T h i s i n v e n t i o n r e l a t e s to n o v e l p r o c a i n a m i d e a n d
N - a c e t y l p r o c a i n a m i d e (NAPA) d e r i v a t i v e s p e r t a i n i n g t o
i m m u n o a s s a y s f o r d e t e r m i n i n g s u c h d r u g s in l i q u i d
m e d i a s u c h as b i o l o g i c a l f l u i d s . The d e r i v a t i v e s
i n c l u d e i m m u n o g e n s u s e d to s t i m u l a t e p r o d u c t i o n o f
a n t i b o d i e s to t h e d r u g s in h o s t a n i m a l s by c o n v e n -
t i o n a l t e c h n i q u e s . Also p r o v i d e d a r e l a b e l e d c o n j u -
g a t e s u s e d as r e a g e n t s , a l o n g w i t h t h e a n t i b o d i e s ,
in p a r t i c u l a r l y p r e f e r r e d i m m u n o a s s a y s . I n t e r -
m e d i a t e s in t h e s y n t h e s i s of t h e a f o r e m e n t i o n e d
i m m u n o g e n s and l a b e l e d c o n j u g a t e s a r e a l s o p r o v i d e d .
The c a r d i a c d e p r e s s a n t d r u g s p r o c a i n a m i d e a n d
N - a c e t y l p r o c a i n a m i d e a re u s e d c l i n i c a l l y to t r e a t
or p r e v e n t c a r d i a c a r r h y t h m i a ( J . K o s h - W e s e r , et aZ3
New E n g l a n d J. Med. 2 8 1 : 1 2 5 3 ( 1 9 6 9 ) ; J. K o c h - W e s e r
and S . W . K l e i n , J. Am. Med. A s s o c . 2 1 5 : 1 4 5 4 ( 1 9 7 1 ) ] .
N - a c e t y l p r o c a i n a m i d e ( a b b r e v i a t e d as NAPA and a l s o
known as a c e c a i n i d e ) is t he m a j o r m e t a b o l i t e o f
p r o c a i n a m i d e in man. The c o n c e n t r a t i o n of t h i s
m e t a b o l i t e in t he p l a s m a of p a t i e n t s r e c e i v i n g p r o -
c a i n a m i d e o f t e n e x c e e d s t h e c o n c e n t r a t i o n of t h e
p a r e n t d r u g i t s e l f . M e t a b o l i s m is by in v ivo a c e t y l i - z a t i o n and much g e n e t i c - b a s e d v a r i a t i o n has been o b s e r v e c
in t h e r a t e a t w h i c h i n d i v i d u a l p a t i e n t s t r a n s f o r m t h e
d r u g to i t s m e t a b o l i t e [D. D r a y e r , e t aZ, Drug M e t a b .
Rev. 1 0 : 2 3 9 ( 1 9 7 9 ) ] . T h i s p h e n o m e n o n i s of i m p o r t a n c e
in t h e c l i n i c a l u s e of t he d r u g s b e c a u s e of t h e l o w e r
i n c i d e n c e of s i d e e f f e c t s a s s o c i a t e d w i t h NAPA. B o t h
t h e t h e r a p e u t i c u s e f u l n e s s and t h e t o x i c i t y o f
t h e d r u g s a r e b e t t e r c o r r e l a t e d w i t h t h e i r b l o o d
l e v e l s t h a n w i t h t h e i r d o s a g e s . The r e l a t i o n s h i p
b e t w e e n t h e a m o u n t of d rug a d m i n i s t e r e d and t h e
b l o o d l e v e l s is q u i t e v a r i a b l e . I t i s i n f l u e n c e d
by c o m p l e t e n e s s of a b s o r p t i o n , d i s t r i b u t i o n c h a r a c -
t e r i s t i c s and r a t e s of m e t a b o l i s m and e x c r e t i o n .
2. DESCRIPTION OF THE PRIOR ART
B e c a u s e of t h e s e c o n s i d e r a t i o n s , n u m e r o u s a n a l y t i -
c a l m e t h o d s h a v e b e e n d e v e l o p e d to d e t e r m i n e t h e
b l o o d l e v e l s of t h e s e d r u g s , i n c l u d i n g h i g h p r e s s u r e
l i q u i d c h r o m a t o g r a p h y (HPLC) [ L . R . S h u k u s , et aZ3 C Z i n .
Chem. 2 3 : 7 0 5 ( 1 9 7 7 ) ] , h o m o g e n e o u s enzyme i m m u n o a s s a y
[ C . B . W a l b e r g , " P r o c . I n t l . Sym. Enzyme L a b e l e d H o r -
mones and D r u g s " , S . B . P a l , e d . , W . d e G r u y t e r & Co .
( B e r l i n , 1 9 7 8 ) , p. 4 2 9 ] , and q u a n t i t a t i v e t h i n l a y e r
c h r o m a t o g r a p h y (TLC) [ B . W e s l e y - H a d z y a and A . M .
M a t t o c k s , J. C h r o m a t . 1 4 3 : 3 0 7 ( 1 9 7 7 ) ] .
The p r e p a r a t i o n of a n t i b o d i e s to p r o c a i n a m i d e
and NAPA f o r u se in i m m u n o a s s a y s to d e t e r m i n e t h e
d r u g s - h a s b e e n a c c o m p l i s h e d in t h e p r i o r a r t by e s s e n -
t i a l l y two d i f f e r e n t a p p r o a c h e s . One a p p r o a c h h a s
b e e n to c o u p l e p r o c a i n a m i d e t h r o u g h t h e r i n g a m i n o
g r o u p by d i a z o t i z a t i o n and s u b s e q u e n t c o n d e n s a t i o n
to an a l b u m i n c a r r i e r [ A . S . R u s s e l , e t a l , CZin. E x p .
ImmunoZ. 3 : 9 0 1 ( 1 9 6 8 ) and M o j a v e r i a n et aZ3 J. P h a r m .
S c i . 6 9 : 7 2 1 ( 1 9 8 0 ) ] . The r e s u l t i n g a n t i b o d i e s s h o w
a h i g h d e g r e e of c r o s s - r e a c t i v i t y w i t h NAPA a n d
w o u l d t h e r e f o r e be u n s u i t e d f o r use in i m m u n o a s s a y s
s p e c i f i c f o r one or t h e o t h e r d r u g .
The s e c o n d a p p r o a c h i n v o l v e s c o u p l i n g of t h e
d r u g s at t h e c o m p l e t e o p p o s i t e end of t h e i r s t r u c -
t u r e s , at t h e N - d i e t h y l a m i n o g r o u p , by m o d i f i c a t i o n
of one of t h e e t h y l s u b s t i t u e n t s f o r s u b s e q u e n t
c o u p l i n g to a c a r r i e r [U .S . P a t . No. 4 , 2 3 5 , 9 6 9 ] . As
a r e s u l t , a n t i b o d i e s a r e r a i s e d a g a i n s t an i m m u n o -
gen in wh ich a m a j o r f u n c t i o n a l g r o u p of t h e d r u g s
has been m o d i f i e d in o r d e r to c o u p l e them to t h e
c a r r i e r .
The s t a t e - o f - t h e - a r t of p r e p a r i n g a n t i b o d i e s
to h a p t e n s s u c h as d r u g s is r e p r e s e n t e d by W e i n r y b
e t a l , Drug M e t a b o l i s m R e v i e w s 1 0 : 2 7 1 ( 1 9 7 9 ) ; P l a y f a i r
e t a l , Br. Med. BuZZ. 3 0 : 2 4 ( 1 9 7 4 ) ; B r o u g h t o n et aZ3
c l i n . Chem. 2 2 : 7 2 6 ( 1 9 7 6 ) ; and B u t l e r , J. I m m u n o Z .
Meth . 7 : 1 ( 1 9 7 5 ) and P h a r m a c o Z . Rev. 2 9 ( 2 ) : 1 0 3 ( 1 9 7 8 ) .
L a b e l e d c o n j u g a t e s , c o m p r i s i n g t h e a n a l y t e o r
a d e r i v a t i v e or o t h e r a n a l o g t h e r e o f , c o u p l e d to a
l a b e l i n g s u b s t a n c e a r e v a r i o u s l y d e s c r i b e d in t h e
l i t e r a t u r e , e . g . , U . S . P a t . Nos. 4 , 2 7 9 , 9 9 2 ; 4 , 1 8 2 , 8 5 6 ;
4 , 2 5 9 , 2 3 3 ; and 4 , 2 9 2 , 4 2 5 w h e r e i n t he l a b e l is t h e
f l u o r o g e n i c enzyme s u b s t r a t e β - g a l a c t o s y l - u m b e l l i f e r o n e .
SUMMARY OF THE INVENTION
The p r e s e n t i n v e n t i o n u n i q u e l y p r o v i d e s r e a g e n t s f o r u se in p r o c a i n a m i d e and NAPA i m m u n o a s s a y s i n v o l v -
ing t h e c o u p l i n g to or d e r i v a t i z a t i o n of t he d r u g
a t t h e a - p o s i t i o n on t h e amide s i d e c h a i n . T h e
i m m u n o g e n of t h e p r e s e n t i n v e n t i o n , c o m p r i s i n g t h e
h a p t e n i c d r u g c o v a l e n t l y l i n k e d a t t h e a - p o s i t i o n t o
an i m m u n o g e n i c c a r r i e r m a t e r i a l , s t i m u l a t e s t h e
p r o d u c t i o n of a n t i b o d i e s to t h e r e s p e c t i v e d r u g s . By
c o u p l i n g t h e d r u g at t h e a - p o s i t i o n w h e r e no s u b s t i -
t u e n t s a p p e a r in t h e p a r e n t d r u g s , t h e immunogen c o n -
j u g a t e is p r e p a r e d w i t h o u t m o d i f y i n g any f u n c t i o n a l
or d i s t i n g u i s h i n g g r o u p s on t h e d r u g s .
