Journal of Immunological Methods, 9 (1976) 231--234
231
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A R A P I D T E C H N I Q U E F O R T H E I S O L A T I O N O F H U M A N IgD MYELOMA PROTEINS EMPLOYING ULTRAGEL AcA34
R. JEFFERIS Departmenl of Experimental Pathology, University of Birmingham Medical School. Birmingham B15 2TJ, England
(Received 14 June 1975, accepted 30,June 1975)
A relatively rapid technique for the isolation of human IgD myeloma proteins from whole sera is described. It is based on the use of the newly available Ultragel AcA34 gel filtration medium which yields a very substantially purified IgD fraction from whole serum. The absence of IgG from this fraction allows further purification on DEAE cellulose under conditions where the IgD protein is not absorbed but other protein contaminants are retained. The overall yield of IgD protein is estimated at >90% and the technique is particularly applicable to the isolation of IgD from small serum volumes.
H u m a n IgD m y e l o m a p r o t e i n s c a n n o t be isolated f r o m serum in pure f o r m b y a n y single step p r o c e d u r e . T h e m o s t widely a d o p t e d separation t e c h n i q u e has e m p l o y e d S e p h a d e x G-200 gel filtration f o l l o w e d b y f u r t h e r purification o f the IgD c o n t a i n i n g f r a c t i o n b y gradient elution f r o m D E A E cellulose ( R o w e and F a h e y , 1 9 6 5 ) . This isolation p r o c e d u r e requires a fairly n a r r o w ' c u t ' to be t a k e n f r o m the S e p h a d e x G-200 separation to yield a pure prep a r a t i o n a f t e r the c h r o m a t o g r a p h i c step and the c o m p r o m i s e b e t w e e n p u r i t y and yield, c o m m o n to almost all p r o t e i n p u r i f i c a t i o n problems, results in a p o o r overall yield o f IgD protein. Since for detailed structural studies relatively large a m o u n t s o f proteins are required we have s o u g h t alternative separation t e c h n i q u e s which might allow high yields o f purified p r o t e i n to be o b t a i n e d . I r e c e n t l y described the application o f isotachophoresis to the isolation o f a h u m a n IgD p r o t e i n (Jefferis, 1 9 7 5 ) . A significantly purified IgD p r e p a r a t i o n was o b t a i n e d in g o o d yield b y this single step p r o c e d u r e . However, a m o r e rapid p r o c e d u r e is required to process the large serum or plasma volumes which m a y be o b t a i n e d following plasmaphoresis. As an alternative to S e p h a d e x G - 2 0 0 (Pharmacia Ltd.) we have investigated the gel filtration p r o p e r t i e s of Ultragel AcA22 and A c A 3 4 ( L K B Ins t r u m e n t s Ltd.) and have f o u n d the latter p r o d u c t to be e x t r e m e l y useful as a first stage p r o c e d u r e in the isolation o f h u m a n IgD proteins. In fig. 1 we illustrate the separation profile o f n o r m a l h u m a n serum on S e p h a d e x G - 2 0 0 and Ultragel A c A 3 4 . T h e f r a c t i o n s indicated were p o o l e d and each concent r a t e d to the original serum v o l u m e applied to the c o l u m n and analysed b y
232 r5
05
50
C~ 0
100
150
200
09
0"6
G.t
~0 ELUTiON
'bl~ VOLUME
~5c
20d
:ml 'l
Fig. I. Elution profiles o f normal h u m a n s e r u m (1 m]) on Ultragel A c A 3 4 (A) and S e p h a d e x G-200 (B). C o l u m n d i m e n s i o n s 60 c m × 8 cm 2 ; elution rate 25 ml/hr.
Fig. 2. I m m u n o e l e c t r o p h o r e t i c analysis of S e p h a d e x G-200 fractions I (B), II (C) and Ill (D); Ultragel A e A 3 4 fractions I (E), II (F) and III (G) and w h o l e h u m a n serum A(tt). A n t i s e r u m is s h e e p anti w h o l e h u m a n serum.
