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I N T E N D E D U S E
T h i s E L I S A ( e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ) k i t i s i n t e n d e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f h u m a n p e p s i n o g e n I l e v e l s i n s e r u m . D e t e r m i n a t i o n o f h u m a n s e r u m p e p s i n o g e n I l e v e l w o u l d b e a u s e f u l t o o l i n t h e a i d o f d i a g n o s i n g t h e f u n c t i o n a l s t a t e s o f a c i d - s e c r e t i n g g a s t r i c m u c o s a .
S U M M A R Y O F P H Y S I O L O G Y
P e p s i n o g e n c o n s i s t s o f a s i n g l e p o l y p e p t i d e c h a i n o f 3 7 5 a m i n o a c i d s w i t h a n a v e r a g e m o l e c u l a r w e i g h t o f 4 2 k D a . P e p s i n o g e n I i s s y n t h e s i z e d a t g a s t r i c c h i e f c e l l s a n d m u c o u s n e c k c e l l s , w h i l e p e p s i n o g e n I I i s p r o d u c e d n o t o n l y b y g a s t r i c c h i e f c e l l s a n d m u c o u s n e c k c e l l s , b u t a l s o b y c l e a r m u c o u s c e l l s o f a n t r u m , e t c . T h e c l i n i c a l a p p l i c a t i o n s o f m e a s u r i n g p e p s i n o g e n I a n d p e p s i n o g e n I I a r e a u s e f u l a i d i n d i a g n o s i n g s e v e r e a t r o p h i c g a s t r i t i s a n d s t o m a c h c a n c e r . I t w a s s u g g e s t e d t h a t t h e m e a s u r e m e n t o f s e r u m p e p s i n o g e n s s e r v e d a s a s e r o l o g i c a l b i o p s y f o r p r e d i c t i n g t h e p r e s e n c e o f a t r o p h i c g a s t r i t i s o r s u p e r f i c i a l g a s t r i t i s .
A t r o p h i c G a s t r i t i s : I t w a s f o u n d t h a t s e r u m p e p s i n o g e n I l e v e l f a l l i n g t o l e s s t h a n 2 0 n g / m L w a s h i g h l y s p e c i f i c f o r s e v e r e a t r o p h i c g a s t r i t i s . I t i s a l s o o b s e r v e d t h a t s e r u m p e p s i n o g e n I l e v e l s f e l l w i t h i n c r e a s i n g s e v e r i t y o f m u c o s a l d a m a g e i n a t r o p h i c g a s t r i t i s . T h e d i a g n o s t i c s e n s i t i v i t y a n d s p e c i f i c i t y o f s e r u m p e p s i n o g e n I l e v e l f o r a d v a n c e d a t r o p h i c c o r p u s g a s t r i t i s a r e a b o u t 9 2 % a n d 9 0 % r e s p e c t i v e l y . O n t h e o t h e r h a n d , t h e d e c r e a s e i n s e r u m p e p s i n o g e n I l e v e l s i n p a t i e n t s w i t h p e r n i c i o u s a n e m i a a n d a t r o p h i c g a s t r i t i s w a s f o u n d t o b e a s s o c i a t e d w i t h n o r m a l o r r a i s e d p e p s i n o g e n I I l e v e l s . T h e r e f o r e , a p e p s i n o g e n I / p e p s i n o g e n I I r a t i o i s s i g n i f i c a n t l y l o w e r t h a n t h o s e w i t h s u p e r f i c i a l g a s t r i t i s o r n o r m a l r e m n a n t m u c o s a .
S t o m a c h C a n c e r : L o w s e r u m p e p s i n o g e n I l e v e l s w e r e f o u n d i n p a t i e n t s w i t h g a s t r i c c a n c e r , w i t h a t h r e e f o l d h i g h e r i n c i d e n c e . O t h e r s t u d i e s h a v e c o n c l u d e d t h a t l o w s e r u m p e p s i n o g e n I l e v e l s m a y i d e n t i f y p e r s o n s a t i n c r e a s e d r i s k f o r i n t e s t i n a l t y p e s o f s t o m a c h c a n c e r .
D u o d e n a l U l c e r : A l o w s e r u m p e p s i n o g e n I l e v e l c a n e x c l u d e a d i a g n o s i s o f d u o d e n a l u l c e r . A l t h o u g h a h i g h p e p s i n o g e n I l e v e l h a s l e s s c l i n i c a l u s e f o r e s t a b l i s h i n g t h e d i a g n o s i s o f a d u o d e n a l u l c e r , t h e c o m b i n a t i o n o f h y p e r g a s t r i n e m i a a n d a h i g h l y e l e v a t e d s e r u m p e p s i n o g e n I s t r o n g l y s u g g e s t s t h e p o s s i b i l i t y o f t h e Z o l l i n g e r - E l l i s o n s y n d r o m e .
