11 Analysis of Pesticide Residues: Immunological Techniques C. D .
ERCEGOVICH
Department of Entomology and Pesticide Research Laboratory, Pennsylvania State University, University Park, P a . 16802
Past investigations
which
on immunological degradation
DDT,
are limited
groups.
in developing
Two
antisera
logical
clearly
or fortified
demonstrate
methods
methods for
couraged.
were
methods
are
were
aminotriazole, detected
screening
and by
immuno-
of as
and
confirmatory
and
disadvantages
analysis
their
results
immunological
The development analysis
None
for the
however,
usefulness
of
several
of investigators.
samples;
pesticide
successful
a metabolite
of such methods
pesticides.
advantages
pesticides
DDA,
readily
residue
for
rapid The
for
a potential
methods for detecting nological
their sepa-
by the third group
had shown the practicality of actual
and
Parathion,
products
methods
conducted
to those of only three
of these groups
specific
and malathion.
degradation
to have been
methods for analyzing
products
rate research
are known
of
immu-
supplemental test is of
ensuch
discussed.
T n k e e p i n g w i t h the t h e m e of this v o l u m e , i d e n t i f i c a t i o n of pesticides at the r e s i d u e ( s u b m i c r o g r a m ) l e v e l , t h e questions of w h a t a p p l i c a t i o n s of i m m u n o l o g i c a l t e c h n i q u e s h a v e b e e n u s e d i n the past, a n d w h a t p o t e n t i a l usefulness d o these t e c h n i q u e s h a v e for these purposes i n t h e f u t u r e are c o n s i d e r e d . T h e t o p i c considers b o t h i m m u n o l o g i c a l a n d s e r o l o g i c a l t e c h n i q u e s . I m m u n o l o g y , i n a r e s t r i c t e d sense, deals w i t h t h e p r o c e d u r e s u s e d a n d the m e c h a n i s m s i n v o l v e d w h e r e b y a host establishes resistance to a disease ( i m m u n e state) after a specific exposure to a f o r e i g n i n f e c tious agent ( a n t i g e n ) .
I n a b r o a d e r sense, i m m u n o l o g y is c o n c e r n e d
w i t h h y p e r s e n s i t i v e b i o l o g i c a l p h e n o m e n a of a l t e r e d tissue r e a c t i v i t y s u c h as allergies, a c q u i r e d tolerances to a n d rejection of f o r e i g n tissue, a n d a u t o i m m u n e diseases.
S e r o l o g y is a b r a n c h of b i o l o g i c a l science 162
con-
11.
ERCEGOVICH
Immunological
163
Techniques
c e r n e d w i t h the d i a g n o s t i c a n d e x p e r i m e n t a l p r o c e d u r e s b y w h i c h a n t i g e n a n t i b o d y reactions c a n b e s t u d i e d .
I t is so n a m e d b e c a u s e
serology
i n v o l v e s the use of s e r u m . S i n c e m a n y i m m u n e reactions are s t u d i e d b y s e r o l o g i c a l t e c h n i q u e s , i t is p e r m i s s i b l e to refer to t h e t o p i c m e r e l y as i m m u n o l o g i c a l techniques. T h e average p e s t i c i d e r e s i d u e c h e m i s t o r toxicologist m a y h a v e n o o r o n l y a s u p e r f i c i a l k n o w l e d g e of i m m u n o l o g y a n d serology. his f a m i l i a r i t y w i t h these sciences is l i m i t e d to a s o p h o m o r i c
Perhaps knowledge
of t h e i r r e l a t i o n to i m m u n i z a t i o n i n disease p r e v e n t i o n , diagnosis
for
allergies, b l o o d t y p i n g , a n d disease resistance. C e r t a i n l y these are a m o n g t h e p r i m a r y concerns of i m m u n o l o g y a n d serology, a n d m o r e
recently
the i m p o r t a n c e of i m m u n i z a t i o n as i t a p p l i e s to the r e j e c t i o n of o r g a n transplants has g i v e n s p e c i a l i m p o r t a n c e to these
fields.
Because
these
sciences are h i g h l y s p e c i a l i z e d , i t m a y b e w o r t h w h i l e to r e v i e w b r i e f l y some of t h e i r u n d e r l y i n g p r i n c i p l e s to assist better the p e s t i c i d e researcher to g a i n a n u n d e r s t a n d i n g a n d a p p r e c i a t i o n of h o w these t e c h n i q u e s m a y a p p l y to his interests. S i n c e o n l y a l i m i t e d d i s c u s s i o n of b a s i c p r i n c i p l e s and methodology
c a n b e p r e s e n t e d here, the r e a d e r is r e f e r r e d to ex-
cellent monographs W e i s e r et ah (4)
b y B u r r o w s et ah
(I),
D a y (2),
Gary (3),
f o r the g e n e r a l p r i n c i p l e s of i m m u n o l o g y ,
c h e m i s t r y , a n d i m m u n e reactions, L a n d s t e i n e r ( 5 ) , Pressman a n d Grossberg (7)
Nezlin
and
immuno(6),
and
for a better u n d e r s t a n d i n g a b o u t t h e b i o -
c h e m i s t r y a n d specificity of a n t i b o d i e s , a n d C a m p b e l l et ah ( 8 ) , K a b a t and Mayer (9), C h a s e (12)
Kwapinski
(10),
Nowotny
(11),
and Williams and
for specific a n d d e t a i l e d i n f o r m a t i o n a b o u t
methodology.
General Principles E s s e n t i a l l y , w h e n a f o r e i g n p r o t e i n , a n t i g e n , is i n j e c t e d i n t o a s u i t a b l e a n i m a l , t h e o r g a n i s m responds b y p r o d u c i n g a specific p r o t e i n c a l l e d a n a n t i b o d y . T h e i m m u n o l o g i c a l response of a n a n i m a l to t h e i n i t i a l i n j e c t i o n of a n a n t i g e n i c substance is not i m m e d i a t e , b u t after a s u i t a b l e i n c u b a t i o n p e r i o d the p r o p e r t i e s of t h e b l o o d s e r u m i n respect to the a n t i g e n are a l t e r e d . T h i s effect is d e m o n s t r a t e d b y the p r o d u c t i o n of a n i m m u n e s e r u m , a n t i s e r u m , w h i c h differs f r o m n o r m a l s e r u m i n t h a t i t reacts e i t h e r in vivo or in vitro w i t h the h o m o l o g o u s a n t i g e n . T h i s p r o p erty of a n t i s e r u m is the result of the presence of a n t i b o d i e s ,
substances
w h i c h are f o r m e d b y the o r g a n i s m i n response to t h e i n j e c t e d a n t i g e n . T h e a n t i b o d y is f o u n d i n the largest a m o u n t a n d most c o n v e n i e n t f o r m i n the b l o o d s e r u m a n d is i n t i m a t e l y associated w i t h the s e r u m p r o t e i n . A n t i s e r u m m a y c o n t a i n f r o m 1 to 5 or e v e n m o r e , m g p e r m l of a n t i b o d y protein.
T h e a n t i b o d y a c t i v i t y g e n e r a l l y is l o c a l i z e d i n t h e g l o b u l i n
f r a c t i o n of the s e r u m p r o t e i n . T h e i m m u n e g l o b u l i n , h o w e v e r , is i n d i s t i n g u i s h a b l e f r o m n o r m a l s e r u m g l o b u l i n b y c h e m i c a l analysis. A n t i b o d y
164
PESTICIDES
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a c t i v i t y of a n t i s e r u m m a y b e d e s t r o y e d b y d e n a t u r a t i o n w i t h heat, a l c o h o l , o r u r e a a n d b y d e g r a d a t i o n w i t h s t r o n g a c i d or a l k a l i . I m m u n e g l o b u l i n reacts w i t h substances s u c h as
1 3 1
I or fluorescent d y e s to l a b e l t h e a n t i b o d y
f o r v a r i o u s e x p e r i m e n t a l purposes w i t h o u t loss of a c t i v i t y . A l t h o u g h the s t i m u l a t i o n of a n t i b o d y p r o d u c t i o n is u s u a l l y o b s e r v e d w i t h p r o t e i n s , c e r t a i n p o l y s a c c h a r i d e s , s u c h as the c a p s u l a r c o m p o n e n t s of p n e u m o c o c c i a n d other m i c r o o r g a n i s m s , are also a n t i g e n i c . T h e a n t i genic
property
of
substances
is v a r i a b l e i n t h a t some antigens
give
m a r k e d i m m u n e response w h i l e others s t i m u l a t e o n l y a l o w g r a d e i m m u n e response as d e m o n s t r a t e d b y the s m a l l a m o u n t of a n t i b o d y p r o d u c t i o n . Substances s u c h as o v a l b u m i n , s e r u m g l o b u l i n , d i p h t h e r i a t o x i n , a n d tetanus toxoids are examples of g o o d antigens w h i l e
hemoglobin,
n u c l e o p r o t e i n s , a n d histones are p o o r antigens. G o o d antigens are g e n e r a l l y n a t u r a l l y o c c u r r i n g substances of r e l a t i v e l y l a r g e m o l e c u l a r size, at least p a r t i a l l y d i g e s t i b l e b y
enzymes,
a n d are f o r e i g n o r
contain
structures w h i c h are f o r e i g n to t h e a n t i b o d y - p r o d u c i n g a n i m a l . T h e n a t u r a l l y o c c u r r i n g proteins w h i c h are g o o d antigens u s u a l l y c o n t a i n a f u l l c o m p l e m e n t of a m i n o acids. A n u m b e r of d e r i v e d p r o t e i n s , n o t a b l y g e l a t i n , are n o t a n t i g e n i c , or o n l y f e e b l y a n t i g e n i c , e v e n t h o u g h they may be of h i g h molecular weight.
