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Specific antisera produced by immunization with precipitin lines
Journal of Immunological Methods, 13 (1976) 125--130 © Elsevier/North-Itolland Biomedical Press
125
S P E C I F I C A N T I S E R A P R O D U C E D BY I M M U N I Z A T I O N W I T H PRECIPITIN LINES
JENS KROLL and METTE M. ANDERSEN The Finsen Laboratory, The Finsen Institute, Strandboulevarden 49, 2100 Copenha~,~en O, Denmark
(Received 27 May 1976, accepted 3 June 1976)
Individual plasma proteins were precipitated, identified and isolated on tile basis of line immunoeleetrophoresis. A monospecific antibody response was induced by immunization of rabbits with less than 50 ng of precipitated antigen. Preservation of monospeeifieity was obtained by reimmunization with precipitates developed against the specific antisora. INTRODUCTION Direct a t t a i n m e n t o f a specific a n t i s e r u m requires a high degree o f p u r i t y o f the i m m u n o g e n . E x t e n s i v e p u r i f i c a t i o n p r o c e d u r e s m a y , h o w e v e r , m o d i f y the m o l e c u l e s resulting in loss o f essential antigenic d e t e r m i n a n t s . Besides, i m m u n i z a t i o n even with highly p u r i f i e d p r o t e i n s m a y result in f o r m a t i o n o f a n t i b o d i e s against t r a c e c o n t a m i n a n t s in the p r e p a r a t i o n . O n e w a y b y w h i c h o n e can o b t a i n native i m m u n o g e n s is to isolate precipitating a n t i g e n ~ t i b o d y c o m p l e x e s and use these as i m m u n i z i n g m a t e r i a l ( S m i t h et al., 1 9 6 4 ; G o u d i e et al., 1 9 6 6 ; Shivers and J a m e s , 1 9 6 7 ; N a n s e n e t al., 1 9 7 1 ; Crowle et al., 1 9 7 2 ; K o c h and Nielsen, 1 9 7 5 ) . A l t h o u g h k n o w n for sew~ral years, the principle has n o t gained wide a p p l i c a t i o n , p r o b a b l y d u e to the d i f f i c u l t y in a t t a i n i n g s u f f i c i e n t a m o u n t s o f p u r e i m m u n o g e n s on the basis o f t r a d i t i o n a l i m m u n o d i f f u s i o n - and i m m u n o e l e c t r o p h o r e t i c m e t h o d s . A r e e v a l u a t i o n o f the principle was c o n s i d e r e d r e l e v a n t as line i m m u n o e l e c t r o p h o r e s i s provides an i m p r o v e d basis for the i d e n t i f i c a t i o n and isolation o f the specific p r e c i p i t a t e s (Kr¢ll and A n d e r s e n , 1 9 7 5 ) . MATERIALS AND METHODS
Reagents
N o r m a l h u m a n p l a s m a was o b t a i n e d f r o m b l o o d d o n o r s . N o r m a l rat p l a s m a was o b t a i n e d f r o m f e m a l e Wistar rats b y h e a r t p u n c t u r e . D N A - b i n d ing rat p l a s m a p r o t e i n s were isolated b y D N A - c e l l u l o s e c h r o m a t o g r a p h y as p r e v i o u s l y d e s c r i b e d (Kr¢ll et al., 1976). Specific and p o l y v a l e n t r a b b i t a n t i s e r a against h u m a n and rat p l a s m a pro-
126
teins were obtained from Dakopatts A/S, Copenhagen or from Behringwerke A.G., Marburg. Antiserum against DNA-binding proteins was produced by immunization of rabbits according to Harboe and Ingild {1973).
Immunoelectrophoresis Line immunoelectrophoresis and crossed immunoelectrophoresis were carried out as previously described (Kr¢ll, 1973; Weeke, 1973). After electrophoresis the gel was blotted with filter paper and washed in 0.9% saline to remove non-precipitated proteins. The precipitates were visualised by darkfield illumination or by staining in a dilute aqueous solution of Coomassie brilliant blue (0.1 g/l).
Identification Line-immunoelectrophoresis against a specific antiserum provided direct identification of the corresponding precipitate. Localized absorption with monospecific antisera {Andersen and Kr¢ll, 1975; Kr¢ll and Andersen, 1975) was used to identify precipitates in complex line-immunoelectrophoretic patterns (fig. 1).
