Immunochemical Methods for Environmental ... - ACS Publications

Chapter 12 An

Enzyme-linked

for

Residue

Immunosorbent

Detection 1

of

Assay

Methoprene

1

2

J. V. Mei , C.-M. Yin , and L. A. Carpino 1

2

Downloaded by UNIV OF ARIZONA on June 10, 2017 | http://pubs.acs.org Publication Date: November 11, 1989 | doi: 10.1021/bk-1989-0442.ch012

Department of Entomology andDepartmentof Chemistry, University of Massachusetts, Amherst, MA 01003

An enzyme-linked immunosorbent assay has been developed for the detection of low levels of methoprene from tobacco samples. The generation of anti-methoprene antibodies needed for such an assay r e l i e d on the preparation of a methoprene -carrier immunogen. Methoprene acid was covalently bound v i a an ester to a spacer group, which i n turn was bound to a protein c a r r i e r . Two activated ester methods were used to prepare the immunogen, one of which forms a water soluble, activated ester of methoprene. The resulting polyclonal antibodies raised against the methoprene immunogen were highly specific for methoprene, and did not cross react with closely related esters such as the insect juvenile hormones. The range of the methoprene ELISA was from 5 to 300 ng/mL, with an I of 50 ng/mL. 50

Through the study of the role of hormones i n the regulation of insect l i f e processes, insect hormone analogs have been developed and used i n insect control programs (1-2). Of special interest to entomologists are those hormones and their analogs that can regulate growth, metamorphosis and reproduction (2). Several types of insect growth regulators (IGRs) have been developed to date which are based on the structure of insect hormones. One of the most thoroughly studied and widely used IGRs i s methoprene (4), which mimics the action of juvenile hormone (JH) and disrupts insect metamorphosis. Methoprene (isopropyl 11-methoxy (Ε, E) 3,7,ll-trimethyl-2,4-dodecadienoate) (Figure 1, Structure 1) i s used in many commercial formulations for the control of a variety of pests including mosquitoes, f l i e s , ants, fleas, aphids and storedproduct pests. As the regulation of pesticides becomes 0097-6156790Λ)442-Ό140$06.00Α) © 1990 American Chemical Society Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

MEI ETAL.

ELISA for Residue Detection of Methoprene

141

ο > 0

^\

Methoprene

1 Downloaded by UNIV OF ARIZONA on June 10, 2017 | http://pubs.acs.org Publication Date: November 11, 1989 | doi: 10.1021/bk-1989-0442.ch012

Ο x

OMe

°"

Juvenile Hormone I (JHI)

2 o ΟΜβ

JHH

2 Ο

mm 4

JHm acid

,-4>^~^^^ .1 p OHl OHI

ΟΜβ

JHm diol

OH

F i g u r e 1. The s t r u c t u r e o f methoprene, t h e j u v e n i l e hormones and JH d e r i v a t i v e s .

Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


142

IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS

more r e s t r i c t i v e , and with the increased use of methoprene p r e d i c t e d f o r the f u t u r e (Edman, J.D.; C l a r k , J.M. Mosquito C o n t r o l Board. Massachusetts Department of Food and A g r i c u l t u r e , under review), a need has a r i s e n f o r the d e t e c t i o n of methoprene r e s i d u e s by an easy, economical, s e n s i t i v e and r e l i a b l e assay. Immunochemical techniques have proven t o be powerful t o o l s i n the a n a l y s i s of t r a c e amounts of o r g a n i c compounds i n a wide spectrum of b i o l o g i c a l and environmental matrices. They have been viewed as a t o o l t o supplement the conventional a n a l y t i c a l techniques of gas chromatography (GC) and h i g h pressure l i q u i d chromatography (HPLC). Although the use of immunoassay has been l a r g e l y l i m i t e d t o medical and biochemical a p p l i c a t i o n s , there has been i n c r e a s e d i n t e r e s t i n the use of immunochemical techniques f o r the d e t e c t i o n of environmental p o l l u t a n t s and p e s t i c i d e residues. Some of the advantages and shortcomings of immunochemical assays have been r e c e n t l y o u t l i n e d (56). One of the key steps i n the development of any immunochemical assay f o r a small organic compound, i s the p r o d u c t i o n of a n t i b o d i e s s p e c i f i c t o t h a t compound. Methoprene i s s t r u c t u r a l l y s i m i l a r t o the JHs (Figure 1, S t r u c t u r e s 2-4) and JH immunogens have been prepared by s e v e r a l groups of r e s e a r c h e r s . These s t u d i e s provided the b a s i s f o r the design and s y n t h e s i s of a methoprene immunogen (7-9). Design of the Methoprene Immunogen Both methoprene and JH are small molecules and by themselves w i l l not e l i c i t an immune response. They must be c o v a l e n t l y bound t o a l a r g e molecule, such as a p r o t e i n , which f a c i l i t a t e s p r e s e n t a t i o n of the small molecule t o a mammal's immune system. Such s m a l l molecules, when bound t o c a r r i e r s f o r immunological purposes, are known as haptens. Two methods of forming JH immunogens were developed. The f i r s t method was based on the h y d r o l y s i s of the methyl e s t e r of JH (Figure 1, S t r u c t u r e 4) t o the f r e e a c i d (7-8) (Figure 1, S t r u c t u r e 5). The a c i d was d i r e c t l y conjugated t o the p r o t e i n , human serum albumin, u s i n g the Nhydroxysuccinimide a c t i v e e s t e r . The r e s u l t i n g immunogen lacked the methyl e s t e r f u n c t i o n of the n a t i v e hormone. A n t i s e r a r a i s e d a g a i n s t t h i s JH a c i d immunogen recognized both JH and JH a c i d . A second method i n v o l v e d the h y d r o l y s i s of the JH epoxide (Figure 1, S t r u c t u r e 4) t o the corresponding d i o l (9) (Figure 1, S t r u c t u r e 6). The d i o l was conjugated t o human serum albumin v i a a s u c c i n y l group,



12. MEI ET A K

ELISA for Residue Detection ofMethoprene

which resulted i n loss of the epoxide feature of the n a t i v e hormone. The a n t i s e r u m r a i s e d a g a i n s t this immunogen r e c o g n i z e d o n l y t h e J H d i o l d e r i v a t i v e . From t h e above r e s u l t s , i t was h y p o t h e s i z e d t h a t i f the methoxy and e s t e r f u n c t i o n s o f methoprene are p r e s e r v e d i n t h e i m m u n o g e n , i t may i n d u c e t h e production of antibodies with higher s p e c i f i c i t y for methoprene. I n t h i s w o r k , a n immunogen f o r m e t h o p r e n e ( F i g u r e 4 , S t r u c t u r e 15) was d e v e l o p e d w h i c h m a i n t a i n e d i t s n a t i v e s t r u c t u r e by i n c o r p o r a t i n g a spacer group between the methoprene m o l e c u l e and the p r o t e i n carrier. A f o u r - c a r b o n s p a c e r g r o u p was l i n k e d t o m e t h o p r e n e as an e s t e r , w h i c h i n t u r n , was l i n k e d t o t h e p r o t e i n as an amide. Thus the methoxy f u n c t i o n remained untouched and the e s t e r f u n c t i o n remained intact. A d d i t i o n a l l y , work u s i n g liposomes t o c a r r y h a p t e n s i n t o a mammalian s y s t e m has shown t h a t a s p a c e r group g r e a t l y increases the immunogenicity of the hapten (10-11). The p a r e n t s p a c e r , 4 - h y d r o x y b u t a n o i c a c i d , spontaneously undergoes l o s s of water under a c i d i c or basic conditions to give 4-butyrolactone. To p r e v e n t c y c l i z a t i o n when t h e s p a c e r i s c o u p l e d t o methoprene, b o t h t h e h y d r o x y l and c a r b o x y l f u n c t i o n s had t o be p r o t e c t e d and s e l e c t i v e l y d e p r o t e c t e d . The methods used are summarized i n F i g u r e 2. The p r o t e c t i o n / d e p r o t e c t i o n c h e m i s t r y d e s c r i b e d was o r i g i n a l l y developed f o r use i n the f i e l d of p e p t i d e synthesis.