T h e p o o r a n t i g e n i c p r o p e r t y of
g e l a t i n is t h o u g h t to b e c a u s e d b y its deficiency i n a r o m a t i c a m i n o a c i d s , t h o u g h t h e r e is l i t t l e d e f i n i t i v e e v i d e n c e f o r this belief.
Large molecular
size i n v a r i a b l y a c c o m p a n i e s the a b i l i t y to s t i m u l a t e a n t i b o d y f o r m a t i o n , t h o u g h a l l l a r g e m o l e c u l e s are not a n t i g e n i c . T h e r e is some c o r r e l a t i o n b e t w e e n r e l a t i v e a n t i g e n i c i t y a n d m o l e c u l a r w e i g h t since n a t u r a l l y occ u r r i n g substances h a v i n g m o l e c u l a r w e i g h t s of less t h a n a b o u t
40,000,
e.g. l y s o z y m e , p r o t a m i n e s , histones, a n d i n s u l i n , are p o o r antigens. sistent w i t h this i n f o r m a t i o n t h e a n t i g e n i c i t y of a p r o t e i n
Con-
disappears
r a p i d l y u p o n e n z y m a t i c h y d r o l y s i s , a n d proteoses a n d p o l y p e p t i d e s
are
n o t a n t i g e n i c . I t is p o s s i b l e t h a t h i g h m o l e c u l a r w e i g h t s y n t h e t i c p o l y m e r s are a n t i g e n i c , b u t w i t h t h e e x c e p t i o n of p o l y v i n y l p y r r o l i d o n e n o n e are f o u n d to b e a n t i g e n i c . A p r e r e q u i s i t e for a n t i g e n i c i t y is t h a t the a n t i g e n b e f o r e i g n a n d d i s t i n g u i s h a b l e as "not-self" b y the a n t i b o d y - p r o d u c i n g organisms.
The
f o r e i g n q u a l i t y of antigens is of v a r y i n g d e g r e e a n d is reflected i n t h e specificity of t h e a n t i g e n - a n t i b o d y r e a c t i o n .
W i d e l y different
antigens
a p p e a r to b e q u i t e u n r e l a t e d i m m u n o l o g i c a l l y w h i l e closely r e l a t e d a n t i gens s h o w cross-reaction of v a r y i n g degree w i t h a p p r o p r i a t e heterologous a n t i b o d y a n d b e h a v e as p o o r antigens w h e n t h e y are closely r e l a t e d to c o n s t i t u e n t antigens of the i m m u n i z e d a n i m a l . A n t i g e n i c
substances,
therefore, f r o m one species of a n i m a l s t i m u l a t e a n t i b o d y f o r m a t i o n i n a n o t h e r a n i m a l species, a n d antigens of p l a n t o r i g i n are a n t i g e n i c i n animals.
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165
Techniques
T h i s specificity is r e a d i l y i l l u s t r a t e d b y t h e f o l l o w i n g e x a m p l e .
Serum
p r o t e i n s , w h i c h are a m o n g t h e most c o m m o n l y u s e d antigens, f r o m sheep, horse, c h i c k e n , m a n , etc., b u t n o t h o m o l o g o u s r a b b i t s e r u m , are a n t i g e n i c i n r a b b i t . S i m i l a r l y t h e s e r u m p r o t e i n f r o m r a b b i t , sheep, horse a n d m a n , b u t not the h o m o l o g o u s c h i c k e n s e r u m p r o t e i n , are a n t i g e n i c i n c h i c k e n . S p e c i f i c i t y is also expressed
i n the r e a c t i o n o f a n t i g e n w i t h
antibody.
F o r instance, a n t i s e r u m to horse s e r u m p r o t e i n p r e p a r e d i n the r a b b i t w i l l r e a c t in vitro o n l y w i t h the h o m o l o g o u s horse s e r u m p r o t e i n a n t i g e n a n d w i l l n o t react w i t h s e r u m p r o t e i n f r o m c h i c k e n , cattle, m a n , r a b b i t , etc.
S p e c i f i c i t y of this n a t u r e is r e f e r r e d t o as species specificity.
the other h a n d , specificity is less s h a r p b e t w e e n r e l a t e d sources.
antigens f r o m
On
closely
T h e a n t i s e r u m to c h i c k e n s e r u m p r o t e i n w i l l also react
w i t h p i g e o n s e r u m p r o t e i n ; a n t i s e r u m to sheep g l o b u l i n w i l l react w i t h beef s e r u m g l o b u l i n , etc., b u t the heterologous r e a c t i o n is w e a k e r t h a n t h a t of the h o m o l o g o u s a n t i g e n . E x p e r i m e n t a l e v i d e n c e e s t a b l i s h e d t h a t the specificity of is d e t e r m i n e d b y t h e i r c h e m i c a l c o m p o s i t i o n .
antigens
Experiments w i t h a variety
of a n t i g e n i c proteins s h o w e d that i m m u n o l o g i c a l l y i d e n t i c a l proteins are, as f a r as c a n b e d e t e r m i n e d , i d e n t i c a l i n c o m p o s i t i o n . differing
from
one
another
i n composition
Antigenic proteins
are also
immunologically
d i s t i n c t w h i l e antigens s h o w i n g some degree of cross-reaction are closely r e l a t e d i n c h e m i c a l structure. T h r o u g h the s t u d y of a l t e r e d specificity and
a r t i f i c i a l antigens
e v i d e n c e has b e e n g a i n e d that
specificity is a p r o p e r t y of m o l e c u l a r c o n f i g u r a t i o n .
immunological
Antigenic protein
m a y b e h e a t e d , p a r t i a l l y d e n a t u r e d , or t r e a t e d w i t h f o r m a l d e h y d e i n s u c h a w a y that p a r t of the o r i g i n a l specificity is lost, b u t species specificity remains, although
somewhat
broadened.
Treatment
of
protein
with
i o d i n e , n i t r i c a c i d , o r n i t r o u s a c i d alters t h e specificity of the a n t i g e n so p r o f o u n d l y that species specificity is d e s t r o y e d . L a n d s t e i n e r a n d colleagues
(5)
h a v e s h o w n that species specificity
of antigens m a y be a l t e r e d i n w a y s other t h a n b y a t t a c k i n g the a r o m a t i c moieties of p r o t e i n as w i t h i o d i n e a n d acids.
T h e a d d i t i o n of
small
r a d i c a l s , a c e t y l , e t h y l , or m e t h y l , to large p r o t e i n molecules b y a c e t y l a t i o n w i t h acetic a n h y d r i d e , esterification w i t h e t h y l a l c o h o l , or m e t h y l a t i o n w i t h d i a z o m e t h a n e , r e s p e c t i v e l y , results i n p r o n o u n c e d changes i n s p e c i ficity
of the o r i g i n a l p r o t e i n m o l e c u l e .
The immunological
properties
of e g g a l b u m i n h a v e b e e n a l t e r e d t h r o u g h p h o s p h o r y l a t i o n . U p to this p o i n t w e h a v e b e e n c o n c e r n e d
primarily with
so-called
c o m p l e t e antigens, substances w h i c h b o t h s t i m u l a t e a n t i b o d y p r o d u c t i o n a n d react w i t h the a n t i b o d y so f o r m e d .
O t h e r substances, h o w e v e r , h a v e
a m o r e l i m i t e d antigenicity—e.g., w h i l e t h e y react specifically w i t h a n t i b o d y , t h e y are u n a b l e to s t i m u l a t e a n t i b o d y f o r m a t i o n . T h e s e
substances
are p a r t i a l antigens o r haptens. T h e use of i m m u n o l o g i c a l a n d serological
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t e c h n i q u e s to detect a n d a n a l y z e pesticides d e p e n d s o n the u n d e r s t a n d i n g of haptens. H a p t e n s are s u b d i v i d e d i n t o t w o groups b y some w o r k ers. O n e of these is m a d e u p of haptens w h i c h react w i t h a n t i b o d y in vitro
to g i v e the u s u a l s e r o l o g i c a l r e a c t i o n s ; the other g r o u p
includes
those substances w h i c h react w i t h a n t i b o d y , b u t w i t h o u t overt e v i d e n c e of t h e r e a c t i o n , a n d the a n t i g e n - a n t i b o d y r e a c t i o n is d e m o n s t r a b l e i n d i r e c t l y as a n interference o r i n h i b i t i o n p h e n o m e n o n .
only
T h e latter is a
c o n s e q u e n c e of the s t r u c t u r e of the h a p t e n a n d is not r e l a t e d t o its a n t i g e n i c specificity.
