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The utility of the macrophage migration inhibition test for invitro titration of tuberculins
.Journal of Biological Standardization ( 1 9 8 5 ) 13, 3 4 5 - 3 5 3
The utility of the macrophage migration inhibition test for invitro titration of tuberculins ~
ft. Nyabendat~. a n d / . G. Veas Bendeck§
T h e p u r p o s e o f t h i s s t u d y was t o e x p l o r e t h e f u n d a m e n t a l p r i n c i p l e o f the p o t e n c y e s t i m a t i o n o f t u b e r c u l i n s , as a p p l i e d in z,itro b y t h e m a c r o p h a g e m i g r a t i o n i n h i b i t i o n test ( M I T ) u n d e r agarose. T h e M 1 T was p e r f o r m e d u s i n g specifically s e n s i t i z e d m o u s e a n d g u i n e a - p i g p e r i t o n e a l m a c r o p h a g e s a n d serial d i l u t i o n s o f t h e a n a l o g o u s P P D (purified p r o t e i n d e r i v a t i v e s o f t u b e r c u l i n ) t u b e r c u l i n s as a n t i g e n . S t a t i s t i c a l s t u d i e s p e r f o r m e d i n c l u d e d (a) t h e s t a n d a r d d e v i a t i o n o f t h e m e a n m i g r a t i o n areas, (b) t h e analysis o f v a r i a n c e , (c) t h e regression a n a l y s i s , (d) its c o r r e s p o n d i n g l i n e a r i t y test a n d (e) the d e t e r m i n a t i o n o f t h e related c o r r e l a t i o n coefficient. It was s h o w n for t h e first t i m e a n d in b o t h a n i m a l species u n d e r s t u d y t h a t t h e r e is c o r r e s p o n d e n c e b e t w e e n t h e log d o s e - - r e s p o n s e r e l a r i o n s h i p o f t h e t u b e r c u l in P P D in M I T u n d e r a g a r o s e a n d t h e w e l l k n o w n in t u b e r c u l i n c u t a n e o u s reaction. T h e M I T m a y therefore s u c c e s s f u l l y r e p l a c e t h e in zqt'o t i t r a t i o n o f t u b e r c u l i n s .
INTRODUCTION T h e e s t i m a t i o n o£ t h e b i o l o g i c a l a c t i v i t y o f t h e t u b e r c u l i n s has b e e n a c h i e v e d h i t h e r t o by c o m p a r i n g t h e i n d u r a t i o n o r t h e e r y t h e m a t o u s area o f t h e c u t a n e o u s r e a c t i o n s e l i c i t e d by serial d'.'lution o f test a n d reference t u b e r c u l i n s , i n t r a d e r m a l l y i n j e c t e d i n t o specifically s e n s i t i z e d g u i n e a - p i g s a n d later i n t o h u m a n s or c a t t l e . C l i n i c a l trials in h u m a n s a n d c a t t l e are, h o w e v e r , o u t s i d e t h e r o u t i n e m e t h o d s o f t i t r a t i o n . I n s p i t e o f t h e fact t h a t t h e p o t e n c y o f t u b e r c u l i n s as e s t i m a t e d in g u i n e a - p i g s has n o t a l w a y s b e e n * R e c e i v e d for p u b l i c a t i o n 12 A p r i l 1985. t L a b o r a t o i r e d e s t u b e r c u l i n e s , I n s t i t u t P a s t e u r d u B r a b a n t , rue d u R e m o r q u e u r 2 8 , 1040 B r u x e l l e s , Belguim. :[: T o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d . § F e l l o w s h i p g r a n t e d f r o m t h e C o m m i s s a r i a t G~n~ral a u x R e l a t i o n s I n t e r n a t i o n a l e s d e la C o m m u n a u t O Frangaise d e B e l g i q u e . 0092-11571851040345 + 10 $03.00/0
(~) 1985 The International Association o£ Biological Standardization
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j. NYA,BENDA ANDJ.
G. VEAS BENDECK
f o u n d to m a t c h t h a t f o u n d in h u m a n s a n d c a t t l e , ,.2 t h e g u i n e a - p i g r e m a i n s t h e a n i m a l m o d e l o f c h o i c e for t h e c o n t r o l o f n e w b a t c h e s for p o t e n c y d u r i n g t u b e r c u l i n production. T h e e s t i m a t i o n o f t h e relative p o t e n c y o f t e s t t u b e r c u l i n s in t e r m s o f a reference relys o n t h e c o r r e l a t i o n o f t h e l o g d o s e - r e s p o n s e o b s e r v e d in t h e c u t a n e o u s t u b e r c u l i n r e a c t i o n s . 3 In o r d e r to increase t h e s t a t i s t i c a l r e l i a b i l i t y o f t h e t e s t , a large n u m b e r o f c u t a n e o u s r e a c t i o n s has to be r e c o r d e d in a s i n g l e valid e x p e r i m e n t . E v e n so, w o r k e r s have e x p e r i e n c e d i m m e n s e d i s c r e p a n c i e s w h e n a s s i g n i n g p o t e n c y values w h e n t h e t i t r a t i o n s are d o n e i n d i f f e r e n t l a b o r a t o r i e s a n d e v e n in t h e s a m e l a b o r a t o r y w h e n t h e e x p e r i m e n t s o c c u r in d i f f e r e n t trials- 4"5 F o r t h e s e reasons a n d d u e to l i m i t e d n u m b e r o f i n t r a d e r m a l i n j e c t i o n s p e r a n i m a l (on b o t h sides o f a g u i n e a - p i g , six to 12 i n j e c t i o n p o i n t s are r e c o m m e n d e d by t h e W H O 6 a n d t h e E u r o p e a n P h a r m a c o p o e i a , 7 it is necessary t o k e e p a l a r g e n u m b e r o f s e n s i t i z e d a n i m a l s in t h o s e l a b o r a t o r i e s u n d e r t a k i n g c o n t r o l o f t u b e r c u l i n s . A s i n g l e t i t r a t i o n u s u a l l y r e q u i r e s e i g h t to 12 g u i n e a - p i g s . T h e m a c r o p h a g e m i g r a t i o n i n h i b i t i o n test ( M I T ) , o n t h e o t h e r h a n d , a n d also t h e l y m p h o c y t e t r a n s f o r m a t i o n test ( L T T ) , have b e e n p r o p o s e d by m a n y a u t h o r s as in vitro counterparts of delayed type skin hypersensitivity (DTH). s-m Some tuberculin-active c a r b o h y d r a t e s , a c o m m o n t u b e r c u l i n c o m p o n e n t , a l t h o u g h i n a c t i v e in t h e L T T , have b e e n f o u n d , h o w e v e r , to i n d u c e t h e D T H a n d t h e M I T in g u i n e a p i g s . z l N i l s s o n & M a g n u s s o n 12 h a v e s u c c e s s f u l l y u s e d t h e L'IW in assessing t h e b i o l o g i c a c t i v i t y o f t u b e r c u l i n s . B e s i d e s t h e n e e d to use r a d i o a c t i v e m a t e r i a l s , t h e L T T r e q u i r e s e x p e n s i v e e q u i p m e n t w h e r e a s t h e M I T u n d e r agarose as d e s c r i b e d by C l a u s e n 13 a n d B r o e n d s t r u p 14 is a n easy a n d i n e x p e n s i v e m e t h o d , q u i t e s u i t a b l e for r o u t i n e use. W e , t h e r e f o r e , i m p r o v e d t h i s t e c h n i q u e by u s i n g m i c e a n d g u i n e a - p i g s p e r i t o n e a l m a c r o p h a g e s , a n d w e h a v e d e m o n s t r a t e d in b o t h t h e species, t h e basic p r i n c i p l e o f t h e l o g d o s e - r e s p o n s e r e l a t i o n s h i p in M I T . MATERIALS
AND
METHODS
Guinea-pig model Guinea-pigs. T h e a n i m a l s u s e d w e r e w h i t e f e m a l e D u n k i n - H a r t l e y Animal Production 600-800 g.
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Immunization. T h e g u i n e a - p i g s w e r e i n o c u l a t e d w i t h 2 - 0 m g o f h e a t k i l l e d d e s s i c a t e d Mycobacterium boris, s t r a i n Vallde, s u s p e n d e d in I n c o m p l e t e F r e u n d ' s A d j u v a n t (IFA) at t h e c o n c e n t r a t i o n o f 4 m g / m l : 0- 5 m l was i n j e c t e d i n t r a m u s c u l a r y i n t o a h i n d leg: T h e a n i m a l s w e r e u s e d for e x p e r i m e n t s b e t w e e n t h r e e a n d f o u r m o n t h s after immunization. Preparation of agarose plates. T h e t e c h n i q u e o f C l a u s e n was u s e d w i t h s o m e modifications. A 1"8% ( w / v ) s o l u t i o n ofagarose (Agarose L.E. Miles Laboratories, Ltd. S t o k e P o g e s , U K ) was c o o l e d to 4 7 ° C , m i x e d w i t h h e a t i n a c t i v a t e d s e r u m f r o m n o n - i m m u n i z e d g u i n e a p i g s ( N G P S ) , p h o s p h a t e b u f f e r saline (PBS), h e p e s b u f f e r a n d a t e n - t i m e s c o n c e n t r a t i o n o f M e d i u m 199 ( f r o m G i b c o , Paisley, U K ) a d j u s t e d t o p H 7-2. T h e final s o l u t i o n c o n t a i n e d 0 " 7 5 % (w/v) a g a r o s e a n d 1 0 % (v/v) N G P S . A n t i b i o r i c - a n t i m y c o t i c s o l u t i o n was a d d e d to a final c o n c e n t r a t i o n o f 100 I U penicillin, 100/zg streptomycin and 0"25 /zg Fungizone per millilitre. The volume of 346
IN VITRO TITRATION
OF TUBERCULINS
2- l m l o f this a g a r o s e m e d i u m was p o u r e d i n t o 35-mr:~ d i a m e t e r d i s p o s a b l e Pezri d i s h e s for cell c u l t u r e ( N u n c , K a m s t r u p , R o s k i l d e , D e n m a r k ) . A f t e r t h e gel h a d set, t h e p l a t e s were s t o r e d in t h e 5 % C O 2 , 9 5 % a t m o s p h e r i c air s a t u r a t e d w i t h w a t e r v a p o r i n c u b a t o r at 3 7 ° C , u n t i l u s e d t h e n e x t day. Five holes o f 2-5 m m d i a m e t e r w e r e c u t in each agarose p l a t e on t h e day o f t h e e x p e r i m e n t , u s i n g a sterile stainless steel p u n c h ( L K B - B r o m m a 11, S w e d e n ) .
