Guidelines for the evaluation of RIA methods for prostaglandins

PROSTAGLANDINS

EDITORIAL Guidelines

for the E v a l u a t i o n

of RIA M e t h o d s

for P R O S T A G L A N D I N S

We w i s h to i m p r o v e the "Methods" s e c t i o n of the p a p e r s p u b l i s h e d in the Journal. The first p r o b l e m to be a d d r e s s e d is the nature of s t a n d a r d s w h i c h w i l l be a p p l i e d to RIA m e t h o d o l o g y . D a t a on c o n v e n t i o n a l RIA s h o u l d be c o n f i n e d to the "Methods". On the o t h e r hand, n e w and u n c o n v e n t i o n a l RIA p r o c e d u r e s w i l l be published as full papers. To a s s i s t the reader, c o n t r i b u t o r and E d i t o r i a l reviewer, we i n c l u d e in this issue a d e f i n i t i v e m i n i r e v i e w by Dr. E l i z a b e t h G r a n s t r o m . In addition, we a p p e n d the E d i t o r i a l Guidelines. These G u i d e l i n e s are s t r a i g h t f o r w a r d and have been w i d e l y c i r c u l a t e d and g e n e r a l l y accepted. GUIDELINES i.

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Originality. The m e t h o d s h o u l d d e s c r i b e the d e v e l o p m e n t and c h a r a c t e r i z a t i o n of a n t i s e r a for PGs, for w h i c h RIA m e t h o d s have not yet b e e n described. If they deal w i t h the c l a s s i c a l PGs, e.g. PGE, PGF, there s h o u l d be a c o n s i d e r a b l e m e t h o d o l o g i c a l advantage, e.g. a new c o u p l i n g p r o c e d u r e , a new s e p a r a t i o n procedure, w h i c h leads to a s i g n i f i c a n t i m p r o v e m e n t in c o m p a r i s o n w i t h other m e t h o d s (for i n s t a n c e more a v i d antibodies, h i g h e r titers of antisera, less d e g r a d a t i o n of s e n s i t i v e c o m p o u n d s d u r i n g coupling, etc). Precision. D a t a on p r e c i s i o n s h o u l d be given, both as interassay and i n t r a a s s a y v a r i a t i o n , p r e f e r a b l y as c o e f f i c i e n t of variation. It is not s u f f i c i e n t to state only one figure for each of these two p a r a m e t e r s , as the c o e f f i c i e n t of v a r i a t i o n varies w i t h the c o n c e n t r a t i o n of the a n a l y z e d compound. Specificity. W h e n t e s t i n g cross r e a c t i o n s w i t h o t h e r compounds, it is of i m p o r t a n c e to c h o o s e c o m p o u n d s w h i c h are k n o w n to occur in s i g n i f i c a n t amounts in the b i o l o g i c a l s y s t e m i n t e n d e d to be studied. Thus, it is not s u f f i c i e n t to test an a n t i s e r u m for a u r i n a r y t e t r a n o r m e t a b o l i t e only for cross r e a c t i v i t y w i t h prim a r y PGs, as the latter o c c u r only in m i n u t e amounts in urine in c o m p a r i s o n w i t h the d e g r a d e d products. If not p r o p e r l y t e s t e d a g a i n s t o t h e r i m p o r t a n t u r i n a r y m e t a b o l i t e s , such an a n t i s e r u m w i l l then e x h i b i t a s e e m i n g l y high d e g r e e of s p e c i f i c i t y . v a l i d a t i o n . One of the m o s t i m p o r t a n t p o i n t s of the m e t h o d s under the e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d is v a l i d a t i o n . This may be done a) by u s i n g a l t e r n a t i v e methods, e.g. GC-MS; i s o l a t i o n of the PG w i t h a l a b e l l e d t r a c e r or by b i o a s s a y , b) by u s i n g c o n d t i o n s k n o w n to s t i m u l a t e or i n h i b i t the PG synthesis; infusions of PGs, c) by a d d i t i o n e x p e r i m e n t s . This last type of e x p e r i m e n t is the only one t h a t is r e g u l a r l y c a r r i e d out in most R I A p u b l i c a t i o n s , but it is by no means s u f f i c i e n t for e v a l u a t i n g an assay. The a d d i t i o n s s h o u l d be r e a s o n a b l e and w i t h i n the e x p e c t e d range under the e x p e r i m e n t a l c o n d i t i o n s employed. N o n - s p e c i f i c binding. The p o s s i b i l i t y of n o n - i m m u n o l o g i c a l inhib i t i o n of the a n t i g e n - a n t i b o d y b i n d i n g m u s t always be c o n s i d e r e d and d i s c u s s e d in the paper. Such i n h i b i t i o n gives the same final results as if the samples h a d c o n t a i n e d a high c o n c e n t r a t i o n of

JANUARY 1978 VOL. 15 NO. 1

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PROSTAGLANDINS

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PG, and this is u n f o r t u n a t e l y h o w the results are m o s t often interpreted. N o n - i m m u n o l o g i c a l i n h i b i t i o n is c a u s e d by a large n u m b e r of b i o l o g i c a l s u b s t a n c e s o c c u r i n g in u n e x t r a c t e d samples, or by c o m p o u n d s i n t r o d u c e d into the s a m p l e s by e x t r a c tion and p u r i f i c a t i o n p r o c e d u r e s . Body fluid c o n c e n t r a t i o n s . F u r t h e r m o r e , the r e p o r t e d levels s h o u l d be r e a s o n a b l e . For i n s t a n c e , some of the r e p o r t e d "normal" p l a s m a levels of PGF2e by RIA in humans are so high that they s h o u l d h a v e b e e n a s s o c i a t e d w i t h side e f f e c t s such as a b d o m i n a l cramps, abortion, etc. The f o c u s i n g on p r i m a r y p r o s t a g l a n d i n s in the p e r i p h e r a l c i r c u l a t i o n is s o m e w h a t d e b a t a b l e , since g r e a t care is r e q u i r e d to p r e v e n t c o n t r i b u tions d u r i n g s a m p l i n g from the f o r m e d elements of the blood. Application. The u s e f u l n e s s of the m e t h o d resides to a g r e a t e r e x t e n t in the p r o p e r t i e s of the antibodies. T h e s e are u s u a l l y not a v a i l a b l e o u t s i d e the l a b o r a t o r y they w e r e g e n e r a t e d in and data on cross r e a c t i v i t y etc. is t h e r e f o r e of l i m i t e d g e n e r a l interest. In m o s t cases it is t h e r e f o r e a p p r o p r i a t e to i n c l u d e the i n f o r m a t i o n about the RIA in the "Methods" s e c t i o n of the first paper d e s c r i b i n g its a p p l i c a t i o n . Erik ~ngg~rd P e t e r W. R a m w e l l

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JANUARY 1978 VOL. 15 NO. 1