In a p r e f e r r e d e m b o d i m e n t , t h e p r e s e n t i n v e n t i o n
p r o v i d e s n o v e l i n t e r m e d i a t e s in t h e p r e p a r a t i o n o f
t h e a - s u b s t i t u t e d d r u g - c a r r i e r i m m u n o g e n s . A l s o
p r o v i d e d a r e an i m p r o v e d i m m u n o a s s a y m e t h o d a n d
r e a g e n t means f o r t h e d e t e r m i n a t i o n of t he d r u g s
w i t h t h e u se of t he n o v e l a n t i b o d i e s of t h e p r e s e n t
i n v e n t i o n . The p r e s e n t i n v e n t i o n a l s o p r o v i d e s
l a b e l e d c o n j u g a t e s f o r p a r t i c u l a r l y p r e f e r r e d e m b o d i -
m e n t s of s u c h i m m u n o a s s a y .
BRIEF DESCRIPTION OF THE DRAWINGS
F i g s . 1 and 2 i l l u s t r a t e p a r t i c u l a r s y n t h e t i c
s c h e m e s f o r p r e p a r i n g a - d e r i v a t i v e s of p r o c a i n a m i d e
and NAPA and f o r p r e p a r i n g a p a r t i c u l a r l y u s e f u l
i n t e r m e d i a t e f o r use in s u c h d e r i v a t i z a t i o n s of t h e
d r u g s .
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The p r e s e n t i n v e n t i o n , in a l l of i t s i n t e r r e l a t e d
e m b o d i m e n t s , is f o c u s e d on p r e p a r a t i o n of a - s u b s t i t u t e d d rug d e r i v a t i v e s w h i c h can t h e n be u s e d
to fo rm i m m u n o g e n s by c o u p l i n g them to c o n v e n t i o n a l
c a r r i e r m a t e r i a l s , and s u b s e q u e n t l y u s e d to o b t a i n
a n t i b o d i e s , or can be u s e d to f o rm l a b e l e d c o n j u g a t e s
w h i c h s e r v e as t he d e t e c t i o n r e a g e n t s in i m m u n o a s s a y s
f o r p r o c a i n a m i d e and NAPA.
The c h e m i c a l s t r u c t u r e s of t h e d r u g s , w i t h t h e
a- and S - p o s i t i o n s on t h e a m i d e s i d e c h a i n i n d i c a t e d ,
a r e r e p r e s e n t e d by the f o r m u l a :
w h e r e i n X is h y d r o g e n f o r p r o c a i n a m i d e and a c e t y l f o r
NAPA. The s y n t h e s i s of a - d e r i v a t i v e s can be a c h i e v e d
by v a r i a t i o n s in t he known s y n t h e s i s of t h e p a r e n t
d r u g s .
Wi th r e f e r e n c e to F i g . 1 of t he d r a w i n g s , a n
a p p r o p r i a t e l y f u n c t i o n a l i z e d ( g r o u p d e s i g n a t e d Z)
N , N - d i e t h y l e t h y l e n e d i a m i n e d e r i v a t i v e (1) i s b e n z o y l -
a t e d by r e a c t i o n w i t h 4 - n i t r o b e n z o y l c h l o r i d e to g i v e
n i t r o - s u b s t i t u t e d i n t e r m e d i a t e s ( 2 ) . R e d u c t i o n o f
t he n i t r o g r o u p p r o d u c e s t h e a - s u b s t i t u t e d p r o c a i n -
a m i d e d e r i v a t i v e s (3) w h i c h upon a c e t y l a t i o n y i e l d
t h e a - s u b s t i t u t e d NAPA d e r i v a t i v e s (4) [ M . Y a m a z o h i , e t a l , J. Pha rm. Soc. J a p a n 7 3 : 2 9 4 ( 1 9 5 3 ) ; R . B a l t z l y ,
e t a l , J. Am. Chem. Soc. 6 4 : 2 2 3 1 ( 1 9 4 1 ) ; and D . E . D r a y e r
et aZ, P r o c . Soc. Exp. B i o l . Med. 1 4 6 : 3 5 8 ( 1 9 7 4 ) ] .
A l t e r n a t i v e l y , t h e NAPA d e r i v a t i v e s (4) can b e
p r o d u c e d d i r e c t l y f rom (1) by r e a c t i o n w i t h 4 -
a c e t a m i d o b e n z o i c a c i d w h i c h has b e e n a c t i v a t e d w i t h
a c a r b o x y l - a c t i v a t i n g r e a g e n t such as t h o s e u s e d i n
amide b o n d - f o r m i n g r e a c t i o n s [ s e e "The P e p t i d e s " ,
v o l , 1, E . G r o s s and J . M e i e n h o f e r , e d s . , A c a d e m i c
P r e s s (New York 1979) pp. 66 et s e q ] .
The key i n t e r m e d i a t e in t h e p r e p a r a t i o n of t h e
a - s u b s t i t u t e d d e r i v a t i v e s is t h e p r i m a r y amine ( 1 ) .
The s y n t h e s i s of t h i s i n t e r m e d i a t e is o u t l i n e d i n
F i g . 2 of t h e d r a w i n g s b e g i n n i n g f rom t h e t e r m i n a l
o l e f i n ( 5 ) , n = 1 - 1 0 . The p r e c u r s o r s to t h e a a n d
β c a r b o n a toms in p r o c a i n a m i d e and NAPA (A) a r e t h e
c a r b o n a toms of t h e d o u b l e bond of ( 5 ) . The d o u b l e
bond is e p o x i d i z e d w i t h 3 - c h l o r o p e r b e n z o i c a c i d t o
g i v e o x i r a n e s (6) w h i c h r e a c t w i t h d i e t h y l a m i n e t o
p r o d u c e t h e amino a l c o h o l s ( 7 ) . R e a c t i o n of (7) w i t h
p h t h a l i m i d e and d i e t h y l a z o d i c a r b o x y l a t e l e a d s to t h e
p h t h a l i m i d o d e r i v a t i v e s (8) w h i c h upon t r e a t m e n t w i t h
h y d r a z i n e g i v e s t h e key p r i m a r y amine r e a g e n t ( 1 )
[ O . M i t s u n o b u , e t a l , J. Am. Chem. Soc. 9 4 : 6 7 9 ( 1 9 7 2 ) ] .
The r e s u l t i n g a - s u b s t i t u t e d p r o c a i n a m i d e and NAPA
d e r i v a t i v e s a r e of t h e f o r m u l a :
w h e r e i n X is as d e f i n e d a b o v e , n is an i n t e g e r f r o m
1 t h r o u g h 10, p r e f e r a b l y 1 t h r o u g h 6, and Z i s a f u n c t i o n a l g r o u p c a p a b l e of b e i n g c o u p l e d to i m m u n o -
g e n i c c a r r i e r m a t e r i a l s or l a b e l i n g r e a g e n t s . F u n c -
t i o n a l g r o u p Z is commonly a m i n o , c a r b o x y l , t h i o l ,
h y d r o x y l , or m a l e i m i d o . Such a - f u n c t i o n a l i z e d
d e r i v a t i v e s a r e p r e p a r e d f rom t h e a p p r o p r i a t e l y f u n c -
t i o n a l i z e d o l e f i n s ( 5 ) . For Z = h y d r o x y l ,
w - h y d r o x y o l e f i n s a r e c o m m e r c i a l l y a v a i l a b l e b a s e d o n
m e t a l h y d r i d e r e d u c t i o n of t he w - o l e f i n i c e s t e r s
[ R . M . S c a r b o r o u g h , et a l , J. Am. Chem. Soc. 1 0 2 :
3 9 0 4 ( 1 9 8 0 ) ] or by p a r t i a l r e d u c t i o n of t h e c o r r e s -
p o n d i n g w - h y d r o x y a c e t y l e n e s . For Z = a m i n o , t h e
c o r r e s p o n d i n g ω - o l e f i n s can be o b t a i n e d by r e a c t i o n
w i t h p h t h a l i m i d e and d i e t h y l a z o d i c a r b o x y l a t e f o l l o w e d
by r e a c t i o n w i t h h y d r a z i n e [ M i t s u n o b u s u p r a ] . D e r i v a -
t i v e s f o r Z = c a r b o x y l a r e o b t a i n e d f rom t h e c o r r e s -
p o n d i n g w - o l e f i n i c e s t e r s [ S c a r b o r o u g h , s u p r a ] . D e -
r i v a t i v e s w i t h Z = m a l e i m i d o can be o b t a i n e d by r e -
a c t i n g a - a m i n a t e d d e r i v a t i v e s w i t h N - s u c c i n i m i d y l
m - m a l e i m i d o b e n z o a t e [ T . K i t a g a w a , et aZ3 J. B i o c h e m .
7 9 : 2 3 3 ( 1 9 7 6 ) ] . F i n a l l y , d e r i v a t i v e s w i t h Z = t h i o
can be p r e p a r e d by r e a c t i o n of t he p r i m a r y amino d e -
r i v a t i v e w i t h SAMSA r e a g e n t [ I . M . K l o t z , et aZ3 A r c h .
B i o c h e m . B i o p h y s . 9 5 : 6 0 5 ( 1 9 6 2 ) ] .