Antigen. B o v i n e P P D f r o m M. boris, s t r a i n Vall6e, was p r e p a r e d in t h e T u b e r c u l i n P r o d u c t i o n L a b o r a t o r y o f t h e P a s t e u r I n s t i t u t e , Brussels. T h e P P D - t u b e r c u l i n was p r e p a r e d by t h e t e c h n i q u e d e s c r i b e d by M a g n u s s o n 15 w i t h m i n o r m o d i f i c a t i o n s . T h e t e c h n i q u e c o n s i s t s o f p r e c i p i t a t i o n by 2 % t r i c h l o r o a c e t i c acid o f p r o t e i n s in a h e a t k i l l e d m y c o b a c t e r i u m c u l t u r e filtrate f o l l o w e d by t h r e e w a s h i n g s o f t h e p r e c i p i t a t e w i t h a c e t o n e a n d a n h y d r o u s e t h e r successively. T h e p r o d u c t is finally d r i e d to p o w d e r . T h e PPD was d i s s o l v e d in 0 - 1 5 M P B S , p H 7 - 2 , at a c o n c e n t r a t i o n o f 2 m g / m l , sterile filtred o n 0 " 2 2 ~ m p o r e size m i l l e x filters ( M i l l i p o r e C o r p o r a t i o n , M a s s a c h u s s e t t s , U S A ) a n d s t o r e d at - - 2 0 ° C u n t i l n e e d e d for use b u t n o t for l o n g e r t h a n six m o n t h s . Migration inhibition test. T h e M I T was p e r f o r m e d as d e s c r i b e d by B r o e n d s t r u p a n d c o w o r k e r s w i t h s o m e m o d i f i c a t i o n s . P e r i t o n e a l e x u d a t e cells (PEC) w e r e o b t a i n e d f r o m t w o g u i n e a - p i g s five d a y s a f t e r an i n t r a - p e r i t o n e a l (i. p . ) i n j e c t i o n o f 20 m l o f sterile l i q u i d paraffin ( B e l g o l a b o , O v e r i j s e , B e l g i u m ) . O n t h e d a y o f t h e assay t h e g u i n e a - p i g s w e r e a n a e s t h e t i z e d w i t h c h o l o r o f o r m , e x a n g u i n a t e d by cardiac p u n c t u r e a n d i n j e c t e d i . p . w i t h 6 0 m l o f H a n k ' s b a l a n c e d salt s o l u t i o n ( H B S S ) ( G i b c o , Paisley, U K ) . A f t e r g e n t l e m a s s a g e , t h e p e r i t o n e u m was o p e n e d u n d e r s t e r i l e c o n d i t i o n s a n d t h e p e r i t o n e a l fluid was a s p i r a t e d a n d c o l l e c t e d in a sterile E r l e n m e y e r flask c o n t a i n i n g 6 0 m l o f H B S S . T h e lavage fluid w a s c e n t r i f u g e d at 3 5 0 g for seven m i n u t e s a n d t h e p e l l e t e d cells w e r e t h e n r e s u s p e n d e d in a s m a l l v o l u m e o f H B S S for transfer to a clean t u b e . F o l l o w i n g t w o m o r e w a s h e s t h e cells w e r e r e s u s p e n d e d , c o u n t e d in C o u l t e r C o u n t e r ( C o u l t e r E l e c t r o n i c s , B e d f o r d s h i r e , U K ) a n d a d j u s t e d to 1 × 10 s c e l l s / m l . T h e v o l u m e o f c o u n t e d cells was d e v i d e d -~to e q u a l v o l u m e s i n t o five conical t u b e s , c e n t r i f u g e d a n d r e s u s p e n d e d in an e q u i v a l e n t v o l u m e o f m e d i u m 199 at p H 7"2, c o n t a i n i n g 2 0 % N G P S , L - g l u t a m i n e , a n t i b i o t i c - a n t i m y c o t i c s o l u t i o n a n d serial doses o f b o v i n e P P D ; o n e t u b e c o n t a i n e d n o P P D as a c o n t r o l a n d t h e o t h e r four t u b e s c o n t a i n e d 1, 10, 5 0 a n d 100 ~ g / m l o f P P D . T h e five t u b e s w e r e p r e i n c u b a t e d in 5 % C O 2 , 9 5 % a t m o s p h e r i c air s a t u r a t e d w i t h w a t e r v a p o r , at 3 7 ° C for 2 0 r a i n . T h e v i a b i l i t y o f t h e cells, m e a s u r e d by t h e n i g r o s i n e d y e e x c l u s i o n t e s t , was n o less t h a n 9 0 % . Migration in agarose. S e v e n m i c r o l i t r e s o f t h e p r e i n c u b a t e d ceils were t r a n s f e r e d to t h e holes o f e i g h t to t e n agarose p l a t e s , all d i l u t i o n s on every p l a t e . T h e agarose p l a t e s w e r e t h e n i n c u b a t e d for 2 0 h in t h e s a m e c o n d i t i o n s as d e s c r i b e d above. A f t e r i n c u b a t i o n , t h e cells u n d e r agarose w e r e fixed a n d s t a i n e d w i t h 0" 1% (w/v) C o o m a s s i e b r i l l i a n t b l u e in e t h a n o l ( 4 5 % , v/v), a c e t i c acid ( 1 0 % , v/v) a n d d i s t i l l e d w a t e r ( 4 5 % , v/v) for 30 r a i n . A f t e r r e m o v a l o f t h e g e l t h e cells w e r e s t a i n e d for a f u r t h e r 15 r a i n a n d finally r i n s e d w i t h d i s t i l l e d w a t e r a n d left to d r y b y e v a p o r a t i o n . T h e i n d i v i d u a l areas o f m i g r a t i o n w e r e p r o j e c t e d t h r o u g h a 2 0 % p h o t o m a g n i f i e r (Asahi P e n t a x B e l l o w s II) o n t o t h e surface o f an e l e c t r o n i c p l a n i m e t e r ( M e s s g e r a t e G m b H , K o n t r o n ) w h e r e t h e h o l e area ( i n s i d e surface) a n d t h e e n t i r e m i g r a t i o n area w e r e r e c o r d e d t h r e e t i m e s 347
j . N Y A B E N D A A N D J . G. VEAS BENDECK successively. T h e d i f f e r e n c e b e t w e e n t h e m e a n s o f these t w o r e c o r d i n g s expresses t h e m i g r a t i o n area.
Mice model Mice. F e m a l e o u t b r e d Swill a l b i n o m i c e , w e i g h i n g 4 0 - 1 2 + 6 - I g , f r o m t h e A n i m a l P r o d u c t i o n U n i t o f xhe P a s t e u r I n s t i t u t e , Brussels, w e r e used.