T h e d e f i n i t i o n of a h a p t e n has v a r i e d since the
w o r d w a s first u s e d b y L a n d s t e i n e r i n 1921 to refer to s i m p l e o r g a n i c residues t h a t react specifically w i t h a n t i b o d i e s .
H o w e v e r , none describe
w h a t is r e a l l y m e a n t as w e l l as the o r i g i n a l G r e e k m e a n i n g , to t o u c h , to grasp, a n d to fasten ( 2 ) .
B y simple immunobiological definition hap-
tens are d e s c r i b e d as p a r t i a l antigens w h i c h i n themselves are i n c a p a b l e of i n d u c i n g a n t i b o d y f o r m a t i o n i n a n i m a l s , b u t w h i c h , w h e n to o r d i n a r y i m m u n o g e n s
s u c h as proteins a n d p o l y s a c c h a r i d e s ,
a n t i b o d y f o r m a t i o n against themselves.
attached induce
O t h e r s u b s t a n c e s — s u c h as c o l -
l o d i o n p a r t i c l e s , sephadex, r e d b l o o d cells, a n d c h a r c o a l — h a v e b e e n u s e d as t h e c a r r i e r m a t e r i a l for haptens.
H a p t e n s r a n g e i n size f r o m s m a l l
o r g a n i c c o m p o u n d s s u c h as p - a m i n o b e n z o i c a c i d (5, 13) c h a i n s (14,
to p o l y p e p t i d e
15).
T h e influence of the a d d i t i o n of r e l a t i v e l y s i m p l e o r g a n i c r a d i c a l s o n the i m m u n o l o g i c a l specificity of a n t i g e n i c proteins has b e e n e x p l a i n e d b y the s t u d y of the i m m u n o l o g i c a l b e h a v i o r of a v a r i e t y of c o m p o u n d s p r e p a r e d f r o m p r o t e i n a n d t h e d i a z o n i u m d e r i v a t i v e s of a n u m b e r
of
m e t h y l , c h l o r o , b r o m o , a n d n i t r o s u b s t i t u t i o n p r o d u c t s of a n i l i n e , o-,
m-,
a n d p - a m i n o b e n z e n e s u l f o n i c a c i d , p - a m i n o p h e n y l a r s e n i c a c i d , a n d o-,
m-,
a n d p - a m i n o c i n n a m i c a c i d as w e l l are the p a r e n t c o m p o u n d
13).
(16, 5,
F r o m studies of this n a t u r e i t b e c a m e a p p a r e n t t h a t t h e i m m u n o l o g i c a l b e h a v i o r of antigens c a n b e m o d i f i e d specifically b y a l t e r i n g a r e l a t i v e l y s m a l l p a r t of the l a r g e p r o t e i n m o l e c u l e a n d t h a t the specificity of the a n t i g e n is d e t e r m i n e d b y t h e c h e m i c a l s t r u c t u r e of this a d d e d p a r t .
The
s p a t i a l a r r a n g e m e n t i n the d e t e r m i n a t i v e groups, as w e l l as t h e i r n a t u r e , is reflected i n i m m u n o l o g i c a l b e h a v i o r .
T h e p o s i t i o n of o r t h o , m e t a , o r
p a r a substitutions i n a r o m a t i c r a d i c a l s a t t a c h e d to proteins p r o d u c e s d i f ferences i n specificity. T h e stereo isomers of t a r t a r i c a c i d a n d p - a m i n o b e n z o y l p h e n y l a c e t i c a c i d y i e l d i m m u n o l o g i c a l l y d i s t i n c t antigens w h e n c o u p l e d w i t h p r o t e i n , a n d the p o s i t i o n of a m i n o acids i n p e p t i d e - a z o p r o teins is a factor i n d e t e r m i n i n g i m m u n o l o g i c a l specificity. T h e i m m u n o l o g i c a l f u n c t i o n s of the haptens n o w are c l e a r i n t h e i r r o l e as d e t e r m i n a n t s of i m m u n o l o g i c a l specificity.
T h e relative impor-
t a n c e of h a p t e n - d e t e r m i n e d specificity is v e r y great since a l a r g e p o r t i o n of n a t u r a l l y o c c u r r i n g a n t i g e n i c substances are c o n j u g a t e d antigens c o n -
11.
ERCEGOVICH
Immunological
167
Techniques
sisting of a p r o t e i n a n d a h a p t e n . T h i s is i l l u s t r a t e d f o r some p n e u m o coccus w h o s e p o l y s a c c h a r i d e c a p s u l a r substance acts as a h a p t e n a n d p r o v i d e s the a n t i g e n i c specificity r e q u i r e d to p r o d u c e the i n j e c t e d m i c r o o r g a n i s m s .
a n i m m u n i t y to
I m m u n e response, o f t e n m a n i f e s t e d as a
h y p e r s e n s i t i v i t y , to l o w m o l e c u l a r w e i g h t substances s u c h as d r u g s , dyes, c u t t i n g oils, a n d s i m i l a r substances r e s p o n s i b l e for o c c u p a t i o n a l d e r m a titis, o c c u r f r e q u e n t l y .
It is p r o b a b l e t h a t these
low-molecular-weight
substances f u n c t i o n as a h a p t e n b y c o m b i n i n g w i t h the host's p r o t e i n , thus a l t e r i n g t h e i r specificity so t h a t t h e y b e c o m e f o r e i g n , substances w h i c h p r o v o k e i m m u n e responses.
antigenic
The antibody formed re-
acts specifically w i t h the h a p t e n i c substance p r o d u c i n g a l l e r g i c reactions. S e n s i t i z a t i o n to p e n i c i l l i n , w h i c h is r e l a t i v e l y often
observed,
is
an
excellent e x a m p l e of this p h e n o m e n o n .
Past Attempted Uses of Immunological Techniques for Pesticide Analysis I m m u n o l o g i c a l t e c h n i q u e s h a v e b e e n u s e d e x t e n s i v e l y i n the of m e d i c i n e to s t u d y the causes a n d t r e a t m e n t of diseases.
field
The protein
a n d e n z y m e c h e m i s t has also u s e d i m m u n o l o g i c a l m e t h o d s to g o o d a d vantage to isolate, p u r i f y , a n d s t u d y proteins. I n a g r i c u l t u r e s u c h m e t h ods h a v e b e e n u s e d b y the v i r o l o g i s t w h o uses a n t i g e n - a n t i b o d y reactions to diagnose p l a n t diseases.
T h e v e t e r i n a r i a n uses these t e c h n i q u e s
to
diagnose a n i m a l diseases a n d to i d e n t i f y p a t h o g e n i c b a c t e r i a a n d viruses. E n t o m o l o g i s t s h a v e u s e d i m m u n o l o g i c a l m e t h o d s to s t u d y the v e n o m of insects a n d to d e v e l o p w a y s of i m m u n i z i n g against s u c h substances. h a v e also u s e d i m m u n o s p e c i f i c i t y as a c r i t e r i o n to s t u d y t h e
They
differences
i n v a r i o u s insect e n z y m e s — e . g . , a c e t y l cholinesterase a n d D D T - d e h y d r o c h l o r i n a s e — t o u n d e r s t a n d the m o d e of a c t i o n of insecticides a n d t h e m e c h a n i s m of insect resistance to insecticides. A t t e m p t s to u t i l i z e the specificity of a n t i g e n - a n t i b o d y r e a c t i o n f o r the analysis of pesticides a p p e a r to h a v e b e e n l i m i t e d to w o r k of C e n t e n o et al. (17)
a n d H a a s a n d G u a r d i a (18).
T h e former group postulated,
because of observations of a l l e r g i c contact s e n s i t i v i t y to m a l a t h i o n a n d scattered reports a b o u t u n u s u a l reactions to D D T , t h a t c o m m o n pesticides m i g h t act as a n t i g e n i c a n d a l l e r g i c d e t e r m i n a n t s , p r o v i d e d t h e y b e c o m e c o u p l e d to a n a p p r o p r i a t e p r o t e i n c a r r i e r . T o test this postulate, t h e y s t u d i e d the i m m u n o g e n i c i t y
of
p r o t e i n conjugates
of
closely
related
d e r i v a t i v e s of D D T a n d m a l a t h i o n , the m e t a b o l i t e D D A , [2,2-bis( p - c h l o r o p h e n y l ) acetic a c i d ] , a n d [ 0 , 0 - d i m e t h y l S - ( l , 2 - b i s c a r b o x y e t h y l ) phorodithioate].
phos-
T h e i r results w e r e h i g h l y significant b e c a u s e t h e y w e r e
a b l e to p r o d u c e a n t i b o d i e s i n r a b b i t s to the a b o v e h a p t e n s w h e n
con-
j u g a t e d to b o v i n e s e r u m a l b u m i n ( B S A ) , D D A - B S A , a n d m a l a t h i o n - B S A , respectively.