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T h i s E L I S A i s d e s i g n e d , d e v e l o p e d a n d p r o d u c e d f o r t h e q u a n t i t a t i v e m e a s u r e m e n t o f h u m a n p e p s i n o g e n I l e v e l i n s e r u m s a m p l e . T h e a s s a y u t i l i z e s t h e t w o - s i t e s a n d w i c h t e c h n i q u e w i t h t w o s e l e c t e d m o n o c l o n a l a n t i b o d i e s t h a t b i n d t o d i f f e r e n t e p i t o p e s o f h u m a n p e p s i n o g e n I w i t h o u t a n y c r o s s - r e a c t i o n t o h u m a n p e p s i n o g e n I I .
A s s a y s t a n d a r d s , c o n t r o l s a n d p a t i e n t s e r u m s a m p l e s c o n t a i n i n g h u m a n p e p s i n o g e n I a r e a d d e d d i r e c t l y t o m i c r o t i t e r w e l l s o f m i c r o p l a t e t h a t w a s c o a t e d w i t h s t r e p t a v i d i n . S i m u l t a n e o u s l y , a b i o t i n y l a t e d a n t i b o d y a n d a h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d a n t i b o d y a r e a d d e d t o e a c h w e l l . A f t e r t h e f i r s t i n c u b a t i o n p e r i o d , o n t h e w a l l o f m i c r o t i t e r w e l l c a p t u r e s t h e b i o t i n y l a t e d a n t i b o d y a s w e l l a s a n i m m u n o c o m p l e x i n z t h e f o r m o f s t r e p t a v i d i n b i o t i n - a n t i b o d y p e p s i n o g e n I H R P - a n t i b o d y . U n b o u n d p r o t e i n s a s w e l l a s u n b o u n d H R P - c o n j u g a t e d a n t i b o d y i n e a c h m i c r o t i t e r w e l l a r e r e m o v e d i n t h e s u b s e q u e n t w a s h i n g s t e p . T h e w e l l i s i n c u b a t e d w i t h a s u b s t r a t e s o l u t i o n i n a t i m e d r e a c t i o n a n d t h e n m e a s u r e d i n a s p e c t r o p h o t o m e t r i c m i c r o p l a t e r e a d e r . T h e e n z y m a t i c a c t i v i t y o f t h e t r a c e r a n t i b o d y b o u n d t o t h e p e p s i n o g e n I o n t h e w a l l o f t h e m i c r o t i t e r w e l l i s d i r e c t l y p r o p o r t i o n a l t o t h e a m o u n t o f p e p s i n o g e n I i n t h e s a m p l e . A s t a n d a r d c u r v e i s g e n e r a t e d b y p l o t t i n g t h e a b s o r b a n c e v e r s u s t h e r e s p e c t i v e h u m a n p e p s i n o g e n I c o n c e n t r a t i o n f o r e a c h s t a n d a r d o n P o i n t - t o - P o i n t , C u b i c S p l i n e o r 4 - P a r a m e t e r p l o t . T h e c o n c e n t r a t i o n o f h u m a n p e p s i n o g e n I i n t e s t s a m p l e s i s d e t e r m i n e d d i r e c t l y f r o m t h i s s t a n d a r d c u r v e .
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T h i s t e s t k i t m u s t b e s t o r e d a t 2 8 C u p o n r e c e i p t . F o r t h e e x p i r a t i o n d a t e o f t h e k i t r e f e r t o t h e l a b e l o n t h e k i t b o x . A l l c o m p o n e n t s a r e s t a b l e u n t i l t h i s e x p i r a t i o n d a t e .
P r i o r t o u s e a l l o w a l l r e a g e n t s t o c o m e t o r o o m t e m p e r a t u r e . R e a g e n t s f r o m d i f f e r e n t k i t l o t n u m b e r s s h o u l d n o t b e c o m b i n e d o r i n t e r c h a n g e d .