Immunization. T h e i n o c u l u m c o n s i s t e d o f h e a t k i l l e d a n d d e s s i c a t e d M. boris, s t r a i n B C G , a n d w a s s u b c u t a n e o u s l y i n j e c t e d in t h e s a m e a m o u n t as in t h e case o f t h e guinea-pigs. Agaroseplates. T h e s e w e r e p r e p a r e d in t h e s a m e w a y as t h o s e u s e d for t h e g u i n e a p i g e x p e r i m e n t s . Foetal c a l f s e r u m (FCS) r e p l a c e d in t h e N G P S as a r e s u l t o f o u r p r e l i m i n a r y e x p e r i e n c e s w h i c h h a d s h o w n e v i d e n t a d v a n t a g e s o f foetal c a l f s e r u m o v e r g u i n e a - p i g a n d h o r s e s e r u m in m o u s e P E C c u l t u r e (results n o t s h o w n ) . Antigen. P P D - B C G w a s p r e p a r e d f r o m a seven w e e k s B C G c u l t u r e , h e a t k i l l e d , filtered a n d p u r i f i e d by t h e t e c h n i q u e d e s c r i b e d above. Migration Inhibition Test. T h e M I T was p e r f o r m e d a n d r e c o r d e d in t h e s a m e w a y as t h e g u i n e a p i g s assays w i t h t h e f o l l o w i n g modifications. P E C w e r e o b t a i n e d f r o m five m i c e t h r e e to f o u r days a f t e r an i.p- i n j e c t i o n o f 1 m l o f IFA. O n t h e day o f t h e assay t h e m i c e w e r e k / l i e d b y a n o v e r d o s e o f c h l o r o f o r m a n d t h e cells h a r v e s t e d in 5 m l o f H B S S i n j e c t e d i . p . after s t r i p i n g b a c k t h e fur. T h e P E C w e r e w a s h e d t w i c e in H B S S by c e n t r i f u g a t i o n at 3 5 0 g for s e v e n m i n u t e s , c o u n t e d a n d a d j u s t e d to 1 × l 0 s c e l l s / m l . T h e cells w e r e e q u a l l y d i v i d e d i n t o f o u r conical t u b e s , c e n t r i f u g e d , a n d t h e s u p e r n a t a n t was d i s c a r d e d a n d r e p l a c e d by a n e q u i v a l e n t v o l u m e o f m e d i u m I 9 9 at p H 7 - 2 , c o n t a i n i n g 209~ FCS a n d , r e s p e c t i v e l y , 0, 1, 5 0 a n d 1 0 0 / x g / m l o f P P D - B C G . T h e p r e i n c u b a t i o n t i m e , v i a b i l i t y test, m i g r a t i o n in a g a r o s e as w e l l as t h e i n c u b a t i o n p e r i o d , s t a i n i n g a n d finally t h e m e a s u r e m e n t o f t h e m i g r a t i o n areas w e r e a c h i e v e d in t h e s a m e w a y as d e s c r i b e d a b o v e for t h e m e t h o d w i t h g u i n e a p i g s . RESULTS
Guinea pigs Table 1 shows the migration and inhibition percentages derived from the data of four i n d e p e n d a n t assays p e r f o r m e d in g u i n e a p i g s . F o r every P P D d i l u t i o n t h e m e a n m i g r a t i o n v a l u e a n d its s t a n d a r d d e v i a t i o n w e r e c a l c u l a t e d f r o m t e n m i g r a t i o n area's each r e c o r d e d t h r e e t i m e s . T h e m e a n v a l u e was c o n s i d e r e d e q u i v a l e n t to 1 0 0 % m i g r a t i o n , a n d a c c o r d i n g l y , to zero i n h i b i t i o n for t h e c o n t r o l d i l u t i o n w i t h o u t P P D a n t i g e n . T h e p e r c e n t a g e m i g r a t i o n for each P P D d i l u t i o n was as follows: (percentage migration ~
m i g r a t i o n w i t h a n t i g e n X I 0 0 ~} ~ o n ~t~-t antigen /
The percentage inhibition being 100 minus the percent migration. T h e r e s u l t s are g r a p h i c a l l y r e p o r t e d in F i g . 1 in w h i c h t h e c o r r e s p o n d i n g regression lines are s h o w n . 348
IN VITRO T I T R A T I O N OF T U B E R C U L I N S
TABLE 1. M a c r o p h a g e m i g r a t i o n i n h i b i t i o n test in gtainea-pigs: the m i g r a t i o n i n h i b i t i o n ratio w i t h d i f f e r e n t doses o f bovine purified p r o t e i n derivatives o f t u b e r c u l i n ( P P D ) in four i n d e p e n d a n t assays Surface o f m i g r a t i o n (ram 2) Dose P P D (/zg/ml)
Mean value
SD
% Migration
% Inhibition
I
Control 1 10 50 100
1 ; - 59 6"48 4-26 3-27 2-42
1-47 2-28 0-80 1-06 0"31
100 56 37 28 21
0 44 63 72 79
II
Control 1 10 50 100
4" 56 2-28 1-5 1 1"13 0"87
0"96 0-54 0-40 0-45 0-24
100 50 33 25 19
0 50 67 75 81
III
Control 1 10 50 100
6- 11 3"59 2"85 2"32 1"77
0-58 0-4I 0-26 0"30 0-4 l
100 59 47 38 29
0 41 53 62 71
IV
Control 1 ]0 50 100
6"63 2"34 1-56 0-72 0-47
0-28 0-44 0-45 0"23 0- 15
100 35 24 11 7
0 65 76 89 93
Assay n u m b e r
2-C
1-7
I-0
0
40
I _|, ,50 GO 7 0 80 9O Migration inhibition ( % )
O0
Fig. 1. Macrophage migration inhibition rate versus log-dose of bovine purified protein derivatives of tuberculin in guinea-pigs. E~, Experiment I; o , experiment II; A, experiment III; e , experiment IV. 349
J. NYABENDA A N D J . G. VEAS BENDECK TABLE 2. Macrophage migration inhibition test in guinea-pigs: variance analysis, correlation coefficient and the linearity test of the bovine purified protein derivatives of tuberculin doses in four independant assays Correlation coefficients
Difference between doses Assay number I II III IV
F 17"3t I7"0~ 48" It 62"3t
P 4 2 8 1-5
X 10 - 7 * * * X 10 - 6 * * * X 10-12"** X 10 - l l * * *
r
t
0"998 0"997 0"986 0"995
26"2 19"8 8-4 I4"5
P 0"001"** 0"002*** 0"01"** 0"005***
Linearity (F test) 0"75* 0"64 0"002"** 0"055**
* Non significant difference. ** Significant difference. *~** Highly significant difference. t F~6. * F~8.
T h e statistical analysis o f t h e d a t a as s h o w n in T a b l e 2, reveals a h i g h l y s i g n i f i c a n t variance r a t i o (F) for ~he r e g r e s s i o n c o m p o n e n t s a t P values o f 4 × 10 - 7 , 2 X 10 - 6 , 8 X 10 -12 , a n d 1-5 x 10 -~1, r e s p e c t i v e l y , for t h e e x p e r i m e n t s I t o IV. H i g h l y s i g n i f i c a n t c o r r e l a t i o n coefficients (r) w e r e also f o u n d for t h e d i f f e r e n t assays (1- = 0 " 9 8 6 t o 0 " 9 9 8 ) (P = 0 - 0 0 1 , 0 - 0 0 2 , 0 - 0 1 a n d 0 - 0 0 5 ) . T h e l i n e a r i t y test p r o v e d satisfactory in t w o e x p e r i m e n t s (I a n d II), in w h i c h n o n significant F tests at P ---- 0 - 7 5 a n d 0 - 6 4 , r e s p e c t i v e l y , w e r e c a l c u l a t e d . In e x p e r i m e n t s III a n d IV, h o w e v e r , h i g h l y s i g n i f i c a n t (P = 0 " 0 0 2 ) a n d s i g n i f i c a n t (P -- 0"055) differences w e r e o b t a i n e d , respectively. T h e dose levels at w h i c h t h e e x p e r i m e n t s w e r e c o n d u c t e d d o p r o b a b l y not a c c o u n t for t h e n o n - l i n e a r i t y f o u n d in e x p e r i m e n t s III a n d IV. T w o o t h e r assays, u s i n g t h e s a m e a m o u n t s o f P P D - t u b e r c u l i n , p r o v e d f u l l y valid. Besides, t h e a b s e n c e o f l i n e a r i t y is s o m e t i m e s o b s e r v e d also in in vivo t u b e r c u l i n t i t r a t i o n tests in g u i n e a pigs w i t h well chosen tuberculin dilutions. I n t h e t u b e r c u l i n p o t e n c y e s t i m a t i o n , t h e c o n s e q u e n c e o f the n o n - l i n e a r i t y as observed i n t h e t w o last e x p e r i m e n t s w o u l d p r o b a b l y i m p a i r the p o t e n c y precision in a way t h a t w o u l d cause it to fall b e y o n d a c c e p t a b l e c o n f i d e n t l i m i t s a n d t h e r e f o r e lead to i n v a l i d a t i o n o f t h e test.
Mice T a b l e 3 s h o w s t h e c a l c u l a t e d m i g r a t i o n versus i n h i b i t i o n p e r c e n t a g e s for t h e t h r e e M I T assays p e r f o r m e d w i t h m i c e PEC. T h e m e a n m i g r a t i o n ~.reas a n d the s t a n d a r d d e v i a t i o n s w e r e c a l c u l a t e d as in t h e g u i n e a - p i g e x p e r i m e n t s . T h e c o r r e s p o n d i n g regression lines o f t h e results are g r a p h i c a l l y s h o w n in Fig. 2. T h e statistical analysis o f the d a t a is s h o w n in T a b l e 4. T h e variances b e t w e e n t h e doses p r e s e n t h i g h l y significant differences at t h e P l e v e l s 8 × 10 - 6 , 1 X 10 - 4 a n d 8 x 10 - 3 , for t h e assays I, II a n d III, r e s p e c t i v e l y . T h e c o r r e l a t i o n coefficients (r) w e r e f o u n d h i g h l y s i g n i f i c a n t ( e x p e r i m e n t II, P ~ 0 - 0 1 ) a n d s i g n i f i c a n t ( e x p e r i m e n t I, P -- 0 - 0 3 ; e x p e r i m e n t III, P = 0-05). T h e n o n - l i n e a r i t y a s s e s s m e n t was r e j e c t e d in t h e t h r e e e x p e r i m e n t s , t h e differences b e i n g , t h r o u g h o u t , n o n - s i g n i f i c a n t . 350
IN VITRO T I T R A T I O N OF T U B E R C U L I N S
TABLE 3. M a c r o p h a g e m i g r a t i o n i n h i b i t i o n t est in mice: T h e m i g r a t i o n i n h i b i t i o n ratio w i t h di f ferent doses o f bovine P P D in triplicate i n d e p e n d a n t assays Surface o f m i g r a t i o n ( r a m 2) Dose P P D (brg/ml)
M e an value
SD
% Migration
% Inhibition
I
Control 1 50 100
3" 54 3"08 2"33 2-24
0"87 0"32 0-41 0"25
100 87 66 63
0 13 34 37
II
Control 1 50 100
3" 34 3"21 2-22 2"06
0"67 0-80 0"39 0"36
100 96 66 62
0 4 34 38
III
Control 1 50 100
5" 03 3" 36 2-46 2" 19
0"36 0-83 0"76 0-40
100 67 49 44
0 33 51 56
Assay N o .
