Journal of Immunological Methods, 10 (1976) 97--98 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
97
Short communication A NEW S C R E E N I N G METHOD FO R ADENOSINE DEAMINASE AND NUCLEOSIDE P H O S P H O R Y L A S E
P.J. BLAKE and J.F. GREALLY Department of Immunology, School of Pathology, Trinity College, Dublin, 2, Ireland
(Received 2 September 1975, accepted 5 September 1975)
An improved screening test for both adenosine deaminase and nucleoside phosphorylase is described. C o m b i n e d i m m u n e deficiency associated with lack of red blood cell adenosine deaminase (ADA) was first n o t e d in 1972 (Giblett, 1972). Subseq u en tly it was f ound t hat a defect at a single ADA locus was almost certainly the cause of one form of c om bi ned i m m une deficiency (Hirschhorn, 1975). More recently there has been a r e p o r t of an association between the absence o f nucleoside phosphorylase (NP) and a severe defect in T-cell i m m u n i t y (Giblett, 1975). In the purine metabolic p a t h w a y the e n z y m e immediately following ADA is NP. Various m e t h o d s have been described for measuring ADA and NP activity (Karker, 1964; H o p k i n s o n et al., 1969). A screening technique which can be p e r f o r m e d on a spot of blood from a heel prick has been described by Moor and Meuwissen (1975). The m e t h o d , based on release of am m oni a from adenosine and the subsequent change in colour of an indicator dye, suffers from a n u m b e r o f disadvantages, i.e. false positives due to diffusion of ammonia, inability to obtain a reading due to fungal contamination. It has also been f o u n d that to prevent the indicator dye turning blue on c o n t a c t with the plate it was necessary to acid wash similar plastic plates to those used in the screening test. T o o v er co me these disadvantages the m e t h o d outlined below was adopted. The m e t h o d is based on the c o m m e n t s o f Parks (1975). The wells in a Bio-cult plastic plate (Model 1S MRC 96, Linbro, 96 cups, 8.5 × 12.8 cm) were filled to just below the surface with a gel of the following co mp o s itio n : Adenosine, final c o n c e n t r a t i o n Agarose gel, final c o n c e n t r a t i o n Nitro Blue T e t r a z o l i u m (NBT), final c o n c e n t r a t i o n
0.01 M 0.1% 0.005%
T h e c o m p o u n d s were dissolved in phos pha te buffer 0.025 M, pH 7.5.
98 T h e b l o o d t o be t e s t e d was s p o t t e d o n W h a t m a n N o . 4 filter p a p e r and dried f o r 10 m i n at 37°C. Discs 6.0 m m in d i a m e t e r w e r e t h e n p u n c h e d f r o m t h e b l o o d s p o t s a n d p l a c e d f i r m l y o n t h e gel and 5 X 10 .3 u n i t s o f x a n t h i n e o x i d a s e (Sigma, G r a d e 1) a d d e d to e a c h disc. T h e p l a t e was sealed w i t h parafilm ( A m e r i c a n Can Co.) a n d i n c u b a t e d o v e r n i g h t u n d e r f l u o r e s c e n t light at r o o m t e m p e r a t u r e . Positive results s h o w e d as a b l a c k disc w h e r e a s the controls w i t h o u t a d e n o s i n e were l i g h t - b r o w n in c o l o u r as were t h e h o r s e e r y t h r o c y t e s used as negative c o n t r o l s . I f p h o s p h a t e is o m i t t e d t h e readings will all b e negative as NP is p h o s p h a t e or a r s e n a t e d e p e n d e n t . I s o l a t e d l y m p h o c y t e s c a n be s c r e e n e d f o r A D A w i t h a d d e d NP (Sigma, 2.1 X 10 -3 units/disc) a n d x a n t h i n e o x i d a s e ( X O D ) using 3 X 104 cells/disc a n d an i n c u b a t i o n t i m e of o n e h o u r at r o o m t e m p e r a t u r e . U n d e r the s a m e c o n d i t i o n s t h e l y m p h o c y t e s m a y be t e s t e d f o r b o t h A D A and NP b y a d d i n g o n l y t h e X O D a n d i n c u b a t i n g t h e p l a t e overnight. T h e m e t h o d d e s c r i b e d has the a d v a n t a g e s t h a t it is relatively fast t h u s o v e r c o m i n g t h e p r o b l e m of c o n t a m i n a t i o n . T h e r e is n o d a n g e r o f false positives d u e to d i f f u s i o n o f a m m o n i a as a positive result is d e p e n d e n t on the rea c t i o n c o n t i n u i n g as far as p r o d u c t i o n o f x a n t h i n e and uric acid w i t h reduct i o n o f t h e a d d e d N B T . T h e b l o o d s p o t s m a y b e l e f t on t h e filter p a p e r at r o o m t e m p e r a t u r e f o r 5 d a y s and still p r o d u c e a positive result. T h e screening s i m u l t a n e o u s l y tests f o r A D A a n d NP d e f i c i e n c y .
