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Immuno-ultracentrifugation: A combination of density gradient centrifugation and immunodiffusion
Journal of Immunological Methods, 14 (1977) 2 5 3 - - 2 5 5
253
© E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
IMMUNO-ULTRACENTRIFUGATION: A COMBINATION OF DENSITY GRADIENT CENTRIFUGATION AND IMMUNODIFFUSION
P.J. Q U I N N I and M.A. F E R N A N D O
Departmenl of Veterinary Microbiology and Immunology and Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada (Received 18 S e p t e m b e r 1976, a c c e p t e d .1 O c t o b e r 1976)
By i n c o r p o r a t i n g a c r y l a m i d e in a sucrose gradient and p h o t o p o l y m e r i z i n g the t u b e s at the end of the run, direct i m m u n o c h e m i c a l and o t h e r analysis can be p e r f o r m e d on the separated material in situ w i t h o u t recovering fractions f r o m the c e n t r i f u g e tube. I m m u n o diffusion p e r f o r m e d on the sliced gel can be e m p l o y e d to locate a n t i b o d y activity in the gel slice or d e t e r m i n e the d i s t r i b u t i o n o f antigens in a sucrose gradient.
Material recovered following density gradient centrifugation can be analysed by physical, chemical and immunological methods. The procedures used to characterize antigenic molecules include electrophoresis, immunodiffusion, immunoelectrophoresis and various modifications of these procedures such as counter-current immunoelectrophoresis and quantitative immunoelectrophoresis (Cordoba et al., 1965; Williams and Chase, 1967; Feinberg and Hill, 1968; Axelsen and Bock, 1972). All these methods, however, require that the separated material be recovered by drop collection, tube slicing or displacement with a dense solution (Trautman, 1965). The present report describes a method for the direct analysis of either antigen or antibody subjected to density gradient centrifugation. This method combines the technique reported by Jolley et al. (1967) and by Cole and Brooks (1968) for immobilization of materials separated on a sucrose gradient, with immunodiffusion. We call this m e t h o d immuno-ultracentrifugation. A 10 to 35% sucrose gradient containing 7.5% acrylamide was used in all the experiments reported in this paper. '/he gradient was prepared manually by layering 0.8 ml of 10 to 35% sucrose-acrylamide solutions in 5% increments and allowed to diffuse for approximately 6 h at 4°C in 1.3 × 5 cm cellulose nitrate tubes. Antiserum or an appropriate dilution of antigen was layered on top in 0.3 ml amounts and the tubes were centrifuged at 35,000 rev/min (100,000 g) in a SW50L rotor in a Spinco model L preparative ultracentrifuge at 4°C. At the end of the run the tubes ~ere photopoly1 Present address, D e p a r t m e n t o f V e t e r i n a r y Medicine, University College Dublin, Veterinary Colh,ge, Dublin, 4, Ireland,
254 m e r i z e d as d e s c r i b e d b y C o l e a n d B r o o k s ( 1 9 6 8 ) . T h e r e s u l t i n g gels w e r e r e m o v e d f r o m t h e t u b e s , p l a c e d i n agar m o u l d s a n d c u t l e n g t h w i s e in 2 m m slices. T h e m o u l d s w e r e p r e p a r e d b y p o u r i n g m o l t e n 2% agar a r o u n d a n i n v e r t e d 1.3 × 6 . 4 c m c e l l u l o s e n i t r a t e t u b e h e l d v e r t i c a l l y i n a l a r g e r c o n t a i n e r w i t h a r e m o v a b l e b a s e . T h e p h o t o p o l y m e r i z e d gel was e x p e l l e d i n t o t h e m o u l d a n d s e a l e d a t e i t h e r e n d w i t h m o l t e n agar b e f o r e s l i c i n g . A s i n g l e slice was p l a c e d i n t h e e e n t e r o f a 6 × 1 0 c m glass slide a n d 1% agar i n B a r b i t a l b u f f e r , p H 8 . 2 a d d e d t o c o v e r t h e slice. T r o u g h s w e r e c u t i n t h e agar
A
Fig. 1. Preeipitin patterns obtained with different antigen antibody systems. The slice of polyaerylamide gel appears as a dark area between the troughs. The rounded end of the slice corresponds to the highest sucrose concentration (bottom of centrifuge tube). A) Bovine serum albumin (BSA) centrifuged, rabbit antiserum to BSA added to troughs. B) Rabbit antiserum to BSA centrifuged, BSA added to troughs. C) Human serum centrifuged, rabbit antiserum to whole human serum added to troughs. D) Human serum centrifuged, rabbit antiserum to human serum albumin added to top trough and goat antiserum to human gamma globulin added to bottom trough.