2-0b7
o o
Io
20
3o
4o
50
6o
M~gration inhibition (©/o}
Fig. 2. Macrophage migration inhibition rate versus of log-dose purified protein derivative of tuberculin-BCG in mice. Q, Experiment I; o , experiment lI; ~, experiment III. TABLE 4. M a c r o p h a g e m i g r a t i o n i n h i b i t i o n test i n mice: Variance analysis, c o r r e l a t i o n coefficient and the linearity test o f t h e purified p r o t e i n derivatives o f t u b e r c u l i n - B C G Difference b e t w e e n doses Assay No. J II III
Correlation coefficients
F
P
r
t
P
Linearity (F test)
18"8t 12"5t 6"2~:
8 X 10 - 6 * * * 1 X 10 - 4 * * * 8 X 10 - 3 * * *
0"999 0"999 0"996
23"3 61"2 11"7
0"03** 0"01"*" 0"05**
0"62* 0"88* 0"58*
- Non significant difference. "* Significant difference. **" Highly significant difference. t F~7351
.J. NYABENDA AND 3. G. VEAS BENDECK DISCUSSION T h e p r e s e n t e x p e r i m e n t s w e r e u n d e r t a k e n in o r d e r to d e t e r m i n e w h e t h e r t h e f u n d a m e n t a l p r i n c i p l e u n d e r l y i n g t h e p o t e n c y e s t i m a t i o n o f t u b e r c u l i n s , n a m e l y , the c o r r e l a t i o n log d o s e - r e s p o n s e , a p p l i e d to t h e in vitro m a c r o p h a g e m i g r a t i o n i n h i b i t i o n test. T h e t e c h n i q u e was r e s t r i c t e d to p e r i t o n e a l e x u d a t e cells as these cells are well k n o w n to be p o t e n t in t h e t r a n s f e r o f t u b e r c u l i n h y p e r s e n s i t i v i t y 16 and o t h e r c e l l - m e d i a t e d i m m u n e s y s t e m s i n c l u d i n g t h e in vitro p r o d u c t i o n o f l y m p h o k i n e s . 17 Peritoneal e x u d a t e cells have also b e e n n o t e d for t h e i r a n t i g e n i c specificity in d e l a y e d h y p e r s e n s i t i v i t y reactions, m. 19 Mycobacterium boris, strain Vall6e, is, besides t h e A N 5 strain, o n e o f t h e t w o strains r e c o m m e n d e d for bovine P P D p r o d u c t i o n . It has been c u l t u r e d in o u r l a b o r a t o r y for m a n y years for t h e p r o d u c t i o n o f t h e b o v i n e P P D used in B e l g i u m . T h i s P P D has been c a l i b r a t e d in g u i n e a - p i g s a n d in t u b e r c u l o u s cattle. 2o T h e in vitro t i t r a t i o n o f this P P D b a t c h u s i n g t h e d e s c r i b e d M I T t e c h n i q u e is n o w in progress. Mycobacterium bowls, strain B C G , s u b s t r a i n I P 1173 has been used fbr t h e p r o d u c t i o n o f t h e r e l a t e d B C G - P P D as d e s c r i b e d in m a t e r i a l s and m e t h o d s . T h e B C G - P P D has been w e i g h e d for clinical use b y G r i e p & Bleiker. 21 T h e n o n p a t h o g e n i c i t y o f B C G ensures safe m a n i p u l a t i o n s in c u l t u r e s a n d in sensitising l a b o r a t o r y a n i m a l s w i t h live bacilli. It s e e m e d to us, t h e r e f o r e , i n t e r e s t i n g e n o u g h t o k n o w t h e b e h a v i o u r o f B C G - P P D in the in vitro t i t r a t i o n assays. T h e r e p l a c e m e n t o f the m a n y t u b e r c u l i n s , d i f f e r e n t in o r i g i n , by a t u b e r c u l i n p r o d u c e d by a u n i q u e well s t a n d a r d i z e d strain such as B C G , w o u l d g r e a t l y c o n t r i b u t e to t h e s t a n d a r d i z a t i o n o f t u b e r c u l i n s . T h e t e c h n i q u e o f t h e M I T u n d e r agarose offers a g r e a t practical a d v a n t a g e over o t h e r c e l l u l a r i m m u n i t y tests s u c h as t h e L T T , a n d e v e n over the i n t r a d e r m a l reaction. It is easy to p e r f o r m , it does n o t n e e d any e x p e n s i v e a p p a r a t u s or m a t e r i a l s , a n d can be p e r f o r m e d m a n y t i m e s w i t h v e r y f e w a n i m a l s . T h e average a m o u n t o f P E C collected f r o m t w o i m m u n i z e d g u i n e a - p i g s a n d five m i c e was a b o u t 1-12 × 108 and 1-25 × t08 cells, respectively. T h i s a m o u n t was e n o u g h for at least 30 Petri dishes w i t h five P P D d i l u t i o n s o n each. T h e fixed ceils after C o o m a s s i e t r e a t m e n t can be k e p t a l l m o s t i n d e f i n i t e l y , p e r m i t t i n g a record at a n y t i m e . M o r e o v e r , t h e fact that m o n o n u c l e a r cells can be frozen a n d successfully c u l t u r e d later, 22 will be o f g r e a t i m p o r t a n c e in e s t a b l i s h i n g t h e biological a c t i v i t y o f reference t u b e r c u l i n s . G u i n e a - p i g s are t h e c o m m o n l y used ~ n i m a l s for t h e t u b e r c u l i n t i t r a t i o n in vivo. T h e i r use in t h e d e v e l o p m e n t o f an in vitro t u b e r c u l i n t i t r a t i o n rest t h u s s e e m s obvious. It was h o w e v e r , i n t e r e s t i n g to d e t e r m i n e w h e t h e r t h e p r i n c i p l e a p p l i e d to o t h e r a n i m a l species; a n d therefore, m i c e w e r e i n c l u d e d in t h e e x p e r i m e n t . In b o t h species u n d e r s t u d y , a n d for b o t h P P D s t h e results clearly s h o w the fairly g o o d log d o s e - r e s p o n s e c o r r e l a t i o n in M I T .
A cknowledgmnents W e wish to express o u r g r a t i t u d e to the Commissariat G6n6ral aux Relations Internationales de la Communaut6 Frangaise de Belgique for the fellowship, Professor D. Dekegel (Instirut Pasteur du Brabant) for mensuration facilities, Dr R. Dobbelaer (Institut d'Hygi~ne et d'Epid6miologie, Brussels) for statistical data analysis, all the colleagues of the Tuberculin Department, especially P. De Backere and E. Bautens for technical assistance, and JM Pirotte for typing the manuscript. 352
I N V I T R O T I T R A T I O N OF T U B E R C U L I N S
REFERENCES 1. Guld J , Bentzon M W , Bleiker MA, Griep W A , Magnusson M, Waaler H. The standardization of a new batch o f purified tuberculin. Bull W H O 1958; 1 9 : 8 4 5 - 9 5 1. 2. Dobbelaer R, O'Reilly LM, Gdnicot A, Haagsma J. The potency o f bovine P P D tuberculins in guinea pigs and in tuberculous cattle. J Biol Stand 1983; 1 1 : 2 1 3 - 2 2 0 . 3- Long DA, Miles AA, Perry LM. The assay of tuberculin. Bull W H O 1954; 10: 9 8 9 - 1 0 0 2 . 4. Comstock G W , Edwards LB, Philip R N , W i n n W A . A comparison in the United States of America of t w o tuberculins, P P D - S and R T 23. Bull W H O 1964; 31: 161-170. 5. Schneider ~0~T, Dobbelaer R, Dam A, J o r g e n s e n J B , Gayot G, A u g i e r J , H a a g s m a J , Rees W H G , Lesslie I W , Nancy H e b e r t C. Collaborative assay of EEC standards for bovine tuberculins. J Biol Stand 1979; 7: 53--66. 6~ Org Mond Santd. Comit6 d'experts de la Standardisation biologique. Sdrie de rapports techniques 1968; 384: 2 3 - 4 1 . 7. Pharmacop6e Europ6enne Vol. 2. Ministate de la Sant6 Publique, Bruxelles 1 9 7 4 : 4 3 6 and 4398 . George M, Vaughan J H . In vitro cell migration as a model for delayed hypersensitivity. Proc Soc Exp Biol Med 1962; 1 1 1 : 5 1 4 - 5 2 1 . 9. David J R . Cell-associated hypersensitivity studied in vitro. Cancer Res 1968; 28: 1387-139110. Miller SD, Jones HE. Correlation o f lymphocyte transformation with tuberculin skin-test sensitivity. Am Rev Respir Dis 1973; 107: 5 3 0 - 5 3 8 . 11. Chaparas SD, Thor DE, Hedrick SR. Comparison of lymphocyte transformation, inhibition ofmacrophage migration and skin-tests using dializable and non-dializable tuberculin fractions from Mycobacteriurn bovis (BCG). J Immunol 1971; 197: 149-153. 12. Nilsson BS, Magnusson M. Comparison of the biologic activity oftuberculins by the use of lymphocyte cultures. A m Rev Respir Dis 1973; 108: 5 6 5 - 5 7 0 . 13. Clausen JE. Tuberculin-induced migration inhibition o f human peripheral leucocytes in agarose medium. Acta Allergol 1971; 26: 5 6 - 8 0 . 14. Broendstrup O, Magnusson M, Maxild J, Werdelin O. Demonstration of delayed-type hypersensitivity in guinea-pigs by the agarose plate technique. Acra Pathol Microbiol Stand It] 1975; 83: 5 2 - 5 8 . 15. Magnusson M, Bentzon M W . Preparation o f purified tuberculin R T 2.3. Bull W H O 1958; 19: 8 2 9 - 8 4 3 . 16. Chase M W . The cellular transfer of cutaneous hypersensitivity to tuberculin. Proc Soc Exp Biol Med 1945; 59: 1 3 4 - 1 3 5 . 17. D u m o n d e DC, Wolstencroft RA, Panayi GS, Mathew M, Morley J , Howson W T . "Lymphokines': N o n - a n t i b o d y mediators o f cellular i m m u n i t y generated by lymphocyte activation. Nature 1969; 224: 3 8 - 4 2 . 18. David J R , A1-Askary S, Lawrence HS, Thomas L. Delayed hypersensitivity in vitro I: The specificity of inhibition o f cell migration by antigens. J Immunol 1964; 93: 2 6 4 - 2 7 3 . 19. David J R , Lawrence HS, Thomas L. Delayed hypersensitivity in vitro III: the specificity o f hapten-protein conjugates in the inhibition o f cell migration. J lmmunol 1964; 93: 279-282. 20. Haagsma J , O'Reilly LM, Dobbelaer R, Murphy TM. A comparison o f the relative potencies o f various bovine P P D tuberculins in naturally infected tuberculous cattle. J Biol Stand 1982; 10: 2 7 3 - 2 8 4 . 21. Gr/ep W A , Bleiker MA. lntracutaneous reactions with tuberculins prepared from Mycobacterium Tuberculosis H u m a n u m and from BCG. Proc Tuberc Res Counc 1960; 47: 2 4 - 3 3 . 22. Harel-Bellan A, Marchiol C, Kaplan C, Muller J Y , Chouaib S, Ythier A, Nowill A, Fradelizi D. Improved culture conditions for quantitative evaluation o f Interleukin 2 production by frozen human lymphocytes. J Immunol Methods 1983; 64: 61--69.