Immunogen

Synthesis

Incorporation of the Spacer Group. 4-Benzyloxybutanoic a c i d ( F i g u r e 3 , S t r u c t u r e 7) w a s p r e p a r e d ( 1 2 ) , u s i n g a b e n z y l e t h e r group as p r o t e c t i o n f o r the h y d r o x y l function. The p r o t e c t e d a c i d was c o u p l e d t o 2 t r i m e t h y l s i l y l e t h a n o l ( F i g u r e 3 , S t r u c t u r e 8) u s i n g d i c y c l o h e x y l c a r b o d i i m i d e ( D C C ) (13.) , t o g i v e c o m p o u n d 9 ( F i g u r e 3 ) , i n w h i c h t h e t r i m e t h y I s i l y l e t h y l (TMSE) group p r o t e c t s the c a r b o x y l i c a c i d f u n c t i o n . The b e n z y l g r o u p was n e x t removed u s i n g c a t a l y t i c h y d r o g é n a t i o n (15) t o p r o v i d e t h e p r o t e c t e d a l c o h o l ( F i g u r e 3, S t r u c t u r e 10). The T M S E - p r o t e c t e d a l c o h o l ( F i g u r e 3, Structure 10) was t h e n c o u p l e d t o m e t h o p r e n e a c i d ( F i g u r e 3 , S t r u c t u r e 11) u s i n g D C C . M e t h o p r e n e a c i d was o b t a i n e d by b a s e - c a t a l y s e d h y d r o l y s i s o f the i s o p r o p y l e s t e r ( F i g u r e 1, S t r u c t u r e 6 ) . The r e s u l t i n g protected m e t h o p r e n e d e r i v a t i v e ( F i g u r e 3 , S t r u c t u r e 12) was d e b l o c k e d v i a t e t r a e t h y l a m m o n i u m f l u o r i d e (13.) t o g i v e the desired methoprene-spacer a c i d (Figure 3, Structure 13). A c i d 13 ( F i g u r e 3) a n d a l l i n t e r m e d i a t e s were


143



F i g u r e 2. Diagramatic scheme f o r the p r o t e c t i o n / d e p r o t e c t i o n steps r e q u i r e d t o p l a c e a spacer group between a hapten and a c a r r i e r .



12.

MEIETAL.


145

11 F i g u r e 3. Synthesis of the p r o t e c t e d four-carbon spacer group ( S t r u c t u r e 9), followed by c o u p l i n g t o methoprene a c i d ( S t r u c t u r e 11) which y i e l d e d S t r u c t u r e 12. D e p r o t e c t i o n of compound 12 gave methoprene-spacer a c i d ( S t r u c t u r e 13).