S t r e p t a v i d i n C o a t e d M i c r o p l a t e ( C a t . N o . 1 0 0 4 0 )
O n e m i c r o p l a t e w i t h 1 2 x e i g h t s t r i p s ( 9 6 w e l l s t o t a l ) c o a t e d w i t h s t r e p t a v i d i n . T h e p l a t e i s f r a m e d a n d s e a l e d i n a f o i l Z i p l o c b a g w i t h a d e s i c c a n t . T h i s r e a g e n t s h o u l d b e s t o r e d a t 2 8 C a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
P e p s i n o g e n I T r a c e r A n t i b o d y ( C a t . N o . 3 0 0 6 1 )
O n e v i a l c o n t a i n s 0 . 6 m L c o n c e n t r a t e d h o r s e r a d i s h p e r o x i d a s e ( H R P ) - c o n j u g a t e d a n t i - h u m a n p e p s i n o g e n I t r a c e r a n t i b o d y i n a s t a b i l i z e d p r o t e i n m a t r i x . T h i s r e a g e n t m u s t b e d i l u t e d w i t h T r a c e r A n t i b o d y D i l u e n t b e f o r e u s e . T h i s r e a g e n t s h o u l d b e s t o r e d a t 2 8 C a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
P e p s i n o g e n I C a p t u r e A n t i b o d y ( C a t . N o . 3 0 0 6 2 )
O n e v i a l c o n t a i n s 0 . 6 m L c o n c e n t r a t e d b i o t i n y l a t e d a n t i - h u m a n p e p s i n o g e n I c a p t u r e a n t i b o d y i n a s t a b i l i z e d p r o t e i n m a t r i x . T h i s r e a g e n t m u s t b e d i l u t e d w i t h T r a c e r A n t i b o d y D i l u e n t b e f o r e u s e . T h i s r e a g e n t s h o u l d b e s t o r e d a t 2 8 C a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
T r a c e r A n t i b o d y D i l u e n t ( C a t . N o . 3 0 0 1 7 )
O n e v i a l c o n t a i n s 1 2 m L r e a d y - t o - u s e b u f f e r . I t s h o u l d b e o n l y u s e d f o r t r a c e r a n t i b o d y d i l u t i o n a c c o r d i n g t o t h e a s s a y p r o c e d u r e s . T h i s r e a g e n t s h o u l d b e s t o r e d a t 2 8 C a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
E L I S A W a s h C o n c e n t r a t e ( C a t . N o . 1 0 0 1 0 )
O n e b o t t l e c o n t a i n s 3 0 m L o f 3 0 - f o l d c o n c e n t r a t e . B e f o r e u s e t h e c o n t e n t s m u s t b e d i l u t e d w i t h 8 7 0 m L o f d i s t i l l e d w a t e r a n d
m i x w e l l . U p o n d i l u t i o n t h i s y i e l d s a w o r k i n g w a s h s o l u t i o n c o n t a i n i n g a s u r f a c t a n t i n p h o s p h a t e b u f f e r e d s a l i n e w i t h a n o n - a z i d e p r e s e r v a t i v e . T h e d i l u t e d s o l u t i o n s h o u l d b e s t o r e d a t r o o m t e m p e r a t u r e a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
E L I S A H R P S u b s t r a t e ( C a t . N o . 1 0 0 2 0 )
O n e b o t t l e c o n t a i n s 1 2 m L o f t e t r a m e t h y l b e n z i d i n e ( T M B ) w i t h h y d r o g e n p e r o x i d e . T h i s r e a g e n t s h o u l d b e s t o r e d a t 2 8 C a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
E L I S A S t o p S o l u t i o n ( C a t . N o . 1 0 0 3 0 )
O n e b o t t l e c o n t a i n s 1 2 m L o f 0 . 5 M s u l f u r i c a c i d . T h i s r e a g e n t s h o u l d b e s t o r e d a t 2 8 C o r r o o m t e m p e r a t u r e a n d i s s t a b l e u n t i l t h e e x p i r a t i o n d a t e o n t h e k i t b o x .
P e p s i n o g e n I S t a n d a r d s ( C a t . N o . 3 0 0 5 3 3 0 0 5 8 )
S i x v i a l s e a c h c o n t a i n l y o p h i l i z e d h u m a n p e p s i n o g e n I i n a b o v i n e s e r u m a l b u m i n - b a s e d m a t r i x w i t h a n o n - a z i d e p r e s e r v a t i v e . R e f e r t o v i a l s f o r e x a c t c o n c e n t r a t i o n f o r e a c h s t a n d a r d . A l l t h e s t a n d a r d s s h o u l d b e r e c o n s t i t u t e d w i t h D I - w a t e r a n d s t o r e d a t - 2 0 C o r b e l o w a f t e r t h e f i r s t u s e w i t h u p t o 3 f r e e z e c y c l e s .
P e p s i n o g e n I C o n t r o l s ( C a t . N o . 3 0 0 5 9 3 0 0 6 0 )
T w o v i a l s e a c h c o n t a i n l y o p h i l i z e d h u m a n p e p s i n o g e n I i n a b o v i n e s e r u m a l b u m i n - b a s e d m a t r i x w i t h a n o n - a z i d e p r e s e r v a t i v e . R e f e r t o v i a l s f o r e x a c t c o n c e n t r a t i o n r a n g e f o r e a c h c o n t r o l . B o t h c o n t r o l s s h o u l d b e r e c o n s t i t u t e d w i t h D I - w a t e r a n d s t o r e d a t - 2 0 C o r b e l o w a f t e r t h e f i r s t u s e w i t h u p t o 3 f r e e z e c y c l e s .