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The utility of the macrophage migration inhibition test for invitro titration of tuberculins ~
ft. Nyabendat~. a n d / . G. Veas Bendeck§
T h e p u r p o s e o f t h i s s t u d y was t o e x p l o r e t h e f u n d a m e n t a l p r i n c i p l e o f the p o t e n c y e s t i m a t i o n o f t u b e r c u l i n s , as a p p l i e d in z,itro b y t h e m a c r o p h a g e m i g r a t i o n i n h i b i t i o n test ( M I T ) u n d e r agarose. T h e M 1 T was p e r f o r m e d u s i n g specifically s e n s i t i z e d m o u s e a n d g u i n e a - p i g p e r i t o n e a l m a c r o p h a g e s a n d serial d i l u t i o n s o f t h e a n a l o g o u s P P D (purified p r o t e i n d e r i v a t i v e s o f t u b e r c u l i n ) t u b e r c u l i n s as a n t i g e n . S t a t i s t i c a l s t u d i e s p e r f o r m e d i n c l u d e d (a) t h e s t a n d a r d d e v i a t i o n o f t h e m e a n m i g r a t i o n areas, (b) t h e analysis o f v a r i a n c e , (c) t h e regression a n a l y s i s , (d) its c o r r e s p o n d i n g l i n e a r i t y test a n d (e) the d e t e r m i n a t i o n o f t h e related c o r r e l a t i o n coefficient. It was s h o w n for t h e first t i m e a n d in b o t h a n i m a l species u n d e r s t u d y t h a t t h e r e is c o r r e s p o n d e n c e b e t w e e n t h e log d o s e - - r e s p o n s e r e l a r i o n s h i p o f t h e t u b e r c u l in P P D in M I T u n d e r a g a r o s e a n d t h e w e l l k n o w n in t u b e r c u l i n c u t a n e o u s reaction. T h e M I T m a y therefore s u c c e s s f u l l y r e p l a c e t h e in zqt'o t i t r a t i o n o f t u b e r c u l i n s .
INTRODUCTION T h e e s t i m a t i o n o£ t h e b i o l o g i c a l a c t i v i t y o f t h e t u b e r c u l i n s has b e e n a c h i e v e d h i t h e r t o by c o m p a r i n g t h e i n d u r a t i o n o r t h e e r y t h e m a t o u s area o f t h e c u t a n e o u s r e a c t i o n s e l i c i t e d by serial d'.'lution o f test a n d reference t u b e r c u l i n s , i n t r a d e r m a l l y i n j e c t e d i n t o specifically s e n s i t i z e d g u i n e a - p i g s a n d later i n t o h u m a n s or c a t t l e . C l i n i c a l trials in h u m a n s a n d c a t t l e are, h o w e v e r , o u t s i d e t h e r o u t i n e m e t h o d s o f t i t r a t i o n . I n s p i t e o f t h e fact t h a t t h e p o t e n c y o f t u b e r c u l i n s as e s t i m a t e d in g u i n e a - p i g s has n o t a l w a y s b e e n * R e c e i v e d for p u b l i c a t i o n 12 A p r i l 1985. t L a b o r a t o i r e d e s t u b e r c u l i n e s , I n s t i t u t P a s t e u r d u B r a b a n t , rue d u R e m o r q u e u r 2 8 , 1040 B r u x e l l e s , Belguim. :[: T o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d . § F e l l o w s h i p g r a n t e d f r o m t h e C o m m i s s a r i a t G~n~ral a u x R e l a t i o n s I n t e r n a t i o n a l e s d e la C o m m u n a u t O Frangaise d e B e l g i q u e . 0092-11571851040345 + 10 $03.00/0
(~) 1985 The International Association o£ Biological Standardization
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j. NYA,BENDA ANDJ.
G. VEAS BENDECK
f o u n d to m a t c h t h a t f o u n d in h u m a n s a n d c a t t l e , ,.2 t h e g u i n e a - p i g r e m a i n s t h e a n i m a l m o d e l o f c h o i c e for t h e c o n t r o l o f n e w b a t c h e s for p o t e n c y d u r i n g t u b e r c u l i n production. T h e e s t i m a t i o n o f t h e relative p o t e n c y o f t e s t t u b e r c u l i n s in t e r m s o f a reference relys o n t h e c o r r e l a t i o n o f t h e l o g d o s e - r e s p o n s e o b s e r v e d in t h e c u t a n e o u s t u b e r c u l i n r e a c t i o n s . 3 In o r d e r to increase t h e s t a t i s t i c a l r e l i a b i l i t y o f t h e t e s t , a large n u m b e r o f c u t a n e o u s r e a c t i o n s has to be r e c o r d e d in a s i n g l e valid e x p e r i m e n t . E v e n so, w o r k e r s have e x p e r i e n c e d i m m e n s e d i s c r e p a n c i e s w h e n a s s i g n i n g p o t e n c y values w h e n t h e t i t r a t i o n s are d o n e i n d i f f e r e n t l a b o r a t o r i e s a n d e v e n in t h e s a m e l a b o r a t o r y w h e n t h e e x p e r i m e n t s o c c u r in d i f f e r e n t trials- 4"5 F o r t h e s e reasons a n d d u e to l i m i t e d n u m b e r o f i n t r a d e r m a l i n j e c t i o n s p e r a n i m a l (on b o t h sides o f a g u i n e a - p i g , six to 12 i n j e c t i o n p o i n t s are r e c o m m e n d e d by t h e W H O 6 a n d t h e E u r o p e a n P h a r m a c o p o e i a , 7 it is necessary t o k e e p a l a r g e n u m b e r o f s e n s i t i z e d a n i m a l s in t h o s e l a b o r a t o r i e s u n d e r t a k i n g c o n t r o l o f t u b e r c u l i n s . A s i n g l e t i t r a t i o n u s u a l l y r e q u i r e s e i g h t to 12 g u i n e a - p i g s . T h e m a c r o p h a g e m i g r a t i o n i n h i b i t i o n test ( M I T ) , o n t h e o t h e r h a n d , a n d also t h e l y m p h o c y t e t r a n s f o r m a t i o n test ( L T T ) , have b e e n p r o p o s e d by m a n y a u t h o r s as in vitro counterparts of delayed type skin hypersensitivity (DTH). s-m Some tuberculin-active c a r b o h y d r a t e s , a c o m m o n t u b e r c u l i n c o m p o n e n t , a l t h o u g h i n a c t i v e in t h e L T T , have b e e n f o u n d , h o w e v e r , to i n d u c e t h e D T H a n d t h e M I T in g u i n e a p i g s . z l N i l s s o n & M a g n u s s o n 12 h a v e s u c c e s s f u l l y u s e d t h e L'IW in assessing t h e b i o l o g i c a c t i v i t y o f t u b e r c u l i n s . B e s i d e s t h e n e e d to use r a d i o a c t i v e m a t e r i a l s , t h e L T T r e q u i r e s e x p e n s i v e e q u i p m e n t w h e r e a s t h e M I T u n d e r agarose as d e s c r i b e d by C l a u s e n 13 a n d B r o e n d s t r u p 14 is a n easy a n d i n e x p e n s i v e m e t h o d , q u i t e s u i t a b l e for r o u t i n e use. W e , t h e r e f o r e , i m p r o v e d t h i s t e c h n i q u e by u s i n g m i c e a n d g u i n e a - p i g s p e r i t o n e a l m a c r o p h a g e s , a n d w e h a v e d e m o n s t r a t e d in b o t h t h e species, t h e basic p r i n c i p l e o f t h e l o g d o s e - r e s p o n s e r e l a t i o n s h i p in M I T . MATERIALS
AND
METHODS
Guinea-pig model Guinea-pigs. T h e a n i m a l s u s e d w e r e w h i t e f e m a l e D u n k i n - H a r t l e y Animal Production 600-800 g.