146



f u l l y c h a r a c t e r i z e d using NMR and IR spectroscopy and elemental a n a l y s i s (15). Synthesis o f A c t i v a t e d E s t e r s . Haptens a r e f r e q u e n t l y conjugated t o c a r r i e r s by means o f a c t i v a t e d e s t e r s . An N-hydroxysuccinimide (NHS) e s t e r d e r i v a t i v e (Figure 4a, S t r u c t u r e 14) o f methoprene-spacer a c i d (Figure 4a, S t r u c t u r e 13) was prepared, f o l l o w i n g methods r e p o r t e d f o r t h e JHs (7^8). The a c t i v a t e d e s t e r (9.2 μπιοί) was allowed t o r e a c t with the p r o t e i n , human serum albumin (0.17 μιηοΐ) , i n organic-aqueous s o l u t i o n a t pH 8.5, which was lower than t h a t used f o r JH-NHS e s t e r - p r o t e i n conjugation r e a c t i o n s . Although t h e p r o t e i n was not f u l l y s o l u b l e under these c o n d i t i o n s , a r e a c t i o n time of 48 hours allowed f o r the i n t r o d u c t i o n o f 53 molecules o f methoprene p e r molecule o f p r o t e i n (Figure 4a, S t r u c t u r e 15), as c a l c u l a t e d by u s i n g a methoprene t r a c e r l a b e l e d with C a t carbon-5. The p r o t e i n conjugate was d i a l y s e d e x h a u s t i v e l y a g a i n s t both phosphate b u f f e r e d s a l i n e (0.05M PO^*^, 0.05M NaCl, pH 7.4) and d i s t i l l e d water, and l y o p h i l y z e d t o g i v e a f l u f f y white s o l i d (90-95 % recovery o f p r o t e i n conjugates). A water s o l u b l e a c t i v a t e d e s t e r of methoprene (Figure 5, S t r u c t u r e 16) was a l s o prepared from sodium l-hydroxy-2-nitro-4-benzene s u l f o n a t e (16). The amount of compund 16 i n crude preparations which contained both compound 16 and the f r e e d i a n i o n (Figure 5, S t r u c t u r e 17) was determined by spectrophotometry i n aqueous s o l u t i o n . Upon h y d r o l y s i s compound 16 y i e l d e d the d i a n i o n (Structure 17) which absorbed v i s i b l e l i g h t a t 406 nm i n t h e presence o f n u c l e o p h i l e s (Figure 5 ) . Two absorbance readings were r e q u i r e d t o determine t h e amount o f compound 16 present i n the crude m a t e r i a l . The f i r s t reading ( A ) measured the amount o f f r e e d i a n i o n i n t h e mixture. Hydroxide was then added t o completely hydrolyze compound 16 t o i t s a c i d (Figure 5, S t r u c t u r e 13) and the d i a n i o n , and a second reading was taken (A,Qg(NaOH)). T h i s determined the amount o f d i a n i o n l i b e r a t e d by the h y d r o l y s i s o f t h i s a c t i v a t e d e s t e r o f methoprene. The percent t o t a l e s t e r i n t h e crude mixture was c a l c u l a t e d u s i n g Equation 1. 1 4

4 0 6

A

4Q6

(NaOH) - A

4 0 6

χ A

406

100

=

Percent e s t e r i n mixture

(1)

(NaOH)

F i g u r e 5 shows t h e UV s p e c t r a before and a f t e r h y d r o l y s i s . T h i s p a r t i c u l a r sample was composed o f about 60% o f the a c t i v e e s t e r . In a s i m i l a r manner the a c y l a t i o n of t h e p r o t e i n c a r r i e r with the HNSA-methoprene a c t i v e e s t e r was


12. MEIETAL.


147


Ο

F i g u r e 4. P r e p a r a t i o n o f the methoprene immunogen ( S t r u c t u r e 15) by two methods: a. The NHS-ester o f methoprene (Structure 14) was conjugated t o human serum albumin (H N-HSA) i n organic/aqueous s o l u t i o n , b. A water s o l u b l e a c t i v e e s t e r o f methoprene ( S t r u c t u r e 16) was prepared by the DCC c o u p l i n g o f methoprene-spacer a c i d ( S t r u c t u r e 13) with l-hydroxy-2-nitro-4-benzene sulfonate. Reaction o f compound 16 with H2N-HSA was c a r r i e d out i n aqueous phosphate b u f f e r e d s a l i n e (PBS). 2

American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036 Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


148


' τ

100

>

1

200

•

1

'

300

1

400

•

1

500

1

w

τ

600

·

1

700

Wavelength (nm)

F i g u r e 5. H y d r o l y s i s r e a c t i o n o f HNSA-ester ( S t r u c t u r e 16) with base gave methoprene-spacer a c i d ( S t r u c t u r e 13) and t h e d i a n i o n ( S t r u c t u r e 17). The spectrum shows the absorbance o f the d i a n i o n before (•) and a f t e r hydrolysis (o).


12.

149


MEIETAL.


followed spectrophotometrically. The r e a c t i o n of e s t e r and p r o t e i n was c a r r i e d out i n aqueous b u f f e r s a t n e u t r a l pH (Figure 4b) due t o the high degree of s o l u b i l i t y of both r e a c t a n t s i n aqueous s o l u t i o n . Generation of the f r e e d i a n i o n was d i r e c t l y p r o p o r t i o n a l t o the amount of a c y l a t i o n of the p r o t e i n . The c a l c u l a t i o n of hapten d e n s i t y was determined by measuring d i a n i o n i n c r e a s e over time. Using t h i s method, and with g r e a t l y reduced r e a c t i o n times, an average of 31 molecules of methoprene were introduced per molecule of p r o t e i n (Table I ) . Hapten conjugates were d i a l y z e d and l y o p h i l i z e d as d e s c r i b e d above.