233
immunoelectrophoresis (fig. 2). It can be seen that the Sephadex G-200 fractions, I, II and III, contain typically the macroglobulins, the '7S' globulins (including IgG) and the albumin fraction respectively. In contrast the Ultragel AcA34 allows the wide separation of the macroglobulins, fractions and I[, from the other serum components present in fraction III (including [gG and albumin). Human IgD proteins elute on Sephadex G-200 in a region spanning the macroglobulin and '7S' globulin fraction and therefore the observation of a widening of this separation zone between these serum fractions on Ultragel AcA34 led us to investigate the elution profile of a human IgD myeloma serum on this medium. The elution profiles obtained in parallel experiments with Sephadex G-200 and Ultragel AcA34 are shown in fig. 3. It can be seen that a 'new' peak is observed and the fraction indicated was pooled and concentrated back to the original serum volume and examined by immunoelectrophoresis (fig. 4). It can be seen that the bulk of this fraction is IgD and that the number of contaminating serum components is low and also are obviously present in low amounts. Therefore this single isolation step allows a substantial purification of the IgD paraprotein and virtually its total recovery. Of particular significance is the absence of contaminating IgG and of other proteins having the mobility of the IgD protein. Thus the IgD could be obtained in pure form from the Ultragel AcA34 fraction by absorption of contaminating proteins with DEAE cellulose equilibrated in 0.035 M phosphate, pH 8.0 (fig. 4). Our routine preparative procedure is to develop the Ultragel AcA34 separation in 0.035 M phosphate, pH 8.0 buffer and to immediately pass the IgD
05 (3
0
50
I00
!50
50
100
150
0-9
03
ELUTION VOLUME ( m l )
Fig. 3. E l u t i o n profiles of a h u m a n IgD m y e l o m a s e r u m (1 ml) o n Ultragel A e A 3 4 (A) a n d S e p h a d e x G - 2 0 0 (B). C o l u m n d i m e n s i o n s (60 c m × 8 c m : ; e l u t i o n rate 25 m l / h r .
234
Fig. 4. Immunoelectrophoretic analysis of normal human serum (A), IgD myeloma serum Ha (B), Ultragel AcA34 IgD fraction (C) and DEAE cellulose pm'ified IgDHa (D). Developing antisera were sheep anti whole human serum (I) and sheep anti human IgDHa (II). c o n t a i n i n g f r a c t i o n s o v e r a D E A E cellulose c o l u m n with t h e e x c h a n g e r also e q u i l i b r a t e d in 0 . 0 3 5 M p h o s p h a t e , p H 8.0. In this w a y we have d e v e l o p e d a s u b s t a n t i a l l y m o r e rapid isolation p r o c e d u r e which allows > 9 0 % r e c o v e r y of the IgD p r o t e i n p r e s e n t in w h o l e s e r u m . T h e m i n i m i s a t i o n o f handling and c o n c e n t r a t i n g p r o c e d u r e s is also i m p o r t a n t since IgD p r o t e i n s are n o t o r i o u s l y subject t o p r o t e o l y s i s during isolation. All b u f f e r s used in the isolation conrained 0.01 M e - a m i n o c a p r o i c acid to p r o t e c t against such p r o t e o l y s i s and all e x p e r i m e n t s were carried o u t at 4 ° C. T h e high yields o b t a i n e d b y this m e t h o d o l o g y is i m p o r t a n t also in allowing t h e isolation of ' w o r k a b l e ' a m o u n t s of p u r e p r o t e i n f r o m small s e r u m v o l u m e s w h i c h are f r e q u e n t l y all t h a t is available. Where the level of I g D p r o t e i n p r e s e n t in t h e starting s e r u m or p l a s m a is low (less t h a n a p p r o x . 3 m g / m l ) an initial p r e c i p i t a t i o n step w i t h 35% a m m o n i u m s u l p h a t e has b e e n f o u n d to be a d v a n t a g e o u s ; the p r o p o r t i o n o f e o n t a m i n a t i n g p r o t e i n s being significantly r e d u c e d in the re-dissolved p r e c i p i t a t e .
REFERENCES JeFfe~'is, R., 1975, Science Tools 22, No. 1. p. 1. Rowe, D.S. and J.L. Fahey, 1965, J. Exp. Med. 121, 185.