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T h e r e a g e n t s m u s t b e u s e d i n r e s e a r c h l a b o r a t o r y a n d i s f o r r e s e a r c h u s e o n l y . R e a g e n t s o f b o v i n e s e r u m a l b u m i n w e r e d e r i v e d i n t h e c o n t i g u o u s 4 8 U n i t e d S t a t e s . I t w a s o b t a i n e d o n l y f r o m d o n o r h e a l t h a n i m a l s m a i n t a i n e d u n d e r v e t e r i n a r y s u p e r v i s i o n a n d f o u n d f r e e o f c o n t a g i o u s d i s e a s e s . W e a r g l o v e s w h i l e p e r f o r m i n g t h i s a s s a y a n d h a n d l e t h e s e r e a g e n t s a s i f t h e y a r e p o t e n t i a l i n f e c t i o u s . A v o i d c o n t a c t w i t h r e a g e n t s c o n t a i n i n g T M B , h y d r o g e n p e r o x i d e , o r s u l f u r i c a c i d . T M B m a y c a u s e i r r i t a t i o n t o s k i n a n d m u c o u s m e m b r a n e s a n d c a u s e a n a l l e r g i c s k i n r e a c t i o n . T M B i s a s u s p e c t e d c a r c i n o g e n . S u l f u r i c a c i d m a y c a u s e s e v e r i r r i t a t i o n o n c o n t a c t w i t h s k i n . D o n o t g e t i n e y e s , o n s k i n , o r o n c l o t h i n g . D o n o t i n g e s t o r i n h a l e f u m e s . O n c o n t a c t , f l u s h w i t h c o p i o u s a m o u n t s o f w a t e r f o r a t l e a s e 1 5 m i n u t e s . U s e G o o d L a b o r a t o r y P r a c t i c e s .
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P r e c i s i o n s i n g l e c h a n n e l p i p e t t e s c a p a b l e o f d e l i v e r i n g 2 0 L , 2 5 L , 1 0 0 L , a n d 1 0 0 0 L , e t c .
R e p e a t i n g d i s p e n s e r s u i t a b l e f o r d e l i v e r i n g 1 0 0 L .
D i s p o s a b l e p i p e t t e t i p s s u i t a b l e f o r a b o v e v o l u m e d i s p e n s i n g .
D i s p o s a b l e 1 2 x 7 5 m m o r 1 3 x 1 0 0 g l a s s t u b e s .
D i s p o s a b l e p l a s t i c 1 0 0 0 m L b o t t l e w i t h c a p s .
A l u m i n u m f o i l .
D e i o n i z e d o r d i s t i l l e d w a t e r .
P l a s t i c m i c r o t i t e r w e l l c o v e r o r p o l y e t h y l e n e f i l m .
E L I S A m u l t i c h a n n e l w a s h b o t t l e o r a u t o m a t i c ( s e m i - a u t o m a t i c ) w a s h i n g s y s t e m .
S p e c t r o p h o t o m e t r i c m i c r o p l a t e r e a d e r c a p a b l e o f r e a d i n g a b s o r b a n c e a t 4 5 0 n m .
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O n l y 5 0 L o f h u m a n s e r u m i s r e q u i r e d f o r h u m a n p e p s i n o g e n I m e a s u r e m e n t i n d u p l i c a t e . N o s p e c i a l p r e p a r a t i o n o f i n d i v i d u a l i s n e c e s s a r y p r i o r t o s p e c i m e n c o l l e c t i o n . H o w e v e r , w e r e c o m m e n d d r a w i n g a 1 0 - h o u r f a s t i n g s e r u m s a m p l e f o r t h e t e s t . W h o l e b l o o d s h o u l d b e c o l l e c t e d a n d m u s t b e a l l o w e d t o c l o t f o r m i n i m u m 3 0 m i n u t e s a t r o o m t e m p e r a t u r e b e f o r e t h e s e r u m i s s e p a r a t e d b y c e n t r i f u g a t i o n ( 8 5 0 1 5 0 0 x g f o r 1 0 m i n u t e s ) . T h e s e r u m s h o u l d b e s e p a r a t e d f r o m t h e c l o t w i t h i n t h r e e h o u r s o f b l o o d c o l l e c t i o n a n d t r a n s f e r r e d t o a c l e a n t e s t t u b e . S e r u m s a m p l e s s h o u l d b e s t o r e d a t 2 0 C o r b e l o w u n t i l m e a s u r e m e n t . A v o i d m o r e t h a n t h r e e f r e e z e - t h a w c y c l e s o f s p e c i m e n .
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1 . R e a g e n t P r e p a r a t i o n
P r i o r t o u s e a l l o w a l l r e a g e n t s t o c o m e t o r o o m t e m p e r a t u r e . R e a g e n t s f r o m d i f f e r e n t k i t l o t n u m b e r s s h o u l d n o t b e c o m b i n e d o r i n t e r c h a n g e d .
E L I S A W a s h C o n c e n t r a t e ( C a t . 1 0 0 1 0 ) m u s t b e d i l u t e d t o w o r k i n g s o l u t i o n p r i o r t o u s e . P l e a s e s e e R E A G E N T S s e c t i o n f o r d e t a i l s .