Aa •
m
. . . . . .
" . ~-?:.~":~ ~ , ...,. , ~
i
.
II I
.
~
.
.
,~.v ~,r
.
~
•1 ii
C~a • D:;...~.~j.
',
: - ~ , ~ ? : : . ~ . ~ -.
Fig. 1. I d e n t i f i c a t i o n a n d isolation of rat f a c t o r XIII precipitate. T h e sample gel S (1 x 5 x 60 m m ) c o n t a i n e d 3% o f n o r m a l rat plasma. In t h e circular well a ( d i a m e t e r 2.5 m m ) was placed 2 pl h u m a n c o a g u l a t i o n f a c t o r XIII a n t i s e r u m . T h e a n t i s e r u m gel A G c o n t a i n e d 5% a n t i s e r u m against rat plasma D N A b i n d i n g p r o t e i n s . I m m u n o e l e c t r o p h o r e s i s was carried o u t at 1.5 V / c m for 16 h, a n o d e at top. S t a i n : C o o m a s s i e b r i l l i a n t blue 0.1 g/l in i s o t o n i c saline. Partial specific a b s o r p t i o n in a causes t h e negative d e f l e c t i o n of t h e f a c t o r XIII line a b o v e t h e well ( a r r o w ) T h e f r a m e d gel s e c t i o n c o n t a i n i n g this p r e c i p i t a t e was c u t o u t a n d used for i m m u n i z a t i o n .
t27
A6
A
B
Fig. 2. Line i m m u n o e l e c t r o p h o r e s i s o f rat p l a s m a and D N A - b i n d i n g plasma p r o t e i n s . In t h e s a m p l e gel A / B (2 X 20 x 50 m m ) s e c t i o n A c o n t a i n e d 3% of a p r o t e i n f r a c t i o n (total p r o t e i n c o n c e n t r a t i o n 0.4 m g / m l ) isolated b y DNA-cellulose c h r o m a t o g r a p h y o f 2 ml of n o r m a l rat plasma (Kr~bll et al., 1976). S e c t i o n B c o n t a i n e d 0.25% o f n o r m a l rat p l a s m a (total p r o t e i n c o n c e n t r a t i o n 62.1 m g / m l ) . T h e a n t i s e r u m gel AG was m o u l e d in t h r e e s e c t i o n s as i n d i c a t e d b y h o r i z o n t a l lines. T h e l o w e r gel section c o n t a i n e d 1%, t h e m i d d l e s e c t i o n 5% and the u p p e r s e c t i o n 10% a n t i s e r u m against rat s e r u m p r o t e i n s . I m m u n o e l e c t r o p h o r e s i s was carried o u t at 1.5 V / c m for 20 h, a n o d e at top. Cooma&sle stain.
12a I s o l a t i o n a n d i m m un iza t ion
The identified precipitates were cut o u t by m e a n s o f a scalpel and dispersed by s o n i c a t i o n in 0.15 M NaC1, 1 ml per em line. 2 0 0 pl o f the h o m o genate representing 1 0 - - 5 0 ng o f antigen was, after a d m i x t u r e o f 50 gl o f F r e u n d ' s adjuvant, used for each i m m u n i z a t i o n o f rabbits a c c o r d i n g to the p r o c e d u r e o f H a r b o e and Ingild ( 1 9 7 3 ) . A f t e r a t t a i n m e n t o f a specific i m m u n e response the animals were r e i m m u n i z e d o n l y at signs o f a decrease in titer, and t h e n o n l y with the precipitate d e v e l o p e d against the specific antisermn. I£ESULTS Tile l i n e - i m m u n o e l e c t r o p h o r e t i c p a t t e r n d e v e l o p e d by rat serum against the p o l y v a l e n t a n t i s e r u m appears in fig. 2. T o o b t a i n a clear separation o f the lines representing the D N A - b i n d i n g proteins it was necessary to isolate these p r o t e i n s or to use a less p o l y v a l e n t antiserum (figs. 1 and 3). R a t c o a g u l a t i o n f a c t o r X I I I was n o t p r e c i p i t a t e d by i m m u n o e l e c t r o phoresis against the h u m a n f a c t o r X I I I a n t i s e r u m . H o w e v e r the presence o f c o m m o n antigenic d e t e r m i n a n t s p e r m i t t e d the use o f this antiserum for
AG
A
¢,
Fig. 3. Crossed immunoelectrophoresis of rat factor XIII. In the circular well s (diameter 2.5 mm) was placed 2 HI normal rat plasma. The first-dimensional run (I) was carried out at 7 V/cm for 1.5 h, anode to the left. The antiserum gel AG contained 5% antiserum obtained by immunization of rabbits v,,ith rat factor XIII precipitate (fig. 1). The immunoclectrophoretic run (II) was carried out at 1.5 V/cm for 15 h, anode at top. Cooma~sie stain.
