- Email: [email protected]
The biological assay of epidermal growth factor in neonatal mice
57
Biochimica et Biophysiea Acta, 354 ( 1 9 7 4 ) 57--.60 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27414
THE BIOLOGICAL ASSAY OF EPIDERMAL GROWTH FACTOR IN NEONATAL MICE
P H I L I P T. B L O S S E a n d E R N E S T L. F E N T O N
Department of Biological Chemistry, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent (Great Britain) (Received January 9th, 1974)
Summary A quantitative m e t h o d for the estimation of the biological activity of epidermal growth factor has been developed, based on the precocious eye opening response in neonatal mice.
Epidermal growth factor was first described by Cohen [1] in 1962 as a substance extracted from the submaxillary glands of adult male mice, causing precocious eye opening and t o o t h eruption when injected into neonatal mice. Subsequently its effects on epidermal growth were shown by histological investigations [2]. The chemical characterisation of epidermal growth factor as a peptide has been established, and the primary structure elucidated [3]. Although eye opening [1] and t o o t h eruption responses [4] are still used as measures of the biological activity of epidermal growth factor preparations, no attempts have been made to use these responses in a strictly quantitative assay for epidermal growth factor activity. A sensitive radioimmunoassay has been described [5] but since this depends on antigen recognition and not on biological activity, it is clearly more subject to misinterpretation. Further, there is no evidence to suggest that antigenicity is a true indication of the biological activity of epidermal growth factor, especially when low molecular weight fragments may be present. For these reasons it was decided to examine the biological responses in vivo with a view to the development of a reliable and quantitative assay procedure. For a typical assay, three litters of nine neonatal mice (Porton albino strain, 1.5--2.0 g body wt) were re
58 that of Savage et al. [3,6], was used in these studies. Epidermal growth factor samples in phosphate-buffered isotonic saline (pH 7.4) were injected subcutaneously in a volume of 0.033 ml/g b o d y weight per day for 7 days. Control animals received an equivalent volume of buffered saline, based on b o d y weight. Daily records were made on b o d y weight, eye opening and t o o t h eruption. Using this procedure, control mice receiving no epidermal growth factor showed eye opening on days 12 to 13 and t o o t h eruption on days 10 to 11, whereas injection of epidermal growth factor for maximum effect resulted in responses on days 6 to 7 for both eye and t o o t h effects. It was found that the t o o t h eruption responses could not be employed satisfactorily for assay purposes in a quantitative application. However, using the eye opening response as the criterion for epidermal growth factor activity, mean responses within the range 7 to 12 days were found to be linearly related to the logarithm of the epidermal growth factor dosage, as shown in Fig. 1. This relationship has been demonstrated in numerous dose response experiments. Failure to obtain such linearity was attributed either to incorrect assay procedure (e.g. inadequate randomnisation of litters, excessively high or low birth weights) or to the use of impure epidermal growth factor preparations showing high toxicity. The slope of the regression line varied between test occasions {e.g. between experiments set up on different days) but within any single experiment the slopes of the regression lines for various epidermal growth factor preparations were found to be similar. This emphasised the importance of eliminating between-litter variations in eye opening responses by the use of randomnised and selected litters. From these observations, it was possible to consider the use of a parallel line bioassay procedure [7,8] for the estimation of the relative potencies of 13 12 11 10
o'.125
o125
I
0.5
11o
2:0
I
4.0
I
8.0
Epidemal growth factor dosage ( ~ j / g body wt. per day, log scale) F i g . 1. L o g d o s e / r e s p o n s e r e g r e s s i o n line f o r e p i d e r m a l g r o w t h f a c t o r b a s e d o n t h e e y e o p e n i n g r e s p o n s e s in neonatal mice. Three randomnised litters were employed in the assay and each response represents the m e a n value f r o m t h r e e m i c e . Six m i c e r e c e i v i n g no e p i d e r m a l g r o w t h f a c t o r ( c o n t r o l g r o u p ) all gave eye opening responses o n d a y 13.