R e c o n s t i t u t e a l l a s s a y s t a n d a r d s a n d c o n t r o l s b y a d d i n g 0 . 5 m L o f d e m i n e r i a l i z e d w a t e r t o t h e v i a l o f s t a n d a r d l e v e l 1 a n d 0 . 5 m L d e m i n e r i a l i z e d w a t e r t o t h e v i a l s o f s t a n d a r d l e v e l s 2 - 6 a n d c o n t r o l s 1 & 2 . A l l o w t h e s t a n d a r d s a n d c o n t r o l s t o s i t u n d i s t u r b e d f o r 1 0 m i n u t e s , a n d t h e n m i x w e l l b y g e n t l e v o r t e x i n g . M a k e s u r e t h a t a l l s o l i d i s d i s s o l v e d c o m p l e t e l y p r i o r t o u s e . T h e s e r e c o n s t i t u t e d s t a n d a r d s a n d c o n t r o l s m u s t b e s t o r e d a t - 1 0 C o r b e l o w . D o n o t e x c e e d 3 f r e e z e - t h a w c y c l e s .
2 . A s s a y P r o c e d u r e
P l a c e a s u f f i c i e n t n u m b e r o f s t r e p t a v i d i n - c o a t e d m i c r o w e l l s t r i p s ( C a t . 1 0 0 4 0 ) i n a h o l d e r t o r u n h u m a n p e p s i n o g e n I s t a n d a r d s , c o n t r o l s a n d u n k n o w n s a m p l e s i n d u p l i c a t e .
T e s t C o n f i g u r a t i o n
R O W S T R I P 1 S T R I P 2 S T R I P 3 A S T D 1 S T D 5 S A M P L E 1 B S T D 1 S T D 5 S A M P L E 1 C S T D 2 S T D 6 S A M P L E 2 D S T D 2 S T D 6 S A M P L E 2 E S T D 3 C 1 S A M P L E 3 F S T D 3 C 1 S A M P L E 3 G S T D 4 C 2 H S T D 4 C 2
P r e p a r e w o r k i n g T r a c e r A n t i b o d y a n d C a p t u r e A n t i b o d y m i x t u r e b y 1 : 2 1 f o l d d i l u t i o n o f t h e P e p s i n o g e n I T r a c e r A n t i b o d y ( C a t . 3 0 0 6 1 ) a n d t h e P e p s i n o g e n I C a p t u r e A n t i b o d y ( C a t . 3 0 0 6 2 ) w i t h t h e T r a c e r A n t i b o d y D i l u e n t ( C a t . 3 0 0 1 7 ) . F o r e a c h s t r i p , i t i s r e q u i r e d t o m i x 1 m L o f T r a c e r A n t i b o d y D i l u e n t w i t h t h e a d d i t i o n o f 5 0 L o f T r a c e r A n t i b o d y a n d 5 0 L C a p t u r e A n t i b o d y i n a c l e a n t e s t t u b e o r v i a l . F o l l o w i n g i s a t a b l e t h a t o u t l i n e s t h e r e l a t i o n s h i p o f s t r i p s u s e d a n d a n t i b o d y m i x p r e p a r e d .
S t r i p n o . T r a c e r A n t i b o d y D i l u e n t T r a c e r A n t i b o d y C a p t u r e A n t i b o d y 1 1 m L 5 0 L 5 0 L 2 2 m L 1 0 0 L 1 0 0 L 3 3 m L 1 5 0 L 1 5 0 L 4 4 m L 2 0 0 L 2 0 0 L 5 5 m L 2 5 0 L 2 5 0 L 6 6 m L 3 0 0 L 3 0 0 L 7 7 m L 3 5 0 L 3 5 0 L 8 8 m L 4 0 0 L 4 0 0 L 9 9 m L 4 5 0 L 4 5 0 L 1 0 1 0 m L 5 0 0 L 5 0 0 L 1 1 1 1 m L 5 5 0 L 5 5 0 L 1 2 1 2 m L 6 0 0 L 6 0 0 L N o t e : t h i s a n t i b o d y m i x s h o u l d b e f r e s h l y p r e p a r e d r i g h t b e f o r e r u n n i n g t h e a s s a y .
A d d 2 5 L o f s t a n d a r d s , c o n t r o l s a n d p a t i e n t s e r u m s a m p l e s i n t o t h e d e s i g n a t e d m i c r o w e l l .
A d d 1 0 0 L o f a b o v e a n t i b o d y m i x t u r e t o e a c h w e l l
M i x g e n t l y a n d c o v e r t h e p l a t e w i t h o n e p l a t e s e a l e r a n d a l s o w i t h a l u m i n u m f o i l t o a v o i d e x p o s u r e t o l i g h t .
I n c u b a t e p l a t e a t r o o m t e m p e r a t u r e f o r 1 h o u r .
R e m o v e t h e a l u m i n u m f o i l a n d p l a t e s e a l e r . A s p i r a t e t h e c o n t e n t s o f e a c h w e l l . W a s h e a c h w e l l 5 t i m e s b y d i s p e n s i n g 3 5 0 L o f w o r k i n g w a s h s o l u t i o n i n t o e a c h w e l l a n d t h e n c o m p l e t e l y a s p i r a t i n g t h e c o n t e n t s . A l t e r n a t i v e l y , a n a u t o m a t e d m i c r o p l a t e w a s h e r c a n b e u s e d .
