r o n s p i n resonance (ESR) probes to a n t i gens and monitoring the ESR band width of the n i t r o x i d e s i g n a l . Metal tagged haptens which are then analyzed by atomic a b s o r p t i o n spectrometry show some promise (47,48) . Lasers are i n c r e a s i n g the s e n s i t i v i t y of t u r b i d i t y methods but, at best, these methods are of moderate s e n s i t i v i t y . O p t i m i z a t i o n of the Assay. As discussed e a r l i e r , competit i v e binding assays are based on the law of mass a c t i o n where the a f f i n i t y of the antibody f o r an antigen i s K = Ab*Ag/(Ab)(Ag) and when 50% of the t o t a l antigen i s bound K = l / ( A b ) . I f an antibody p o p u l a t i o n were homogeneous, the mathematics used to d e s c r i b e the binding would be r a t h e r s t r a i g h t f o r w a r d . However, the h e t e r o g e n i c i t y of the antibody p o p u l a t i o n complicates the s i t u a t i o n so that there i s no mathematical treatment that w i l l completely d e s c r i b e a l l antibody-antigen i n t e r a c t i o n s . The mathematical bases of immunoassay have been discussed by a number of workers (49-54), and work i n t h i s d i r e c t i o n i s c o n t i n u i n g with a trend towards the development of computer programs u n i v e r s a l l y adaptable to data from a v a r i e t y of immunoassays Ç53-56). One can optimize an assay based on a l o g i c a l p r o g r e s s i o n of e x p e r i ments using the p h y s i c a l constants i n t r i n s i c to the assay (46, a

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56), or one can approach the optimum assay c o n d i t i o n s e m p i r i c a l l y by determining the amount of antibody needed to g i v e 35-50% binding of vL0,000 CPM of the r a d i o l a b e l e d a n t i g e n at e x p e r i mentally determined i n c u b a t i o n times (4,46). Most assays a r e run under c o n d i t i o n s approaching or at e q u i l i b r i u m . However, the t h e o r e t i c a l assay s e n s i t i v i t y and assay speed can be increased by u s i n g n o n e q u i l i b r i u m c o n d i t i o n s . Nonequilibrium c o n d i t i o n s are o f t e n used i n automated procedures. Numerous methods e x i s t f o r p l o t t i n g competitive b i n d i n g d a t a . In choosing a method, one should keep i n mind that the s e l e c t i o n of an optimum p l o t t i n g technique may s i m p l i f y data handling or f a c i l i t a t e q u a l i t y c o n t r o l e v a l u a t i o n s , but p l o t t i n g methods cannot enhance the i n t r i n s i c s e n s i t i v i t y or accuracy of an assay. The amount of antibody bound ligand i s u s u a l l y measured because small changes i n antigen c o n c e n t r a t i o n w i l l y i e l d l a r g e r r e l a t i v e changes i n the antibody bound r a d i o a c t i v i t y than i n the unbound r a d i o a c t i v i t y . In theory, assay p r e c i s i o n should be enhanced by monitoring both bound and f r e e a n t i g e n , but t h i s course i s seldom followed. Parker (4) p r a g m a t i c a l l y suggests p l o t t i n g counts p r e c i p i tated on the o r d i n a t e a g a i n s t the logarithm of the t o t a l unl a b e l e d a n t i g e n c o n c e n t r a t i o n on the a b c i s s a r a t h e r than spending an i n o r d i n a t e amount of time i n s e l e c t i n g the "optimum p l o t t i n g procedure. The standard curve can u s u a l l y be made more l i n e a r by using l o g i t , p r o b i t , or a r c s i n e f u n c t i o n s (52) . Unless automated data r e d u c t i o n i s used, such p l o t s provide adequate standard curves f o r most assays. There are numerous commercial products f o r RIA data r e d u c t i o n as w e l l as a v a r i e t y of published programs. With constant random e r r o r , the p r e c i s i o n of an RIA i n c r e a s e s as the slope of the dose-response curve increases and decreases as the e r r o r i n c r e a s e s with constant s l o p e . Future a v a i l a b i l i t y of monoclonal a n t i b o d i e s may g r e a t l y i n c r e a s e the steepness and improve the shape of the r e s u l t i n g dose-response curve. As w i t h any a n a l y t i c a l techniques, i t i s c r u c i a l to a p p r e c i a t e the confidence i n t e r v a l s which one has at v a r i o u s p o i n t s of the dose-response curve, i n a d d i t i o n to the many measurement and c o l l e c t i o n e r r o r s which may be made before the immunoassay i s employed. 11

1 4

Choice of R a d i o l i g a n d . A C r a d i o l a b e l w i l l probably e x i s t f o r most p e s t i c i d e s which w i l l be considered f o r radioimmunoassay development. Such an i n t r i n s i c r a d i o l a b e l w i l l prove very v a l u a b l e i n t i t e r i n g a n t i s e r a and p o s s i b l y i n numerous other steps from a n t i g e n s y n t h e s i s through assay development. Unfort u n a t e l y , f o r the a c t u a l assay, the commonly a v a i l a b l e C r a d i o l a b e l s may not be of high enough s p e c i f i c a c t i v i t y . The t h e o r e t i c a l l i m i t on the s p e c i f i c a c t i v i t y of a s i n g l e carbon atom i s ^63 mCi/mmole, and few p e s t i c i d e s have a s p e c i f i c a c t i v i t y of over 50 mCi/mmole even when they a r e l a b e l e d i n 1 4