168
PESTICIDES
IDENTIFICATION
A method for ascertaining the antibody titer of the respective a n t i sera w a s d e v e l o p e d u s i n g a b i s ( d i a z o t i z e d - b e n z i d i n e ) system.
hemagglutination
T h o u g h significant titers to these conjugates w e r e d e t e c t e d
a l l of t h e a n i m a l s , t h e c o n j u g a t e d
in
malathion antigen elicited antibody
response i n m u c h h i g h e r t i t e r a n d after f e w e r injections t h a n d i d t h e D D A a n t i g e n . G e l d i f f u s i o n tests e s t a b l i s h e d t h e specificity of r e a c t i o n b e t w e e n a n t i s e r a to D D A - B S A a n d m a l a t h i o n - B S A w i t h t h e i r respective homologous B S A antigen.
T h e antisera to m a l a t h i o n - B S A g a v e n o
de-
t e c t a b l e p r e c i p i t i n r e a c t i o n w i t h b o v i n e s e r u m a l b u m i n , i n d i c a t i n g that the c a r r i e r p r o t e i n u n d e r w e n t m a r k e d c o n f i g u r a t i o n a l changes d u r i n g c o n j u g a t i o n o r t h a t some of its a n t i g e n i c d e t e r m i n a n t g r o u p s w e r e m a s k e d . C e n t e n o et al. (17)
also stress that t h e D D A - B S A a n d m a l a t h i o n - B S A
conjugates d i d n o t react w i t h a n t i s e r a to b o v i n e s e r u m a l b u m i n , i n d i c a t i n g t h a t t h e y w e r e a n t i g e n i c a l l y different f r o m t h e o r i g i n a l c a r r i e r p r o t e i n . T h e i r s t u d y d e m o n s t r a t e d that a n t i b o d i e s to D D T a n d m a l a t h i o n m e t a b o lites are r e a d i l y p r o d u c e d , a n d t h e y suggest t h a t these a n t i b o d i e s
serve
for t h e d e v e l o p m e n t of sensitive h i s t o l o g i c a l p r o c e d u r e s a l o n g w i t h r a d i o immunochemical and
fluorescein
l a b e l l i n g t e c h n i q u e s , to d e t e r m i n e
the
l o c a l i z a t i o n of these c h e m i c a l s i n a n i m a l tissues. T h e r e p o r t b y H a a s a n d G u a r d i a (18)
p e r t a i n s to t h e i r efforts
to
a p p l y i m m u n o l o g i c a l m e t h o d s f o r the assay of p e s t i c i d e residues, a n d expresses t h e i r a i m to test t h e s u i t a b i l i t y of m e t h o d s for field a n a l y t i c a l purposes.
H a a s a n d G u a r d i a also u s e d D D T a n d m a l a t h i o n to represent
t w o of the most i m p o r t a n t classes of i n s e c t i c i d e s , c h l o r i n a t e d h y d r o c a r bons a n d organophosphorus
compounds.
T h e y first a t t e m p t e d t o p r e p a r e
i n s e c t i c i d e - p r o t e i n antigens i n w h i c h enzymes w e r e u s e d as t h e p r o t e i n c a r r i e r . A n t i s e r u m of r a b b i t s i n j e c t e d w i t h D D A - c a r b o n i c a n h y d r a s e or m a l a t h i o n - c h y m o t r y p s i n f a i l e d t o s h o w t h e presence o f t h e
respective
antibodies. T h e y e x p e r i m e n t e d w i t h f o u r o t h e r proteins as c a r r i e r s : r a b b i t s e r u m albumin, bovine serum albumin, bovine
fibrinogen
fraction I, a n d bovine
/ ^ - g l o b u l i n f r a c t i o n I I I . T h e s t r u c t u r a l l y r e l a t e d d e r i v a t i v e s of D D T a n d malathion, D D A , and 0 , 0 - d i m e t h y l S-carboxy-carboxyethyl
phosphoro-
d i t h i o a t e ( m a l a t h i o n h a l f e s t e r ) , r e s p e c t i v e l y , w e r e u s e d as t h e
specific
haptens a t t a c h e d to these c a r r i e r proteins. T h e s e c o m p o u n d s c o n t a i n free carboxyl groups, w h i c h w h e n they reacted w i t h thionylchloride, provide a means of c o u p l i n g of the h a p t e n to the a m i n o groups of the p r o t e i n c a r r i e r . U n l i k e C e n t e n o et al. (17)
H a a s a n d G u a r d i a c o u l d not s h o w t h e
p r e s e n c e of a n t i b o d i e s i n t h e a n t i s e r u m of r a b b i t s i m m u n i z e d w i t h the h a p t e n conjugates of b o v i n e s e r u m a l b u m i n . T h e results w i t h ^ - g l o b u l i n conjugates w e r e ester
inconsistent, w h e r e a s
conjugates w i t h
fibrinogen
the D D A a n d m a l a t h i o n h a l f -
g a v e the
best
antigenic
responses.
T h r o u g h the s e r o l o g i c a l test m e t h o d s of p r e c i p i t a t i o n , t a n n e d c e l l h e m a g -
11.
ERCEGOVICH
Immunological
169
Techniques
glutination, and tanned cell hemagglutination inhibition they s t r a t e d t h e presence fibrinogen
demon-
of a n t i b o d i e s to D D A - f i b r i n o g e n a n d m a l a t h i o n -
i n t h e a n t i s e r u m of the r e s p e c t i v e l y i m m u n i z e d r a b b i t s .
U s i n g t h e t a n n e d c e l l h e m a g g l u t i n a t i o n i n h i b i t i o n test, t h e y d e t e r m i n e d t h a t t h e s e n s i t i v i t y of t h i s m e t h o d w a s b e t w e e n 0.1 a n d 1.0 ftgram f o r D D A a n d m a l a t h i o n . D D T c o u l d n o t b e d e t e c t e d i n this m a n n e r . Neither D D T , D D A , nor malathion could be detected b y direct reaction i n t h e p r e c i p i t i n or t a n n e d c e l l h e m a g g l u t i n a t i o n tests.
T h e antibodies
p r o d u c e d against t h e v a r i o u s conjugates w e r e specific f o r t h e p a r t i c u l a r p r o t e i n c a r r i e r since the i m m u n e s e r u m f r o m r a b b i t s i n o c u l a t e d w i t h D D A - f i b r i n o g e n d i d not a g g l u t i n a t e t h e D D A - r a b b i t s e r u m a l b u m i n c o n jugate a n d v i c e versa. A n t i h a p t e n a n t i b o d i e s a p p e a r e d to b e t r a n s i t o r y . M a x i m u m titer was obtained approximately 6 weeks
after t h e
initial
i n o c u l a t i o n a n d n o r e a c t i o n w a s d e t e c t a b l e 3 w e e k s later. T h e h a p t e n i c antisera w e r e u n s t a b l e after a f e w days of storage at 4 ° o r — 1 0 ° C .
The
a n t i b o d y a c t i v i t y a p p e a r e d to b e m a i n t a i n e d i f the a n t i s e r a w e r e
first
f r o z e n w i t h d r y i c e a n d stored at — 30 ° C . P r i o r to l e a r n i n g a b o u t t h e a f o r e m e n t i o n e d g r o u p s ' efforts i n v e s t i g a tions w e r e b e g u n i n the a u t h o r s l a b o r a t o r y t o test t h e usefulness
of
i m m u n o l o g i c a l m e t h o d s f o r d e t e c t i n g a n d a n a l y z i n g pesticides a n d p e s t i c i d a l d e g r a d a t i o n p r o d u c t s i n b i o l o g i c a l specimens. these studies c a n b e p r e s e n t e d h e r e since o u r
O n l y a s u m m a r y of
findings
are
considered
p r e l i m i n a r y a n d specific details w i l l b e p u b l i s h e d s u b s e q u e n t l y elsewhere. U n l i k e the other w o r k e r s w h o selected c o m p o u n d s
possessing
car-
b o n y l g r o u p s for c o u p l i n g purposes to t h e p r o t e i n carriers, o u r i n v e s t i g a tions c e n t e r e d o n p e s t i c i d e c o m p o u n d s
w h i c h possess either a n a m i n e
group or a nitro group, w h i c h can be reduced readily to an amine. included
the h e r b i c i d e
pesticide
degradation
aminotriazole, the
products
insecticide
These
parathion, and
nitrophenol, 4-chloronitrotoluene,
and
a n i l i n e . H a p t e n — p r o t e i n conjugates o f a l l of these m a t e r i a l s w e r e r e a d i l y p r e p a r e d b y m o d i f y i n g s l i g h t l y the m e t h o d d e s c r i b e d b y W i l l i a m s a n d C h a s e (12).
A n u n s u c c e s s f u l a t t e m p t to use e g g a l b u m i n as t h e c a r r i e r
p r o t e i n l e d to t h e use of b o v i n e p l a s m a p r o t e i n . T h e s e c a r r i e r p r o t e i n s w e r e u n s a t i s f a c t o r y b e c a u s e t h e y l a c k e d specificity a n d l o w titer f o r m a t i o n i n the a n t i s e r u m . T h e h i g h degree of heterogenous
antibody pro-
d u c t i o n c r e a t e d p r o b l e m s of cross r e a c t i o n w h i c h c o u l d not b e adequately through antiserum-antigen adsorption techniques. the u n d e s i r a b l e results w i t h these t w o p r o t e i n carriers, b o v i n e
corrected Following fibrinogen
f r a c t i o n I w a s tested f o r this p u r p o s e at t h e suggestion of H a a s ( p e r s o n a l communication)
(19).