A d d 1 0 0 L o f E L I S A H R P S u b s t r a t e ( C a t . 1 0 0 2 0 ) i n t o e a c h o f t h e w e l l s .
C o v e r t h e p l a t e w i t h o n e n e w p l a t e s e a l e r a n d a l s o w i t h a l u m i n u m f o i l t o a v o i d e x p o s u r e t o l i g h t .
I n c u b a t e p l a t e a t r o o m t e m p e r a t u r e f o r 2 0 m i n u t e s . ( T h i s i n c u b a t i o n p e r i o d m a y b e r e d u c e d t o 8 1 5 m i n i f a l o w e r O D r e a d i n g i s d e m a n d e d t o f i t t o t h e p l a t e r e a d e r s s p e c i f i c a t i o n . )
R e m o v e t h e a l u m i n u m f o i l a n d p l a t e s e a l e r . A d d 1 0 0 L o f E L I S A S t o p S o l u t i o n ( C a t . 1 0 0 3 0 ) i n t o e a c h o f t h e w e l l s . M i x g e n t l y .
R e a d t h e a b s o r b a n c e a t 4 5 0 n m w i t h i n 1 0 m i n u t e s i n a m i c r o p l a t e r e a d e r .
P R O C E D U R A L N O T E S
I t i s r e c o m m e n d e d t h a t a l l s t a n d a r d s , c o n t r o l s a n d u n k n o w n s a m p l e s b e a s s a y e d i n d u p l i c a t e . T h e a v e r a g e a b s o r b a n c e r e a d i n g o f e a c h d u p l i c a t e s h o u l d b e u s e d f o r d a t a r e d u c t i o n a n d t h e c a l c u l a t i o n o f r e s u l t s .
K e e p l i g h t - s e n s i t i v e r e a g e n t s i n t h e o r i g i n a l b o t t l e s a n d a v o i d u n n e c e s s a r y e x p o s u r e t o t h e l i g h t .
S t o r e a n y u n u s e d a n t i b o d y - c o a t e d s t r i p s i n t h e f o i l Z i p l o c b a g w i t h d e s i c c a n t t o p r o t e c t f r o m m o i s t u r e .
C a r e f u l t e c h n i q u e a n d u s e o f p r o p e r l y c a l i b r a t e d p i p e t t i n g d e v i c e s a r e n e c e s s a r y t o e n s u r e r e p r o d u c i b i l i t y o f t h e t e s t .
I n c u b a t i o n t i m e s o r t e m p e r a t u r e s o t h e r t h a n t h o s e s t a t e d i n t h i s i n s e r t m a y a f f e c t t h e r e s u l t s .
A v o i d a i r b u b b l e s i n t h e m i c r o w e l l a s t h i s c o u l d r e s u l t i n l o w e r b i n d i n g e f f i c i e n c y a n d h i g h e r C V % o f d u p l i c a t e r e a d i n g .
A l l r e a g e n t s s h o u l d b e m i x e d g e n t l y a n d t h o r o u g h l y p r i o r u s e . A v o i d f o a m i n g .
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C a l c u l a t e t h e a v e r a g e a b s o r b a n c e f o r e a c h p a i r o f d u p l i c a t e t e s t r e s u l t s .
S u b t r a c t t h e a v e r a g e a b s o r b a n c e o f t h e S T D 1 ( 0 n g / m L ) f r o m t h e a v e r a g e a b s o r b a n c e o f a l l o t h e r r e a d i n g s t o o b t a i n c o r r e c t e d a b s o r b a n c e .
T h e s t a n d a r d c u r v e i s g e n e r a t e d b y t h e c o r r e c t e d a b s o r b a n c e o f a l l s t a n d a r d l e v e l s o n t h e o r d i n a t e a g a i n s t t h e s t a n d a r d c o n c e n t r a t i o n o n t h e a b s c i s s a u s i n g p o i n t - t o - p o i n t o r l o g - l o g p a p e r . A p p r o p r i a t e c o m p u t e r a s s i s t e d d a t a r e d u c t i o n p r o g r a m s m a y a l s o b e u s e d f o r t h e c a l c u l a t i o n o f r e s u l t s .
I t i s r e c o m m e n d e d t o u s e f o l l o w i n g c u r v e f i t s : ( 1 ) P o i n t - t o - P o i n t , o r ( 2 ) 4 - P a r a m e t e r o r ( 3 ) C u b i c S p l i n e .
T h e h u m a n p e p s i n o g e n I c o n c e n t r a t i o n s f o r t h e c o n t r o l s a n d p a t i e n t s a m p l e s a r e r e a d d i r e c t l y f r o m t h e s t a n d a r d c u r v e u s i n g t h e i r r e s p e c t i v e c o r r e c t e d a b s o r b a n c e .