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numerous p o s i t i o n s . Although many f a c t o r s i n f l u e n c e r a d i o immunoassay s e n s i t i v i t y , assay s e n s i t i v i t y g e n e r a l l y increases with the square root of the s p e c i f i c a c t i v i t y of the r a d i o l i g a n d . Thus, H and I are commonly used. H may be incorporated i n t o the s t r u c t u r e of a p e s t i c i d e d i r e c t l y (an i n t r i n s i c r a d i o l i g a n d ) . High l e v e l s of i n c o r p o r a t i o n are p o s s i b l e by a wide v a r i e t y of procedures, and c a r r i e r f r e e t r i t i u m w i l l y i e l d about 29 C i / mmol/atom incorporated. Isotope e f f e c t s are much more common with than with C , and seemingly t r i v i a l r a d i o syntheses may become very d i f f i c u l t when high s p e c i f i c a c t i v i t i e s are d e s i r e d . I t i s not necessary that the t r a c e r or r a d i o l i g a n d i s s t r u c t u r a l l y i d e n t i c a l to the p e s t i c i d e of i n t e r e s t . The same cons i d e r a t i o n s used i n d e c i d i n g where to a t t a c h a hapten to a p r o t e i n should be applied to a t t a c h i n g a hapten to e i t h e r a commercially a v a i l a b l e labeled compound or to a compound which i s e a s i l y labeled i n a subsequent step. For instance, the conjugat i o n of the hemisuccinate of S ^ - b i o a l l e t h r i n to commercially a v a i l a b l e H tyramine ( j 3 - [ 2-aminoethyl]phenol) led to a u s e f u l r a d i o l i g a n d (.6,7). The most common isotope used i n radioimmunoassays i s I. Incorporation of a s i n g l e atom of I will result in a specific a c t i v i t y of ^2400 Ci/mmol. Since i n t r o d u c t i o n of an i o d i n e w i l l u s u a l l y cause a tremendous change i n a hapten, the i o d i n e i s u s u a l l y introduced on a separate moiety such as histamine, t y r o s i n e , or tyramine which i s attached to the hapten. I offers many advantages over H as a t r a c e r . I t i s r e l a t i v e l y easy and inexpensive to introduce, and i t s high s p e c i f i c a c t i v i t y leads to greater t h e o r e t i c a l assay s e n s i t i v i t y . As a gamma emitter i t i s seldom subject to quench, and i t can be e f f i c i e n t l y detected with a s o l i d s c i n t i l l a t i o n counter. S o l i d s c i n t i l l a t i o n counters are u s u a l l y l e s s expensive than l i q u i d s c i n t i l l a t i o n counters of similar sophistication. d t r e q u i r e the use of s c i n t i l l a t i o n s o l u t i o n which makes assays e a s i e r , cheaper, and f a s t e r . However, as a gamma emitter which can undergo b i o accumulation, I must be handled very c a r e f u l l y , and i t s 2month h a l f - l i f e (vs 12 y r s f o r H) n e c e s s i t a t e s repeated r a d i o syntheses. An a d d i t i o n a l problem i s that many l a b o r a t o r i e s new to radioimmunoassay do not have s o l i d s c i n t i l l a t i o n counters even though l i q u i d s c i n t i l l a t i o n counters are commonly a v a i l a b l e . There are numerous commercial adapters which increase the e f f i c i e n c y of l i q u i d s c i n t i l l a t i o n counters f o r gamma e m i t t e r s . A m o d i f i c a t i o n of a suggestion by Beckman Instruments has proven quite useful i n t h i s laboratory. Thin-walled g l a s s tubes were permanently attached through a hole i n the cap of a standard s c i n t i l l a t i o n v i a l with epoxy-cement. The v i a l was f i l l e d with a standard s c i n t i l l a t i o n s o l u t i o n such as Omniflour® or 1.5% b u t y l PBD i n toluene c o n t a i n i n g , i n a d d i t i o n , t e t r a e t h y l or t e t r a b u t y l l e a d . For the c o n d i t i o n s i n our l a b o r a t o r y , 3% v/v of t e t r a e t h y l lead i n the s c i n t i l l a t i o n s o l u t i o n r e s u l t e d i n >55% counting efficiency for I on the H window of a Beckman LS230 f o r 3

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samples added i n 6 χ 50 mm g l a s s t e s t tubes. The s c i n t i l l a t i o n v i a l s were permanently sealed under Ν 2 i n order to avoid the decomposition of the s o l u t i o n and the r e l e a s e of t o x i c vapors. There a r e s e v e r a l other suggestions f o r the counting of I in l i q u i d s c i n t i l l a t i o n systems (57,58). Iodine i s o f t e n introduced ortho to the phenol of t y r o s i n e or a t y r o s i n e - l i k e m a t e r i a l or i n t o histamine attached to a hapten under mild o x i d i z i n g c o n d i t i o n s . Chloramine Τ (N-chloro-p_-toluenesulfonamide sodium s a l t ) or l a c t o p e r o x i d a s e - H 2 0 2 are o f t e n used as o x i d i z i n g agents. A s o l i d phase chloroamide has been r e c e n t l y reported (59). These procedures are o n l y s u i t a b l e i f the molecule i s s t a b l e to o x i d i z ing c o n d i t i o n s . A l t e r n a t i v e l y , a separate molecule may be l a b e l e d with i o d i n e and then attached to the hapten (18). Some such compounds a r e commercially a v a i l a b l e and d e t a i l e d procedures are a v a i l a b l e from most s u p p l i e r s of r a d i o a c t i v e i o d i n e . The same p h i l o s o p h i e s which apply to the c h o i c e of a r a d i o l a b e l f o r radioimmunoassay g e n e r a l l y apply to the attachment of any t r a c e r or i n d i c a t o r molecule. These i n d i c a t o r s may i n c l u d e such t h i n g s as a f l u o r e s c e n t , e l e c t r o n s p i n resonance, m e t a l l i c , or enzymatic markers. I f an i n t r i n s i c r a d i o l a b e l i s not used, the method by which the l a b e l i s introduced may e f f e c t the assay s p e c i f i c i t y and s e n s i t i v i t y , j u s t as the c h o i c e of hapten does ( F i g . 1 ) . I f the same hapten d e r i v a t i v e i s used f o r preparing the a n t i g e n and the r a d i o l i g a n d , the r e s u l t i n g antibody may have a higher a f f i n i t y f o r the r a d i o l i g a n d than the molecule to be assayed. T h i s s i t u a t i o n w i l l reduce the t h e o r e t i c a l s e n s i t i v i t y of the assay (see F i g u r e 5).