T h e first results o b t a i n e d f r o m tests u s i n g
fibrino-
g e n as t h e p r o t e i n c a r r i e r r e v e a l e d a h i g h degree of specificity t o w a r d the homologous azoprotein
antigens.
T h i s specificity w a s
somewhat
greater, h o w e v e r , d u r i n g t h e first hours of i n c u b a t i o n a n d decreased as a
170
PESTICIDES
IDENTIFICATION
f u n c t i o n of t i m e w i t h a l i m i t i n g v a l u e of a p p r o x i m a t e l y 24 h o u r s . V e r y l i t t l e , i f a n y , cross-reactions w e r e o b s e r v e d i n t h e e a r l y h o u r s o f i n c u b a t i o n , b u t heterogenous p r e c i p i t a t e s b e g a n to f o r m after a b o u t e i g h t hours of i n c u b a t i o n of t h e p r e c i p i t i n tests. w i t h the v a r i o u s h a p t e n
fibrinogen
I n a second i m m u n i z a t i o n trial
a z o p r o t e i n s , s o m e w h a t h i g h e r titers
of antisera w e r e p r o d u c e d b u t w i t h a greater loss of specificity as e v i d e n c e d f r o m the d e c r e a s e d l e n g t h of t i m e i n v o l v e d for to
cross-reactions
develop. I n a t h i r d a t t e m p t to use
fibrinogen
as the c a r r i e r p r o t e i n a different
i m m u n i z a t i o n s c h e d u l e w a s f o l l o w e d . W h e r e a s i n the earlier experiments the animals, N e w Zealand male a n d female rabbits, were b l e d for antis e r u m r e c o v e r y f o u r days after the last of seven injections, i n t e r v a l s of t w o days after t h e last i n j e c t i o n of t h e h a p t e n - p r o t e i n conjugates, w e r e tried.
R e - i m m u n i z a t i o n of the r a b b i t s w a s r e p e a t e d 30 days a f t e r w a r d s
i n s t e a d of the f o r m e r s i x - d a y i n t e r v a l .
A n t i s e r a of h i g h e r t i t e r
were
p r o d u c e d i n a l l cases, h o w e v e r , the shorter p e r i o d of i m m u n i z a t i o n d i d not p r e v e n t t h e p r o d u c t i o n of
r e l a t i v e l y h i g h titers of
heterogenous
a n t i b o d i e s as e v i d e n c e d b y the h i g h degree of cross reactions. I n a d d i t i o n to the l a c k of specificity a n d cross-reactions w i t h t h e
fibrinogen
conjugates this p r o t e i n w a s less d e s i r a b l e because of t h e d i f f i c u l t y exp e r i e n c e d i n the p r e p a r a t i o n of w o r k solutions of its h a p t e n - a z o p r o t e i n conjugates.
T o dissolve s u c h a z o p r o t e i n s a s o l u t i o n of 8 M u r e a a n d
sodium hydroxide was used.
U r e a , a m i l d d e n a t u r i n g agent, d i s r u p t s
the s e c o n d a r y structure of p r o t e i n s b y c a u s i n g the p r o t e i n c h a i n to b e c o m e stretched, thus e x p o s i n g a n e x t r e m e l y large n u m b e r of a n t i g e n i c sites.
possible
O n c e injected into a n animal such chains apparently
are easily d e g r a d e d i n t o s m a l l e r fragments w h i c h are also c a p a b l e inducing antibody formation.
This phenomenon
m a y explain the lack
of specificity a n d h i g h p r o d u c t i o n of heterogenous antisera as d e m o n s t r a t e d b y p r e c i p i t i n tests.
of
antibodies i n our
It is v i r t u a l l y i m p o s s i b l e
to r e m o v e these i n t e r f e r r i n g heterogenous a n t i b o d i e s f r o m t h e a n t i s e r u m b y a d s o r p t i o n t e c h n i q u e s w i t h a n t i g e n since the r e s p o n s i b l e w e r e f o r m e d in vivo azoprotein.
antigens
o w i n g to d e g r a d a t i o n of the o r i g i n a l l y i n j e c t e d
Adsorption with
fibrinogen
alone
removed
f r a c t i o n of the o v e r a l l i n t e r f e r r i n g heterogenous
only a
small
antibodies.
B o v i n e s e r u m a l b u m i n p r o v e d to b e a m u c h better p r o t e i n c a r r i e r of t h e haptens u s e d i n o u r i n v e s t i g a t i o n . T h i s p r o t e i n w a s also u s e d b y C e n t e n o et al. (17)
w h o s e p r o d u c t i o n of a n t i s e r a to the D D A - B S A a n d
m a l a t h i o n - B S A conjugates is d i s c u s s e d i n p r e c e d i n g p a r a g r a p h s . B o v i n e s e r u m a l b u m i n is r e l a t i v e l y s m a l l e r i n m o l e c u l a r size t h a n
fibrinogen.
T h e a z o p r o t e i n conjugates p r o d u c e d w i t h i t c a n b e s o l u b i l i z e d b y less erratic p r o c e d u r e s w h i c h cause less s t r u c t u r a l alterations of t h e c a r r i e r p r o t e i n , therefore
producing fewer
heterogenous
antibodies.
The
ex-
11.
ERCEGOVICH
Immunological
171
Techniques
traneous a n t i b o d i e s w h i c h d o o c c u r w h e n b o v i n e s e r u m a l b u m i n is u s e d as t h e h a p t e n c a r r i e r is r e m o v e d r e a d i l y f r o m the a n t i s e r u m b y a d s o r p t i o n w i t h the unconjugated protein. A digest of the l i m i t e d a m o u n t of i n f o r m a t i o n a v a i l a b l e a b o u t the subject causes this r e v i e w e r to b e s o m e w h a t o p t i m i s t i c a b o u t t h e p o t e n t i a l usefulness of i m m u n o l o g i c a l m e t h o d s f o r t h e analysis of pesticides. E x periences g a i n e d f r o m i n v e s t i g a t i o n s c o n d u c t e d i n o u r l a b o r a t o r y r e v e a l that m e t h o d o l o g y exists w h i c h c a n b e u s e d t o i m p l e m e n t t h e d e v e l o p m e n t of i m m u n o l o g i c a l p r o c e d u r e s for p e s t i c i d e analysis. T h e three who
are k n o w n to h a v e tested i m m u n o l o g i c a l m e t h o d s
for
groups
detecting
pesticides w e r e a l l successful i n d e v e l o p i n g antisera f o r s o m e v e r y c o m m o n pesticides a n d t h e i r d e g r a d a t i o n p r o d u c t s .
Two
of
the
groups
d e m o n s t r a t e d t h a t i t w a s p o s s i b l e to detect s u b m i c r o g r a m q u a n t i t i e s o f these c h e m i c a l s . T h e same w o r k e r s h a v e s h o w n t h a t w h i l e i t m a y not b e p o s s i b l e to detect these pesticides b y d i r e c t s e r o l o g i c a l tests—e.g., p r e cipitin a n d hemagglutination reactions—these chemicals can be detected i n t r a c e a m o u n t s b y i n d i r e c t s e r o l o g i c a l tests w h i c h are r e l a t i v e l y s i m p l e to c o n d u c t .
T w o of the m o s t u s e f u l i n d i r e c t m e t h o d s w h i c h c a n b e u s e d
to detect the u n c o n j u g a t e d f o r m of the pesticides are t h e h a p t e n i n h i b i t i o n of p r e c i p i t a t i o n a n d passive h e m a g g l u t i n a t i o n i n h i b i t i o n test. T h e p r i n c i p l e of h a p t e n i n h i b i t i o n i n v o l v e s the r e a c t i o n of a n a n t i b o d y w i t h free h a p t e n a n d o b s e r v i n g a decrease i n the p r e c i p i t a t i o n of the a n t i b o d y w i t h the a d d i t i o n of t h e a p p r o p r i a t e antigen—i.e., the h a p t e n c o n j u g a t e d precipitating protein.
T h e a m o u n t of p r e c i p i t a t i o n is d i r e c t l y p r o p o r -
t i o n a l to the a m o u n t of h a p t e n present to c o m p e t e for a c t i v e sites o n t h e a n t i b o d y . T h e h a p t e n i n h i b i t i o n of p r e c i p i t a t i o n m e t h o d is m o r e specific t h a n the passive h e m a g g l u t i n a t i o n i n h i b i t i o n m e t h o d b u t is less a d a p t a b l e for r e l i a b l e q u a n t i t a t i v e d a t a .
Prognosis for the Future Use of Immunological Methods for Pesticide Analysis T h e p e s t i c i d e c h e m i s t w i l l q u e s t i o n w h a t the p r a c t i c a l a p p l i c a t i o n s of i m m u n o l o g i c a l t e c h n i q u e s for r e s i d u e analysis of pesticides are.