231
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A R A P I D T E C H N I Q U E F O R T H E I S O L A T I O N O F H U M A N IgD MYELOMA PROTEINS EMPLOYING ULTRAGEL AcA34
R. JEFFERIS Departmenl of Experimental Pathology, University of Birmingham Medical School. Birmingham B15 2TJ, England
(Received 14 June 1975, accepted 30,June 1975)
A relatively rapid technique for the isolation of human IgD myeloma proteins from whole sera is described. It is based on the use of the newly available Ultragel AcA34 gel filtration medium which yields a very substantially purified IgD fraction from whole serum. The absence of IgG from this fraction allows further purification on DEAE cellulose under conditions where the IgD protein is not absorbed but other protein contaminants are retained. The overall yield of IgD protein is estimated at >90% and the technique is particularly applicable to the isolation of IgD from small serum volumes.
H u m a n IgD m y e l o m a p r o t e i n s c a n n o t be isolated f r o m serum in pure f o r m b y a n y single step p r o c e d u r e . T h e m o s t widely a d o p t e d separation t e c h n i q u e has e m p l o y e d S e p h a d e x G-200 gel filtration f o l l o w e d b y f u r t h e r purification o f the IgD c o n t a i n i n g f r a c t i o n b y gradient elution f r o m D E A E cellulose ( R o w e and F a h e y , 1 9 6 5 ) . This isolation p r o c e d u r e requires a fairly n a r r o w ' c u t ' to be t a k e n f r o m the S e p h a d e x G-200 separation to yield a pure prep a r a t i o n a f t e r the c h r o m a t o g r a p h i c step and the c o m p r o m i s e b e t w e e n p u r i t y and yield, c o m m o n to almost all p r o t e i n p u r i f i c a t i o n problems, results in a p o o r overall yield o f IgD protein. Since for detailed structural studies relatively large a m o u n t s o f proteins are required we have s o u g h t alternative separation t e c h n i q u e s which might allow high yields o f purified p r o t e i n to be o b t a i n e d . I r e c e n t l y described the application o f isotachophoresis to the isolation o f a h u m a n IgD p r o t e i n (Jefferis, 1 9 7 5 ) . A significantly purified IgD p r e p a r a t i o n was o b t a i n e d in g o o d yield b y this single step p r o c e d u r e . However, a m o r e rapid p r o c e d u r e is required to process the large serum or plasma volumes which m a y be o b t a i n e d following plasmaphoresis. As an alternative to S e p h a d e x G - 2 0 0 (Pharmacia Ltd.) we have investigated the gel filtration p r o p e r t i e s of Ultragel AcA22 and A c A 3 4 ( L K B Ins t r u m e n t s Ltd.) and have f o u n d the latter p r o d u c t to be e x t r e m e l y useful as a first stage p r o c e d u r e in the isolation o f h u m a n IgD proteins. In fig. 1 we illustrate the separation profile o f n o r m a l h u m a n serum on S e p h a d e x G - 2 0 0 and Ultragel A c A 3 4 . T h e f r a c t i o n s indicated were p o o l e d and each concent r a t e d to the original serum v o l u m e applied to the c o l u m n and analysed b y
232 r5
05
50
C~ 0
100
150
200
09
0"6
G.t
~0 ELUTiON
'bl~ VOLUME
~5c
20d
:ml 'l
Fig. I. Elution profiles o f normal h u m a n s e r u m (1 m]) on Ultragel A c A 3 4 (A) and S e p h a d e x G-200 (B). C o l u m n d i m e n s i o n s 60 c m × 8 cm 2 ; elution rate 25 ml/hr.
Fig. 2. I m m u n o e l e c t r o p h o r e t i c analysis of S e p h a d e x G-200 fractions I (B), II (C) and Ill (D); Ultragel A e A 3 4 fractions I (E), II (F) and III (G) and w h o l e h u m a n serum A(tt). A n t i s e r u m is s h e e p anti w h o l e h u m a n serum.