1

2

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Enzyme-linked Immunosorbent Assay. A promising a l t e r n a t i v e to the RIA procedure i s an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a f u n c t i o n a l enzyme to e i t h e r an a n t i g e n or antibody. The amount of enzyme present i n a c o m p e t i t i v e b i n d i n g assay i s quantitated instead of the amount of r a d i o l a b e l e d compound. The c o n c e n t r a t i o n of the enzyme can be determined through i t s subsequent r e a c t i o n with a sub s t r a t e which r e s u l t s i n a measurable s p e c t r o s c o p i c change. Conjugation of enzymes to antigens or a n t i b o d i e s were f i r s t developed f o r h i s t o c h e m i c a l techniques and were used f o r l o c a l i z a t i o n of antigens and a n t i b o d i e s i n t i s s u e s e c t i o n s (60). Enzymes were q u i c k l y adapted f o r immunoassays, and E n g v a l l and Perlmann (61) developed a procedure f o r the q u a n t i t a t i o n of an a n t i g e n . A l k a l i n e phosphatase was conjugated v i a glutaraldehyde to r a b b i t IgG ( a n t i g e n ) . Sheep antibody a g a i n s t r a b b i t IgG was coupled to m a c r o c r y s t a l l i n e c e l l u l o s e by cyanogen bromide and the amount of a n t i g e n binding to the antibody was a d i r e c t r e l a t i o n ship w i t h the amount of phosphate e s t e r cleaved by the coupled enzyme i n a given period of time. T h i s technique was widely adapted f o r the q u a n t i t a t i o n of v a r i o u s p r o t e i n s and i n f e c t i o u s agents (62-69). A number of enzymes have been used with immunoassays. These



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i n c l u d e l a c t i c dehydrogenase, mushroom t y r o s i n a s e , glucose oxidase, a c i d phosphatase, a l k a l i n e phosphatase, and h o r s e - r a d i s h peroxidase. The l a t t e r two enzymes have received most of the a t t e n t i o n and peroxidase i s u s u a l l y p r e f e r r e d because of i t s low cost (70) although other r e a c t i o n s may be more s e n s i t i v e and r e p r o d u c i b l e (68,69). B i - or m u l t i - f u n c t i o n a l reagents have been used to l i n k enzymes to other p r o t e i n s . These i n c l u d e v a r i o u s carbodiimides, b i s d i a z o t i z e d amines, cyanuric c h l o r i d e and glutaraldehyde. Enzymes l i n k e d to r a b b i t IgG a n t i b o d i e s from sheep, goats, and horses are commercially a v a i l a b l e and g r e a t l y f a c i l i t a t e the ELISA procedure. The p r e p a r a t i o n of the antigen and the develop ment of the corresponding r a b b i t antibody (IgG f r a c t i o n ) have been described p r e v i o u s l y . The ELISA procedure r e c e n t l y has been used f o r the a n a l y s i s of p a r a t h i o n (31). Since t h i s procedure has c o n s i d e r a b l e poten t i a l a more d e t a i l e d d e s c r i p t i o n of the a n a l y s i s of p a r a t h i o n i s i n order. The conjugation procedure using amino p a r a t h i o n (AP) was described e a r l i e r ( F i g . 3, Rn 9), and t h i s conjugate was then administered to r a b b i t s f o r development of a p o p u l a t i o n of s p e c i f i c a n t i b o d i e s (Ab^) against BSA or AP. Ab^ demonstrated immunological a c t i v i t y only f o r the hapten when AP was conjugated to r a b b i t serum albumin (RSA). T h i s antigen (RSA-AP) was render ed i n s o l u b l e v i a attachment to the p o l y s t y r e n e surface of microt i t e r p l a t e s under b a s i c c o n d i t i o n s ( F i g . 6.1). Following the removal of excess antigen the s p e c i f i c a n t i serum (Abi) was allowed to r e a c t with the s u r f a c e bound antigen ( F i g . 6.2). A f t e r washing away excess antiserum, an enzyme (E, h o r s e - r a d i s h peroxidase) conjugated to goat γ-globulin (Ab2) produced a g a i n s t r a b b i t γ-globulin of the antiserum ( A b i ) , was added ( F i g . 6.3). The binding of the enzyme complex to the s o l i d phase was a measure of the amount of bound RSA-AP and Ab^. The enzyme c o n c e n t r a t i o n was measured s p e c t r o p h o t o m e t r i c a l l y by means of i t s c a t a l y z i n g the o x i d a t i o n of hydrogen peroxide i n the presence of 5 - a m i n o s a l i c y c l i c acid i n a given period of time ( F i g . 6.4). A n a l y s i s of parathion by t h i s technique i s based on the com p e t i t i o n between the f r e e form of p a r a t h i o n (P) and i t s conjuga ted form (AP) f o r the binding s i t e s on the f i r s t antibody (Abi) ( F i g . 6.5). Due to t h i s competition, there i s a decrease i n the binding of the conjugated form as the c o n c e n t r a t i o n of the f r e e form (P) i n c r e a s e s . Complete i n h i b i t i o n of the binding of Abi f o r BSA-AP may r e s u l t when Ρ i s present i n greater q u a n t i t y than Ab^. The c o n c e n t r a t i o n of the parathion i n an unknown sample can then be determined by comparing the degree of antibody i n h i b i t i o n caused by the a d d i t i o n of the sample e x t r a c t with that r e s u l t i n g from the a d d i t i o n of known amounts of the same substance. For p a r a t h i o n a n a l y s i s no cleanup of the e x t r a c t s of f r u i t s or vege t a b l e s were necessary. Various parameters of the ELISA procedure need to be 5


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7mm

7f\


55mm

Figure 5. Cross section of a scintillation vial illustrating a system for counting I in a liquid scintillation system. 125

The sample to be counted is inserted into the 7 X 55 mm glass well immersed in heavy metal charged scintillation cocktail in a permanently sealed vial.

(I)

•

•

RSA^-AP

k-RSA~AP

(2)

RSAÂPÂb,

(4)

(5)

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R S A ~ A P ^ A b , + X(P~Ab,)

(excess)

Figure 6.