I t w i l l be e v i d e n t to one of o r d i n a r y s k i l l i n
t h e a r t t h a t t h e a b o v e - d e s c r i b e d s y n t h e t i c s c h e m e s ,
or s i m p l e m o d i f i c a t i o n s t h e r e o f , can be a p p l i e d t o
t h e d e r i v a t i z a t i o n of o t h e r m o l e c u l e s , p a r t i c u l a r l y
d r u g s , w h i c h p o s s e s s t h e same amide s i d e c h a i n a s
p r o c a i n a m i d e and NAPA w i t h o u t d e p a r t i n g f rom t h e
i n v e n t i v e c o n c e p t p r e s e n t e d h e r e i n . The r e s u l t i n g
a - s u b s t i t u t e d d e r i v a t i v e s can t h e n of c o u r s e be u s e d
to c o u p l e to c a r r i e r m o l e c u l e s or l a b e l i n g r e a g e n t s
a s w i l l be d e s c r i b e d more f u l l y b e l o w . E x a m p l e s o f
s u c h o t h e r m o l e c u l e s to w h i c h t h e p r e s e n t i n v e n t i o n
can be a p p l i e d e q u i v a l e n t l y a r e m e t o c l o p r a m i d e
[Merck I n d e x , 9 t h ed. ( 1 9 7 6 ) p. 801] and d i b u c a i n e
[ i b i d , p. 3 9 9 ] .
I m m u n o g e n s
The a b o v e - d e s c r i b e d a - f u n c t i o n a l i z e d d e r i v a t i v e s
can be c o v a l e n t l y l i n k e d by any n u m b e r of c o n v e n t i o n a l
t e c h n i q u e s to i m m u n o g e n i c c a r r i e r m a t e r i a l s to y i e l d
i m m u n o g e n s c o m p r i s i n g one or more r e s i d u e s of t h e
f o r m u l a :
w h e r e i n X is as d e f i n e d a b o v e .
More p a r t i c u l a r l y s u c h i m m u n o g e n s w i l l have t h e
f o r m u l a :
w h e r e i n n is d e f i n e d a b o v e , Z' is t h e r e s i d u e f r o m
f u n c t i o n a l g r o u p Z r e m a i n i n g a f t e r t h e c o u p l i n g
r e a c t i o n , R' is a bond or an a p p r o p r i a t e l i n k i n g
g r o u p , C a r r i e r is an i m m u n o g e n i c c a r r i e r m a t e r i a l , and p i s t h e number of h a p t e n m o i e t i e s c o n j u g a t e d
to t h e c a r r i e r . The n u m b e r p is s o m e t i m e s r e f e r r e d
to as t h e e p i t o p i c d e n s i t y of t h e immunogen and i s
l i m i t e d o n l y by t he number of a v a i l a b l e c o u p l i n g s i t e s on t h e c a r r i e r m o l e c u l e . H o w e v e r , in t h e u s u a l
s i t u a t i o n w h e r e t he c a r r i e r i s a p r o t e i n such a s
a l b u m i n , p w i l l be on t he a v e r a g e f rom 1 to a b o u t 5 0 ,
more n o r m a l l y f rom 1 to a b o u t 25. O p t i m a l e p i t o p i c
d e n s i t i e s in such u s u a l c a s e , c o n s i d e r i n g t h e e a s e a n d
r e p r o d u c i b i l i t y of s y n t h e s i s of t h e immunogen and a n t i -
body r e s p o n s e , f a l l b e t w e e n a b o u t 2 and a b o u t 2 0 ,
more u s u a l l y b e t w e e n 5 and 1 5 .
The i m m u n o g e n i c c a r r i e r m a t e r i a l can be s e l e c t e d
f rom any of t h o s e c o n v e n t i o n a l l y known. In m o s t
c a s e s , t h e c a r r i e r w i l l be a p r o t e i n or p o l y p e p t i d e ,
a l t h o u g h o t h e r m a t e r i a l s s u c h as c a r b o h y d r a t e s ,
p o l y s a c c h a r i d e s , l i p o p o l y s a c c h a r i d e s , n u c l e i c a c i d s
and t h e l i k e of s u f f i c i e n t s i z e and i m m u n o g e n i c i t y
can l i k e w i s e be u s e d . For t h e mos t p a r t , i m m u n o g e n i c
p r o t e i n s and p o l y p e p t i d e s w i l l h ave m o l e c u l a r w e i g h t s
b e t w e e n 5 , 0 0 0 and 1 0 , 0 0 0 , 0 0 0 , p r e f e r a b l y g r e a t e r t h a n
1 5 , 0 0 0 , and more u s u a l l y g r e a t e r t h a n 5 0 , 0 0 0 . G e n e r a l l y ,
p r o t e i n s t a k e n f rom one a n i m a l s p e c i e s w i l l be i m m u n o g e n i c
when i n t r o d u c e d i n t o t he b l o o d s t r e a m of a n o t h e r s p e c i e s .
P a r t i c u l a r l y u s e f u l p r o t e i n s a r e a l b u m i n s , g l o b u l i n s ,
e n z y m e s , h e m o c y a n i n s , g l u t e l i n s , p r o t e i n s h a v i n g s i g n i -
f i c a n t n o n p r o t e i n a c e o u s c o n s t i t u e n t s , e . g . , g l y c o -
p r o t e i n s , and t he l i k e . The a l b u m i n s and g l o b u l i n s o f
m o l e c u l a r w e i g h t b e t w e e n 3 0 , 0 0 0 and 2 0 0 , 0 0 0 a r e p a r t i c u -
l a r l y p r e f e r r e d . F u r t h e r r e f e r e n c e f o r t h e s t a t e - o f -
t h e - a r t c o n c e r n i n g c o n v e n t i o n a l i m m u n o g e n i c c a r r i e r
m a t e r i a l s and t e c h n i q u e s f o r c o u p l i n g h a p t e n s t h e r e t o
may be had to t he f o l l o w i n g : P a r k e r , R a d i o i m m u n o a s s a y
o f B i o l o g i c a l y A c t i v e C o m p o u n d s , P r e n t i c e - H a l l ( E n g l e - wood C l i f f s , New J e r s e y USA, 1 9 7 6 ) ; B u t l e r J. ImmunoZ.
Me th . 7 : 1 - 2 4 ( 1 9 7 5 ) ; W e i n r y b and S h r o f f , Drug M e t a b .
Rev. 1 0 : 2 7 1 - 2 8 3 ( 1 9 7 5 ) ; B r o u g h t o n and S t r o n g , C Z i n .
Chem. 2 2 : 7 2 6 - 7 3 2 ( 1 9 7 6 ) ; and P l a y f a i r et a l , Br. Med.
BuZZ. 3 0 : 2 4 - 3 1 ( 1 9 7 4 ) .
R e s i d u e Z' w i l l v a r y a c c o r d i n g to t h e f u n c t i o n a l
g r o u p Z in a - d e r i v a t i v e (B) u s e d , and p r e f e r a b l y w i l l
be i m i n o , c a r b o x y l , s u l f o , or o x y .
P r e f e r a b l y , g r o u p R' is a bond to an a p p r o p r i a t e f u n c t i o n a l g r o u p in t he c a r r i e r m a t e r i a l . A p p r o p r i a t e
a - s u b s t i t u t e d d rug d e r i v a t i v e s a r e c o u p l a b l e to i m m u n o -
g e n i c c a r r i e r m a t e r i a l s a c c o r d i n g to w e l l known t e c h -
n i q u e s . For e x a m p l e , t h e amino d e r i v a t i v e s can b e
a t t a c h e d d i r e c t l y to t h e c a r r i e r by t h e f o l l o w i n g
m e a n s . The amino g r o u p of t h e d r u g m o i e t y can b e
a t t a c h e d to a m i n o - c o n t a i n i n g c a r r i e r s ( e . g . , p r o t e i n
or p o l y p e p t i d e c a r r i e r s ) by t o l u e n e - 2 , 4 - d i i s o c y a n a t e
[ A . F . S c h i c k and S . J . S i n g e r , J. B ioZ . Chem. 2 3 6 : 2 4 7 7
( 1 9 6 1 ) ] ; 4 , 4 ' - d i f l u o r o - 3 , 3 ' - d i n i t r o d i p h e n y l s u l f o n e
[ P . S . C u a t r e c a s a s , et aZ3 J. B ioZ. Chem. 2 4 4 : 4 0 6
( 1 9 6 9 ) ] ; g l u t a r a l d e h y d e [ L . A . F r o h m a n , et a l , E n d o -
c r i n o l . 8 7 : 1 0 5 5 ( 1 9 7 0 ) ] ; b i s - i m i d a t e s [A. D u t t o n ,
e t aZ, B i o c h e m . B i o p h y s . Res . Comm. 2 3 : 7 3 0 ( 1 9 6 6 ) ] ;
and c h l o r o t r i a z i n e [ T . L a n g , e t a l , J . C . S . P e r k i n 4 :
2189 ( 1 9 7 7 ) ] . A l s o , t h e amino d e r i v a t i v e s can b e
c o u p l e d to c a r b o x y l - b e a r i n g c a r r i e r s ( e . g . , a g a i n ,
p r o t e i n or p o l y p e p t i d e c a r r i e r s ) by common p e p t i d e
b o n d - f o r m i n g r e a c t i o n s by means of m i x e d a n h y d r i d e s ,
a c t i v a t e d e s t e r s , a c y l a z i d e f o r m a t i o n , c a r b o d i i m i d e s ,
e t c . , see P e p t i d e s , ed. Goodman and M e i n h o f e r , J o h n
W i l e y S o n s (New Y o r k , 1977) p. 6 et s e q , and T h e
P e p t i d e s , A n a l y s i s , S y n t h e s i s , B i o l o g y , V o l . 1 ,
A c a d e m i c P r e s s (New York 1 9 7 9 ) . The same m e t h o d s
a p p l y l i k e w i s e f o r a t t a c h i n g c a r b o x y l a t e d d e r i v a t i v e s
to a m i n o - b e a r i n g c a r r i e r s .