He
w i l l b e c o n c e r n e d a b o u t the specificity, q u a n t i t a t i v e aspects, a n d a d v a n tages these t e c h n i q u e s h a v e o v e r the e x i s t i n g c o l o r i m e t r i c , c h r o m a t o g r a p h i c , s p e c t r o p h o t o m e t r i c , a n d bioassay m e t h o d s w h i c h w o r k for h i m . B e c a u s e of t h e stringent r e q u i r e m e n t s to amass a l l of t h e
necessary
t o x i c o l o g i c a l a n d r e s i d u e d a t a n e e d e d to o b t a i n r e g i s t r a t i o n for the sale of a p e s t i c i d e , one of the first objectives of a w o u l d - b e p r o d u c e r is to d e v e l o p r e l i a b l e m e t h o d s to a n a l y z e his p r o d u c t .
F o r this reason there
exist a d e q u a t e m e t h o d s for a n a l y z i n g f o o d p r o d u c t s , c o m p o n e n t s of t h e e n v i r o n m e n t , a n d p a t h o l o g i c a l specimens for specific pesticides a n d t h e i r
172
PESTICIDES
degradation products.
IDENTIFICATION
T h e s e m e t h o d s , h o w e v e r g o o d t h e y are f o r a n a -
l y z i n g specific c o m p o u n d s , are not a d e q u a t e to a n a l y z e efficiently a n d m e a n i n g f u l l y the g e n e r a l f o o d s u p p l y , a n d a l l of the other samples of c o n c e r n , f o r the c o m p r e h e n s i v e q u a l i t a t i v e a n d q u a n t i t a t i v e i n f o r m a t i o n w h i c h is d e s i r e d a b o u t the pesticides t h a t m a y b e present. P e s t i c i d e laboratories of f o o d i n d u s t r i e s a n d r e g u l a t o r y agencies are c o n t i n u a l l y f a c e d w i t h the p r o b l e m of a n a l y z i n g samples w h o s e h i s t o r y of exposure to pesticides is u n k n o w n . M o r e t h a n one p e s t i c i d e m a y b e present i n a n y of these samples a n d the r e s i d u e of e a c h m a y h a v e to b e determined.
T o h e l p solve t h i s p r o b l e m of a n a l y z i n g d i v e r s e s a m p l e
types f o r exposure to different types of pesticides, effort has b e e n m a d e b y t h e F D A scientists, a m o n g others, to d e v e l o p m e t h o d s for the m u l t i p l e analysis of pesticides. T h e subject of m e t h o d s f o r the m u l t i p l e analysis of pesticides has been adequately reviewed recently b y B u r k e (20).
A c c o r d i n g to B u r k e ,
h o w e v e r , a definite p r o c e d u r e f o r c o n f i r m i n g the i d e n t i t y of a g i v e n p e s t i c i d e r e s i d u e has n o t b e e n established. T h i n - l a y e r c h r o m a t o g r a p h y , gas c h r o m a t o g r a p h y o n c o l u m n s that g i v e different r e t e n t i o n patterns, a n d t h e p-values of B o w m a n a n d B e r o z a (21, 22)
are p r o b a b l y t h e m o s t
universally applicable confirmatory techniques.
D e r i v a t i z a t i o n of
the
r e s i d u e i n q u e s t i o n f o l l o w e d b y gas c h r o m a t o g r a p h y of the d e r i v a t i v e seems a n excellent a p p r o a c h , a n d s e v e r a l p r o c e d u r e s are d e s c r i b e d i n t h e recent l i t e r a t u r e (23, 24).
M u l t i r e s i d u e m e t h o d s h a v e a serious l i m -
i t a t i o n since m a n y p e s t i c i d e c h e m i c a l s are not d e t e r m i n e d a n d n o t a l l s a m p l e types c a n b e h a n d l e d i n a r o u t i n e f a s h i o n . A l s o , as p r e v i o u s l y m e n t i o n e d , there is n o c o m p l e t e s c h e m e f o r c o n f i r m i n g the i d e n t i t y of m a n y pesticides. T h e s e l e c t i o n of m e t h o d s of a n a l y s i s , therefore, d e p e n d s e x t e n s i v e l y u p o n the pesticides a n d n a t u r e of the s a m p l e to b e a n a l y z e d , t h e e q u i p m e n t a n d p e r s o n n e l r e q u i r e d , a n d the s i m p l i c i t y , speed, cost, a c c u r a c y , a n d r e p r o d u c i b i l i t y of the m e t h o d .
T i m e frequently plays a
p r e d o m i n a n t r o l e i n t h e selection of m e t h o d s to b e u s e d b y t h e f o o d processor,
who
must comply
to p e s t i c i d e
tolerances,
and regulatory
agencies, w h o m u s t enforce t h e m . A p p a r e n t l y , a c o m b i n a t i o n of s c r e e n i n g a n d specific m e t h o d s is n e e d e d b y s u c h o r g a n i z a t i o n s since n e i t h e r types of m e t h o d s alone c a n satisfy t h e i r a n a l y t i c a l r e q u i r e m e n t s . S o m e t e c h n i q u e s m a y offer selective s c r e e n i n g as w e l l as specificity —e.g., m i c r o c o u l o m e t r i c m e t h o d s d e s c r i b e d b y C o u l s o n et al. (24).
This
t e c h n i q u e consists of a c o m b i n a t i o n of gas c h r o m a t o g r a p h y , c o m b u s t i o n , a n d continuous coulometric titration for chlorine or sulfur. T h e developm e n t of the flame p h o t o m e t r i c detector offers a s i m i l a r p o t e n t i a l f o r t h e selective s c r e e n i n g a n d s p e c i f i c i t y of pesticides w h i c h c o n t a i n p h o s p h o r u s o r s u l f u r (25).
E v e n so, o n e o r m o r e tests i n a d d i t i o n to the i n i t i a l
11.
ERCEGOVICH
Immunological
173
Techniques
analysis m a y b e r e q u i r e d to i d e n t i f y p r o p e r l y the pesticides present i n a sample. A c r i t i c a l e v a l u a t i o n of the l i m i t e d a m o u n t of i n f o r m a t i o n a v a i l a b l e a b o u t the d e t e c t i o n of pesticides b y i m m u n o l o g i c a l m e t h o d s enables one, w i t h o u t too m u c h o p t i m i s m , to r e p l y i n the affirmative to t h e r e s i d u e chemist's q u e s t i o n r e g a r d i n g specificity. B o t h C e n t e n o et ah a n d H a a s a n d G u a r d i a w e r e successful i n p r o d u c i n g a n t i s e r a w h i c h w e r e specific f o r d e r i v a t i v e s of D D T a n d m a l a t h i o n , D D A a n d m a l a t h i o n esters, r e spectively.
A n t i s e r a w h i c h w e r e specific f o r p a r a t h i o n , a m i n o t r i a z o l e ,
p-nitrophenol
(a
degradation product
of
parathion), and
n i t r o t o l u e n e h a v e b e e n p r o d u c e d i n the a u t h o r s l a b o r a t o r y .
4-chloro-2The pro-
d u c t i o n o f these antisera w a s a c c o m p l i s h e d b y c o n v e n t i o n a l i m m u n o l o g i c a l techniques.
C o n f i r m a t i o n of these a n t i b o d i e s w a s r e a d i l y d e m o n s t r a t e d
b y w e l l k n o w n serological methods. T h e relation between
a n t i g e n a n d a n t i b o d y is h i g h l y specific, as
i n d i c a t e d . I n a sense the s e r o l o g i c a l methods u s e d to demonstrate a n t i g e n a n t i b o d y reactions are a n a l y t i c a l m e t h o d s , sensitive, s e m i - q u a n t i t a t i v e , a n d h i g h l y specific. l o g i c a l specificity.
T h e r e is a l i m i t , h o w e v e r , to the p r e c i s i o n of seroW h e n different h o m o l o g o u s antigens are u s e d
to
i m m u n i z e a n i m a l s , the a n t i b o d i e s f o r one a n t i g e n react also w i t h o t h e r antigens, t h o u g h less strongly. T h e s e are c a l l e d cross-reactions.
Saying
t h a t a n a n t i b o d y is specific f o r a p a r t i c u l a r a n t i g e n is a r e l a t i v e t e r m because i t is b a s e d o n t h e degree to w h i c h a n a n t i b o d y reacts w i t h v a r i o u s antigens, not r e q u i r i n g i t to react solely w i t h o n l y one of t h e m . It is also r e l a t i v e because t h e a n t i b o d y m a y not h a v e b e e n tested against a l l p o s s i b l e antigens; thus there c o u l d b e others w i t h w h i c h i t m i g h t react. A n t i s e r u m to a n a z o p r o t e i n or to other types of c o n j u g a t e d p r o t e i n is p r o d u c e d p r i m a r i l y to o b t a i n a n t i b o d i e s against k n o w n structures. F o r some u n k n o w n reason h a p t e n i c groups are n o t v e r y i m m u n o g e n i c , r e gardless of t h e p r o t e i n c a r r i e r , a n d a n t i s e r u m u s u a l l y contains a r e l a t i v e l y l o w c o n c e n t r a t i o n of a n t i b o d y against the c o n j u g a t e d h a p t e n . I n w o r k i n g w i t h c o n j u g a t e d p r o t e i n s it s h o u l d b e r e m e m b e r e d that a n t i b o d i e s p r o d u c e d to i t m a y e x h i b i t three types of s p e c i f i c i t y : one d i r e c t e d t o w a r d the h a p t e n , one t o w a r d the p r o t e i n c a r r i e r , a n d one t o w a r d the h a p t e n p r o t e i n conjugate.