129 identification of the rat factor XIII line (fig. 1). A specific antibody response was obtained by immunization with the isolated precipitate of rat factor XIII (fig. 3). Similarly, immunization with precipitates obtained by line immunoelectrophoresis of human serum against commercially available specific antisera resulted in a directly specific antibody response against the proteins considered (albumin, a ~-antitrypsin, haptoglobin and transferrin). Monospecificity of the antibody response have been preserved for more than one year by reimmunization according to the present procedure. DISCUSSION The present investigation demonstrates that precipitin lines representing plasma trace proteins can be isolated on the basis of line immunoelectrophoresis in amounts and at a purity sufficient for the elicitation of a directly specific antibody response. Furthermore the results suggest that precipitates formed against specific antisera can be used for reimmunization to preserve specificity. The a m o u n t of i m m u n o c o m p l e x e d antigen used for immunization was less than 10 -2 of the doses generally recommended for non-polymer proteins (Mitchison, 1972; Herbert, 1973; Harboe and Ingild, 1973), and probably was far from the lower level needed for antibody response against immunocomplexed antigen (Crowle et al., 1972). The reason for the strong immune response against immunocomplexed antigens is unknown; it might represent an analogy to the general increase in antigenicity caused by polymerization or coprecipitation of weak antigens {Gill, 1972). Howew~r, the use of antigen--antibody complexes as immunogens is preferable to chemical polymerization procedures involving a risk for modification of the antigen. Secondly, for the recognition and isolation of some plasma trace components there may be no alternative method. The above procedure for making pure and native immunogens thus tends to eliminate two of the major difficulties in the production of monospecific antisera, i.e. the frequently troublesome conventional antigen purification and antibody absorption procedures. ACKNOWLEDGEMENTS This investigation was supported by a grant from Den Lmgevidenskabelige Forskningsfond for StorkCbenhavn, Faer¢erne og GrSnland. The skilful technical assistance of Miss Susanne Vissing, Mrs. Birthe I,arsen and Mrs. Lene Ahrenst is gratefully acknowledged.
REFERENCES Andersen, M.M. and J. Kr~bll, 1975, Scand. J. Immunol. 4, ~uppl. 2, 163. Crowle, A.J., G.J. Revis.and K. Jarrett, 1972, Immunol. Commun. 1, ;325.
130 Harboe, N. and A. Ingild, 1973, Stand. J. Immunol. 2, suppl. 1, 161. (i-ill, T.J., 1972, in: Immunogenicity, ed. F. Borek (North-lTolland, Amsterdam) p. 5. (]-oudie, R.B., C.H.W. Home and P.C. Wilkinson, 1966, Lancet, ii 122,i. Herbert, W.J., 1973, in: Handbook of experimental immunology, ed. D.M. Weir (Blackwell, Oxford) p. A2, 1. Koch, C. and tt.E. Nielsen, 1975, Scand. J. Immunol. 4, suppl. 2, 121. Kr~bll, J., 1973, Scand. J. Immunol. 2, suppl. I, 61. Kr~bll, J. and M.M. Andersen, 1975, J. Immunol. Methods 9, 14 I. Kr~bll, J., J.K. Larsen, If. Loft, M. Ezban, K. Wallevik and M. Faber, 1976, Biochem. Biophys. Acta (in press). Mitcbison, N.A., 1972, in: Immunogenicity, ed. F. Borek (North-Holland, Amsterdam)p. 87. Nansen, P., T. Flagstad and K.B. Pedersen, 197l, Acta Pathol. Microbiol. Scand. 79, ,159. Shivers, C.A. and J.M. James, 1967, Immunology 13,547. Smith, H., R.C. Gallop and B.T. Tozer, 1964, Immunology 7, l 11. Weeke, B., 1973, Scand. J. Immunol. 2, suppl. 1, 47.