T h i o l a t e d d e r i v a t i v e s can be a t t a c h e d to t h i o l -
c o n t a i n i n g p o l y m e r s (IgG or t h i o l a t e d p r o t e i n s ) by t h e
d i s u l f i d e e x c h a n g e p r o c e d u r e [J . M a r t i n , et aZ3 B i o c h e m .
2 0 : 4 2 2 9 ( 1 9 8 1 ) ] . A l t e r n a t e l y , an a m i n o - c o n t a i n i n g
p o l y m e r can be r e a c t e d w i t h t he r e a g e n t MBS and t h e
p r o d u c t c o u p l e d to t h i o l - c o n t a i n i n g d e r i v a t i v e s b y t h e p r o c e s s d e s c r i b e d by T. K i t a g a w a and T. A i k a w a , J. B i o c h e m . 7 9 : 2 3 3 ( 1 9 7 6 ) . M a l e i m i d e d e r i v a t i v e s
can s i m i l a r l y be c o u p l e d to t h i o l - c o n t a i n i n g c a r r i e r s
[ i b i d ] . H y d r o x y d e r i v a t i v e s can be a t t a c h e d t o c a r r i e r s u s i n g t r i c h l o r o t r i a z i n e [G.Kay and E . M . C r o o k ,
N a t u r e 2 1 8 : 5 1 4 ( 1 9 6 7 ) ] .
A m u l t i t u d e of o t h e r c o u p l i n g t e c h n i q u e s a r e a v a i l a b l e to t h o s e of o r d i n a r y s k i l l in t h e a r t f o r
j o i n i n g t he v a r i o u s a - s u b s t i t u t e d d e r i v a t i v e s of t h e
p r e s e n t i n v e n t i o n w i t h c o n v e n t i o n a l i m m u n o g e n i c
c a r r i e r m a t e r i a l s . For e x a m p l e , one s k i l l e d in t h e
a r t can r e a c t an a p p r o p r i a t e a - s u b s t i t u t e d d e r i v a t i v e
(B) w i t h a b i f u n c t i o n a l r e a g e n t s u c h t h a t one e n d
t h e r e o f c o v a l e n t l y c o u p l e s w i t h t h e d e r i v a t i v e a n d
t h e o t h e r end has a f u n c t i o n a l g r o u p f o r c o u p l i n g t o
c a r r i e r s as d e s c r i b e d above ( e . g . , a m i n o , c a r b o x y l ,
t h i o l , h y d r o x y l , and m a l e i m i d o ) . For e x a m p l e , b i -
f u n c t i o n a l c o u p l i n g r e a g e n t s a re w e l l known f o r
c o u p l i n g amine d e r i v a t i v e s to a m i n o - c o n t a i n i n g
m a c r o m o l e c u l e s ( e . g . , p r o t e i n s and p o l y p e p t i d e s ) ,
i n c l u d i n g b i s - i m i d a t e s , b i s - i s o c y a n a t e s , a n d
g l u t a r a l d e h y d e [ I m m u n o c h e m . 6 : 5 3 ( 1 9 6 9 ) ] . O t h e r
u s e f u l c o u p l i n g r e a c t i o n s a r e t h o r o u g h l y d i s c u s s e d
in t h e l i t e r a t u r e [ s e e K o p p l e , P e p t i d e s and A m i n o
A c i d s , W. A. B e n j a m i n , I n c . (New York 1 9 6 6 ) ; Lowe
and Dean , A f f i n i t y C h r o m a t o g r a p h y , J o h n W i l e y & S o n s
(New York 1 9 7 4 ) ; Means and F e e n e y , C h e m i c a Z M o d i f i c a -
t i o n o f P r o t e i n s , H o l d e n - D a y (San F r a n c i s c o 1 9 7 1 ) ;
and G l a z e r e t a l , C h e m i c a l M o d i f i c a t i o n o f P r o t e i n s ,
E l s e v i e r (New York 1 9 7 5 ) ] .
As a r e s u l t , l i n k i n g g r o u p R' and r e s i d u e Z' c a n
be c o n s i d e r e d as a s i n g l e b r i d g e g r o u p R, and t h u s
t he i m m u n o g e n s have t he g e n e r a l f o r m u l a :
w h e r e i n R c o m p r i s e s t h e r e s i d u e - Z ' - R ' - and may v a r y
w i d e l y , i t s e x a c t c h e m i c a l s t r u c t u r e no t b e i n g c r i t i c a l
so l o n g as i t s e r v e s t h e p u r p o s e of l i n k i n g t h e h a p t e n
r e s i d u e w i t h o u t i n t e r f e r i n g w i t h t h e i m m u n o g e n i c
p r o p e r t i e s of t he r e s u l t i n g i m m u n o g e n . For e x a m p l e , R can c o m p r i s e l i n e a r or b r a n c h e d a l k y l e n e c o m p r i s i n g
f rom 1 to as many as 15, more u s u a l l y 10 or l e s s ,
and n o r m a l l y l e s s t h a n 6, c a r b o n a t oms ( e . g . , m e t h y -
l e n e , e t h y l e n e , n - p r o p y l e n e , i s o - p r o p y l e n e , n -
b u t y l e n e , and so f o r t h ) . In a d d i t i o n , such a l k y l e n e
can c o n t a i n o t h e r s u b s t i t u e n t g r o u p s such as c y a n o , amino ( i n c l u d i n g s u b s t i t u t e d a m i n o ) , a c y l a m i n o , h a l o -
gen , t h i o l , h y d r o x y l , c a r b o n y l g r o u p s , c a r b o x y l
( i n c l u d i n g s u b s t i t u t e d c a r b o x y l s s u c h as e s t e r s ,
a m i d e s , and s u b s t i t u t e d a m i d e s ) . B r i d g e g r o u p R c a n
a l s o c o n t a i n or c o n s i s t of s u b s t i t u t e d or u n s u b s t i t u t e d
a r y l , a r a l k y l , or h e t e r o a r y l g r o u p s ( e . g . , p h e n y l e n e ,
p h e n e t h y l e n e , and so f o r t h ) . A d d i t i o n a l l y , s u c h
l i n k a g e s can c o n t a i n one or more h e t e r o a t o m s s e l e c t e d
from n i t r o g e n , s u l f u r and o x y g e n in t he form of e t h e r ,
e s t e r , a m i d o , a m i n o , t h i o e t h e r , a m i d i n o , s u l f o n e , o r
s u l f o x i d e . P r e f e r a b l y R w i l l be a c h a i n , u s u a l l y
a l i p h a t i c c o m p r i s i n g b e t w e e n 1 and a b o u t 20 a t o m s ,
more u s u a l l y b e t w e e n 1 and 10, e x c l u d i n g h y d r o g e n , of w h i c h b e t w e e n 0 and 5 a r e h e t e r o a t o m s s e l e c t e d
f rom n i t r o g e n , o x y g e n , and s u l f u r . A c c o r d i n g l y , t h e
c h o i c e of b r i d g e g r o u p R is n o t c r i t i c a l to t h e
p r e s e n t i n v e n t i o n and may be v a r i e d w i d e l y by one o f
o r d i n a r y s k i l l in t he a r t .
P a r t i c u l a r l y p r e f e r r e d a r e t h e i m m u n o g e n s o f
t h e f o r m u l a :
w h e r e i n X is as d e f i n e d a b o v e , Y i s an amide g r o u p , i . e . , -NHCO-, C a r r i e r is an i m m u n o g e n i c p r o t e i n o r
p o l y p e p t i d e , n is an i n t e g e r f rom 1 t h r o u g h 1 0 ,
p r e f e r a b l y f rom 1 t h r o u g h 6, and p is on t h e a v e r a g e f rom 1 to t h e n u m b e r of a v a i l a b l e a m i d e c o u p l i n g s i t e s
on t h e c a r r i e r m a t e r i a l and p r e f e r a b l y is as d e f i n e d
a b o v e . The a m i d e c o u p l i n g g r o u p can be o r i e n t e d i n
e i t h e r of t h e two p o s s i b l e w a y s , w i t h t h e n i t r o g e n
atom in t h e amide g r o u p b e i n g f rom c a r r i e r a m i n o
g r o u p s and t h e c a r b o n atom b e i n g f rom an a p p r o p r i a t e a - s u b s t i t u t e d d e r i v a t i v e ( e . g . , a c a r b o x y l i c a c i d ) ,
w i t h p t h e n r e p r e s e n t i n g t h e a v e r a g e number o f
c o u p l e d amino g r o u p s in t h e c a r r i e r (and p r e f e r a b l y
is as d e f i n e d a b o v e ) , or w i t h t h e n i t r o g e n a t o m
b e i n g f rom an a p p r o p r i a t e a - s u b s t i t u t e d d e r i v a t i v e
( e . g . , an amino d e r i v a t i v e ) and t h e c a r b o n atom b e i n g
f rom c a r r i e r c a r b o x y l g r o u p s , w i t h p t h e n r e p r e s e n t -
ing t h e a v e r a g e number of c o u p l e d c a r b o x y l g r o u p s i n
t h e c a r r i e r ( and p r e f e r a b l y is a g a i n as d e f i n e d a b o v e ) .