A s a r u l e a n a n t i s e r u m against a g i v e n
p r o t e i n contains a d i v e r s i t y of a n t i b o d i e s .
conjugated
B e c a u s e of this m i x t u r e of
a n t i b o d y m o l e c u l e s , a s e r o l o g i c a l test for h a p t e n a n t i b o d y m u s t b e m a d e w i t h t h e h a p t e n a t t a c h e d to t h e p r o t e i n m o l e c u l e h a v i n g n o cross-reaction w i t h the one u s e d f o r i m m u n i z a t i o n . T h e i n t e r f e r r i n g a n t i b o d i e s , w h i c h g i v e rise to cross-reactions, h o w e v e r , are r e m o v e d f r o m a n t i s e r u m b y a process k n o w n as a d s o r p t i o n . T h i s process i n v o l v e s i n c u b a t i n g t h e a n t i s e r u m w i t h respective antigens w h i c h are r e q u i r e d to react w i t h the extraneous a n t i b o d i e s .
T h e resulting a n t i g e n - a n t i b o d y complexes can
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PESTICIDES
IDENTIFICATION
then be removed by centrifugation, leaving a specificity-enriched antiserum. T h e e x p l o r a t o r y investigations w h i c h h a v e b e e n
conducted
using
i m m u n o l o g i c a l a n d s e r o l o g i c a l t e c h n i q u e s to a n a l y z e pesticides h a v e b e e n r e s t r i c t e d to a t y p i c a l s a m p l e c o n d i t i o n s .
T h r o u g h t h e use of t a n n e d c e l l
h e m a g g l u t i n a t i o n i n h i b i t i o n tests, H a a s a n d G u a r d i a c o u l d detect q u a n tities of 0.1 a n d 1.0 /mgram of D D A a n d m a l a t h i o n . H o w e v e r , b y this same t e c h n i q u e t h e y w e r e u n a b l e to detect D D T w i t h the a n t i s e r u m w h i c h w a s p r o d u c e d i n response to i m m u n i z a t i o n w i t h D D A - f i b r i n o g e n . These
findings
are significant because t h e y demonstrate specificity for
one of the m a j o r d e g r a d a t i o n p r o d u c t s of D D T a n d t h a t s u c h p r o c e d u r e s c a n detect m i c r o g r a m q u a n t i t i e s of t w o i m p o r t a n t p e s t i c i d a l l y r e l a t e d c h e m i c a l s . It is of interest to a t t e m p t to conjugate D D T itself to a c a r r i e r p r o t e i n b y m a k i n g a n arsenate d e r i v a t i v e of i t , or a c o m p o u n d
more
closely r e l a t e d s t r u c t u r a l l y to D D T t h a n D D A . S u c h a c o m p o u n d w o u l d be Kelthane, [ l , l - b i s ( p-chlorophenyl )-2,2,2-trichloroethanol]. P r e l i m i n a r y trials u s i n g a s i m i l a r t y p e of passive h e m a g g l u t i n a t i o n techniques
enabled Babish (personal communication)
to
(26)
detect
12 n g r a m s of p a r a t h i o n a n d 150 n g r a m s of a m i n o t r i a z o l e . N e i t h e r g r o u p of w o r k e r s , h o w e v e r , a t t e m p t e d to detect a n y of these c o m p o u n d s
in
c r u d e or f o r t i f i e d extracts of s o i l , p l a n t , or a n i m a l tissue. T h e r e f o r e , t h e q u e s t i o n a b o u t the s u i t a b i l i t y a n d effectiveness
of d e t e c t i n g residues of
these pesticides i n a c t u a l samples r e m a i n s to b e a n s w e r e d . these
findings
Nonetheless,
suggest t h a t a c o m b i n a t i o n of i m m u n o l o g i c a l a n d
sero-
l o g i c a l t e c h n i q u e s h a v e the p o t e n t i a l of q u a n t i t a t i o n t h a t the r e s i d u e c h e m i s t r e q u i r e s a n d s h o u l d b e e x a m i n e d m o r e seriously f o r
pesticide
r e s i d u e analysis. T h e r e are a n u m b e r of o b v i o u s advantages to the use of i m m u n o l o g i c a l t e c h n i q u e s for p e s t i c i d e analysis, b u t the converse is also t r u e . N o inference is m a d e
that s u c h t e c h n i q u e s
c o u l d ever r e p l a c e t h e
s o p h i s t i c a t e d m e t h o d s of analysis p r e s e n t l y b e i n g u s e d . gested
t h a t t h e y h a v e p o t e n t i a l to s u p p l e m e n t
more
It is o n l y s u g -
rather than supplant
e x i s t i n g m e t h o d s of analysis. T h e i r greatest usefulness is for the r a p i d screening of a large n u m b e r of samples for the presence of specific types of pesticides or t h r e s h o l d q u a n t i t i e s . T h e s e m e t h o d s are also i d e a l for c o n f i r m a t o r y tests.
T h r o u g h t h e use of p r o p e r m e t h o d s
degree of specificity for v a r i o u s c o m p o u n d s oped. R
f
a very high
could ultimately be devel-
S u c h c o n f i r m a t o r y tests w o u l d b e i n d e p e n d e n t of r e l i a n c e u p o n
values, r e t e n t i o n times, a n d other h i g h l y e m p i r i c a l indexes p r e s e n t l y
u s e d i n c o n j u n c t i o n w i t h gas, l i q u i d , a n d t h i n l a y e r
chromatography.
P e r h a p s one of the greatest potentials for i m m u n o l o g i c a l m e t h o d s p e s t i c i d e r e s i d u e analysis is t h e i r use i n d e v e l o p i n g countries.
for
Because
n o h i g h l y s p e c i a l i z e d or expensive e q u i p m e n t is r e q u i r e d , e x c e p t f o r a
11.
ERCEGOVICH
Immunological
175
Techniques
s u i t a b l e centrifuge. P e r s o n n e l r e q u i r e m e n t s w o u l d n o t b e c r i t i c a l , except for a senior m e m b e r w h o has the p r o p e r t r a i n i n g a n d u n d e r s t a n d i n g of immunological methods.
T h e o p e r a t i n g t e c h n i c i a n s w o u l d not h a v e to
b e h i g h l y s k i l l e d i n electronics a n d other d i s c i p l i n e s w h i c h are r e q u i r e d for
the p r o p e r
operation
of
gas
chromatographic
and
spectrometric
equipment. The
o v e r a l l advantages
of
i m m u n o l o g i c a l methods
for
pesticide
analysis are t h a t t h e y c a n b e p e r f o r m e d r e l a t i v e l y fast, e c o n o m i c a l l y , a n d s i m p l y w i t h a h i g h degree of a c c u r a c y a n d r e p r o d u c i b i l i t y . S i n c e
no
h i g h cost elaborate e q u i p m e n t is r e q u i r e d , there w o u l d b e n o great i n i t i a l i n v e s t m e n t , subsequent
replacement,
a n d s e r v i c i n g costs.
Technician
r e q u i r e m e n t s i n t r a i n i n g a n d s p e c i a l i z a t i o n w o u l d b e m i n i m a l as w e l l as the n u m b e r of p e r s o n n e l n e e d e d . I n c o m p a r i s o n w i t h t h e 20 to 40 analyses w h i c h a t e c h n i c i a n c a n p e r f o r m o n one gas c h r o m a t o g r a p h d u r i n g a n e i g h t - h o u r w o r k d a y his p r o d u c t i v i t y w i t h s e r o l o g i c a l m e t h o d s c o u l d b e increased many-fold. E v e n t h o u g h the p r o b l e m s o f p r o d u c i n g h i g h l y specific a n t i s e r a c a n b e o v e r c o m e , these m e t h o d s m a y s t i l l h a v e c e r t a i n disadvantages.
The
foremost d i s a d v a n t a g e concerns the source a n d a v a i l a b i l i t y of the respect i v e a n t i b o d i e s w h i c h w o u l d b e n e e d e d f o r analysis. T h i s p r o b l e m is not i n s u r m o u n t a b l e f o r i t is not e x p e c t e d
t h a t e a c h l a b o r a t o r y w i s h i n g to
use s u c h t e c h n i q u e s w o u l d p r o d u c e its o w n a n t i s e r a , except i n those f e w cases i n w h i c h the v o l u m e a n d t y p e of o p e r a t i o n w o u l d j u s t i f y d o i n g so. If a n d w h e n i m m u n o l o g i c a l t e c h n i q u e s are p r o v e d r e l i a b l e a n d w o r t h y for the r o u t i n e analysis of p e s t i c i d e residues, one w o u l d expect t h a t specific antisera w o u l d e v e n t u a l l y b e c o m e a v a i l a b l e f r o m
commercial
sources as is n o w t r u e for a w i d e v a r i e t y of antisera. O n e s u p p l i e r of antisera p r e s e n t l y fists a n u m b e r of antisera of the n a t u r e d i s c u s s e d i n this c h a p t e r f o r t h e f o l l o w i n g h a p t e n s : 3-indoleacetic a c i d , d i n i t r o p h e n o l , azobenzenearsonate,
acid, gibberellic
thymidine, and uridine. E v e n
i n the l o w q u a n t i t y i n w h i c h these antisera m u s t b e p r o d u c e d , o w i n g to t h e i r v e r y l i m i t e d use, t h e i r cost is c o m p e t i t i v e w i t h present
methods
of analysis.