Table I. Number of methoprene molecules introduced per molecule of p r o t e i n u s i n g the HNSA a c t i v a t e d e s t e r

Reaction Time (min)

Crude Ester (mg)

15 30 180

10.52 8.11 8.50

Protein

(μπιοί)

(μπιοί)

21.86 16.86 17.66

0.072 0.064 0.068

Dianion Liberated

Residues Reacted

A

*406

0

0.750 0.579 0.607

0.837 0.640 0.656

C

1

4

34 32 27

° 31 28 28

a: Spectrophotometric absorbance reading taken a t 406 nm t o determine the amount of f r e e d i a n i o n i n the mixture a t time = 0 min. b: Second reading taken t o determine the amount of d i a n i o n l i b e r a t e d by r e a c t i o n of the a c t i v e e s t e r w i t h p r o t e i n a t the time i n d i c a t e d . c: The spectrophotometric method was used t o determine hapten d e n s i t y . d: 5-l C-methoprene was used t o determine hapten density. 4

The two a c t i v e e s t e r methods f o r hapten conjugation are compared i n Table I I . The NHS-ester method r e l i e s on a d j u s t i n g pH, s o l v e n t and time i n order t o o b t a i n maximum hapten d e n s i t y . The HNSA-ester method i s c a r r i e d out i n aqueous s o l u t i o n and i s monitored e a s i l y t o determine hapten d e n s i t y . Both a c t i v a t e d e s t e r methods produced s u i t a b l e immunogens.


d

I M M U N O C H E M I C A L METHODS F O R ENVIRONMENTAL ANALYSIS

150

Table I I . Comparison of hapten d e n s i t y by d i f f e r e n t a c t i v e e s t e r methods

Residues

Method

Trial


NHS-Ester

HNSA-Ester

Reaction Time

1 2 3 1 2

24 48 48 15 30

3

180

h h h min min min

Reacted

A

21 53 48 31 28 28

4 0 6

NA NA NA 34 32 27

NA: Not a p p l i c a b l e . NHS: N-hydroxysuccinimide. HNSA: l - H y d r o x y - 2 - n i t r o - 4 - b e n e z e n e s u l f o n a t e . See Table I for explanation of abbreviations.

ELISA Development Female, New Zealand white r a b b i t s were each immunized with an emulsion o f t h e methoprene immunogen ( 2 0 0 nq per animal, 5 3 molecules o f methoprene per molecule o f p r o t e i n ) i n 2 5 0 M L phosphate b u f f e r e d s a l i n e and 2 5 0 μ,Ι, Freund's complete adjuvant. A booster shot o f t h e immunogen ( 2 0 0 pq per animal) i n Freund's incomplete adjuvant was g i v e n t o each animal one month a f t e r t h e i n i t i a l immunization and again two weeks a f t e r t h e f i r s t booster shot. Rabbit anti-methoprene antiserum was c o l l e c t e d and used t o develop a competition enzymel i n k e d immunosorbent assay o r cELISA f o r methoprene. The competition assay was designed which f o l l o w e d the standard i n d i r e c t ELISA format ( 1 7 - 1 8 ) . The methoprene conjugate was bound t o a s o l i d support i n the form o f a m i c r o t i t e r p l a t e . Free methoprene i n methanol ( 5 liL) was added t o the pre-coated w e l l s f o l l o w e d by methoprene-specific antiserum. The a n t i b o d i e s were allowed t o compete f o r both immunogenbound and f r e e methoprene. Enzyme-conjugated, goata n t i r a b b i t antibody was added, f o l l o w e d by s u b s t r a t e , and t h e c o l o r was allowed t o develop. The absorbance of s u b s t r a t e over a range o f methoprene c o n c e n t r a t i o n s can be drawn as a standard curve, which i s presented as percent i n h i b i t i o n of t h e assay (Figure 6 ) . The 5 0 % inhibition (15η) methoprene was a t a c o n c e n t r a t i o n of approximately 5 0 ng/mL. Cross r e a c t i v i t y o f t h e methoprene antiserum was t e s t e d u s i n g JH I and JH I I I , and methoprene o

f


12.