Schematic representation of ELI S A.

(O) polystyrene surface; (RSA) rabbit serum albumin; (AP) conjugated aminoparathion; (Abj) first antibody (rabbit anti-parathion); (Ab ) second antibody (goat anti-rabbit); (E) enzyme (horse-radish peroxidase); (S) substrate; and (P) free hapten (parathion). 2



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optimized f o r each a n a l y s i s . These parameters i n c l u d e the c o n c e n t r a t i o n of a n t i b o d i e s , time of incubations, temperature, and the composition of the washing f l u i d s . Optimum time of incubations f o r a n a l y s i s of parathion ranged from 2-3 hours. A s o l u t i o n of 0.9% NaCl and Tween 20 was determined to be most s u i t a b l e f o r washing the m i c r o t i t e r p l a t e i n a l l steps of the procedure. The antiserum exhibited high s p e c i f i c i t y f o r the f u n c t i o n a l i t i e s of p a r a t h i o n , e.g., 58 ng/ml of p a r a t h i o n produced 50% i n h i b i t i o n of the o x i d a t i o n of 5 - a m i n o s a l i c y c l i c a c i d . Changing of the e t h y l groups to methyl groups as i n methyl p a r a t h i o n (2000 ng per 50% i n h i b i t i o n ) or replacement of the s u l f u r atom with oxygen as i n paraoxon (1850 ng per 50% i n h i b i t i o n , 23), g r e a t l y reduced the competitive b i n d i n g . Amino p a r a t h i o n d i d e x h i b i t a s i g n i f i c a n t cross r e a c t i o n (275 ng per 50% i n h i b i t i o n , 23) but p - n i t r o p h e n o l bound poorly to the antibody (5000 ng per 50% i n h i b i t i o n ) . The lower l i m i t of d e t e c t i o n of p a r a t h i o n by the ELISA procedure was found to be 5.0-10.0 ng/ml which corresponded to 0.025-0.05 ppm i n crude e x t r a c t s of f r u i t , v e g e t a b l e s , and human serum. The procedure gave good r e p r o d u c i b i l i t y as expressed i n the c o e f f i c i e n t of v a r i a t i o n (CV%) of r e s u l t s of between-run (6.28.6) and w i t h i n - r u n (4.8-6.5) v a r i a t i o n s . Accuracy of the ELISA procedure was tested by comparing r e s u l t s of p a r a t h i o n a n a l y s i s i n e x t r a c t s of f o r t i f i e d and f i e l d samples with r e s u l t s obtained by a GLC method. C o r r e l a t i o n c o e f f i c i e n t s ranged i n almost a l l cases between 0.93-0.99. The ELISA procedure f o r the a n a l y s i s of p a r a t h i o n as desc r i b e d above r e q u i r e s n e a r l y eight hours, although many samples can be simultaneously assayed. However, i n c u b a t i o n times can be shortened to one-half hour, i n most cases, r e s u l t i n g i n only a 10% r e d u c t i o n i n s e n s i t i v i t y . A l s o the p o l y s t y r e n e m i c r o t i t e r p l a t e s c o n t a i n i n g bound RSA-AP can be mass produced and stored i n a f r e e z e r . Since the enzyme-linked antibody can be purchased, the l i m i t i n g f a c t o r of the a p p l i c a b i l i t y of the ELISA procedure, as w e l l as the RIA procedures, f o r other p e s t i c i d e s i s the development of the antiserum to the p e s t i c i d e . The ELISA procedure shares many of the advantages of RIA, and i t has a d d i t i o n a l advantages of r e q u i r i n g only inexpensive equipment and of being w e l l adapted to automated or p a r t i a l l y automated methods. For instance, Ruitenberg et a l . , (71) has mechanized the ELISA procedure f o r screening of 4000 sera samples d a i l y . A number of disadvantages of the ELISA procedure a l s o can be s i t e d . These i n c l u d e the n o n s t a b i l i t y of the developed c o l o r r e q u i r i n g d a i l y a n a l y s i s (not necessary i n the RIA method), nonl i n e a r i t y of c o l o r development, and l e s s s e n s i t i v i t y than some other immunochemical methods. Other Immunoassay Methods. Other immunoassay methods can be used to q u a n t i t a t e the hapten; these i n c l u d e homogeneous enzyme