125
S P E C I F I C A N T I S E R A P R O D U C E D BY I M M U N I Z A T I O N W I T H PRECIPITIN LINES
JENS KROLL and METTE M. ANDERSEN The Finsen Laboratory, The Finsen Institute, Strandboulevarden 49, 2100 Copenha~,~en O, Denmark
(Received 27 May 1976, accepted 3 June 1976)
Individual plasma proteins were precipitated, identified and isolated on tile basis of line immunoeleetrophoresis. A monospecific antibody response was induced by immunization of rabbits with less than 50 ng of precipitated antigen. Preservation of monospeeifieity was obtained by reimmunization with precipitates developed against the specific antisora. INTRODUCTION Direct a t t a i n m e n t o f a specific a n t i s e r u m requires a high degree o f p u r i t y o f the i m m u n o g e n . E x t e n s i v e p u r i f i c a t i o n p r o c e d u r e s m a y , h o w e v e r , m o d i f y the m o l e c u l e s resulting in loss o f essential antigenic d e t e r m i n a n t s . Besides, i m m u n i z a t i o n even with highly p u r i f i e d p r o t e i n s m a y result in f o r m a t i o n o f a n t i b o d i e s against t r a c e c o n t a m i n a n t s in the p r e p a r a t i o n . O n e w a y b y w h i c h o n e can o b t a i n native i m m u n o g e n s is to isolate precipitating a n t i g e n ~ t i b o d y c o m p l e x e s and use these as i m m u n i z i n g m a t e r i a l ( S m i t h et al., 1 9 6 4 ; G o u d i e et al., 1 9 6 6 ; Shivers and J a m e s , 1 9 6 7 ; N a n s e n e t al., 1 9 7 1 ; Crowle et al., 1 9 7 2 ; K o c h and Nielsen, 1 9 7 5 ) . A l t h o u g h k n o w n for sew~ral years, the principle has n o t gained wide a p p l i c a t i o n , p r o b a b l y d u e to the d i f f i c u l t y in a t t a i n i n g s u f f i c i e n t a m o u n t s o f p u r e i m m u n o g e n s on the basis o f t r a d i t i o n a l i m m u n o d i f f u s i o n - and i m m u n o e l e c t r o p h o r e t i c m e t h o d s . A r e e v a l u a t i o n o f the principle was c o n s i d e r e d r e l e v a n t as line i m m u n o e l e c t r o p h o r e s i s provides an i m p r o v e d basis for the i d e n t i f i c a t i o n and isolation o f the specific p r e c i p i t a t e s (Kr¢ll and A n d e r s e n , 1 9 7 5 ) . MATERIALS AND METHODS
Reagents
N o r m a l h u m a n p l a s m a was o b t a i n e d f r o m b l o o d d o n o r s . N o r m a l rat p l a s m a was o b t a i n e d f r o m f e m a l e Wistar rats b y h e a r t p u n c t u r e . D N A - b i n d ing rat p l a s m a p r o t e i n s were isolated b y D N A - c e l l u l o s e c h r o m a t o g r a p h y as p r e v i o u s l y d e s c r i b e d (Kr¢ll et al., 1976). Specific and p o l y v a l e n t r a b b i t a n t i s e r a against h u m a n and rat p l a s m a pro-
126
teins were obtained from Dakopatts A/S, Copenhagen or from Behringwerke A.G., Marburg. Antiserum against DNA-binding proteins was produced by immunization of rabbits according to Harboe and Ingild {1973).
Immunoelectrophoresis Line immunoelectrophoresis and crossed immunoelectrophoresis were carried out as previously described (Kr¢ll, 1973; Weeke, 1973). After electrophoresis the gel was blotted with filter paper and washed in 0.9% saline to remove non-precipitated proteins. The precipitates were visualised by darkfield illumination or by staining in a dilute aqueous solution of Coomassie brilliant blue (0.1 g/l).
Identification Line-immunoelectrophoresis against a specific antiserum provided direct identification of the corresponding precipitate. Localized absorption with monospecific antisera {Andersen and Kr¢ll, 1975; Kr¢ll and Andersen, 1975) was used to identify precipitates in complex line-immunoelectrophoretic patterns (fig. 1).
Aa •
m
. . . . . .