Unit
of the
Pasteur
Institute
of Brabant.
Each
from the weighed
Immunization. T h e g u i n e a - p i g s w e r e i n o c u l a t e d w i t h 2 - 0 m g o f h e a t k i l l e d d e s s i c a t e d Mycobacterium boris, s t r a i n Vallde, s u s p e n d e d in I n c o m p l e t e F r e u n d ' s A d j u v a n t (IFA) at t h e c o n c e n t r a t i o n o f 4 m g / m l : 0- 5 m l was i n j e c t e d i n t r a m u s c u l a r y i n t o a h i n d leg: T h e a n i m a l s w e r e u s e d for e x p e r i m e n t s b e t w e e n t h r e e a n d f o u r m o n t h s after immunization. Preparation of agarose plates. T h e t e c h n i q u e o f C l a u s e n was u s e d w i t h s o m e modifications. A 1"8% ( w / v ) s o l u t i o n ofagarose (Agarose L.E. Miles Laboratories, Ltd. S t o k e P o g e s , U K ) was c o o l e d to 4 7 ° C , m i x e d w i t h h e a t i n a c t i v a t e d s e r u m f r o m n o n - i m m u n i z e d g u i n e a p i g s ( N G P S ) , p h o s p h a t e b u f f e r saline (PBS), h e p e s b u f f e r a n d a t e n - t i m e s c o n c e n t r a t i o n o f M e d i u m 199 ( f r o m G i b c o , Paisley, U K ) a d j u s t e d t o p H 7-2. T h e final s o l u t i o n c o n t a i n e d 0 " 7 5 % (w/v) a g a r o s e a n d 1 0 % (v/v) N G P S . A n t i b i o r i c - a n t i m y c o t i c s o l u t i o n was a d d e d to a final c o n c e n t r a t i o n o f 100 I U penicillin, 100/zg streptomycin and 0"25 /zg Fungizone per millilitre. The volume of 346
IN VITRO TITRATION
OF TUBERCULINS
2- l m l o f this a g a r o s e m e d i u m was p o u r e d i n t o 35-mr:~ d i a m e t e r d i s p o s a b l e Pezri d i s h e s for cell c u l t u r e ( N u n c , K a m s t r u p , R o s k i l d e , D e n m a r k ) . A f t e r t h e gel h a d set, t h e p l a t e s were s t o r e d in t h e 5 % C O 2 , 9 5 % a t m o s p h e r i c air s a t u r a t e d w i t h w a t e r v a p o r i n c u b a t o r at 3 7 ° C , u n t i l u s e d t h e n e x t day. Five holes o f 2-5 m m d i a m e t e r w e r e c u t in each agarose p l a t e on t h e day o f t h e e x p e r i m e n t , u s i n g a sterile stainless steel p u n c h ( L K B - B r o m m a 11, S w e d e n ) .
Antigen. B o v i n e P P D f r o m M. boris, s t r a i n Vall6e, was p r e p a r e d in t h e T u b e r c u l i n P r o d u c t i o n L a b o r a t o r y o f t h e P a s t e u r I n s t i t u t e , Brussels. T h e P P D - t u b e r c u l i n was p r e p a r e d by t h e t e c h n i q u e d e s c r i b e d by M a g n u s s o n 15 w i t h m i n o r m o d i f i c a t i o n s . T h e t e c h n i q u e c o n s i s t s o f p r e c i p i t a t i o n by 2 % t r i c h l o r o a c e t i c acid o f p r o t e i n s in a h e a t k i l l e d m y c o b a c t e r i u m c u l t u r e filtrate f o l l o w e d by t h r e e w a s h i n g s o f t h e p r e c i p i t a t e w i t h a c e t o n e a n d a n h y d r o u s e t h e r successively. T h e p r o d u c t is finally d r i e d to p o w d e r . T h e PPD was d i s s o l v e d in 0 - 1 5 M P B S , p H 7 - 2 , at a c o n c e n t r a t i o n o f 2 m g / m l , sterile filtred o n 0 " 2 2 ~ m p o r e size m i l l e x filters ( M i l l i p o r e C o r p o r a t i o n , M a s s a c h u s s e t t s , U S A ) a n d s t o r e d at - - 2 0 ° C u n t i l n e e d e d for use b u t n o t for l o n g e r t h a n six m o n t h s . Migration inhibition test. T h e M I T was p e r f o r m e d as d e s c r i b e d by B r o e n d s t r u p a n d c o w o r k e r s w i t h s o m e m o d i f i c a t i o n s . P e r i t o n e a l e x u d a t e cells (PEC) w e r e o b t a i n e d f r o m t w o g u i n e a - p i g s five d a y s a f t e r an i n t r a - p e r i t o n e a l (i. p . ) i n j e c t i o n o f 20 m l o f sterile l i q u i d paraffin ( B e l g o l a b o , O v e r i j s e , B e l g i u m ) . O n t h e d a y o f t h e assay t h e g u i n e a - p i g s w e r e a n a e s t h e t i z e d w i t h c h o l o r o f o r m , e x a n g u i n a t e d by cardiac p u n c t u r e a n d i n j e c t e d i . p . w i t h 6 0 m l o f H a n k ' s b a l a n c e d salt s o l u t i o n ( H B S S ) ( G i b c o , Paisley, U K ) . A f t e r g e n t l e m a s s a g e , t h e p e r i t o n e u m was o p e n e d u n d e r s t e r i l e c o n d i t i o n s a n d t h e p e r i t o n e a l fluid was a s p i r a t e d a n d c o l l e c t e d in a sterile E r l e n m e y e r flask c o n t a i n i n g 6 0 m l o f H B S S . T h e lavage fluid w a s c e n t r i f u g e d at 3 5 0 g for seven m i n u t e s a n d t h e p e l l e t e d cells w e r e t h e n r e s u s p e n d e d in a s m a l l v o l u m e o f H B S S for transfer to a clean t u b e . F o l l o w i n g t w o m o r e w a s h e s t h e cells w e r e r e s u s p e n d e d , c o u n t e d in C o u l t e r C o u n t e r ( C o u l t e r E l e c t r o n i c s , B e d f o r d s h i r e , U K ) a n d a d j u s t e d to 1 × 10 s c e l l s / m l . T h e v o l u m e o f c o u n t e d cells was d e v i d e d -~to e q u a l v o l u m e s i n t o five conical t u b e s , c e n t r i f u g e d a n d r e s u s p e n d e d in an e q u i v a l e n t v o l u m e o f m e d i u m 199 at p H 7"2, c o n t a i n i n g 2 0 % N G P S , L - g l u t a m i n e , a n t i b i o t i c - a n t i m y c o t i c s o l u t i o n a n d serial doses o f b o v i n e P P D ; o n e t u b e c o n t a i n e d n o P P D as a c o n t r o l a n d t h e o t h e r four t u b e s c o n t a i n e d 1, 10, 5 0 a n d 100 ~ g / m l o f P P D . T h e five t u b e s w e r e p r e i n c u b a t e d in 5 % C O 2 , 9 5 % a t m o s p h e r i c air s a t u r a t e d w i t h w a t e r v a p o r , at 3 7 ° C for 2 0 r a i n . T h e v i a b i l i t y o f t h e cells, m e a s u r e d by t h e n i g r o s i n e d y e e x c l u s i o n t e s t , was n o less t h a n 9 0 % . Migration in agarose. S e v e n m i c r o l i t r e s o f t h e p r e i n c u b a t e d ceils were t r a n s f e r e d to t h e holes o f e i g h t to t e n agarose p l a t e s , all d i l u t i o n s on every p l a t e . T h e agarose p l a t e s w e r e t h e n i n c u b a t e d for 2 0 h in t h e s a m e c o n d i t i o n s as d e s c r i b e d above. A f t e r i n c u b a t i o n , t h e cells u n d e r agarose w e r e fixed a n d s t a i n e d w i t h 0" 1% (w/v) C o o m a s s i e b r i l l i a n t b l u e in e t h a n o l ( 4 5 % , v/v), a c e t i c acid ( 1 0 % , v/v) a n d d i s t i l l e d w a t e r ( 4 5 % , v/v) for 30 r a i n . A f t e r r e m o v a l o f t h e g e l t h e cells w e r e s t a i n e d for a f u r t h e r 15 r a i n a n d finally r i n s e d w i t h d i s t i l l e d w a t e r a n d left to d r y b y e v a p o r a t i o n . T h e i n d i v i d u a l areas o f m i g r a t i o n w e r e p r o j e c t e d t h r o u g h a 2 0 % p h o t o m a g n i f i e r (Asahi P e n t a x B e l l o w s II) o n t o t h e surface o f an e l e c t r o n i c p l a n i m e t e r ( M e s s g e r a t e G m b H , K o n t r o n ) w h e r e t h e h o l e area ( i n s i d e surface) a n d t h e e n t i r e m i g r a t i o n area w e r e r e c o r d e d t h r e e t i m e s 347
j . N Y A B E N D A A N D J . G. VEAS BENDECK successively. T h e d i f f e r e n c e b e t w e e n t h e m e a n s o f these t w o r e c o r d i n g s expresses t h e m i g r a t i o n area.