59 A 12
-8 ,o
o
6 0,25
o15
11o
21o
41o
Epidemal growth factor dosage (iJglg body wt. per day, log scale ) Fig. 2. Parallel line b i o a s s a y for t w o p r e p a r a t i o n s of e p i d e r m a l g r o w t h f a c t o r . T h e e x p e r i m e n t a l p o i n t s are derived from data a s s e m b l e d in Table I. A---A p r e p n A; e - - 4 , p r e p n B.
epidermal growth factor preparations, and a typical example is shown graphically in Fig. 2 from data assembled in Table I. The regression lines for epidermal growth factor preparations A and B were derived from t w o dosages of each, equally spaced on the logarithmic scale. Although the regression line of an extended dose range has been shown to be linear within the limits of 7 to 12 days, for assay purposes greater sensitivity and reliability was obtained if a restricted range from 8 to 11 days was employed. Greater precision may also be obtained by increasing the number of mice in each dose group from three to four or even greater. For calculation of relative potency, the procedure described by Emmens [9] has been employed. This value may be obtained graphically by simple calculation of the difference between log doses on the abscissa (Fig. 2) for a TABLE I B I O L O G I C A L D A T A F O R T H E E V A L U A T I O N O F R E L A T I V E P O T E N C Y O F TWO E P I D E R M A L GROWTH FACTOR PREPARATIONS If, f o r e a c h p r e p a r a t i o n , there are t w o dose g r o u p s a n d a n e q u a l d i f f e r e n c e b e t w e e n log doses, t h e f o l l o w i n g e q u a t i o n c a n be applied (assuming non-significant departure f r o m parallelism). L o g r a t i o o f p o t e n c y , M = I D / B w h e r e I is the log dose r a t i o ( d i f f e r e n c e b e t w e e n log doses); D is the d i f f e r e n c e in m e a n r e s p o n s e s b e t w e e n p r e p a r a t i o n s ; B is the d i f f e r e n c e in m e a n r e s p o n s e b e t w e e n high and l o w doses. T h e r e f o r e , M = I X (A 1 + A2) - (B 1 + B 2 ) / ( A 1 + B 1 ) - (A 2 + B2 ) = 0 . 6 0 2 X ( 2 0 . 2 5 - 1 7 . 7 5 ) / ( 2 0 . 7 5 - 1 7 , 2 5 ) = 0 . 4 3 . Relative p o t e n c y , R, is given b y antflog M = a n t f l o g 0.43 = 2.7. T h e r e f o r e , r e l a t i v e p o t e n c y o f A: B is 1: 2.7. Epidermal growth factor preparation
Dosage (//g/g p e r d a y )
Log dosage
Eye opening r e s p o n s e s (days)
Mean response (days)
A
0 0.5 2.0 0 0.5 2.0
-1.6990 0.3010 _-1.6990 0.3010
13, 11, 9, 13, 9, 8,
13.0 1 1 . 0 (A1) 9 . 2 5 (A2) 13.0 9 . 7 5 (B1) 8.0 (B2)
B
13 11, 11, 11 9, 9, 10 13 10, 10, 10 8, 8, 8
60 given eye opening response on the ordinate, preferably the mean response value for all groups. The antilog of the log difference will give the relative potencies of the two samples. However, ideally this value is obtained by direct calculation, using the equation shown in Table I. In either situation, the calculation is invalid if significant departure from parallelism is present and this can be easily evaluated from the standard errors by conventional statistical analysis [10]. However, in suitably designed experiments, significant departure from parallelism is not normally obtained. In most assay procedures it is convenient to use one dose group for a reference preparation and this may be included in other assays to form an epidermal growth factor standard. In this way, meaningful comparisons can be obtained b e t w e e n epidermal growth factor preparations assayed on different test occasions, thereby minimising the apparent variations in activity obtained without a reference sample. If highly purified native epidermal growth factor or an equally active derivative is available, this may form an ideal standard for such assays in the absence of a recognised unit of epidermal growth factor activity, and the specific activity of other preparations such as lower molecular weight fragments could be meaningfully expressed in terms of the standard activity. The assay described offers a reliable and quantitative m e t h o d for the estimation of epidermal growth factor activity based on its biological effects in vivo. The assay is n o t as rapid or as sensitive as the radioimmunoassay but on the other hand it does not require the specialised materials such as radioactively labelled epidermal growth factor or anti-epidermal growth factor serum essential for the latter technique. Also, the fact that it relies upon an observation b y which epidermal growth factor activity was defined and not upon antigen recognition, makes it more reliable. It is considered therefore that this biological assay will be of value in the measurement of epidermal growth factor activity, especially in studies on the biological activity of lower molecular weight derivatives or analogues of epidermal growth factor. References 1 2 3 4 5 6 7 S 9