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immunoassay (EMIT), r a d i a l immunodiffusion, immunoelectrophoresis and p a s s i v e hemagglutination t e s t s . These techniques are o f t e n used to c h a r a c t e r i z e the antibody, but they a l s o can be used to q u a n t i t a t e the hapten through i n h i b i t i o n experiments. The EMIT procedure i n v o l v e s d i r e c t i n h i b i t i o n of enzyme a c t i v i t y when an antibody binds to a hapten conjugated near the enzyme's a c t i v e s i t e . Thus, i t i s p a r t i c u l a r l y u s e f u l with small molecules, and i t i s very r a p i d because no s e p a r a t i o n steps are required (69). T h i s technique i s promising, but i t has not been widely used and i t i s o f t e n of lower s e n s i t i v i t y than ELISA. R a d i a l immunodiffusion procedures are v a r i e d but a l l depend upon the d i f f u s i o n of the antigen or antibody i n a g e l producing a p r e c i p i t a t e which i s p r o p o r t i o n a l to the q u a n t i t y of r e a c t a n t s ( o f t e n s e n s i t i v e to 25 ng p r o t e i n with v i s u a l methods (32,72, 73)). M o d i f i c a t i o n s u s i n g r a d i o l a b e l e d antigens or a n t i b o d i e s may i n c r e a s e the s e n s i t i v i t y f i f t y - f o l d (74,75). I f the antibody i s f i r s t mixed with the hapten, the c o n c e n t r a t i o n of f r e e unbound antibody w i l l decrease p r o p o r t i o n a t e l y and r e s u l t i n a decrease i n the p r e c i p i t a t e formed with the antigen which can be observed v i s u a l l y or with r a d i o l a b e l e d methods. The hemagglutination t e s t i s o f t e n used to express antibody t i t e r , but i t a l s o can be used to q u a n t i t a t e the hapten. This t e s t i s based on the f a c t that e r y t h r o c y t e s , when treated with a d i l u t e s o l u t i o n of t a n n i c a c i d , a c q u i r e the property of being a b l e to adsorb p r o t e i n (the conjugated a n t i g e n ) . Such p r o t e i n coated red blood c e l l s are a g g l u t i n a t e d by s p e c i f i c antiserum d i r e c t e d a g a i n s t the hapten. The a g g l u t i n a t i o n t i t e r i s expressed as the r e c i p r o c a l of the highest d i l u t i o n of the serum that causes a g g l u t i n a t i o n of the red blood c e l l s . The antiserum can be incubated with the sample e x t r a c t c o n t a i n i n g the s p e c i f i c hapten p r i o r to conducting the hemagglutination t e s t . The amount of unbound antiserum i s reduced by the amount equivalent to the f r e e hapten and consequently the d i l u t i o n of antiserum necessary to produce a g g l u t i n a t i o n i s i n v e r s e l y p r o p o r t i o n a l to the amount of hapten i n the sample (32). Monoclonal Antibody Technology. Based on p i o n e e r i n g work by Kdhler and M i l s t e i n (76) a new technology i s e v o l v i n g which may g r e a t l y improve the a v a i l a b i l i t y , s p e c i f i c i t y , and s e n s i t i v i t y of a n t i s e r a . Monoclonal antibody technology has been the subject of a t e c h n i c a l compendium (43) and a n o n t e c h n i c a l review (45). S i m p l i s t i c a l l y , spleen lymphocytes Immunized i n v i v o or i n v i t r o are fused with a myeloma c e l l l i n e , and the r e s u l t i n g h y b r i d s are s e l e c t e d on the b a s i s of n u t r i t i o n a l requirements and then cloned. Those clones producing monoclonal a n t i b o d i e s of the d e s i r e d s p e c i f i c i t y are i n j e c t e d i n t o mice where the r e s u l t i n g a s c i t e s tumor f l u i d may c o n t a i n gram q u a n t i t i e s of a monoclonal antibody. A l t e r n a t i v e l y , a n t i b o d i e s can be c o l l e c t e d d i r e c t l y from a c e l l c u l t u r e medium. S i n c e c l o n e s are used, i t i s not necessary f o r the antigen to be h i g h l y pure and a n t i b o d i e s



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s e l e c t i v e f o r o p t i c a l isomers c o n c e i v a b l y could be obtained from a racemic hapten. Although i n i t s infancy, monoclonal antibody technology may o f f e r many advantages. As e a r l i e r d i s c u s s e d , the serum of an animal contains a l a r g e p o p u l a t i o n of a n t i b o d i e s with v a r y i n g s p e c i f i c i t i e s and a f f i n i t i e s . With monoclonal a n t i b o d i e s one or more antibody types of high a f f i n i t y and optimum s p e c i f i c i t y can be selected and propagated f o r use i n immunoassays. Immunoassays u s i n g monoclonal a n t i b o d i e s are c h a r a c t e r i z e d by a very steep dose-response curve which t r a n s l a t e s as g r e a t l y enhanced assay p r e c i s i o n . Although the c l o n a l hybrids may have an u n s t a b l e chromosome complement, with proper t e c h n i c a l c a r e the l i n e s could be considered "immortal". Thus, a s i n g l e , uniform antibody reagent of defined s p e c i f i c i t y could be provided to many l a b o r a t o r i e s from frozen c e l l l i n e s which are o c c a s i o n a l l y thawed to produce a n t i b o d i e s . On the negative s i d e , monoclonal t e c h nology i s new and has not been widely applied to the a n a l y s i s of haptens. R e l a t i v e l y simple c e l l c u l t u r e f a c i l i t i e s are needed and s i n c e c u l t u r i n g must c u r r e n t l y be done i n a n t i b i o t i c - f r e e medium, a high l e v e l of t e c h n i c a l s k i l l i s necessary. S e v e r a l companies have r e c e n t l y entered the f i e l d , and i t may soon be p o s s i b l e to o b t a i n a n t i b o d i e s on a c o n t r a c t b a s i s . Some of the p o t e n t i a l advantages and disadvantages of monoclonal technology as a p p l i e d to r e s i d u e a n a l y s i s are l i s t e d i n Table I I . Table I I P o t e n t i a l Advantages and Disadvantages of Monoclonal A n t i b o d i e s as Applied to P e s t i c i d e Residue A n a l y s i s Advantages C e l l l i n e s "immortal" Very h i g h s p e c i f i c i t y possible Steep dose-response curve (very p r e c i s e assays) Technology advancing r a p i d l y Large amount of a n t i b o d i e s Extraordinary high t i t e r Uniform a n t i b o d i e s A c t i v a t e jLn v i v o or _in v i t r o Pure a n t i g e n not necessary

Disadvantages Chromasome complement o f t e n unstable S p e c i f i c i t y too great Dose-response curve too steep (small l i n e a r region) New technology Not widely used with haptens Simple c e l l c u l t u r e f a c i l i t i e s needed A n t i b i o t i c - f r e e medium used P r e c i p i t a t i o n assays d i f f i c u l t

A t t r i b u t e s and L i m i t a t i o n s of Immunoassay Immunoassay S e n s i t i v i t y . Yalow (2) p o i n t s out that as l i t t l e as 0.1 picogram (0.05 picomolar) g a s t r i n can e a s i l y be detected by immunoassay i n a m i l l i l i t e r of i n c u b a t i o n medium. Immunoassays to small l i p o p h i l i c molecules are g e n e r a l l y l e s s