Mice model Mice. F e m a l e o u t b r e d Swill a l b i n o m i c e , w e i g h i n g 4 0 - 1 2 + 6 - I g , f r o m t h e A n i m a l P r o d u c t i o n U n i t o f xhe P a s t e u r I n s t i t u t e , Brussels, w e r e used.
Immunization. T h e i n o c u l u m c o n s i s t e d o f h e a t k i l l e d a n d d e s s i c a t e d M. boris, s t r a i n B C G , a n d w a s s u b c u t a n e o u s l y i n j e c t e d in t h e s a m e a m o u n t as in t h e case o f t h e guinea-pigs. Agaroseplates. T h e s e w e r e p r e p a r e d in t h e s a m e w a y as t h o s e u s e d for t h e g u i n e a p i g e x p e r i m e n t s . Foetal c a l f s e r u m (FCS) r e p l a c e d in t h e N G P S as a r e s u l t o f o u r p r e l i m i n a r y e x p e r i e n c e s w h i c h h a d s h o w n e v i d e n t a d v a n t a g e s o f foetal c a l f s e r u m o v e r g u i n e a - p i g a n d h o r s e s e r u m in m o u s e P E C c u l t u r e (results n o t s h o w n ) . Antigen. P P D - B C G w a s p r e p a r e d f r o m a seven w e e k s B C G c u l t u r e , h e a t k i l l e d , filtered a n d p u r i f i e d by t h e t e c h n i q u e d e s c r i b e d above. Migration Inhibition Test. T h e M I T was p e r f o r m e d a n d r e c o r d e d in t h e s a m e w a y as t h e g u i n e a p i g s assays w i t h t h e f o l l o w i n g modifications. P E C w e r e o b t a i n e d f r o m five m i c e t h r e e to f o u r days a f t e r an i.p- i n j e c t i o n o f 1 m l o f IFA. O n t h e day o f t h e assay t h e m i c e w e r e k / l i e d b y a n o v e r d o s e o f c h l o r o f o r m a n d t h e cells h a r v e s t e d in 5 m l o f H B S S i n j e c t e d i . p . after s t r i p i n g b a c k t h e fur. T h e P E C w e r e w a s h e d t w i c e in H B S S by c e n t r i f u g a t i o n at 3 5 0 g for s e v e n m i n u t e s , c o u n t e d a n d a d j u s t e d to 1 × l 0 s c e l l s / m l . T h e cells w e r e e q u a l l y d i v i d e d i n t o f o u r conical t u b e s , c e n t r i f u g e d , a n d t h e s u p e r n a t a n t was d i s c a r d e d a n d r e p l a c e d by a n e q u i v a l e n t v o l u m e o f m e d i u m I 9 9 at p H 7 - 2 , c o n t a i n i n g 209~ FCS a n d , r e s p e c t i v e l y , 0, 1, 5 0 a n d 1 0 0 / x g / m l o f P P D - B C G . T h e p r e i n c u b a t i o n t i m e , v i a b i l i t y test, m i g r a t i o n in a g a r o s e as w e l l as t h e i n c u b a t i o n p e r i o d , s t a i n i n g a n d finally t h e m e a s u r e m e n t o f t h e m i g r a t i o n areas w e r e a c h i e v e d in t h e s a m e w a y as d e s c r i b e d a b o v e for t h e m e t h o d w i t h g u i n e a p i g s . RESULTS
Guinea pigs Table 1 shows the migration and inhibition percentages derived from the data of four i n d e p e n d a n t assays p e r f o r m e d in g u i n e a p i g s . F o r every P P D d i l u t i o n t h e m e a n m i g r a t i o n v a l u e a n d its s t a n d a r d d e v i a t i o n w e r e c a l c u l a t e d f r o m t e n m i g r a t i o n area's each r e c o r d e d t h r e e t i m e s . T h e m e a n v a l u e was c o n s i d e r e d e q u i v a l e n t to 1 0 0 % m i g r a t i o n , a n d a c c o r d i n g l y , to zero i n h i b i t i o n for t h e c o n t r o l d i l u t i o n w i t h o u t P P D a n t i g e n . T h e p e r c e n t a g e m i g r a t i o n for each P P D d i l u t i o n was as follows: (percentage migration ~
m i g r a t i o n w i t h a n t i g e n X I 0 0 ~} ~ o n ~t~-t antigen /
The percentage inhibition being 100 minus the percent migration. T h e r e s u l t s are g r a p h i c a l l y r e p o r t e d in F i g . 1 in w h i c h t h e c o r r e s p o n d i n g regression lines are s h o w n . 348
IN VITRO T I T R A T I O N OF T U B E R C U L I N S
TABLE 1. M a c r o p h a g e m i g r a t i o n i n h i b i t i o n test in gtainea-pigs: the m i g r a t i o n i n h i b i t i o n ratio w i t h d i f f e r e n t doses o f bovine purified p r o t e i n derivatives o f t u b e r c u l i n ( P P D ) in four i n d e p e n d a n t assays Surface o f m i g r a t i o n (ram 2) Dose P P D (/zg/ml)
Mean value
SD
% Migration
% Inhibition
I
Control 1 10 50 100
1 ; - 59 6"48 4-26 3-27 2-42
1-47 2-28 0-80 1-06 0"31
100 56 37 28 21
0 44 63 72 79
II
Control 1 10 50 100
4" 56 2-28 1-5 1 1"13 0"87
0"96 0-54 0-40 0-45 0-24
100 50 33 25 19
0 50 67 75 81
III
Control 1 10 50 100
6- 11 3"59 2"85 2"32 1"77
0-58 0-4I 0-26 0"30 0-4 l
100 59 47 38 29
0 41 53 62 71
IV
Control 1 ]0 50 100
6"63 2"34 1-56 0-72 0-47
0-28 0-44 0-45 0"23 0- 15
100 35 24 11 7
0 65 76 89 93
Assay n u m b e r
2-C
1-7
I-0
0
40
I _|, ,50 GO 7 0 80 9O Migration inhibition ( % )
O0
Fig. 1. Macrophage migration inhibition rate versus log-dose of bovine purified protein derivatives of tuberculin in guinea-pigs. E~, Experiment I; o , experiment II; A, experiment III; e , experiment IV. 349
J. NYABENDA A N D J . G. VEAS BENDECK TABLE 2. Macrophage migration inhibition test in guinea-pigs: variance analysis, correlation coefficient and the linearity test of the bovine purified protein derivatives of tuberculin doses in four independant assays Correlation coefficients
Difference between doses Assay number I II III IV
F 17"3t I7"0~ 48" It 62"3t
P 4 2 8 1-5
X 10 - 7 * * * X 10 - 6 * * * X 10-12"** X 10 - l l * * *
r
t
0"998 0"997 0"986 0"995
26"2 19"8 8-4 I4"5
P 0"001"** 0"002*** 0"01"** 0"005***
Linearity (F test) 0"75* 0"64 0"002"** 0"055**
* Non significant difference. ** Significant difference. *~** Highly significant difference. t F~6. * F~8.
T h e statistical analysis o f t h e d a t a as s h o w n in T a b l e 2, reveals a h i g h l y s i g n i f i c a n t variance r a t i o (F) for ~he r e g r e s s i o n c o m p o n e n t s a t P values o f 4 × 10 - 7 , 2 X 10 - 6 , 8 X 10 -12 , a n d 1-5 x 10 -~1, r e s p e c t i v e l y , for t h e e x p e r i m e n t s I t o IV. H i g h l y s i g n i f i c a n t c o r r e l a t i o n coefficients (r) w e r e also f o u n d for t h e d i f f e r e n t assays (1- = 0 " 9 8 6 t o 0 " 9 9 8 ) (P = 0 - 0 0 1 , 0 - 0 0 2 , 0 - 0 1 a n d 0 - 0 0 5 ) . T h e l i n e a r i t y test p r o v e d satisfactory in t w o e x p e r i m e n t s (I a n d II), in w h i c h n o n significant F tests at P ---- 0 - 7 5 a n d 0 - 6 4 , r e s p e c t i v e l y , w e r e c a l c u l a t e d . In e x p e r i m e n t s III a n d IV, h o w e v e r , h i g h l y s i g n i f i c a n t (P = 0 " 0 0 2 ) a n d s i g n i f i c a n t (P -- 0"055) differences w e r e o b t a i n e d , respectively. T h e dose levels at w h i c h t h e e x p e r i m e n t s w e r e c o n d u c t e d d o p r o b a b l y not a c c o u n t for t h e n o n - l i n e a r i t y f o u n d in e x p e r i m e n t s III a n d IV. T w o o t h e r assays, u s i n g t h e s a m e a m o u n t s o f P P D - t u b e r c u l i n , p r o v e d f u l l y valid. Besides, t h e a b s e n c e o f l i n e a r i t y is s o m e t i m e s o b s e r v e d also in in vivo t u b e r c u l i n t i t r a t i o n tests in g u i n e a pigs w i t h well chosen tuberculin dilutions. I n t h e t u b e r c u l i n p o t e n c y e s t i m a t i o n , t h e c o n s e q u e n c e o f the n o n - l i n e a r i t y as observed i n t h e t w o last e x p e r i m e n t s w o u l d p r o b a b l y i m p a i r the p o t e n c y precision in a way t h a t w o u l d cause it to fall b e y o n d a c c e p t a b l e c o n f i d e n t l i m i t s a n d t h e r e f o r e lead to i n v a l i d a t i o n o f t h e test.