255 on e a c h side o f t h e slice w i t h an i m m u n o e l e c t r o p h o r e s i s gel p u n c h . Antis e r u m or an a p p r o p r i a t e d i l u t i o n o f antigen was a d d e d to t h e t r o u g h s and the slides w e r e placed in a m o i s t c h a m b e r at r o o m t e m p e r a t u r e . Precipitin lines d e v e l o p e d in 24 t o 48 h. Fig. 1 s h o w s t h e results o f a t y p i c a l e x p e r i m e n t . In (A) b o v i n e s e r u m a l b u m i n (BSA) was c e n t r i f u g e d a n d r a b b i t a n t i s e r m n to BSA was a d d e d t o t h e t r o u g h s , while in (B) r a b b i t a n t i s e r u m t o BSA was c e n t r i f u g e d and BSA was a d d e d t o t h e t r o u g h s . I t m a y be seen t h a t in (B) a n t i b o d y activity of t h e s e r u m is c o n f i n e d t o the 7S region. Where c o m p l e x a n t i g e n s are c e n t r i f u g e d specific a n t i s e r a allow i d e n t i f i c a t i o n o f the s e p a r a t e d c o m p o n e n t s . Fig. 1C s h o w s t h e p a t t e r n o b t a i n e d w h e n h u m a n s e r u m was c e n t r i f u g e d as d e s c r i b e d and r a b b i t a n t i s e r u m t o w h o l e h u m a n s e r u m was a d d e d t o t h e t r o u g h s . T o i d e n t i f y specific c o m p o n e n t s p r e s e n t in such c o m p l e x antigenic m a t e r i a l , a n t i s e r a specific f o r t h e s e c o m p o n e n t s w e r e e m p l o y e d (fig. 1D). As 5 or 6 slices are usually o b t a i n e d f r o m a single gel, t h e s e p a r a t e d m a t e r i a l can be f u r t h e r c h a r a c t e r i z e d b y a p p r o p r i a t e staining procedures. Whereas i m m u n o e l e c t r ~ p h o r e s i s allows i d e n t i f i c a t i o n o f antigens separ a t e d essentially o n t h e basis o f t h e i r charge, i m m u n o - u l t r a c e n t r i f u g a t i o n relies on t h e i r s e d i m e n t a t i o n in a d e n s i t y gradient. In the l a t t e r m e t h o d antigen as well as a n t i b o d y m a y be s e p a r a t e d a n d c h a r a c t e r i z e d . T h e m a i n a d v a n t a g e o f this p r o c e d u r e is the e f f e c t i v e s e p a r a t i o n o f 19S and 7S g a m m a globulins w i t h t h e s u b s e q u e n t d e m o n s t r a t i o n o f a n t i b o d y a c t i v i t y in situ, e l i m i n a t i n g the i n t e r m e d i a t e s t e p o f f r a c t i o n c o l l e c t i o n . F u r t h e r m o r e , since several slices c o n t a i n i n g t h e s e p a r a t e d m a t e r i a l m a y be o b t a i n e d f r o m a single gel, c o m plex antigens m a y be c h a r a c t e r i z e d b y a p p r o p r i a t e h i s t o c h e m i c a l m e t h o d s in a d d i t i o n to i m m u n o c h e m i c a l analysis with specific antisera. ACKNOWLEDGEMENT We t h a n k Mr. E. E w e r t f o r his t e c h n i c a l assistance. REFERENCES Axelsen, N.H. and E. Bock, 1972, J. Immunol. Methods 1, 109. Cole, T.A. and T.W. Brooks, Jr., 1968, Science 161, 386. Cordoba, F., C. Gonzalez and P. Rivera, 1965, Nature (Lond.) 205,565. Feinberg, J.G. and C.W. Hill, 1968. Int. Arch. Allergy Appl. Immunol. 33, 120. Jolley, W.B., H.W. Allen and O.M. Griffith, 1967. Anal. Biochem., 21, 454. Trautman, R., 1965, in: Instrumental methods in experimental Biology, ed. D.W. Newman (Macmillan Company, New York) p. 211. Williams, C.A. and M.W. Chase (Eds.), 1967, Methods in Immunology and Immunochemistry, Vol II, (Academic Press, New York and London) p. 82.