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s e n s i t i v e than those to p r o t e i n s and peptides, and molecules having s e v e r a l p o l a r f u n c t i o n a l i t i e s separated by nonpolar areas o f t e n lend themselves most r e a d i l y to h i g h l y s e n s i t i v e r a d i o immunoassays ( 4 ) . With s t e r o i d s , s e n s i t i v i t i e s on the order of 1 picogram are not uncommon (44) and u s e f u l assays f o r s t e r o i d s and drugs have been devised at much lower s e n s i t i v i t i e s . As d i s cussed e a r l i e r , the a c t u a l assay s e n s i t i v i t y depends upon the a f f i n i t y of the serum, the i n c u b a t i o n volume, and the amount of t r a c e r and antibody used (which t r a n s l a t e , i n p a r t , to the s p e c i f i c a c t i v i t y of the t r a c e r ) . The s e n s i t i v i t y of the o v e r a l l a n a l y t i c a l procedure depends upon many f a c t o r s o b v i o u s l y i n c l u d i n g the type of sample to be analyzed and the s k i l l of the a n a l y t i c a l chemist. I f an immunoassay i s used to measure the amount of p e s t i c i d e i n a water sample by adding the water sample d i r e c t l y to the immunoassay, very high s e n s i t i v i t y may not be obtained although the assay w i l l r e q u i r e very l i t t l e time to perform. A l t e r n a t i v e l y , i f the water sample i s extracted and the immunoassay i s employed only a f t e r s e v e r a l h i g h l y e f f i c i e n t cleanup steps, phenomenal s e n s i t i v i t y may be obtained a t the expense of a l a r g e investment i n time. In some s i t u a t i o n s , immunochemical methods may decrease the l i m i t of d e t e c t a b i l i t y of a p e s t i c i d e r e s i d u e (77) , but more importantly they may, i n some cases, decrease the time and c o s t needed to reach a l e v e l of d e t e c t a b i l i t y as has been demonstrated with p a r a t h i o n (31). S p e c i f i c i t y . The s p e c i f i c i t y of an immunoassay i s r e l a t e d i n some r e s p e c t s to the s e n s i t i v i t y . The high s p e c i f i c i t y of immunoassays o f t e n allows samples to be analyzed with a minimum of cleanup. The remarkable s p e c i f i c i t y of antigen-antibody i n t e r a c t i o n s has been reviewed i n the c l a s s i c t e x t by Landsteiner (30). More r e c e n t l y , Al-Rubae (31) demonstrated a high l e v e l of s p e c i f i c i t y i n an ELISA procedure f o r p a r a t h i o n . As p r e v i o u s l y d i s c u s s e d , p a r a t h i o n could be e a s i l y d i s t i n g u i s h e d from methyl p a r a t h i o n and j>-nitrophenol and the assay demonstrated l i t t l e or no c r o s s r e a c t i v i t y with a number of other p e s t i c i d e s . A r a d i o immunoassay f o r S>-bioallethrin showed no cross r e a c t i o n f o r any of s e v e r a l other p y r e t h r o i d s tested (except p y r e t h r i n I ) , and i t was capable of d i s t i n g u i s h i n g S - b i o a l l e t h r i n (lR,3R,4 S) from the other 7 o p t i c a l and geometrical isomers of a l l e t h r i n . Since b i o l o g i c a l a c t i v i t y and biodégradation may depend upon the conf i g u r a t i o n of an i n s e c t i c i d e (6,2.) the a b i l i t y of immunoassays to d i s t i n g u i s h among c h i r a l isomers (78,79) may become of great importance to f u t u r e p e s t i c i d e metabolism and r e s i d u e i n v e s t i g a t i o n s . The high p o t e n t i a l s p e c i f i c i t y of immunochemical methods may prove very u s e f u l , i n c o n j u n c t i o n with other methods, i n the c o n f i r m a t i o n of the presence of r e s i d u e s . Monoclonal technology i s l i k e l y to a l l o w p e s t i c i d e a n a l y s i s based on immunochemistry to be even more s p e c i f i c and s e n s i t i v e . Although one must u l t i m a t e l y r e l y upon the immune system of f



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an animal to determine the s p e c i f i c i t y of a given antibody popu l a t i o n , methods were discussed e a r l i e r which can be used to p r e d i c t the antibody s p e c i f i c i t y when a given hapten i s used. A n t i s e r a , which w i l l d e t e c t parent p e s t i c i d e p l u s t o x i c metabo l i t e s , could be used i n combination with one or more h i g h l y s p e c i f i c a n t i s e r a to q u a n t i t a t e s e v e r a l molecules of i n t e r e s t . An assay of moderate s p e c i f i c i t y w i l l be of g r e a t e r use i n some a n a l y t i c a l a p p l i c a t i o n s than a h i g h l y s p e c i f i c assay. The more general assay may be v e r y u s e f u l i n screening f o r the presence of a c l a s s of compounds or the presence of a s p e c i f i c f u n c t i o n a l i t y i n a m e t a b o l i t e . Such assays can be used v e r y e f f e c t i v e l y by coupling them with chromatographic techniques such as t h i n - l a y e r chromatography (TLC) or open column chromatography. The use of immunochemical t e s t s as s e n s i t i v e d e t e c t o r s f o r h i g h performance l i q u i d chromatography c e r t a i n l y o f f e r s promise i n p e s t i c i d e a n a l y s i s . A n i c e example of such a procedure i s the a n a l y s i s of Ν,Ν-dimethylindolealkylamines i n b i o l o g i c a l f l u i d s (80). Speed of A n a l y s i s . The speed with which many immunochemical analyses can be completed i l l u s t r a t e s a major advantage of immunochemical procedures. Immunochemical assays are most time and c o s t e f f e c t i v e when the sample load i s l a r g e . Parker (4) estimated that a s i n g l e t e c h n i c i a n could perform 100-5000 r a d i o immunoassays per day with l i t t l e or no assay automation i n com p a r i s o n to 20-40 GLC assays ( 3 ) . Numerous inexpensive systems are a v a i l a b l e to decrease a n a l y s i s time. These systems may i n c l u d e s o l i d phase s e p a r a t i o n techniques, automatic d i s p e n s e r s , t e s t tube racks which w i l l f i t d i r e c t l y into a c e n t r i f u g e and/or s c i n t i l l a t i o n counter, and data handling systems. Alternatively, there a r e f u l l y automated systems based on RIA or ELISA which r e q u i r e very l i t t l e operator a t t e n t i o n and which handle 25-240 samples/hr. Gochman and Bowie (81) have o u t l i n e d the b a s i s of o p e r a t i o n and summarized the f e a t u r e s of automated RIA systems and e x t e n s i v e l i t e r a t u r e i s a v a i l a b l e from the manufacturers. As with many a n a l y t i c a l procedures, the most time-consuming part of the assay i s sample p r e p a r a t i o n . The h i g h s p e c i f i c i t y and s e n s i t i v i t y of immunoassays may tremendously reduce the workup needed before a c t u a l a n a l y s i s of the sample. For example, a n a l y s i s of a l l e t h r i n i n m i l k by the accepted a n a l y t i c a l p r o cedure based on e l e c t r o n capture GLC required 4-8 hour per sample i n our hands (82). S i m i l a r s e n s i t i v i t y and higher s p e c i f i c i t y could be r e a l i z e d u s i n g an immunochemical assay r e q u i r i n g 15-30 minutes per sample. With some l o s s of s e n s i t i v i t y , immunoassays may be v e r y r a p i d . T u r b i d i t y measurements can be made so q u i c k l y and q u a n t i t a t i v e l y that they may be v e r y u s e f u l f o r f i e l d analyses of p e s t i c i d e s . Such r a p i d procedures might prove v e r y u s e f u l i n determining p e s t i c i d e coverage on s p e c i f i c areas of a plant immediately a f t e r a p p l i c a t i o n , d e t e c t i n g d r i f t , or moni t o r i n g the s a f e t y of a f i e l d f o r worker r e e n t r y . In t h i s chapter we have discussed the advantages of immuno-