Mice T a b l e 3 s h o w s t h e c a l c u l a t e d m i g r a t i o n versus i n h i b i t i o n p e r c e n t a g e s for t h e t h r e e M I T assays p e r f o r m e d w i t h m i c e PEC. T h e m e a n m i g r a t i o n ~.reas a n d the s t a n d a r d d e v i a t i o n s w e r e c a l c u l a t e d as in t h e g u i n e a - p i g e x p e r i m e n t s . T h e c o r r e s p o n d i n g regression lines o f t h e results are g r a p h i c a l l y s h o w n in Fig. 2. T h e statistical analysis o f the d a t a is s h o w n in T a b l e 4. T h e variances b e t w e e n t h e doses p r e s e n t h i g h l y significant differences at t h e P l e v e l s 8 × 10 - 6 , 1 X 10 - 4 a n d 8 x 10 - 3 , for t h e assays I, II a n d III, r e s p e c t i v e l y . T h e c o r r e l a t i o n coefficients (r) w e r e f o u n d h i g h l y s i g n i f i c a n t ( e x p e r i m e n t II, P ~ 0 - 0 1 ) a n d s i g n i f i c a n t ( e x p e r i m e n t I, P -- 0 - 0 3 ; e x p e r i m e n t III, P = 0-05). T h e n o n - l i n e a r i t y a s s e s s m e n t was r e j e c t e d in t h e t h r e e e x p e r i m e n t s , t h e differences b e i n g , t h r o u g h o u t , n o n - s i g n i f i c a n t . 350
IN VITRO T I T R A T I O N OF T U B E R C U L I N S
TABLE 3. M a c r o p h a g e m i g r a t i o n i n h i b i t i o n t est in mice: T h e m i g r a t i o n i n h i b i t i o n ratio w i t h di f ferent doses o f bovine P P D in triplicate i n d e p e n d a n t assays Surface o f m i g r a t i o n ( r a m 2) Dose P P D (brg/ml)
M e an value
SD
% Migration
% Inhibition
I
Control 1 50 100
3" 54 3"08 2"33 2-24
0"87 0"32 0-41 0"25
100 87 66 63
0 13 34 37
II
Control 1 50 100
3" 34 3"21 2-22 2"06
0"67 0-80 0"39 0"36
100 96 66 62
0 4 34 38
III
Control 1 50 100
5" 03 3" 36 2-46 2" 19
0"36 0-83 0"76 0-40
100 67 49 44
0 33 51 56
Assay N o .
2-0b7
o o
Io
20
3o
4o
50
6o
M~gration inhibition (©/o}
Fig. 2. Macrophage migration inhibition rate versus of log-dose purified protein derivative of tuberculin-BCG in mice. Q, Experiment I; o , experiment lI; ~, experiment III. TABLE 4. M a c r o p h a g e m i g r a t i o n i n h i b i t i o n test i n mice: Variance analysis, c o r r e l a t i o n coefficient and the linearity test o f t h e purified p r o t e i n derivatives o f t u b e r c u l i n - B C G Difference b e t w e e n doses Assay No. J II III
Correlation coefficients
F
P
r
t
P
Linearity (F test)
18"8t 12"5t 6"2~:
8 X 10 - 6 * * * 1 X 10 - 4 * * * 8 X 10 - 3 * * *
0"999 0"999 0"996
23"3 61"2 11"7
0"03** 0"01"*" 0"05**
0"62* 0"88* 0"58*
- Non significant difference. "* Significant difference. **" Highly significant difference. t F~7351
.J. NYABENDA AND 3. G. VEAS BENDECK DISCUSSION T h e p r e s e n t e x p e r i m e n t s w e r e u n d e r t a k e n in o r d e r to d e t e r m i n e w h e t h e r t h e f u n d a m e n t a l p r i n c i p l e u n d e r l y i n g t h e p o t e n c y e s t i m a t i o n o f t u b e r c u l i n s , n a m e l y , the c o r r e l a t i o n log d o s e - r e s p o n s e , a p p l i e d to t h e in vitro m a c r o p h a g e m i g r a t i o n i n h i b i t i o n test. T h e t e c h n i q u e was r e s t r i c t e d to p e r i t o n e a l e x u d a t e cells as these cells are well k n o w n to be p o t e n t in t h e t r a n s f e r o f t u b e r c u l i n h y p e r s e n s i t i v i t y 16 and o t h e r c e l l - m e d i a t e d i m m u n e s y s t e m s i n c l u d i n g t h e in vitro p r o d u c t i o n o f l y m p h o k i n e s . 17 Peritoneal e x u d a t e cells have also b e e n n o t e d for t h e i r a n t i g e n i c specificity in d e l a y e d h y p e r s e n s i t i v i t y reactions, m. 19 Mycobacterium boris, strain Vall6e, is, besides t h e A N 5 strain, o n e o f t h e t w o strains r e c o m m e n d e d for bovine P P D p r o d u c t i o n . It has been c u l t u r e d in o u r l a b o r a t o r y for m a n y years for t h e p r o d u c t i o n o f t h e b o v i n e P P D used in B e l g i u m . T h i s P P D has been c a l i b r a t e d in g u i n e a - p i g s a n d in t u b e r c u l o u s cattle. 2o T h e in vitro t i t r a t i o n o f this P P D b a t c h u s i n g t h e d e s c r i b e d M I T t e c h n i q u e is n o w in progress. Mycobacterium bowls, strain B C G , s u b s t r a i n I P 1173 has been used fbr t h e p r o d u c t i o n o f t h e r e l a t e d B C G - P P D as d e s c r i b e d in m a t e r i a l s and m e t h o d s . T h e B C G - P P D has been w e i g h e d for clinical use b y G r i e p & Bleiker. 21 T h e n o n p a t h o g e n i c i t y o f B C G ensures safe m a n i p u l a t i o n s in c u l t u r e s a n d in sensitising l a b o r a t o r y a n i m a l s w i t h live bacilli. It s e e m e d to us, t h e r e f o r e , i n t e r e s t i n g e n o u g h t o k n o w t h e b e h a v i o u r o f B C G - P P D in the in vitro t i t r a t i o n assays. T h e r e p l a c e m e n t o f the m a n y t u b e r c u l i n s , d i f f e r e n t in o r i g i n , by a t u b e r c u l i n p r o d u c e d by a u n i q u e well s t a n d a r d i z e d strain such as B C G , w o u l d g r e a t l y c o n t r i b u t e to t h e s t a n d a r d i z a t i o n o f t u b e r c u l i n s . T h e t e c h n i q u e o f t h e M I T u n d e r agarose offers a g r e a t practical a d v a n t a g e over o t h e r c e l l u l a r i m m u n i t y tests s u c h as t h e L T T , a n d e v e n over the i n t r a d e r m a l reaction. It is easy to p e r f o r m , it does n o t n e e d any e x p e n s i v e a p p a r a t u s or m a t e r i a l s , a n d can be p e r f o r m e d m a n y t i m e s w i t h v e r y f e w a n i m a l s . T h e average a m o u n t o f P E C collected f r o m t w o i m m u n i z e d g u i n e a - p i g s a n d five m i c e was a b o u t 1-12 × 108 and 1-25 × t08 cells, respectively. T h i s a m o u n t was e n o u g h for at least 30 Petri dishes w i t h five P P D d i l u t i o n s o n each. T h e fixed ceils after C o o m a s s i e t r e a t m e n t can be k e p t a l l m o s t i n d e f i n i t e l y , p e r m i t t i n g a record at a n y t i m e . M o r e o v e r , t h e fact that m o n o n u c l e a r cells can be frozen a n d successfully c u l t u r e d later, 22 will be o f g r e a t i m p o r t a n c e in e s t a b l i s h i n g t h e biological a c t i v i t y o f reference t u b e r c u l i n s . G u i n e a - p i g s are t h e c o m m o n l y used ~ n i m a l s for t h e t u b e r c u l i n t i t r a t i o n in vivo. T h e i r use in t h e d e v e l o p m e n t o f an in vitro t u b e r c u l i n t i t r a t i o n rest t h u s s e e m s obvious. It was h o w e v e r , i n t e r e s t i n g to d e t e r m i n e w h e t h e r t h e p r i n c i p l e a p p l i e d to o t h e r a n i m a l species; a n d therefore, m i c e w e r e i n c l u d e d in t h e e x p e r i m e n t . In b o t h species u n d e r s t u d y , a n d for b o t h P P D s t h e results clearly s h o w the fairly g o o d log d o s e - r e s p o n s e c o r r e l a t i o n in M I T .
A cknowledgmnents W e wish to express o u r g r a t i t u d e to the Commissariat G6n6ral aux Relations Internationales de la Communaut6 Frangaise de Belgique for the fellowship, Professor D. Dekegel (Instirut Pasteur du Brabant) for mensuration facilities, Dr R. Dobbelaer (Institut d'Hygi~ne et d'Epid6miologie, Brussels) for statistical data analysis, all the colleagues of the Tuberculin Department, especially P. De Backere and E. Bautens for technical assistance, and JM Pirotte for typing the manuscript. 352
I N V I T R O T I T R A T I O N OF T U B E R C U L I N S
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