MEIETAL.


intermediates used t o prepare the immunogen. JH I and JH I I I a t c o n c e n t r a t i o n s up t o 800 ng/mL d i d not c r o s s r e a c t w i t h the methoprene antiserum. By c o n t r a s t , methoprene a c i d and methoprene-spacer a c i d were good competitors f o r the antiserum (Figure 7 ) . A p p l i c a t i o n of the Methoprene cELISA


Methoprene r e s i d u e s are u s u a l l y determined by standard a n a l y t i c a l techniques such as HPLC (19-20) and GC (2122). These techniques, however, remain c o s t l y , time consuming, and r e q u i r e e x t e n s i v e sample clean-up and preparation. E x t r a c t i o n of Tobacco. Methoprene i s used on tobacco a g a i n s t the c i g a r e t t e b e e t l e and the tobacco moth. Methoprene-treated tobacco samples were e x t r a c t e d f o l l o w i n g a procedure f o r the e x t r a c t i o n of p l a n t m a t e r i a l s f o r determining methoprene r e s i d u e s by GC (21). Known amounts of methoprene i n 1 mL methanol were added t o 1 g p o r t i o n s of shredded tobacco, mixed w e l l and allowed t o thoroughly a i r dry. The s p i k e d tobacco was then s t i r r e d with a 25 mL mixture of a c e t o n i t r i l e / w a t e r / C e l i t e 45 (250 mL/30 mL/10 g ) . The mixture was f i l t e r e d by s u c t i o n and the f i l t e r cake was washed with a c e t o n i t r i l e / w a t e r . The f i l t r a t e was e x t r a c t e d with ether, d i s t i l l e d water, and sodium chloride. Ether e x t r a c t s were combined and washed t h r e e times with d i s t i l l e d water, d r i e d , f i l t e r e d and the s o l v e n t removed. The r e s i d u e was taken up i n t o methanol (1 mL) and a p p l i e d t o the pre-coated m i c r o t i t e r p l a t e s (5 μL methanol/well), f o l l o w e d by the anti-methoprene antibody as d e s c r i b e d above. Use of the cELISA t o determine methoprene content of tobacco r e s i d u e s prepared as d e s c r i b e d above, showed t h a t the e x t r a c t s contained m a t e r i a l s which i n t e r f e r e d with the assay (Figure 8 ) . E x t r a c t s of tobacco not only contained methoprene, but a l s o p l a n t substances t h a t i n t e r f e r e d with the assay. Thin-layer chromatography (TLC) was then used t o c l e a n up crude e x t r a c t s from tobacco samples t o reduce and/or eliminate interference. Tobacco r e s i d u e s were resuspended i n methanol (20 M L ) and a p p l i e d t o TLC p l a t e s (2 μΐ,/lane) along with a methoprene standard. The p l a t e s were developed with e t h y l acetate:dichloromethane:hexane (1:2:7). The lane c o n t a i n i n g the standard was cut from the p l a t e and developed i n i o d i n e . Regions c o n t a i n i n g methoprene ( r f = 0.46) were scraped from the other lanes (approx. 1 cm /lane), and a f t e r adding hexane (400 M L ) the s c r a p i n g s were vortexed (1 min) and c e n t r i f u g e d (8000 χ g, 15 min). The supernatants were removed t o c l e a n v i a l s , evaporated by a stream of a i r , and resuspended 2


151


152


Methoprene

(ng/mL)

F i g u r e 6. Percent i n h i b i t i o n o f the antiserum by methoprene. 50% i n h i b i t i o n o f t h e assay can be seen a t about 50 ng/mL. V e r t i c a l bars i n d i c a t e standard e r r o r (n=3 f o r each p o i n t ) . Β

Methoprene

0 I I I llllllf .1 1

I I I Tlliq I I I lllllj 1*1 I ÎIIH 10 100 1000

Inhlbitior

(ng/mL)

F i g u r e 7. Cross r e a c t i v i t y o f the methoprene antiserum with methoprene d e r i v a t i v e s and j u v e n i l e hormones. The JHs showed v i r t u a l l y no c r o s s r e a c t i o n with t h e antiserum. Due t o the complexity o f the f i g u r e , standard e r r o r bars were omitted (n=4 f o r each p o i n t ) .