233
immunoelectrophoresis (fig. 2). It can be seen that the Sephadex G-200 fractions, I, II and III, contain typically the macroglobulins, the '7S' globulins (including IgG) and the albumin fraction respectively. In contrast the Ultragel AcA34 allows the wide separation of the macroglobulins, fractions and I[, from the other serum components present in fraction III (including [gG and albumin). Human IgD proteins elute on Sephadex G-200 in a region spanning the macroglobulin and '7S' globulin fraction and therefore the observation of a widening of this separation zone between these serum fractions on Ultragel AcA34 led us to investigate the elution profile of a human IgD myeloma serum on this medium. The elution profiles obtained in parallel experiments with Sephadex G-200 and Ultragel AcA34 are shown in fig. 3. It can be seen that a 'new' peak is observed and the fraction indicated was pooled and concentrated back to the original serum volume and examined by immunoelectrophoresis (fig. 4). It can be seen that the bulk of this fraction is IgD and that the number of contaminating serum components is low and also are obviously present in low amounts. Therefore this single isolation step allows a substantial purification of the IgD paraprotein and virtually its total recovery. Of particular significance is the absence of contaminating IgG and of other proteins having the mobility of the IgD protein. Thus the IgD could be obtained in pure form from the Ultragel AcA34 fraction by absorption of contaminating proteins with DEAE cellulose equilibrated in 0.035 M phosphate, pH 8.0 (fig. 4). Our routine preparative procedure is to develop the Ultragel AcA34 separation in 0.035 M phosphate, pH 8.0 buffer and to immediately pass the IgD
05 (3
0
50
I00
!50
50
100
150
0-9
03
ELUTION VOLUME ( m l )
Fig. 3. E l u t i o n profiles of a h u m a n IgD m y e l o m a s e r u m (1 ml) o n Ultragel A e A 3 4 (A) a n d S e p h a d e x G - 2 0 0 (B). C o l u m n d i m e n s i o n s (60 c m × 8 c m : ; e l u t i o n rate 25 m l / h r .
234
Fig. 4. Immunoelectrophoretic analysis of normal human serum (A), IgD myeloma serum Ha (B), Ultragel AcA34 IgD fraction (C) and DEAE cellulose pm'ified IgDHa (D). Developing antisera were sheep anti whole human serum (I) and sheep anti human IgDHa (II). c o n t a i n i n g f r a c t i o n s o v e r a D E A E cellulose c o l u m n with t h e e x c h a n g e r also e q u i l i b r a t e d in 0 . 0 3 5 M p h o s p h a t e , p H 8.0. In this w a y we have d e v e l o p e d a s u b s t a n t i a l l y m o r e rapid isolation p r o c e d u r e which allows > 9 0 % r e c o v e r y of the IgD p r o t e i n p r e s e n t in w h o l e s e r u m . T h e m i n i m i s a t i o n o f handling and c o n c e n t r a t i n g p r o c e d u r e s is also i m p o r t a n t since IgD p r o t e i n s are n o t o r i o u s l y subject t o p r o t e o l y s i s during isolation. All b u f f e r s used in the isolation conrained 0.01 M e - a m i n o c a p r o i c acid to p r o t e c t against such p r o t e o l y s i s and all e x p e r i m e n t s were carried o u t at 4 ° C. T h e high yields o b t a i n e d b y this m e t h o d o l o g y is i m p o r t a n t also in allowing t h e isolation of ' w o r k a b l e ' a m o u n t s of p u r e p r o t e i n f r o m small s e r u m v o l u m e s w h i c h are f r e q u e n t l y all t h a t is available. Where the level of I g D p r o t e i n p r e s e n t in t h e starting s e r u m or p l a s m a is low (less t h a n a p p r o x . 3 m g / m l ) an initial p r e c i p i t a t i o n step w i t h 35% a m m o n i u m s u l p h a t e has b e e n f o u n d to be a d v a n t a g e o u s ; the p r o p o r t i o n o f e o n t a m i n a t i n g p r o t e i n s being significantly r e d u c e d in the re-dissolved p r e c i p i t a t e .
REFERENCES JeFfe~'is, R., 1975, Science Tools 22, No. 1. p. 1. Rowe, D.S. and J.L. Fahey, 1965, J. Exp. Med. 121, 185.