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chemical methods as a supplement to more c l a s s i c a l a n a l y t i c a l techniques. P o s s i b l y among the most important c o n t r i b u t i o n s of immunochemistry to f u t u r e p e s t i c i d e a n a l y s i s w i l l be i t s use as a t o o l to open new areas of p e s t i c i d e a n a l y t i c a l chemistry. I f very r a p i d , inexpensive assays can be developed, p e s t i c i d e a n a l y s i s may be i n c r e a s i n g l y employed to enhance e f f e c t i v e p e s t i c i d e use r a t h e r than as simply an enforcement or r e s i d u e t o o l . Cost Ef f e c t i v e n e s s . As with the other advantages of immunochemical a n a l y s i s , cost may be q u i t e v a r i a b l e . Reagent c o s t s f o r s e v e r a l automated systems have been estimated at under $1.25 per sample. The cost i s o b v i o u s l y much lower f o r l e s s s o p h i s t i c a t e d assay systems, e s p e c i a l l y i f some reagents a r e prepared i n house. A major c o n s i d e r a t i o n i s the expense of new instrumentation. For dedicated or automated instrumentation f o r e i t h e r RIA or ELISA procedures, the cost may be $50-100,000. However, most a n a l y t i c a l l a b o r a t o r i e s a l r e a d y have the b a s i c instrumentation needed f o r immunoassays. Moderate s e n s i t i v i t y can be obtained through the use of numerous procedures such as r a d i a l immunodiffusion and hemagglutination. These procedures r e q u i r e no expensive equipment or reagents and they may be very u s e f u l i n areas where equipment a c q u i s i t i o n or maintenance i s a problem. The expense of an a n a l y t i c a l procedure depends upon much more than the cost of the f i n a l a n a l y s i s . Much of the expense of an assay i s r e l a t e d to sample p r e p a r a t i o n , and f o r many a p p l i c a t i o n s immunoassays have tremendously reduced the time needed f o r sample p r e p a r a t i o n . Another c o n s i d e r a t i o n i s the amount of time needed f o r the development of an assay. The a d d i t i o n a l e x p e r t i s e which must be developed i n an a n a l y t i c a l l a b o r a t o r y before immunoassays can be used with confidence may seem formidable, and w a i t i n g f o r an animal to develop a n t i b o d i e s may lead to unaccept a b l e delays i n assay development. On the other hand, once a usable antibody t i t e r i s obtained, the development of a workable assay i s u s u a l l y s t r a i g h t f o r w a r d . I t i s a l s o l i k e l y , i f immunoassays become accepted f o r some aspects of p e s t i c i d e a n a l y s i s , immunoassay k i t s or at l e a s t c r i t i c a l reagents w i l l become comm e r c i a l l y a v a i l a b l e . Such k i t s already e x i s t f o r many pharmac e u t i c a l products and hormones, and numerous companies w i l l supply a n t i b o d i e s to a user supplied hapten on a c o n t r a c t b a s i s (83)· A p p l i c a b i l i t y . Parker (4) p o i n t s out that one can assume that workable radioimmunoassays can be developed "with a l l except the s m a l l e s t or most u n s t a b l e molecules." Once a u s e f u l antibody t i t e r i s obtained, o f t e n only very small changes i n a g e n e r a l i z e d procedure are needed to o b t a i n a workable assay. Although immunoassays would appear to be g e n e r a l l y a p p l i c a b l e to p e s t i c i d e a n a l y t i c a l problems they may be most u s e f u l i n s o l v i n g s p e c i f i c problems which appear i n t r a c t a b l e when c l a s s i c a l procedures are used. Immunoassays a r e o f t e n most s e n s i t i v e and s p e c i f i c when


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s e v e r a l p o l a r f u n c t i o n a l i t i e s e x i s t . Such compounds may be r a t h e r n o n v o l a t i l e or heat l a b i l e and d i f f i c u l t to analyze by c l a s s i c a l methods. Although s e n s i t i v e , s p e c i f i c immunoassays have been developed f o r nonpolar compounds, such compounds may be most r e a d i l y analyzed by GLC procedures. For l a b o r a t o r i e s not i n t e r e s t e d i n the development of t h e i r own a n t i s e r a , each F a l l "Lab World (83) l i s t s s u p p l i e r s of immunochemical reagents.