12. MEIETAL.


153

i n methanol (1 mL) . A l i q u o t s (5 μΙ>) of the p u r i f i e d r e s i d u e were a p p l i e d t o the methoprene cELISA. F i g u r e 9 shows the curve f o r tobacco r e s i d u e s p u r i f i e d by TLC before a n a l y s i s by the assay. I t i s c l e a r t h a t the i n t e r f e r e n c e seen p r e v i o u s l y was p r a c t i c a l l y e l i m i n a t e d . However, such extensive sample p r e p a r a t i o n makes use of t h i s assay i n i t s present form cumbersome, a t best. We are p r e s e n t l y i n v e s t i g a t i n g an a l t e r n a t i v e form of immunoassay, the enzyme immunoassay (EIA) (f>, 21) · In t h i s assay methoprene i s conjugated d i r e c t l y t o an enzyme and the anti-methoprene antibody i s bound t o the s o l i d support. Free methoprene and methoprene-enzyme conjugate are i n s o l u t i o n and compete f o r immobilized antibody b i n d i n g s i t e s . Unbound methoprene i s washed from the assay p r i o r t o a d d i t i o n of s u b s t r a t e . P r e l i m i n a r y r e s u l t s under these c o n d i t i o n s i n d i c a t e t h a t tobacco e x t r a c t s of a c e t o n i t r i l e / w a t e r (9:1) do not r e q u i r e f u r t h e r p u r i f i c a t i o n steps p r i o r t o a p p l i c a t i o n t o the EIA. Summary The adaptation of immunochemical methods f o r many c l a s s e s of s m a l l compounds has made i t p o s s i b l e t o develop assays f o r p e s t i c i d e r e s i d u e d e t e c t i o n . A cELISA was developed f o r methoprene, an analog of i n s e c t j u v e n i l e hormone. Because of i t s s i z e , methoprene does not e l i c i t an immune response by itself. However, by conjugating methoprene t o a c a r r i e r p r o t e i n i t was made immunogenic i n animals. A four-carbon spacer group was i n c o r p o r a t e d between methoprene and the c a r r i e r p r o t e i n . The spacer was f i r s t coupled t o methoprene a c i d by a s e r i e s of p r o t e c t i o n / d e p r o t e c t i o n steps. Two d i f f e r e n t a c t i v a t e d e s t e r methods were used t o conjugate methoprene t o a p r o t e i n . Both the NHS-ester and the HNSA-ester methods produced immunogens of s u i t a b l e hapten d e n s i t y , however the HNSA-ester method can be c a r r i e d out i n aqueous s o l u t i o n and allows f o r the spectrophotometric determination of hapten d e n s i t y . Rabbit p o l y c l o n a l antiserum r a i s e d a g a i n s t the methoprene immunogen was used i n a cELISA t o d e t e c t f r e e methoprene i n the 5-300 ng/mL range. When methoprene was e x t r a c t e d from t r e a t e d tobacco samples, the crude e x t r a c t s contained m a t e r i a l s which i n t e r f e r e d with the cELISA. P u r i f i c a t i o n of tobacco r e s i d u e s by TLC was found t o s u f f i c i e n t l y e l i m i n a t e the interference. Acknowledgments T h i s r e s e a r c h was funded, i n p a r t , by the R.J. Reynolds Tobacco Company, c o n t r i b u t i o n no. 2970 from the Massachusetts Experiment S t a t i o n .