REFERENCES Giblett, E.R., G.E. Anderson, F. Cohen, B. Pollara and H.G. Meuwissen, 1972, Lancet 2, 1067. Giblett, E., A.G. Amman, D. Wara, R. Sandman and L.K. Diamond, 1975, Lancet 1, 1010. Hirschhorn, R., 1975, J. Clin. Invest. 55, 661. Hopkinson, D.A., P. Cook and H. Harris, 1969, Ann. Hum. Genet. 32, 361. Karker, H. (1964) Scand. J. Clin. Lab. Invest. 16,570. Moore, E.C. and H.G. Meuwissen, 1975, in: Combined immunodeficiency disease and adenosine deaminase deficiency. A molecular defect, eds. H.G. Meuwissen, R.G. Pickering, B. Pollara and J.H. Porter (Academic Press Inc., New York, San Francisco and London) p. 219. Parks, R.E., Jr., 1975, in: Combined immunodeficiency disease and adenosine deaminase deficiency. A molecular defect, eds. H.G. Meuwissen, R.G. Pickering, B. Pollara and J.H. Porter (Academic Press Inc., New York, San Francisco and London) p. 302.
97
Short communication A NEW S C R E E N I N G METHOD FO R ADENOSINE DEAMINASE AND NUCLEOSIDE P H O S P H O R Y L A S E
P.J. BLAKE and J.F. GREALLY Department of Immunology, School of Pathology, Trinity College, Dublin, 2, Ireland
(Received 2 September 1975, accepted 5 September 1975)
An improved screening test for both adenosine deaminase and nucleoside phosphorylase is described. C o m b i n e d i m m u n e deficiency associated with lack of red blood cell adenosine deaminase (ADA) was first n o t e d in 1972 (Giblett, 1972). Subseq u en tly it was f ound t hat a defect at a single ADA locus was almost certainly the cause of one form of c om bi ned i m m une deficiency (Hirschhorn, 1975). More recently there has been a r e p o r t of an association between the absence o f nucleoside phosphorylase (NP) and a severe defect in T-cell i m m u n i t y (Giblett, 1975). In the purine metabolic p a t h w a y the e n z y m e immediately following ADA is NP. Various m e t h o d s have been described for measuring ADA and NP activity (Karker, 1964; H o p k i n s o n et al., 1969). A screening technique which can be p e r f o r m e d on a spot of blood from a heel prick has been described by Moor and Meuwissen (1975). The m e t h o d , based on release of am m oni a from adenosine and the subsequent change in colour of an indicator dye, suffers from a n u m b e r o f disadvantages, i.e. false positives due to diffusion of ammonia, inability to obtain a reading due to fungal contamination. It has also been f o u n d that to prevent the indicator dye turning blue on c o n t a c t with the plate it was necessary to acid wash similar plastic plates to those used in the screening test. T o o v er co me these disadvantages the m e t h o d outlined below was adopted. The m e t h o d is based on the c o m m e n t s o f Parks (1975). The wells in a Bio-cult plastic plate (Model 1S MRC 96, Linbro, 96 cups, 8.5 × 12.8 cm) were filled to just below the surface with a gel of the following co mp o s itio n : Adenosine, final c o n c e n t r a t i o n Agarose gel, final c o n c e n t r a t i o n Nitro Blue T e t r a z o l i u m (NBT), final c o n c e n t r a t i o n
0.01 M 0.1% 0.005%
T h e c o m p o u n d s were dissolved in phos pha te buffer 0.025 M, pH 7.5.