C o h e n , S. ( 1 9 6 2 ) J. Biol. C h e m . 2 3 7 , 1 5 5 5 - - 1 5 6 2 C o h e n , S. a n d E l l i o t t , G . A . ( 1 9 6 3 ) J. Invest. D e r m a t o l . 4 0 , 1 - - 5 S a v a g e , C . R . , I n a g a m i , T. a n d C o h e n , S. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 6 1 2 - - 7 6 2 1 Covelli, I., Rossi, R . , Mozzi, R. a n d F r a t i , L. ( 1 9 7 2 ) E u r . J. B i o c h e m . 2 7 , 2 2 5 - - 2 3 0 B y n n y , R . L . , O r t h , D . N . a n d C o h e n , S. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 1 2 6 1 - - 1 2 6 6 S a v a g e , C . R . a n d C o h e n , S. ( 1 9 7 2 ) J. Biol. C h e m . , 2 4 7 , 7 6 0 9 - - 7 6 1 1 Bliss, C.I. a n d M a r k s , H.P. ( 1 9 3 9 ) Q. J. P h a r m . P h a r m a c o l . 1 2 , 8 2 - - 1 1 0 Bliss, C.I. a n d M a r k s , H.P. ( 1 9 3 9 ) Q . J. P h a r m . P h a r m a c o l . 1 2 , 1 8 2 - - 2 1 1 E m m e n s , C.W. ( 1 9 5 0 ) in S t a t i s t i c a l M e t h o d s . H o r m o n e A s s a y ( E m m e n s , C.W., e d . ) , p p . 1 8 - - 1 9 , A c a d e m i c Press, N e w Y o r k 1 0 E m m e n s , C.W. ( 1 9 4 6 ) in P r i n c i p l e s o f B i o l o g i c a l A s s a y ( E m m e n s , C.W., e d . ) , p p . 8 8 - - 0 7 , C h a p m a n a n d Hall, L o n d o n
Biochimica et Biophysiea Acta, 354 ( 1 9 7 4 ) 57--.60 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27414
THE BIOLOGICAL ASSAY OF EPIDERMAL GROWTH FACTOR IN NEONATAL MICE
P H I L I P T. B L O S S E a n d E R N E S T L. F E N T O N
Department of Biological Chemistry, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent (Great Britain) (Received January 9th, 1974)
Summary A quantitative m e t h o d for the estimation of the biological activity of epidermal growth factor has been developed, based on the precocious eye opening response in neonatal mice.
Epidermal growth factor was first described by Cohen [1] in 1962 as a substance extracted from the submaxillary glands of adult male mice, causing precocious eye opening and t o o t h eruption when injected into neonatal mice. Subsequently its effects on epidermal growth were shown by histological investigations [2]. The chemical characterisation of epidermal growth factor as a peptide has been established, and the primary structure elucidated [3]. Although eye opening [1] and t o o t h eruption responses [4] are still used as measures of the biological activity of epidermal growth factor preparations, no attempts have been made to use these responses in a strictly quantitative assay for epidermal growth factor activity. A sensitive radioimmunoassay has been described [5] but since this depends on antigen recognition and not on biological activity, it is clearly more subject to misinterpretation. Further, there is no evidence to suggest that antigenicity is a true indication of the biological activity of epidermal growth factor, especially when low molecular weight fragments may be present. For these reasons it was decided to examine the biological responses in vivo with a view to the development of a reliable and quantitative assay procedure. For a typical assay, three litters of nine neonatal mice (Porton albino strain, 1.5--2.0 g body wt) were re