M S - 1 2 7 0
A n t i b o d i e s
P r e p a r a t i o n of s p e c i f i c a n t i b o d i e s u s i n g t h e
p r e s e n t i m m u n o g e n c o n j u g a t e s may f o l l o w any c o n v e n -
t i o n a l t e c h n i q u e . N u m e r o u s t e x t s a r e a v a i l a b l e d e s -
c r i b i n g t h e f u n d a m e n t a l a s p e c t s 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 , f o r e x a m p l e r e f e r e n c e may be made t o
P a r k e r , R a d i o i m m u n o a s s a y of B i o l o g i c a l l y A c t i v e Com-
p o u n d s , P r e n t i c e - H a l l ( E n g l e w o o d C l i f f s , New J e r s e y
USA, 1 9 7 6 ) . In the u s u a l c a s e , a h o s t a n i m a l s u c h
as a r a b b i t , g o a t , m o u s e , g u i n e a p i g , or h o r s e i s
i n j e c t e d a t one or more of a v a r i e t y of s i t e s w i t h
the i m m u n o g e n c o n j u g a t e , n o r m a l l y in m i x t u r e w i t h a n
a d j u v a n t . F u r t h e r i n j e c t i o n s a r e made at t he s a m e
s i t e or d i f f e r e n t s i t e s a t r e g u l a r or i r r e g u l a r i n t e r -
v a l s t h e r e a f t e r w i t h b l e e d i n g s b e i n g t a k e n to a s s e s s
a n t i b o d y t i t e r u n t i l i t is d e t e r m i n e d t h a t o p t i m a l
t i t e r has b e e n r e a c h e d . The h o s t a n i m a l is b l e d t o
y i e l d a s u i t a b l e v o l u m e of s p e c i f i c a n t i s e r u m . W h e r e
d e s i r a b l e , p u r i f i c a t i o n s t e p s may be t a k e n to r e m o v e
u n d e s i r e d m a t e r i a l such as n o n s p e c i f i c a n t i b o d i e s
b e f o r e t h e a n t i s e r u m is c o n s i d e r e d s u i t a b l e f o r u s e
in p e r f o r m i n g a c t u a l a s s a y s .
The a n t i b o d i e s can a l s o be o b t a i n e d by s o m a t i c
c e l l h y b r i d i z a t i o n t e c h n i q u e s , s u c h a n t i b o d i e s b e i n g
commonly r e f e r r e d to as m o n o c l o n a l a n t i b o d i e s . R e -
v i e w s of s u c h m o n o c l o n a l a n t i b o d y t e c h n i q u e s a r e
f o u n d in L y m p h o c y t e H y b r z d o m a s , ed. M e l c h e r s e t a l ,
S p r i n g e r - V e r l a g (New York 1 9 7 8 ) , N a t u r e 2 6 6 : 4 9 5 ( 1 9 7 7 ) ,
S c i e n c e 2 0 8 : 6 9 2 ( 1 9 8 0 ) , and M e t h o d s in E n z y m o l o g y 73
( P a r t B ) : 3 - 4 6 ( 1 9 8 1 ) .
I m m u n o a s s a y T e c h n i q u e s
The a n t i b o d i e s p r e p a r e d f rom t h e i m m u n o g e n s of t he p r e s e n t i n v e n t i o n can be u s e d in any immuno-
a s s a y m e t h o d , and t h e c o r r e s p o n d i n g r e a g e n t s y s t e m , f o r d e t e r m i n i n g p r o c a i n a m i d e and NAPA, i n c l u d i n g
a g g l u t i n a t i o n t e c h n i q u e s , r a d i o i m m u n o a s s a y s , h e t e r o -
g e n e o u s enzyme i m m u n o a s s a y s ( c f . U .S . P a t . No.
3 , 6 5 4 , 0 9 0 ) , h e t e r o g e n e o u s f l u o r e s c e n t i m m u n o a s s a y s
( c f . U.S . P a t s . Nos. 4 , 2 0 1 , 7 6 3 ; 4 , 1 7 1 , 3 1 1 ; 4 , 1 3 3 , 6 3 9 and 3 , 9 9 2 , 6 3 1 ) , and h o m o g e n e o u s ( s e p a r a t i o n - f r e e )
i m m u n o a s s a y s . The l a t t e r - m o s t a r e p a r t i c u l a r l y
p r e f e r r e d and i n c l u d e s u c h t e c h n i q u e s as f l u o r e s c e n c e
q u e n c h i n g or e n h a n c e m e n t ( c f . U.S. P a t . No. 4 , 1 6 0 , 0 1 6 ) , f l u o r e s c e n c e p o l a r i z a t i o n ( c f . J. Exp. Med. 1 2 2 : 1 0 2 9
( 1 9 6 5 ) , enzyme s u b s t r a t e - l a b e l e d i m m u n o a s s a y ( c f .
U .S . P a t . No. 4 , 2 7 9 , 9 9 2 and U.K. P a t . S p e c . 1 , 5 5 2 , 6 0 7 ) ,
p r o s t h e t i c g r o u p - l a b e l e d i m m u n o a s s a y ( c f . U .S . P a t .
No. 4 , 2 3 8 , 5 6 5 ) , enzyme m o d u l a t o r - l a b e l e d i m m u n o a s s a y ,
e . g . , u s i n g i n h i b i t o r l a b e l s ( c f . U .S . P a t s . N o s .
4 , 1 3 4 , 7 9 2 and 4 , 2 7 3 , 8 6 6 ) , e n z y m e - l a b e l e d i m m u n o a s s a y
( c f . U .S . P a t . No. 3 , 8 1 7 , 8 3 7 ) , e n e r g y t r a n s f e r
i m m u n o a s s a y ( c f . U .S . P a t . No. 3 , 9 9 6 , 3 4 5 ) , and d o u b l e
a n t i b o d y s t e r i c h i n d r a n c e i m m u n o a s s a y ( c f . U.S . P a t s .
Nos. 3 , 9 3 5 , 0 7 4 and 3 , 9 9 8 , 9 4 3 ) . H o m o g e n e o u s i m m u n o -
a s s a y s a re t y p i c a l l y p e r f o r m e d by s e t t i n g up c o m p e t i -
t i o n b e t w e e n t h e a n a l y t e and t h e l a b e l e d c o n j u g a t e o f
t h e d rug f o r b i n d i n g to a n t i b o d y and a re c h a r a c t e r i z e d
by t h e f a c t t h a t t h e d e t e c t a b l e l a b e l p r o p e r t y i s
a l t e r e d when t h e l a b e l e d c o n j u g a t e i s b o u n d by a n t i b o d y .
M o r e o v e r , t h e a - s u b s t i t u t e d d e r i v a t i v e s of t h e
p r e s e n t i n v e n t i o n can be u s e d to p r e p a r e t h e l a b e l e d
c o n j u g a t e s n e e d e d to p e r f o r m t h e v a r i o u s i m m u n o a s s a y s
d e s c r i b e d a b o v e . A p p r o p r i a t e d e r i v a t i v e s can b e
r a d i o - l a b e l e d or l a b e l e d w i t h f l u o r e s c e n t m o i e t i e s i n
a c c o r d a n c e w i t h s t a n d a r d m e t h o d s . L i k e w i s e t h e
a p p r o p r i a t e l a b e l i n g m o i e t y f o r t h e p r e f e r r e d h o m o -
g e n e o u s t e c h n i q u e s , e . g . , an enzyme s u b s t r a t e , a
p r o s t h e t i c g r o u p , an enzyme m o d u l a t o r , or an e n z y m e
( w h i c h is a p r o t e i n and can be c o u p l e d s i m i l a r l y t o
t h e i m m u n o g e n i c c a r r i e r as d e s c r i b e d a b o v e ) can b e
c o u p l e d to t h e a - s u b s t i t u t e d d e r i v a t i v e s to y i e l d
l a b e l e d c o n j u g a t e s .
P a r t i c u l a r l y p r e f e r r e d l a b e l e d c o n j u g a t e s a r e
t h e β - g a l a c t o s y l - u m b e l l i f e r o n e - p r o c a i n a m i d e / N A P A
c o n j u g a t e s of t h e f o r m u l a :
w h e r e i n - (CO)βGU i s
and n is an i n t e g e r f rom 1 t h r o u g h 10. Such c o n j u -
g a t e s a re p r e p a r e d by s t a n d a r d p e p t i d e c o n d e n s a t i o n s
of β - g a l a c t o s y l - u m b e l l i f e r o n e c a r b o x y l i c a c i d ( U . S .
P a t . No. 4 , 2 2 6 , 9 7 8 ) w i t h t h e a p p r o p r i a t e a - a m i n o
d e r i v a t i v e .
The r e a g e n t s y s t e m or means of t h e p r e s e n t i n v e n -
t i o n c o m p r i s e s a l l of t h e e s s e n t i a l c h e m i c a l e l e m e n t s
r e q u i r e d to c o n d u c t a d e s i r e d p r o c a i n a m i d e or NAPA
i m m u n o a s s a y m e t h o d e n c o m p a s s e d by t h e p r e s e n t i n v e n -
t i o n . The r e a g e n t s y s t e m is p r e s e n t e d in a c o m m e r -
c i a l l y p a c k a g e d f o r m , as a c o m p o s i t i o n or a d m i x t u r e
w h e r e t h e c o m p a t i b i l i t y of t h e r e a g e n t s w i l l a l l o w , in a t e s t d e v i c e c o n f i g u r a t i o n , or as a t e s t k i t ,
i . e . , a p a c k a g e d c o m b i n a t i o n of one or more c o n t a i n e r s
h o l d i n g t h e n e c e s s a r y r e a g e n t s . I n c l u d e d in t h e
r e a g e n t s y s t e m a r e t h e r e a g e n t s a p p r o p r i a t e f o r t h e
b i n d i n g r e a c t i o n s y s t e m d e s i r e d , e . g . , an a n t i b o d y and l a b e l e d c o n j u g a t e of t h e p r e s e n t i n v e n t i o n . Of
c o u r s e , t h e r e a g e n t s y s t e m can i n c l u d e o t h e r m a t e r i a l s
as a r e known in t h e a r t and w h i c h may be d e s i r a b l e
f rom a c o m m e r c i a l and u s e r s t a n d p o i n t , such as b u f f e r s ,
d i l u e n t s , s t a n d a r d s , and so f o r t h . P a r t i c u l a r l y
p r e f e r r e d is a t e s t k i t f o r t h e h o m o g e n e o u s c o m p e t i t i v e
b i n d i n g i m m u n o a s s a y of t h e p r e s e n t i n v e n t i o n c o m p r i s -
ing (a) an a n t i b o d y of t h e p r e s e n t i n v e n t i o n and ( b )
a l a b e l e d c o n j u g a t e w h i c h has a d e t e c t a b l e p r o p e r t y w h i c h i s a l t e r e d when b o u n d w i t h t h e a n t i b o d y . A l s o
p r e f e r r e d is a t e s t d e v i c e c o m p r i s i n g a r e a g e n t c o m p o - s i t i o n i n c l u d i n g an a n t i b o d y of t h e p r e s e n t i n v e n t i o n
and a l a b e l e d c o n j u g a t e w h i c h has a d e t e c t a b l e
p r o p e r t y w h i c h is a l t e r e d when b o u n d w i t h t he a n t i b o d y ,
and a s o l i d c a r r i e r member i n c o r p o r a t e d w i t h t h e
r e a g e n t c o m p o s i t i o n . The v a r i o u s f o r m s of such t e s t
d e v i c e a r e d e s c r i b e d in U . S . P a t . A p p l n . S e r . N o .