253
© E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
IMMUNO-ULTRACENTRIFUGATION: A COMBINATION OF DENSITY GRADIENT CENTRIFUGATION AND IMMUNODIFFUSION
P.J. Q U I N N I and M.A. F E R N A N D O
Departmenl of Veterinary Microbiology and Immunology and Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada (Received 18 S e p t e m b e r 1976, a c c e p t e d .1 O c t o b e r 1976)
By i n c o r p o r a t i n g a c r y l a m i d e in a sucrose gradient and p h o t o p o l y m e r i z i n g the t u b e s at the end of the run, direct i m m u n o c h e m i c a l and o t h e r analysis can be p e r f o r m e d on the separated material in situ w i t h o u t recovering fractions f r o m the c e n t r i f u g e tube. I m m u n o diffusion p e r f o r m e d on the sliced gel can be e m p l o y e d to locate a n t i b o d y activity in the gel slice or d e t e r m i n e the d i s t r i b u t i o n o f antigens in a sucrose gradient.
Material recovered following density gradient centrifugation can be analysed by physical, chemical and immunological methods. The procedures used to characterize antigenic molecules include electrophoresis, immunodiffusion, immunoelectrophoresis and various modifications of these procedures such as counter-current immunoelectrophoresis and quantitative immunoelectrophoresis (Cordoba et al., 1965; Williams and Chase, 1967; Feinberg and Hill, 1968; Axelsen and Bock, 1972). All these methods, however, require that the separated material be recovered by drop collection, tube slicing or displacement with a dense solution (Trautman, 1965). The present report describes a method for the direct analysis of either antigen or antibody subjected to density gradient centrifugation. This method combines the technique reported by Jolley et al. (1967) and by Cole and Brooks (1968) for immobilization of materials separated on a sucrose gradient, with immunodiffusion. We call this m e t h o d immuno-ultracentrifugation. A 10 to 35% sucrose gradient containing 7.5% acrylamide was used in all the experiments reported in this paper. '/he gradient was prepared manually by layering 0.8 ml of 10 to 35% sucrose-acrylamide solutions in 5% increments and allowed to diffuse for approximately 6 h at 4°C in 1.3 × 5 cm cellulose nitrate tubes. Antiserum or an appropriate dilution of antigen was layered on top in 0.3 ml amounts and the tubes were centrifuged at 35,000 rev/min (100,000 g) in a SW50L rotor in a Spinco model L preparative ultracentrifuge at 4°C. At the end of the run the tubes ~ere photopoly1 Present address, D e p a r t m e n t o f V e t e r i n a r y Medicine, University College Dublin, Veterinary Colh,ge, Dublin, 4, Ireland,
254 m e r i z e d as d e s c r i b e d b y C o l e a n d B r o o k s ( 1 9 6 8 ) . T h e r e s u l t i n g gels w e r e r e m o v e d f r o m t h e t u b e s , p l a c e d i n agar m o u l d s a n d c u t l e n g t h w i s e in 2 m m slices. T h e m o u l d s w e r e p r e p a r e d b y p o u r i n g m o l t e n 2% agar a r o u n d a n i n v e r t e d 1.3 × 6 . 4 c m c e l l u l o s e n i t r a t e t u b e h e l d v e r t i c a l l y i n a l a r g e r c o n t a i n e r w i t h a r e m o v a b l e b a s e . T h e p h o t o p o l y m e r i z e d gel was e x p e l l e d i n t o t h e m o u l d a n d s e a l e d a t e i t h e r e n d w i t h m o l t e n agar b e f o r e s l i c i n g . A s i n g l e slice was p l a c e d i n t h e e e n t e r o f a 6 × 1 0 c m glass slide a n d 1% agar i n B a r b i t a l b u f f e r , p H 8 . 2 a d d e d t o c o v e r t h e slice. T r o u g h s w e r e c u t i n t h e agar
A
Fig. 1. Preeipitin patterns obtained with different antigen antibody systems. The slice of polyaerylamide gel appears as a dark area between the troughs. The rounded end of the slice corresponds to the highest sucrose concentration (bottom of centrifuge tube). A) Bovine serum albumin (BSA) centrifuged, rabbit antiserum to BSA added to troughs. B) Rabbit antiserum to BSA centrifuged, BSA added to troughs. C) Human serum centrifuged, rabbit antiserum to whole human serum added to troughs. D) Human serum centrifuged, rabbit antiserum to human serum albumin added to top trough and goat antiserum to human gamma globulin added to bottom trough.