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A t y p i c a l a b s o r b a n c e d a t a a n d t h e r e s u l t i n g s t a n d a r d c u r v e f r o m h u m a n p e p s i n o g e n I E L I S A a r e r e p r e s e n t e d . T h i s c u r v e s h o u l d n o t b e u s e d i n l i e u o f s t a n d a r d c u r v e r u n w i t h e a c h a s s a y .
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S e v e n t y - t h r e e n o r m a l a d u l t s e r a w e r e m e a s u r e d w i t h t h i s h u m a n p e p s i n o g e n I E L I S A . T h e e x p e c t e d n o r m a l r a n g e i s l i s t e d i n t h e f o l l o w i n g t a b l e w i t h d i f f e r e n t p e r c e n t i l e c u t - o f f a n d t h e m e d i a n l e v e l o f t h i s g r o u p o f p o p u l a t i o n i s 6 2 . 8 n g / m L .
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I t i s h i g h l y r e c o m m e n d e d t h a t e a c h l a b o r a t o r y e s t a b l i s h t h e i r o w n n o r m a l r a n g e f o r p e p s i n o g e n I b a s e d o n l o c a l p o p u l a t i o n s .
P a t i e n t s w i t h a t r o p h i c g a s t r i t i s , a s w e l l a s p a t i e n t s w i t h s t o m a c h c a n c e r m a y h a v e a p e p s i n o g e n I l e v e l b e l o w 2 0 n g / m L . H o w e v e r , g a s t r o e n d o s c o p e a n d t i s s u e b i o p s y s h o u l d b e u s e d a s f i n a l a n d c o n f i r m a t i v e d i a g n o s t i c m e t h o d .
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S i n c e t h e r e i s n o G o l d S t a n d a r d c o n c e n t r a t i o n a v a i l a b l e f o r h u m a n p e p s i n o g e n I m e a s u r e m e n t , t h e v a l u e s o f a s s a y s t a n d a r d s w e r e e s t a b l i s h e d b y d i l u t i n g a h i g h l y p u r i f i e d h u m a n p e p s i n o g e n I i n a p r o t e i n m a t r i x .
F o r u n k n o w n s a m p l e v a l u e r e a d d i r e c t l y f r o m t h e a s s a y t h a t i s g r e a t e r t h a n 3 0 0 n g / m L , i t i s r e c o m m e n d e d t o m e a s u r e a f u r t h e r d i l u t e d s a m p l e f o r m o r e a c c u r a t e m e a s u r e m e n t .
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T o a s s u r e t h e v a l i d i t y o f t h e r e s u l t s e a c h a s s a y s h o u l d i n c l u d e a d e q u a t e c o n t r o l s w i t h k n o w n p e p s i n o g e n I l e v e l s . W e r e c o m m e n d t h a t a l l a s s a y s i n c l u d e t h e l a b o r a t o r y s o w n h u m a n s e r u m b a s e d p e p s i n o g e n I c o n t r o l s i n a d d i t i o n t o t h o s e p r o v i d e d w i t h t h i s k i t .
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T h e s e n s i t i v i t y o f t h i s h u m a n p e p s i n o g e n I E L I S A i s 0 . 1 n g / m L a s d e t e r m i n e d b y m e a s u r i n g z e r o s t a n d a r d 1 6 t i m e s i n t h e s a m e a s s a y a n d c a l c u l a t i n g t h e d e t e c t i o n l i m i t a t 3 s t a n d a r d d e v i a t i o n s a b o v e t h e p e p s i n o g e n I z e r o s t a n d a r d . T h e a s s a y a n a l y t i c a l s e n s i t i v i t y i s a p p r o x i m a t e l y 0 . 5 n g / m L .
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1 . K u i p e r s E J . I n t h r o u g h t h e o u t d o o r : s e r o l o g y f o r a t r o p h i c g a s t r i t i s . E u r J G a s t r o e n t e r o l H e p a t o l . 2 0 0 3 A u g ; 1 5 ( 8 ) : 8 7 7 - 9 .
2 . M i k i K , M o r i t a M , S a s a j i m a M , H o s h i n a R , K a n d a E , U r i t a Y . U s e f u l n e s s o f g a s t r i c c a n c e r s c r e e n i n g u s i n g t h e s e r u m p e p s i n o g e n t e s t m e t h o d . A m J G a s t r o e n t e r o l . 2 0 0 3 A p r ; 9 8 ( 4 ) : 7 3 5 - 9 .
3 . M i k i K . [ S e r u m p e p s i n o g e n I / I I r a t i o t e s t ] N i p p o n R i n s h o . 2 0 0 3 J a n ; 6 1 ( 1 ) : 9 2 - 5 . J a p a n e s e .