" . ~-?:.~":~ ~ , ...,. , ~
i
.
II I
.
~
.
.
,~.v ~,r
.
~
•1 ii
C~a • D:;...~.~j.
',
: - ~ , ~ ? : : . ~ . ~ -.
Fig. 1. I d e n t i f i c a t i o n a n d isolation of rat f a c t o r XIII precipitate. T h e sample gel S (1 x 5 x 60 m m ) c o n t a i n e d 3% o f n o r m a l rat plasma. In t h e circular well a ( d i a m e t e r 2.5 m m ) was placed 2 pl h u m a n c o a g u l a t i o n f a c t o r XIII a n t i s e r u m . T h e a n t i s e r u m gel A G c o n t a i n e d 5% a n t i s e r u m against rat plasma D N A b i n d i n g p r o t e i n s . I m m u n o e l e c t r o p h o r e s i s was carried o u t at 1.5 V / c m for 16 h, a n o d e at top. S t a i n : C o o m a s s i e b r i l l i a n t blue 0.1 g/l in i s o t o n i c saline. Partial specific a b s o r p t i o n in a causes t h e negative d e f l e c t i o n of t h e f a c t o r XIII line a b o v e t h e well ( a r r o w ) T h e f r a m e d gel s e c t i o n c o n t a i n i n g this p r e c i p i t a t e was c u t o u t a n d used for i m m u n i z a t i o n .
t27
A6
A
B
Fig. 2. Line i m m u n o e l e c t r o p h o r e s i s o f rat p l a s m a and D N A - b i n d i n g plasma p r o t e i n s . In t h e s a m p l e gel A / B (2 X 20 x 50 m m ) s e c t i o n A c o n t a i n e d 3% of a p r o t e i n f r a c t i o n (total p r o t e i n c o n c e n t r a t i o n 0.4 m g / m l ) isolated b y DNA-cellulose c h r o m a t o g r a p h y o f 2 ml of n o r m a l rat plasma (Kr~bll et al., 1976). S e c t i o n B c o n t a i n e d 0.25% o f n o r m a l rat p l a s m a (total p r o t e i n c o n c e n t r a t i o n 62.1 m g / m l ) . T h e a n t i s e r u m gel AG was m o u l e d in t h r e e s e c t i o n s as i n d i c a t e d b y h o r i z o n t a l lines. T h e l o w e r gel section c o n t a i n e d 1%, t h e m i d d l e s e c t i o n 5% and the u p p e r s e c t i o n 10% a n t i s e r u m against rat s e r u m p r o t e i n s . I m m u n o e l e c t r o p h o r e s i s was carried o u t at 1.5 V / c m for 20 h, a n o d e at top. Cooma&sle stain.
12a I s o l a t i o n a n d i m m un iza t ion
The identified precipitates were cut o u t by m e a n s o f a scalpel and dispersed by s o n i c a t i o n in 0.15 M NaC1, 1 ml per em line. 2 0 0 pl o f the h o m o genate representing 1 0 - - 5 0 ng o f antigen was, after a d m i x t u r e o f 50 gl o f F r e u n d ' s adjuvant, used for each i m m u n i z a t i o n o f rabbits a c c o r d i n g to the p r o c e d u r e o f H a r b o e and Ingild ( 1 9 7 3 ) . A f t e r a t t a i n m e n t o f a specific i m m u n e response the animals were r e i m m u n i z e d o n l y at signs o f a decrease in titer, and t h e n o n l y with the precipitate d e v e l o p e d against the specific antisermn. I£ESULTS Tile l i n e - i m m u n o e l e c t r o p h o r e t i c p a t t e r n d e v e l o p e d by rat serum against the p o l y v a l e n t a n t i s e r u m appears in fig. 2. T o o b t a i n a clear separation o f the lines representing the D N A - b i n d i n g proteins it was necessary to isolate these p r o t e i n s or to use a less p o l y v a l e n t antiserum (figs. 1 and 3). R a t c o a g u l a t i o n f a c t o r X I I I was n o t p r e c i p i t a t e d by i m m u n o e l e c t r o phoresis against the h u m a n f a c t o r X I I I a n t i s e r u m . H o w e v e r the presence o f c o m m o n antigenic d e t e r m i n a n t s p e r m i t t e d the use o f this antiserum for
AG
A
¢,
Fig. 3. Crossed immunoelectrophoresis of rat factor XIII. In the circular well s (diameter 2.5 mm) was placed 2 HI normal rat plasma. The first-dimensional run (I) was carried out at 7 V/cm for 1.5 h, anode to the left. The antiserum gel AG contained 5% antiserum obtained by immunization of rabbits v,,ith rat factor XIII precipitate (fig. 1). The immunoclectrophoretic run (II) was carried out at 1.5 V/cm for 15 h, anode at top. Cooma~sie stain.