2 0 2 , 3 7 8 , f i l e d O c t o b e r 30, 1980 , w h i c h is i n c o r p o r a t e d
h e r e i n by r e f e r e n c e and w h i c h has p u b l i s h e d as E u r o -
p e a n p a t e n t a p p l i c a t i o n 5 1 , 2 1 3 . The s p e c i f i c l a b e l
u s e d in t h e p r e f e r r e d t e s t k i t a n d t e s t d e v i c e w i l l
d e p e n d on t h e t e c h n i q u e f o l l o w e d , as d e s c r i b e d h e r e i n -
a b o v e .
The p r e s e n t i n v e n t i o n w i l l now be i l l u s t r a t e d , bu t is n o t i n t e n d e d to be l i m i t e d , by t h e f o l l o w i n g
e x a m p l e s :
EXAMPLES
R e a g e n t s
I t a l i c i z e d n u m b e r s a p p e a r i n g a f t e r c h e m i c a l n a m e s r e f e r to t h e s t r u c t u r a l f o r m u l a e i d e n t i f i e d in t h e
t e x t a b o v e a n d / o r in t h e d r a w i n g s .
A. P r e p a r a t i o n of d r u g d e r i v a t i v e
N - ( 1 - H e x e n - 6 - y l ) p h t h a l i m i d e , ( 5 ) , Z = p h t h a l i m i d o , n = 4 .
To a s t i r r e d s o l u t i o n of 1 - h e x e n e - 6 - o l [ R . E . L y l e ,
et aZ3 J. Org. Chem. 2 1 : 6 1 ( 1 9 5 6 ) in 1 l i t e r (L) o f
e t h e r c o n t a i n i n g 40 .5 g rams (g) [0 .4 m o l e s ) ( m o l ) ] o f
t r i e t h y l a m i n e was added d r o p w i s e a s o l u t i o n of 4 5 . 8 g (0 .4 mol) of m e t h a n e s u l f o n y l c h l o r i d e in 100 m i l l i l i t e r s
(mL) of e t h e r . A f t e r 3 h o u r s at room t e m p e r a t u r e , t h e
m i x t u r e was f i l t e r e d and e v a p o r a t e d to g i v e an o i l .
I t was c o m b i n e d w i t h 600 mL of dry d i m e t h y l f o r m a m i d e
(DMF) and 75 g ( 0 . 4 mol) of p o t a s s i u m p h t h a l i m i d e a n d
h e a t e d at 100°C f o r 12 h o u r s . The s o l v e n t w a s r e m o v e d
u n d e r vacuum and t he r e s i d u e p a r t i t i o n e d b e t w e e n e t h y l
a c e t a t e and H20. The o r g a n i c p h a s e was d r i e d o v e r
a n h y d r o u s m a g n e s i u m s u l f a t e (MgSO)4, f i l t e r e d , a n d
e v a p o r a t e d to l e a v e 80 g of an o i l y s o l i d . I t w a s
t a k e n up in h e x a n e , f i l t e r e d , and the f i l t r a t e f r a c -
t i o n a l l y d i s t i l l e d to g i v e 65 g (75% y i e l d ) of N-
( 1 - h e x e n - 6 - y l ) p h t h a l i m i d e as a c l e a r o i l , b o i l i n g
p o i n t (bp) 1 2 8 - 1 3 5 ° C ( 0 . 0 1 t o r r ) .
N - ( 5 - E p o x y h e x y l ) p h t h a l i m i d e , ( 6 ) , Z = p h t h a l i m i d o , n = 4 .
A s o l u t i o n of 2 .3 g [10 m i l l i m o l e s (mmol) ] of t h e
a b o v e p h t h a l i m i d e in 50 mL of m e t h y l e n e c h l o r i d e
(CH2Cl2) was s t i r r e d w i t h 1 . 2 9 g (15 mmol) of s o d i u m
b i c a r b o n a t e (NaHC03) w h i l e a s o l u t i o n of 2 .6 g ( 1 5
mmol) of 3 - c h l o r o p e r b e n z o i c a c i d in 25 mL of CH2C12 w a s
a d d e d . A f t e r s t i r r i n g f o r 3 h o u r s a t room t e m p e r a t u r e ,
e x c e s s p e r a c i d was d e s t r o y e d by t h e a d d i t i o n of 20 mL
of 10% s o d i u m s u l f i t e (Na2S03) s o l u t i o n . The o r g a n i c
p h a s e was s e p a r a t e d , w a s h e d w i t h s a t u r a t e d NaHCO3 s o l u -
t i o n , w a s h e d w i t h s a t u r a t e d s o d i u m c h l o r i d e (NaCl) s o l u -
t i o n , t h e n d r i e d o v e r a n h y d r o u s MgS04. I t was f i l t e r e d
and e v a p o r a t e d l e a v i n g a r e s i d u e w h i c h was f r a c t i o n a l l y
d i s t i l l e d to g i v e 1 .6 g (64% y i e l d ) of t h e e p o x i d e a s
an o i l , bp 1 5 6 - 1 6 0 ° C ( 0 . 0 1 t o r r ) .
A n a l y s i s : C a l c u l a t e d f o r C14H15NO3: C, 6 8 . 5 5 ; H, 6 . 1 6 ; N, 5 . 7 1
F o u n d : C, 6 8 . 8 3 ; H, 6 . 2 7 ; N, 5 . 7 0
N - ( 6 - D i e t h y l a m i n o - 5 - h y d r o x y h e x y l ) p h t h a l i m i d e , ( 7 ) , Z = p h t h a l i m i d o , n = 4 .
A m i x t u r e of 24 .5 g ( 0 . 1 mol) of t h e p r e c e e d i n g
e p o x i d e , 22 g ( 0 . 3 mol) of d i e t h y l a m i n e , and 50 mL o f
e t h a n o l was h e a t e d a t 100°C in a s t e e l a u t o c l a v e f o r
7 h o u r s . When c o o l , t h e a u t o c l a v e was o p e n e d and t h e
c o n t e n t s r e m o v e d and e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e .
The r e s i d u e was p a r t i t i o n e d b e t w e e n e t h e r and 10%
h y d r o c h l o r i c a c i d . The a q u e o u s p h a s e was s e p a r a t e d ,
made b a s i c w i t h s o d i u m h y d r o x i d e (NaOH) s o l u t i o n , a n d
e x t r a c t e d w i t h e t h e r . Remova l of t he e t h e r l e f t a n
o i l y r e d i s u e w h i c h was f r a c t i o n a l l y d i s t i l l e d to g i v e
1 3 . 6 g (43% y i e l d ) of t he p h t h a l i m i d e as a c l e a r o i l ,
bp 1 8 0 - 1 8 5 ° C ( 0 . 1 t o r r ) .
A n a l y s i s : C a l c u l a t e d f o r C 1 8 H 2 6 N 2 O 3 : C, 6 7 , 8 9 ; H, 8 . 2 3 ; N, 8 . 8 0
F o u n d : C, 6 8 . 0 9 ; H, 8 . 3 0 ; N, 9 . 4 2
6 - A m i n o - l - d i e t h y l a m i n o - 2 - h y d r o x y h e x a n e , ( 7 ) , Z = a m i n o , n = 4 .
The p r e c e e d i n g p h t h a l i m i d e ( 9 . 2 1 g, 29 mmol) w a s d i s s o l v e d in 150 mL of m e t h a n o l c o n t a i n i n g 1 .8 g ( 3 6
mmol) of h y d r a z i n e h y d r a t e and r e f l u x e d f o r 3 h o u r s .
The r e a c t i o n was c o o l e d , f i l t e r e d , and t h e f i l t r a t e
f r a c t i o n a l l y d i s t i l l e d to g i v e 4 . 2 4 g (78% y i e l d ) o f
t he d i a m i n e as a c l e a r o i l , bp 9 2 - 9 6 ° C ( 0 . 0 1 t o r r ) .
A n a l y s i s : C a l c u l a t e d f o r C10H24N2O: C, 6 3 . 7 8 ; H, 1 2 . 8 5 ; N, 1 4 , 8 8
6 - ( t e r t - B u t y l o x y c a r b o n y l a m m o ) - 1 - d i e t h y l a m i n o - 2 - ( N - p h t h a l i m i d o ) h e x a n e , ( 8 ) , Z = p r o t e c t e d a m i n o , n = 4 .