154


1

100

10

1

Inhibitor

1000

(ng/g)

F i g u r e 8. Methoprene treated-tobacco samples were e x t r a c t e d and the r e s u l t i n g r e s i d u e s were a p p l i e d d i r e c t l y t o the assay. The e x t r a c t s contained m a t e r i a l s which caused c o n s i d e r a b l e i n t e r f e r e n c e with the assay. The competition of methoprene, i n the absence of tobacco, was a l s o p l o t t e d f o r comparison. V e r t i c a l bars i n d i c a t e standard e r r o r (n=4 f o r each point).

Inhibitor

(ng/mL)

F i g u r e 9. P u r i f i c a t i o n of methoprene-treated tobacco r e s i d u e s by t h i n l a y e r chromatography reduced the i n t e r f e r e n c e seen i n F i g u r e 8. V e r t i c a l bars i n d i c a t e standard e r r o r (n=4 f o r each p o i n t ) .


12. MEI ET AL. Literature 1. 2. 3. 4.

5.


6.

7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

ELISA for Residue Detection of Methoprene Cited

Williams, C.M. Sci. Am. 1967, 217, 13-17. Bowers, W.S. E n t . Exp. Appl. 1982, 31, 3-14. Staal,G.B. Ent. Exp. Appl. 1982, 31, 15-33. Wakabayashi, N.; Waters, R.M. In Handbook of Natural Pesticides; Morgan, E.D.; Manduva, N.B. (Eds.); CRC Press: Boca Raton, FL, 1985; Vol. 3, pp. 87-151. Herman, B. W. In Immunological Techniques in Insect Biology; Gilbert, L.I.; Miller, T.A. (Eds.); Springer-Verlag, NY, 1988; Chapter 5. Nakamura, R.M., Voller Α., Bidwell, D.E. In Immunochemistry: Weir, D.M., Herzenberg, L.A., Blackwell, C. (Eds.); Blackwell Scientific Publications, Oxford, 1986; Chapter 27. Lauer, R.C.; Solomon, Ρ.Η.; Nakanishi, K; Erlanger, B.F. Experientia. 1974, 30, 558-560. Baehr, J.C.; Pradelles, P.; Lebreux, C.; Cassier, P.; Dray, F. FEBS Lett. 1976, 69, 123-128. Strambi, C.; Strambi, A; De Reggi, M.L.; Hirn, M.H.; Delaage, M.A. Eur. J. Biochem. 1981, 118. 401-406. Kinsky, S.C.; Hashimoto, K.; Loader, J.E.; Benson, A. L. Biochim. Biophys. Acta. 1984, 769, 543-550. Scott, D.; Nitecki, D.E.; Kindler, H.; Goodman, J.W. Mol. Immunol. 1984, 21, 1055-1060. Sudo, R.; Kaneda, A.; Itoh, N. J. Org. Chem. 1967, 32, 1844-1846. Sieber, P.; Andreatta, R.H.; Eisler, K.; Kamber, B.; Riniker, B.; Rink, H. In Peptides; Goodman, M.; Meienhofer, J., Eds.; Proceedings of the 5th Peptide Symposium: John Wiley and Sons, Inc.: New York, 1977; p. 543. van Duzee, E.M.; Adkins, H. J. Am. Chem. Soc. 1935, 57, 147-151. Mei, J.V. Masters of Science Thesis, University of Massachusetts, Amherst, MA, 1988. Aldwin,L.; Nitecki, D.E. Anal. Biochem. 1987, 164, 494-501. Voller, Α.; Bidwell, D.E.; Bartlett, A. The Enzyme Linked Immunosorbent Assay (ELISA); Dynatech Laboratories, Inc.: Alexandria, VA, 1979; p.35. DeToma, F.J.; MacDonald, B.A. Experimental Immunology: A Guidebook; MacMillan Publishing Co.: New York, 1987; p. 121. Chamberlain, S.J. Analyst. 1985, 110, 879-880. Heckman, R.A., Conner T.R. LC-GC. 1989, 7, 855-856. Wright, J.E.; Jones, R.L. Bull. Environ. Contam. Toxicol. 1976, 15, 525-529. Miller, W.W.; Wilkins, J.S.; Dunham, L.L. J. AOAC. 1975, 58, 10-14. Kurstak, E. In Enzyme Immunodiagnosis: Academic Press: New York, 1986. Chapters 3 and 4.

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