129 identification of the rat factor XIII line (fig. 1). A specific antibody response was obtained by immunization with the isolated precipitate of rat factor XIII (fig. 3). Similarly, immunization with precipitates obtained by line immunoelectrophoresis of human serum against commercially available specific antisera resulted in a directly specific antibody response against the proteins considered (albumin, a ~-antitrypsin, haptoglobin and transferrin). Monospecificity of the antibody response have been preserved for more than one year by reimmunization according to the present procedure. DISCUSSION The present investigation demonstrates that precipitin lines representing plasma trace proteins can be isolated on the basis of line immunoelectrophoresis in amounts and at a purity sufficient for the elicitation of a directly specific antibody response. Furthermore the results suggest that precipitates formed against specific antisera can be used for reimmunization to preserve specificity. The a m o u n t of i m m u n o c o m p l e x e d antigen used for immunization was less than 10 -2 of the doses generally recommended for non-polymer proteins (Mitchison, 1972; Herbert, 1973; Harboe and Ingild, 1973), and probably was far from the lower level needed for antibody response against immunocomplexed antigen (Crowle et al., 1972). The reason for the strong immune response against immunocomplexed antigens is unknown; it might represent an analogy to the general increase in antigenicity caused by polymerization or coprecipitation of weak antigens {Gill, 1972). Howew~r, the use of antigen--antibody complexes as immunogens is preferable to chemical polymerization procedures involving a risk for modification of the antigen. Secondly, for the recognition and isolation of some plasma trace components there may be no alternative method. The above procedure for making pure and native immunogens thus tends to eliminate two of the major difficulties in the production of monospecific antisera, i.e. the frequently troublesome conventional antigen purification and antibody absorption procedures. ACKNOWLEDGEMENTS This investigation was supported by a grant from Den Lmgevidenskabelige Forskningsfond for StorkCbenhavn, Faer¢erne og GrSnland. The skilful technical assistance of Miss Susanne Vissing, Mrs. Birthe I,arsen and Mrs. Lene Ahrenst is gratefully acknowledged.
REFERENCES Andersen, M.M. and J. Kr~bll, 1975, Scand. J. Immunol. 4, ~uppl. 2, 163. Crowle, A.J., G.J. Revis.and K. Jarrett, 1972, Immunol. Commun. 1, ;325.
130 Harboe, N. and A. Ingild, 1973, Stand. J. Immunol. 2, suppl. 1, 161. (i-ill, T.J., 1972, in: Immunogenicity, ed. F. Borek (North-lTolland, Amsterdam) p. 5. (]-oudie, R.B., C.H.W. Home and P.C. Wilkinson, 1966, Lancet, ii 122,i. Herbert, W.J., 1973, in: Handbook of experimental immunology, ed. D.M. Weir (Blackwell, Oxford) p. A2, 1. Koch, C. and tt.E. Nielsen, 1975, Scand. J. Immunol. 4, suppl. 2, 121. Kr~bll, J., 1973, Scand. J. Immunol. 2, suppl. I, 61. Kr~bll, J. and M.M. Andersen, 1975, J. Immunol. Methods 9, 14 I. Kr~bll, J., J.K. Larsen, If. Loft, M. Ezban, K. Wallevik and M. Faber, 1976, Biochem. Biophys. Acta (in press). Mitcbison, N.A., 1972, in: Immunogenicity, ed. F. Borek (North-Holland, Amsterdam)p. 87. Nansen, P., T. Flagstad and K.B. Pedersen, 197l, Acta Pathol. Microbiol. Scand. 79, ,159. Shivers, C.A. and J.M. James, 1967, Immunology 13,547. Smith, H., R.C. Gallop and B.T. Tozer, 1964, Immunology 7, l 11. Weeke, B., 1973, Scand. J. Immunol. 2, suppl. 1, 47.