98 T h e b l o o d t o be t e s t e d was s p o t t e d o n W h a t m a n N o . 4 filter p a p e r and dried f o r 10 m i n at 37°C. Discs 6.0 m m in d i a m e t e r w e r e t h e n p u n c h e d f r o m t h e b l o o d s p o t s a n d p l a c e d f i r m l y o n t h e gel and 5 X 10 .3 u n i t s o f x a n t h i n e o x i d a s e (Sigma, G r a d e 1) a d d e d to e a c h disc. T h e p l a t e was sealed w i t h parafilm ( A m e r i c a n Can Co.) a n d i n c u b a t e d o v e r n i g h t u n d e r f l u o r e s c e n t light at r o o m t e m p e r a t u r e . Positive results s h o w e d as a b l a c k disc w h e r e a s the controls w i t h o u t a d e n o s i n e were l i g h t - b r o w n in c o l o u r as were t h e h o r s e e r y t h r o c y t e s used as negative c o n t r o l s . I f p h o s p h a t e is o m i t t e d t h e readings will all b e negative as NP is p h o s p h a t e or a r s e n a t e d e p e n d e n t . I s o l a t e d l y m p h o c y t e s c a n be s c r e e n e d f o r A D A w i t h a d d e d NP (Sigma, 2.1 X 10 -3 units/disc) a n d x a n t h i n e o x i d a s e ( X O D ) using 3 X 104 cells/disc a n d an i n c u b a t i o n t i m e of o n e h o u r at r o o m t e m p e r a t u r e . U n d e r the s a m e c o n d i t i o n s t h e l y m p h o c y t e s m a y be t e s t e d f o r b o t h A D A and NP b y a d d i n g o n l y t h e X O D a n d i n c u b a t i n g t h e p l a t e overnight. T h e m e t h o d d e s c r i b e d has the a d v a n t a g e s t h a t it is relatively fast t h u s o v e r c o m i n g t h e p r o b l e m of c o n t a m i n a t i o n . T h e r e is n o d a n g e r o f false positives d u e to d i f f u s i o n o f a m m o n i a as a positive result is d e p e n d e n t on the rea c t i o n c o n t i n u i n g as far as p r o d u c t i o n o f x a n t h i n e and uric acid w i t h reduct i o n o f t h e a d d e d N B T . T h e b l o o d s p o t s m a y b e l e f t on t h e filter p a p e r at r o o m t e m p e r a t u r e f o r 5 d a y s and still p r o d u c e a positive result. T h e screening s i m u l t a n e o u s l y tests f o r A D A a n d NP d e f i c i e n c y .
REFERENCES Giblett, E.R., G.E. Anderson, F. Cohen, B. Pollara and H.G. Meuwissen, 1972, Lancet 2, 1067. Giblett, E., A.G. Amman, D. Wara, R. Sandman and L.K. Diamond, 1975, Lancet 1, 1010. Hirschhorn, R., 1975, J. Clin. Invest. 55, 661. Hopkinson, D.A., P. Cook and H. Harris, 1969, Ann. Hum. Genet. 32, 361. Karker, H. (1964) Scand. J. Clin. Lab. Invest. 16,570. Moore, E.C. and H.G. Meuwissen, 1975, in: Combined immunodeficiency disease and adenosine deaminase deficiency. A molecular defect, eds. H.G. Meuwissen, R.G. Pickering, B. Pollara and J.H. Porter (Academic Press Inc., New York, San Francisco and London) p. 219. Parks, R.E., Jr., 1975, in: Combined immunodeficiency disease and adenosine deaminase deficiency. A molecular defect, eds. H.G. Meuwissen, R.G. Pickering, B. Pollara and J.H. Porter (Academic Press Inc., New York, San Francisco and London) p. 302.