A m i x t u r e of 4 g (21 mmol) of t h e p r e c e e d i n g
d i a m i n e , 4 . 6 g (21 mmol) of d i - t e r t - b u t y l d i c a r b o n a t e ,
and 100 mL of d ry CH2C12 was s t i r r e d f o r 12 h o u r s a t
room t e m p e r a t u r e . S o l v e n t and v o l a t i l e b y - p r o d u c t s
we re r e m o v e d by e v a p o r a t i o n u n d e r r e d u c e d p r e s s u r e ,
l e a v i n g 6 g (100% y i e l d ) of 6 - ( t e r t - b u t y l o x y c a r b o n y l a m i n o )
- 1 - d i e t h y l a m i n o - 2 - h y d r o x y h e x a n e as an o i l w h i c h w a s
n o t f u r t h e r p u r i f i e d . I t was d i s s o l v e d in 100 mL o f
d r y t e t r a h y d r o f u r a n (THF) a l o n g w i t h 3 .27 g (22 mmol )
of p h t h a l i m i d e , 5 .77 g (22 mmol) of t r i p h e n y l p h o s p h i n e ,
and 3 . 8 3 g (22 mmol) of d i e t h y l a z o d i c a r b o x y l a t e (DEAD).
The s o l u t i o n was s t i r r e d at room t e m p e r a t u r e f o r 12
h o u r s a t w h i c h p o i n t , to c o m p l e t e t h e r e a c t i o n , a n
a d d i t i o n a l 1 . 6 4 g (11 mmol) of p h t h a l i m i d e and 1 . 9 2 g (11 mmol) of DEAD were a d d e d and s t i r r i n g c o n t i n u e d f o r
1 h o u r . S o l v e n t was r e m o v e d and t h e r e s i d u a l o i l
p a r t i t i o n e d b e t w e e n e t h e r and 1% h y d r o c h l o r i c a c i d .
The a q u e o u s p h a s e was s e p a r a t e d , made b a s i c w i t h NaOH
s o l u t i o n , and e x t r a c t e d w i t h e t h e r . The e t h e r e x t r a c t
was d r i e d o v e r a n h y d r o u s MgSO4, f i l t e r e d and e v a p o r a t e d .
A p a l e y e l l o w o i l r e s u l t e d w h i c h was d r i e d u n d e r h i g h
v a c u u m to a f f o r d 9 .2 g of t h e p r o t e c t e d a m i n o - p h t h a l i m i d e
as a p a l e y e l l o w o i l .
N - [ 1 - ( 4 - t e r t - B u t y l o x y c a r b o n y l a m i n o b u t y l ) - 2 - ( d i e t h y l a m i n o ) e t h y l ] - 4 - n i t r o b e n z a m i d e , ( 2 ) , Z = p r o t e c t e d a m i n o , n = 4 .
The p r e c e e d i n g o i l was c o n v e r t e d to t he f r e e
amine [ ( 1 ) , Z = a m i n o , n = 4] by r e f l u x i n g i t f o r 2
h o u r s in 200 mL of e t h a n o l c o n t a i n i n g 1 .2 g (24 m m o l )
of h y d r a z i n e h y d r a t e . The s o l u t i o n was c o o l e d , f i l t e r e d , and t h e s o l v e n t r e m o v e d u n d e r r e d u c e d p r e s s u r e to g i v e
an o i l w h i c h was p a r t i t i o n e d b e t w e e n e t h e r and 10% NaOH
s o l u t i o n . The o r g a n i c p h a s e was s e p a r a t e d , w a s h e d w i t h
H20, s a t u r a t e d NaC1 s o l u t i o n , and d r i e d ove r a n h y d r o u s
MgS04. F i l t r a t i o n and e v a p o r a t i o n of t h e f i l t r a t e g a v e 5 .53 g of t h e p r i m a r y amine as a c l e a r o i l . T h i s s o l u -
t i o n was d i s s o l v e d in 200 mL of d ry THF. To t h e s o l u -
t i o n was a d d e d 20 g of a n h y d r o u s K2CO3 and 7 .14 g ( 3 8
mmol) of 4 - n i t r o b e n z o y l c h l o r i d e . A f t e r s t i r r i n g f o r
12 h o u r s at room t e m p e r a t u r e , s o l v e n t was r e m o v e d u n d e r
r e d u c e d p r e s s u r e , and t he r e s i d u e p a r t i t i o n e d b e t w e e n
e t h e r and 10% NaOH s o l u t i o n . The o r g a n i c p h a s e w a s
s e p a r a t e d , w a s h e d w i t h a d d i t i o n a l 10% NaOH s o l u t i o n ,
t h e n e x t r a c t e d w i t h 1% h y d r o c h l o r i c a c i d . The a q u e o u s a c i d p h a s e was s e p a r a t e d , made b a s i c w i t h NaOH, a n d
e x t r a c t e d w i t h e t h e r . The e t h e r p h a s e was d r i e d o v e r
a n h y d r o u s MgS04, f i l t e r e d , and e v a p o r a t e d . The o i l y
r e s i d u e was c r y s t a l l i z e d f rom e t h e r to g i v e 4 .6 g o f
t he n i t r o b e n z a m i d e as a s o l i d , mp 1 0 9 - 1 1 1 ° C .
A n a l y s i s : C a l c u l a t e d f o r C 2 2 H 3 6 N 4 O 5 :
C, 6 0 , 5 3 ; H, 8 . 3 1 ; N, 1 2 , 8 4
F o u n d : C, 6 0 . 5 0 ; H, 8 . 4 4 ; N, 1 2 . 5 6
N - [ l - ( 4 - A m i n o b u t y l ) - 2 - ( d i e t h y l a m i n o ) e t h y l ] - 4 - a m i n o b e n z a m i d e , ( 3 ) , Z = a m i n o , n = 4 .
A s o l u t i o n of 4 .5 g (10 mmol) of t h e p r e c e e d i n g n i t r o b e n z a m i d e in 100 mL of e t h a n o l was h y d r o g e n a t e d at 50 p o u n d s p e r s q u a r e i n c h h y d r o g e n ( p s i H2) o v e r 500 mg of 10% P a l a d i u m on c a r b o n ( P d / C ) . I t was f i l -
t e r e d and e v a p o r a t e d to g i v e 3 . 4 5 g of t h e a r o m a t i c
amine as an o i l . T h i s was d i s s o l v e d in 50 mL o f
a n h y d r o u s t r i f l u o r o a c e t i c a c i d and a l l o w e d to s t a n d
1 h o u r a t 0°C. E x c e s s t r i f l u o r o a c e t i c a c i d was r e - moved u n d e r r e d u c e d p r e s s u r e and t h e r e s i d u e was t a k e n
up in H20. I t was f i l t e r e d and t h e f i l t r a t e s a t u r a t e d
w i t h KC1, and made b a s i c w i t h NaOH. The s o l u t i o n w a s e x t r a c t e d w i t h CHCl3 and t h e e x t r a c t d r i e d , f i l t e r e d ,
and e v a p o r a t e d . T h i s l e f t 1 .4 g of t h e a m i n o -
f u n c t i o n a l i z e d p r o c a i n a m i d e d e r i v a t i v e ( 3 ) , Z = NH2, n = 4 as a c l e a r o i l .
B. P r e p a r a t i o n of i m m u n o g e n
The a m i n o - f u n c t i o n a l i z e d p r o c a i n a m i d e d e r i v a t i v e
p r e p a r e d a b o v e [9 .5 mg (35 µ m o l ) ] was d i s s o l v e d i n
0 .5 mL of d i s t i l l e d w a t e r in a t e s t t u b e . A s m a l l
m a g n e t i c s t i r b a r was a d d e d and t h e pH of t h e s o l u t i o n
was a d j u s t e d to pH 4 .0 w i t h 0 .1 n o r m a l (N) HCl. T h e
s o l u t i o n was c o o l e d in an i c e b a t h and 9 .6 m i l l i g r a m s
(50 µ m o l e s ) of l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l )
c a r b o d i i m i d e was a d d e d a n d d i s s o l v e d . B o v i n e s e r u m
a l b u m i n (25 mg, 35 µ m o l e s ) d i s s o l v e d in 0 .5 mL o f
d i s t i l l e d H2O was s l o w l y a d d e d t o t he s o l u t i o n c o n t a i n -
ing t h e p r o c a i n a m i d e d e r i v a t i v e and c a r b o d i i m i d e .
The r e a c t i o n i n c u b a t e d f o r 24 h o u r s at 4°C, and t h e n
was d i a l y z e d a g a i n s t one l i t e r of d i s t i l l e d w a t e r w h i c h
was c h a n g e d t h r e e t i m e s . Sodium a z i d e was a d d e d to a f i n a l c o n c e n t r a t i o n of 0 .1%. The p r o t e i n c o n c e n t r a t i o n
of t h e immunogen was a d j u s t e d to a c o n c e n t r a t i o n o f
1 mg/mL w i t h s a l i n e (0 .85% N a C l ) .
Six m i l l i l i t e r s of immunogen (1 mg/mL) was com- b i n e d w i t h 12 mL of F r u e n d s C o m p l e t e A d j u v a n t a n d
6 mL of s a l i n e . R a b b i t s were i m m u n i z e d s i m u l t a n e o u s l y e a c h w i t h 2 mL of t h i s m i x t u r e . Four weeks l a t e r
t h e y were r e i m m u n i z e d w i t h t he same m i x t u r e p r e p a r e d w i t h i n c o m p l e t e F r u e n d s a d j u v a n t . The b o o s t e r i m m u n i -
z a t i o n s were r e p e a t e d e v e r y two w e e k s . T e s t b l e e d i n g s
were t a k e n one week a f t e r t he b o o s t e r s . A n t i s e r u m
w i t h s u i t a b l e t i t e r s w e r e o b t a i n e d by f o u r m o n t h s a f t e r
t h e i n i t i a l i m m u n i z a t i o n .