Greater
M o s t of these antisera are p r o d u c e d
i n rabbits.
q u a n t i t i e s at s u b s t a n t i a l l y l o w e r prices c o u l d e a s i l y b e p r o d u c e d to m e e t h i g h v o l u m e d e m a n d b y u s i n g l a r g e r a n i m a l s s u c h as goats, cattle, a n d horses.
O n e m i g h t also expect that the c o m m e r c i a l s u p p l i e r of antisera
w o u l d also s u p p l y i d e n t i f i c a t i o n kits w h i c h c o n t a i n b o t h the
specific
a n t i g e n a n d specific a n t i b o d y as controls a n d f o r p o s i t i v e i d e n t i f i c a t i o n . A n t i s e r a m a y b e h i g h l y p e r i s h a b l e substances i n c o m p a r i s o n
with
the c h e m i c a l s a n d solvents n o r m a l l y u s e d b y the p e s t i c i d e chemist. H a a s and
G u a r d i a suggested
that a p r o b l e m existed i n this respect.
Their
p r e l i m i n a r y results s h o w e d t h a t t h e a n t i s e r u m to D D A - f i b r i n o g e n a n d m a l a t h i o n - f i b r i n o g e n c o u l d b e p r e s e r v e d u p to o n e m o n t h i f f r o z e n w i t h
176
PESTICIDES
IDENTIFICATION
d r y i c e a n d k e p t at — 3 0 ° C . M o r e w o r k o n the s t a b i l i t y of s u c h a n t i b o d i e s w o u l d h a v e to b e c o n d u c t e d , h o w e v e r , b e f o r e g e n e r a l i z a t i o n s a b o u t t h e i r shelf l i f e c a n b e m a d e .
S u p p l i e r s of a n u m b e r of antisera c l a i m shelf
lives of u p to o n e y e a r i f t h e p r o d u c t is m a i n t a i n e d u n d e r t h e r e c o m m e n d e d storage c o n d i t i o n s . T h e d i s a d v a n t a g e i n t h i s respect m a y a m o u n t m o s t l y to a m a t t e r of m o r e c r i t i c a l p l a n n i n g i n regards to needs a n d inventory. I m m u n o l o g i c a l m e t h o d s w o u l d not h a v e the q u a n t i t a t i v e p r e c i s i o n of
gas c h r o m a t o g r a p h i c ,
c o l o r i m e t r i c , or s p e c t r o p h o t o m e t r i c
methods.
However, i n their proper manipulation they can provide semiquantitative data comparable w i t h that obtained b y t h i n layer chromatography.
The
q u a l i t a t i v e i n f o r m a t i o n d e r i v e d f r o m i m m u n o l o g i c a l m e t h o d s w o u l d not necessarily b e a b s o l u t e b u t c e r t a i n l y w o u l d b e m o r e r e l i a b l e t h a n s u c h information based
on retention time a n d R
f
values.
The qualitative
aspects of i m m u n o l o g i c a l m e t h o d s w i l l d e p e n d u p o n specificity, w h i c h w i l l b e i n f l u e n c e d b y the q u a l i t y of the a n t i s e r u m a n d the n a t u r e of t h e antigen (the residue sample).
I n this r e g a r d t h e q u e s t i o n a b o u t s a m p l e
p r e p a r a t i o n a n d c l e a n u p arises. U n f o r t u n a t e l y , d e t a i l e d studies i n this c o n n e c t i o n h a v e not b e e n
conducted.
O n e reason for d e f e r r i n g s u c h
studies i n preference to w o r k i n g w i t h p u r e solutions has b e e n to establ i s h t h e f e a s i b i l i t y of s u c h m e t h o d s a n d t h a t o n a t h e o r e t i c a l basis one expects a lesser d e g r e e of i n t e r f e r e n c e w i t h s e r o l o g i c a l m e t h o d s
than
w i t h m o r e c o n v e n t i o n a l m e t h o d s of analysis. T h e structures of m o s t of the p e s t i c i d a l c o m p o u n d s
differ so g r e a t l y f r o m the n o r m a l constituents
of p l a n t , s o i l , a n d a n i m a l tissues t h a t i t w o u l d b e u n l i k e l y to e x p e c t m a n y of these substances to fit the steric r e q u i r e m e n t s of h i g h l y p u r i f i e d a n d specific a n t i s e r u m . A t most one w o u l d expect t h a t c l e a n u p u s e d f o r gas a n d t h i n l a y e r c h r o m a t o g r a p h y
w o u l d be
procedures
sufficient
for
s e r o l o g i c a l d e t e c t i o n . H o w e v e r , i f l i t t l e or n o c l e a n u p at a l l w o u l d suffice, the p r o d u c t i v i t y p e r t e c h n i c i a n w o u l d b e greater. A c e r t a i n a m o u n t of s p e c i a l i z e d t r a i n i n g w i l l b e r e q u i r e d f o r other m e t h o d s of analysis. P r o p e r u n d e r s t a n d i n g of i m m u n o l o g y a n d serology w o u l d b e r e q u i r e d of t h e senior i n v e s t i g a t o r to i n t e r p r e t the results p r o p e r l y , b u t this r e q u i r e m e n t is n o m o r e of a n i m p o s i t i o n or deterrent f o r these t e c h n i q u e s t h a n i t is for other m e t h o d s of analysis.
Literature Cited (1) (2) (3)
Burrows, W . , Moulder, J. W., Lewert, R. M., "Testbook of Microbiology," 18th ed., W . B . Saunders, Philadelphia, 1965. D a y , E . D . , "Foundations of Immunochemistry," W i l l i a m s & W i l k i n s , Baltimore, 1966. Gary, D . F . , "Immunology," 2 n d ed., Cheshire, Melbourne, Australia, 1970.
11.
ERCEGOVICH
Immunological
Techniques
177
(4) Weiser, R. S., Myrvik, Q. N . , Pearsall, N . N . , "Fundamentals of Immunology," Lea & Febiger, Philadelphia, 1970. (5) Lansteiner, K., " T h e Specificity of Serological Reactions," Dover, New York, 1962. (6) Nezlin, R. S., "Biochemistry of Antibodies," Plenum Press, New York, 1970. (7) Pressman, D . , Grossberg, A . L . , "The Structural Basis of Antibody Specificity," New York, 1968. (8) Campbell, H . , Garvey, J. S., Cremer, N . E., Sussdorf, D . H . , "Methods in Immunology," W . A . Benjamin, New York, 1964. (9) Kabat, E . A . , Mayer, M . , "Experimental Immunochemistry," Charles Thomas, Springfield, 1966. (10) Kwapinski, J. B., "Methods in Serological Research," Wiley & Sons, New York, 1965. (11) Nowotny, A . , "Basic Exercises in Immunochemistry," Springer-Verlag, New York, 1969. (12) Williams, C. A . , Chase, M . W., "Methods in Immunology and Immunochemistry," Academic Press, New York, 1967. (13) Mattioli, C. A., Yazi, A . , Pressman, B., J. Immunol. (1968) 101, 939. (14) Gill, J. T., Boty, P., J. Biol Chem. (1961) 236, 2677. (15) Talano, R. C., Haber, E . , Austen, K. F . , J. Immunol. (1968) 101, 333. (16) Haurowitz, F . J., Immunology (1942) 43, 311. (17) Centeno, E . R., Johnson, W . J., Sehon, A . H . , Int. Arch. Allergy Appl. Immunol. (1970) 37, 1. (18) Haas, G . J., Guardia, E . J., Proc. Soc. Exp. Biol. Med. (1968) 129, 546. (19) Haas, G . J., personal communication (1971). (20) Burke, J. A . , Residue Rev. (1971) 34, 59. (21) Beroza, M . , Inscoe, M . N . , Bowman, M . C., Residue Rev. (1969) 30, 1. (22) Bowman, M . C., Beroza, M . , J. Assoc. Offic. Agr. Chem. (1965) 48, 943. (23) Chau, A . S. Y., Cochrane, W . P.,J.Assoc. Offic. Anal. Chem. (1969) 52, 1220. (24) Coulson, D . M . , Cavanagh, L . A., DeVries, J. E., Walther, B., J. Agr. Food Chem. (1960) 8, 399. (25) Brody, S. S., Chaney, J. E., J. Gas Chromatogr. (1966) 4, 42. (26) Babish, J. G., personal communication (1971). RECEIVED
July 16,
1971.
PESTICIDES
IDENTIFICATION