4 . S o J B , Y e o h K G , M o o c h a l a S , C h a c h l a n i N , H o J , W o n g W K , M a c k P , G o h P M . S e r u m p e p s i n o g e n l e v e l s i n g a s t r i c c a n c e r p a t i e n t s a n d t h e i r r e l a t i o n s h i p w i t h H e l i c o b a c t e r p y l o r i i n f e c t i o n : a p r o s p e c t i v e s t u d y . G a s t r i c C a n c e r . 2 0 0 2 ; 5 ( 4 ) : 2 2 8 - 3 2 .
5 . K o r s t a n j e A , d e n H a r t o g G , B i e m o n d I , L a m e r s C B . T h e s e r o l o g i c a l g a s t r i c b i o p s y : a n o n - e n d o s c o p i c a l d i a g n o s t i c a p p r o a c h i n m a n a g e m e n t o f t h e d y s p e p t i c p a t i e n t : s i g n i f i c a n c e f o r p r i m a r y c a r e b a s e d o n a s u r v e y o f t h e l i t e r a t u r e . S c a n d J G a s t r o e n t e r o l S u p p l . 2 0 0 2 ; ( 2 3 6 ) : 2 2 - 6 . R e v i e w .
6 . S i p p o n e n P , H a r k o n e n M , A l a n k o A , S u o v a n i e m i O . D i a g n o s i s o f a t r o p h i c g a s t r i t i s f r o m a s e r u m s a m p l e . C l i n L a b . 2 0 0 2 ; 4 8 ( 9 - 1 0 ) : 5 0 5 - 1 5 . R e v i e w .
7 . T a b a t a H , F u c h i g a m i T , K o b a y a s h i H , S a k a i Y , N a k a n i s h i M , T o m i o k a K , N a k a m u r a S , M a t s u m o t o T , F u j i s h i m a M . D i f f e r e n c e i n d e g r e e o f m u c o s a l a t r o p h y b e t w e e n e l e v a t e d a n d d e p r e s s e d t y p e s o f g a s t r i c e p i t h e l i a l t u m o r s . S c a n d J G a s t r o e n t e r o l . 2 0 0 1 N o v ; 3 6 ( 1 1 ) : 1 1 3 4 - 4 0 .
8 . V a r i s K , S i p p o n e n P , L a x e n F , S a m l o f f I M , H u t t u n e n J K , T a y l o r P R , H e i n o n e n O P , A l b a n e s D , S a n d e N , V i r t a m o J , H a r k o n e n M .
I m p l i c a t i o n s o f s e r u m p e p s i n o g e n I i n e a r l y e n d o s c o p i c d i a g n o s i s o f g a s t r i c c a n c e r a n d d y s p l a s i a . H e l s i n k i G a s t r i t i s S t u d y G r o u p . S c a n d J G a s t r o e n t e r o l . 2 0 0 0 S e p ; 3 5 ( 9 ) : 9 5 0 - 6 .
9 . F e r n a n d e z R , V i z o s o F , R o d r i g u e z J C , M e r i n o A M , G o n z a l e z L O , Q u i n t e l a I , A n d i c o e c h e a A , T r u a n N , D i e z M C . E x p r e s s i o n a n d p r o g n o s t i c s i g n i f i c a n c e o f p e p s i n o g e n C i n g a s t r i c c a r c i n o m a . A n n S u r g O n c o l . 2 0 0 0 A u g ; 7 ( 7 ) : 5 0 8 - 1 4 .
1 0 . K a l i n o v s k i i V P , G a m a i u n o v a V B , S h u m a k o v A P , K h a n s o n K P .
[ R a d i o i m m u n o a s s a y o f s e r u m p e p s i n o g e n I i n c h r o n i c g a s t r i t i s a n d s t o m a c h c a n c e r ] V o p r O n k o l . 2 0 0 0 ; 4 6 ( 2 ) : 1 5 3 - 5 . R u s s i a n .
1 1 . S h u m a k o v A R , F e d o r o v S N , K a l i n o v s k i i V P , K h a n s o n K P .
[ E v a l u a t i o n o f p e p s i n o g e n A e x p r e s s i o n i n s t o m a c h c a n c e r ] V o p r O n k o l . 1 9 9 9 ; 4 5 ( 3 ) : 2 3 8 - 4 0 . R u s s i a n .
1 2 . K i t a h a r a F , K o b a y a s h i K , S a t o T , K o j i m a Y , A r a k i T , F u j i n o M A .
A c c u r a c y o f s c r e e n i n g f o r g a s t r i c c a n c e r u s i n g s e r u m p e p s i n o g e n c o n c e n t r a t i o n s . G u t . 1 9 9 9 M a y ; 4 4 ( 5 ) : 6 9 3 - 7 .
1 3 . S a m l o f f I M a n d T a g g a r t R T . P e p s i n o g e n s , p e p s i n s , a n d p e p t i c u l c e r . C l i n i c a l a n d I n v e s t i g a t i v e M e d i c i n e 1 9 8 7 ; 1 0 : 2 1 5 - 2 2 1
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A d d 1 0 0 L o f E L I S A H R P S u b s t r a t e i n t o e a c h o f t h e w e l l s .
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