C. P r e p a r a t i o n of l a b e l e d c o n j u g a t e
N - [ 4 - ( 4 - A m i n o b e n z a m i d o ) - 4 - ( 2 - d i e t h y l a m i n o e t h y l ) b u t y l ] - 7 - S - g a l a c t o s y l c o u m a r i n - 3 - c a r b o x a m i d e .
To a s t i r r e d s u s p e n s i o n of 1 .4 g ( 3 . 8 mmol) o f
7 - P - g a l a c t o s y l c o u m a r i n - 3 - c a r b o x y l i c a c i d [ J . F . B u r d , . e t a l , CZin. Chem. 2 3 : 1 4 0 2 ( 1 9 7 7 ) ] in 40 mL of d r y
DMF was added 400 mg (4 mmol) t r i e t h y l a m i n e . The m i x -
t u r e was s t i r r e d u n t i l h o m o g e n e o u s , c o o l e d to - 1 0 ° C
u n d e r a r g o n , and c o m b i n e d w i t h 510 mg (4 mmol) of i s o -
b u t y l c h l o r o f o r m a t e . The r e a c t i o n was s t i r r e d at t h i s
t e m p e r a t u r e f o r 30 m i n u t e s to c o m p l e t e t h e f o r m a t i o n
of t he mixed a n h y d r i d e . A s o l u t i o n of t h e p r e c e e d i n g
d i a m i n e , ( 3 ) , Z = a m i n o , n = 4 [ 1 . 1 5 g, 3 .8 mmol] i n
25 mL of DMF was a d d e d and t h e r e a c t i o n a l l o w e d t o
s t a n d at 0°C f o r 12 h o u r s . C o n c e n t r a t i o n on a r o t a r y
e v a p o r a t o r a t t a c h e d to a vacuum pump l e f t a g l a s s y
r e s i d u e wh ich was t a k e n up in H20 and f i l t e r e d . T h e
pH of t he f i l t r a t e was a d j u s t e d to 8 .0 w i t h NaHC03
s o l u t i o n , 20 g of s i l i c a ge l was a d d e d , and the s o l v e n t
r e m o v e d u n d e r r e d u c e d p r e s s u r e . The i m p r e g n a t e d a d s o r b e n t
was p l a c e d a t o p a c o l u m n of 100 g of s i l i c a ge l made u p in e t h y l a c e t a t e . The c o l u m n was w a s h e d w i t h 1 l i t e r
of e t h y l a c e t a t e ( d i s c a r d e d ) , t h e n e l u t e d w i t h e t h a n o l .
T w e n t y mL f r a c t i o n s we re c o l l e c t e d . F r a c t i o n s 5 0 - 2 2 1
were p o o l e d and e v a p o r a t e d to g i v e 330 mg (13% y i e l d ) of t h e l a b e l e d p r o c a i n a m i d e c o n j u g a t e as a w h i t e s o l i d ,
mp 1 7 5 - 1 7 7 ° C .
I m m u n o a s s a y M e t h o d
A h o m o g e n e o u s s u b s t r a t e - l a b e l e d f l u o r e s c e n t
i m m u n o a s s a y ( S L F I A - U .S . P a t . No. 4 , 2 7 9 , 9 9 2 ) f o r
p r o c a i n a m i d e was e s t a b l i s h e d as f o l l o w s :
A. R e a g e n t s
1. A n t i b o d y / E n z y m e R e a g e n t - 50 mM B i c i n e
b u f f e r [ N , N - b i s - ( 2 - h y d r o x y e t h y l ) g l y c i n e ] ,
( C a l b i o c h e m - B e h r i n g C o r p . , L a J o l l a , CA) ,
pH 8 . 3 , c o n t a i n i n g 0 . 0 0 1 5 u n i t s / m L β -
g a l a c t o s i d a s e , s u f f i c i e n t a n t i s e r u m r a i s e d
a g a i n s t t h e p r o c a i n a m i d e i m m u n o g e n t o
d e c r e a s e f l u o r e s c e n c e to 22% of t h a t i n
t h e a b s e n c e of a n t i s e r u m and 1 5 . 4 nM s o d i u m
a z i d e .
2. C o n j u g a t e R e a g e n t - 30 mM f o r m a t e b u f f e r ,
pH 3 . 5 , c o n t a i n i n g 0.01% ( v / v ) Tween 20
d e t e r g e n t ( F i s h e r S c i e n t i f i c , F a i r l a w n , N J ,
USA) and 0 . 0 2 0 A343 u n i t s of S - G U -
p r o c a i n a m i d e .
3. P r o c a i n a m i d e S t a n d a r d s - USP r e f e r e n c e
s t a n d a r d p r o c a i n a m i d e a d d e d to n o r m a l
human s e r u m ; d i l u t e d 5 1 - f o l d w i t h 50 mM
B i c i n e b u f f e r , c o n t a i n i n g 1 5 . 4 mM s o d i u m
a z i d e .
B. I n h i b i t i o n of H y d r o l y s i s of β - G U - P r o c a i n a m i d e b y A n t i s e r u m to P r o c a i n a m i d e
I n c r e a s i n g a m o u n t s of a n t i s e r u m were added to 1 . 5 mL of B i c i n e b u f f e r c o n t a i n i n g 0 . 0 0 1 5 u n i t s / m L β -
g a l a c t o s i d a s e . The r e a c t i o n was i n i t i a t e d w i t h 50 µL of t h e C o n j u g a t e R e a g e n t a d d e d to e a c h c u v e t t e w i t h
m i x i n g . A f t e r t w e n t y m i n u t e s t h e f l u o r e s c e n c e i n t e n -
s i t y was m e a s u r e d in e a c h c u v e t t e ( e x c i t a t i o n 400 n m , e m i s s i o n 450 nm). The r e s u l t s a r e p r e s e n t e d i n T a b l e 1 .
C. A s s a y M e t h o d and R e s u l t s
To 1 .5 mL of t h e A n t i b o d y / E n z y m e R e a g e n t i n
c u v e t t e s was a d d e d 50 µL ( m i c r o l i t e r s ) of t he d i l u t e d
p r o c a i n a m i d e s t a n d a r d s . Then to b e g i n t h e r e a c t i o n ,
50 µL of t h e C o n j u g a t e R e a g e n t was a d d e d to e a c h
c u v e t t e w i t h m i x i n g . A f t e r 20 m i n u t e s t he f l u o r e s -
c e n c e i n t e n s i t y was m e a s u r e d in e a c h c u v e t t e ( e x c i t a -
t i o n 400 nm, e m i s s i o n 450 n m ) .
P e r f o r m a n c e of t h e a s s a y y i e l d e d the r e s u l t s
shown in T a b l e 2 .
The i m m u n o a s s a y c o u l d be u s e d to d e t e r m i n e p r o - c a i n a m i d e c o n c e n t r a t i o n s in s e r u m s a m p l e s .
1. An i m m u n o g e n c o m p r i s i n g one or m o r e
r e s i d u e s of t h e f o r m u l a :
w h e r e i n X is h y d r o g e n or a c e t y l , c o v a l e n t l y l i n k e d
to an i m m u n o g e n i c c a r r i e r m a t e r i a l .
2. The i m m u n o g e n of C l a i m 1 w h e r e i n s a i d
c a r r i e r m a t e r i a l i s a p r o t e i n or a p o l y p e p t i d e .
3. The immunogen of C l a i m 1 w h e r e i n s a i d o n e
or more r e s i d u e s a r e l i n k e d to s a i d c a r r i e r m a t e r i a l
by a c h a i n c o m p r i s i n g b e t w e e n 1 a n d 20 a t o m s , e x c l u d -
ing h y d r o g e n .
4. An i m m u n o g e n of t h e f o r m u l a :
w h e r e i n X is h y d r o g e n or a c e t y l , Y is an amide g r o u p ,
C a r r i e r i s an i m m u n o g e n i c p r o t e i n or p o l y p e p t i d e c a r r i e r
m a t e r i a l , n i s an i n t e g e r f rom 1 t h r o u g h 10, and p i s
on t h e a v e r a g e f r o m 1 to t h e n u m b e r of a v a i l a b l e a m i d e
c o u p l i n g s i t e s on s a i d c a r r i e r m a t e r i a l .
5 . An a n t i b o d y p r e p a r e d a g a i n s t t h e i m m u n o g e n
of C l a i m 1 .
6. A n a n t i b o d y p r e p a r e d a g a i n s t t h e i m m u n o g e n
of C l a i m 4 .
7. I m m u n o a s s a y p r o c e s s f o r d e t e r m i n i n g p r o c a i n a m i d e
or N - a c e t y l p r o c a i n a m i d e ,
t h e i m p r o v e m e n t w h i c h c o m p r i s e s e m p l o y i n g t h e a n t i b o d y
of C l a i m 5 .
8. In a r e a g e n t s y s t e m f o r d e t e r m i n i n g p r o c a i n a m i d e
or N - a c e t y l p r o c a i n a m i d e by i m m u n o a s s a y ,
t h e i m p r o v e m e n t w h i c h c o m p r i s e s e m p l o y i n g t h e a n t i b o d y
of C l a i m 5 .
9 . A c o m p o u n d of t h e f o r m u l a :
w h e r e i n X is h y d r o g e n or a c e t y l , n i s an i n t e g e r f r o m 1 t h r o u g h 10, and Z is c a r b o x y l or a m i n o .
10. A c o m p o u n d of t h e f o r m u l a :
w h e r e i n n i s an i n t e g e r f r o m 1 t h r o u g h 6 and Z i s c a r b o x y l or a m i n o .