11

Problems w i t h Immunoassays. As with any a n a l y t i c a l t e c h nique, there are numerous problems associated with the use of immunochemical technology. Most of these problems are common to any a n a l y t i c a l procedure, but some are r e l a t i v e l y unique to immunoassay and have been covered by Parker ( 4 ) . The parameters which should be monitored to maintain q u a l i t y c o n t r o l of the assay have been discussed by Rodbard et a l . (50). A major concern discussed e a r l i e r i s c r o s s r e a c t i v i t y or i n t e r f e r e n c e , e s p e c i a l l y i f i t i s unexpected. One can guard a g a i n s t t h i s problem by employing w e l l c h a r a c t e r i z e d antiserum, by u s i n g sample blanks, and by running standard curves i n the presence of e x t r a c t s . One must r e l y upon the equipment and reagents used i n a n a l y t i c a l procedures. A n t i b o d i e s are c e r t a i n l y not as s t a b l e as many chemical reagents; however, the guaranteed s h e l f l i f e of many commercial l y o p h i l i z e d p r e p a r a t i o n s i s over 5 years a t 4°C. The i n t e g r i t y of the reagents must be p e r i o d i c a l l y r e e s t a b l i s h e d , e s p e c i a l l y i f the assays are only performed s p o r a d i c a l l y . Immunoassays lend themselves to the p r o c e s s i n g of a l a r g e number of samples. The same number of c o n t r o l and standard assays a r e required whether one or a l a r g e number of samples a r e assayed. For an a n a l y t i c a l l a b o r a t o r y faced w i t h a n a l y z i n g a l a r g e number of samples f o r the presence of a few p e s t i c i d e s , immunochemical procedures are l i k e l y to o f f e r many advantages over some more c l a s i c a l a n a l y t i c a l methods. I f the same l a b o r a t o r y were faced with q u a n t i t a t i n g the r e s i d u e s of a l a r g e number of d i f f e r i n g chemicals i n a few samples, immunochemical p r o cedures a r e l i k e l y to be l e s s c o s t and time e f f i c i e n t than an e q u a l l y s e n s i t i v e GLC based assay. P o s s i b l e C o n t r i b u t i o n s of Immunochemical Methods to P e s t i c i d e A n a l y s i s . As E r c e g o v i c h (3) pointed out, i t i s unl i k e l y that immunochemical methods w i l l r e p l a c e c u r r e n t , establ i s h e d a n a l y t i c a l methods of p e s t i c i d e a n a l y s i s . However, the a n a l y t i c a l chemist who c a r e f u l l y compares the a t t r i b u t e s and d e f i c i e n c i e s of immunochemical methods of a n a l y s i s with other procedures i s l i k e l y to f i n d a p p l i c a t i o n s f o r which immunochemical methods o f f e r d i s t i n c t advantages. In many cases, those compounds which are most d i f f i c u l t to assay by c l a s s i c a l procedures because of numerous polar f u n c t i o n a l i t i e s and poor v o l a t i l i t y are the very compounds which lend themselves most r e a d i l y to immunochemical a n a l y s i s ( 4 ) . One can a l s o p r e d i c t that the number of p e s t i c i d e s marketed with a high

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degree of o p t i c a l p u r i t y w i l l i n c r e a s e , and immunochemical methods lend themselves to the a n a l y s i s of c h i r a l i t y at the r e s i d u e l e v e l (6,7). Thus, there w i l l probably be some p e s t i c i d e s f o r which immunochemical methods w i l l provide the f u t u r e enforcement procedures of c h o i c e f o r r e s i d u e a n a l y s i s . I t i s envisioned that immunochemical procedures can be more commonly used as a supplement to c l a s s i c a l methods of p e s t i c i d e a n a l y s i s . S i n c e samples can o f t e n be analyzed without expensive and time-consuming cleanup procedures u s u a l l y required of most methods, the immunological assays can r a p i d l y screen many samples at s i g n i f i c a n t l y lower c o s t . When the immunoassays i n d i c a t e that samples c o n t a i n a p p r e c i a b l e p e s t i c i d e r e s i d u e s , the samples can be f u r t h e r analyzed by GLC or other methods. A l t e r n a t i v e l y , an immunochemical assay may p r o v i d e a confirmatory t e s t . Specific a n t i s e r a a l s o can be used to concentrate p e s t i c i d e s and to c l e a n up e x t r a c t s by means of a f f i n i t y chromatography procedures, thereby, p e r m i t t i n g greater s e n s i t i v i t i e s of GLC, HPLC or other methods. I t i s expected that immunochemical and e s p e c i a l l y the ELISA procedures may c o n t r i b u t e to f i e l d r e e n t r y and human exposure problems where simple, r a p i d , inexpensive procedures a r e d e s i r e d . F i n a l l y , the p o s s i b l e usage of these methods i n developing c o u n t r i e s could be of p r a c t i c a l importance due to the s i m p l i c i t y of the procedures, the ease w i t h which they are i n t e r f a c e d w i t h t h i n - l a y e r a n a l y s i s , and the use of r e l a t i v e l y simple l a b o r a t o r y apparatus. Acknowledgements T h i s review i s dedicated to the memory of C. D. E r c e g o v i c h , who pioneered the a p p l i c a t i o n of immunochemical methods to p e s t i c i d e r e s i d u e a n a l y s i s . A. Karu (Dept. of Biochemistry, U n i v e r s i t y of C a l i f o r n i a , R i v e r s i d e ) provided advice on monoclonal antibody technology, and Siong Wie (UCR) c r i t i c a l l y reviewed the manuscript. The o r i g i n a l r e s e a r c h presented here was supported by EPA Grant R806447-01, NIEHS Grant 5 ROI ES01260-03 and the C a l i f o r n i a and Pennsylvania A g r i c u l t u r a l Experiment S t a t i o n s . B.D. Hammock was supported by NIEHS Research Career Development Award 1 K04 ES00046-01. T h i s i s a p u b l i c a t i o n from the Penns y l v a n i a A g r i c u l t u r a l Experiment S t a t i o n .


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