255 on e a c h side o f t h e slice w i t h an i m m u n o e l e c t r o p h o r e s i s gel p u n c h . Antis e r u m or an a p p r o p r i a t e d i l u t i o n o f antigen was a d d e d to t h e t r o u g h s and the slides w e r e placed in a m o i s t c h a m b e r at r o o m t e m p e r a t u r e . Precipitin lines d e v e l o p e d in 24 t o 48 h. Fig. 1 s h o w s t h e results o f a t y p i c a l e x p e r i m e n t . In (A) b o v i n e s e r u m a l b u m i n (BSA) was c e n t r i f u g e d a n d r a b b i t a n t i s e r m n to BSA was a d d e d t o t h e t r o u g h s , while in (B) r a b b i t a n t i s e r u m t o BSA was c e n t r i f u g e d and BSA was a d d e d t o t h e t r o u g h s . I t m a y be seen t h a t in (B) a n t i b o d y activity of t h e s e r u m is c o n f i n e d t o the 7S region. Where c o m p l e x a n t i g e n s are c e n t r i f u g e d specific a n t i s e r a allow i d e n t i f i c a t i o n o f the s e p a r a t e d c o m p o n e n t s . Fig. 1C s h o w s t h e p a t t e r n o b t a i n e d w h e n h u m a n s e r u m was c e n t r i f u g e d as d e s c r i b e d and r a b b i t a n t i s e r u m t o w h o l e h u m a n s e r u m was a d d e d t o t h e t r o u g h s . T o i d e n t i f y specific c o m p o n e n t s p r e s e n t in such c o m p l e x antigenic m a t e r i a l , a n t i s e r a specific f o r t h e s e c o m p o n e n t s w e r e e m p l o y e d (fig. 1D). As 5 or 6 slices are usually o b t a i n e d f r o m a single gel, t h e s e p a r a t e d m a t e r i a l can be f u r t h e r c h a r a c t e r i z e d b y a p p r o p r i a t e staining procedures. Whereas i m m u n o e l e c t r ~ p h o r e s i s allows i d e n t i f i c a t i o n o f antigens separ a t e d essentially o n t h e basis o f t h e i r charge, i m m u n o - u l t r a c e n t r i f u g a t i o n relies on t h e i r s e d i m e n t a t i o n in a d e n s i t y gradient. In the l a t t e r m e t h o d antigen as well as a n t i b o d y m a y be s e p a r a t e d a n d c h a r a c t e r i z e d . T h e m a i n a d v a n t a g e o f this p r o c e d u r e is the e f f e c t i v e s e p a r a t i o n o f 19S and 7S g a m m a globulins w i t h t h e s u b s e q u e n t d e m o n s t r a t i o n o f a n t i b o d y a c t i v i t y in situ, e l i m i n a t i n g the i n t e r m e d i a t e s t e p o f f r a c t i o n c o l l e c t i o n . F u r t h e r m o r e , since several slices c o n t a i n i n g t h e s e p a r a t e d m a t e r i a l m a y be o b t a i n e d f r o m a single gel, c o m plex antigens m a y be c h a r a c t e r i z e d b y a p p r o p r i a t e h i s t o c h e m i c a l m e t h o d s in a d d i t i o n to i m m u n o c h e m i c a l analysis with specific antisera. ACKNOWLEDGEMENT We t h a n k Mr. E. E w e r t f o r his t e c h n i c a l assistance. REFERENCES Axelsen, N.H. and E. Bock, 1972, J. Immunol. Methods 1, 109. Cole, T.A. and T.W. Brooks, Jr., 1968, Science 161, 386. Cordoba, F., C. Gonzalez and P. Rivera, 1965, Nature (Lond.) 205,565. Feinberg, J.G. and C.W. Hill, 1968. Int. Arch. Allergy Appl. Immunol. 33, 120. Jolley, W.B., H.W. Allen and O.M. Griffith, 1967. Anal. Biochem., 21, 454. Trautman, R., 1965, in: Instrumental methods in experimental Biology, ed. D.W. Newman (Macmillan Company, New York) p. 211. Williams, C.A. and M.W. Chase (Eds.), 1967, Methods in Immunology and Immunochemistry, Vol II, (Academic Press, New York and London) p. 82.