58 that of Savage et al. [3,6], was used in these studies. Epidermal growth factor samples in phosphate-buffered isotonic saline (pH 7.4) were injected subcutaneously in a volume of 0.033 ml/g b o d y weight per day for 7 days. Control animals received an equivalent volume of buffered saline, based on b o d y weight. Daily records were made on b o d y weight, eye opening and t o o t h eruption. Using this procedure, control mice receiving no epidermal growth factor showed eye opening on days 12 to 13 and t o o t h eruption on days 10 to 11, whereas injection of epidermal growth factor for maximum effect resulted in responses on days 6 to 7 for both eye and t o o t h effects. It was found that the t o o t h eruption responses could not be employed satisfactorily for assay purposes in a quantitative application. However, using the eye opening response as the criterion for epidermal growth factor activity, mean responses within the range 7 to 12 days were found to be linearly related to the logarithm of the epidermal growth factor dosage, as shown in Fig. 1. This relationship has been demonstrated in numerous dose response experiments. Failure to obtain such linearity was attributed either to incorrect assay procedure (e.g. inadequate randomnisation of litters, excessively high or low birth weights) or to the use of impure epidermal growth factor preparations showing high toxicity. The slope of the regression line varied between test occasions {e.g. between experiments set up on different days) but within any single experiment the slopes of the regression lines for various epidermal growth factor preparations were found to be similar. This emphasised the importance of eliminating between-litter variations in eye opening responses by the use of randomnised and selected litters. From these observations, it was possible to consider the use of a parallel line bioassay procedure [7,8] for the estimation of the relative potencies of 13 12 11 10
o'.125
o125
I
0.5
11o
2:0
I
4.0
I
8.0
Epidemal growth factor dosage ( ~ j / g body wt. per day, log scale) F i g . 1. L o g d o s e / r e s p o n s e r e g r e s s i o n line f o r e p i d e r m a l g r o w t h f a c t o r b a s e d o n t h e e y e o p e n i n g r e s p o n s e s in neonatal mice. Three randomnised litters were employed in the assay and each response represents the m e a n value f r o m t h r e e m i c e . Six m i c e r e c e i v i n g no e p i d e r m a l g r o w t h f a c t o r ( c o n t r o l g r o u p ) all gave eye opening responses o n d a y 13.
59 A 12
-8 ,o
o
6 0,25
o15
11o
21o
41o
Epidemal growth factor dosage (iJglg body wt. per day, log scale ) Fig. 2. Parallel line b i o a s s a y for t w o p r e p a r a t i o n s of e p i d e r m a l g r o w t h f a c t o r . T h e e x p e r i m e n t a l p o i n t s are derived from data a s s e m b l e d in Table I. A---A p r e p n A; e - - 4 , p r e p n B.
epidermal growth factor preparations, and a typical example is shown graphically in Fig. 2 from data assembled in Table I. The regression lines for epidermal growth factor preparations A and B were derived from t w o dosages of each, equally spaced on the logarithmic scale. Although the regression line of an extended dose range has been shown to be linear within the limits of 7 to 12 days, for assay purposes greater sensitivity and reliability was obtained if a restricted range from 8 to 11 days was employed. Greater precision may also be obtained by increasing the number of mice in each dose group from three to four or even greater. For calculation of relative potency, the procedure described by Emmens [9] has been employed. This value may be obtained graphically by simple calculation of the difference between log doses on the abscissa (Fig. 2) for a TABLE I B I O L O G I C A L D A T A F O R T H E E V A L U A T I O N O F R E L A T I V E P O T E N C Y O F TWO E P I D E R M A L GROWTH FACTOR PREPARATIONS If, f o r e a c h p r e p a r a t i o n , there are t w o dose g r o u p s a n d a n e q u a l d i f f e r e n c e b e t w e e n log doses, t h e f o l l o w i n g e q u a t i o n c a n be applied (assuming non-significant departure f r o m parallelism). L o g r a t i o o f p o t e n c y , M = I D / B w h e r e I is the log dose r a t i o ( d i f f e r e n c e b e t w e e n log doses); D is the d i f f e r e n c e in m e a n r e s p o n s e s b e t w e e n p r e p a r a t i o n s ; B is the d i f f e r e n c e in m e a n r e s p o n s e b e t w e e n high and l o w doses. T h e r e f o r e , M = I X (A 1 + A2) - (B 1 + B 2 ) / ( A 1 + B 1 ) - (A 2 + B2 ) = 0 . 6 0 2 X ( 2 0 . 2 5 - 1 7 . 7 5 ) / ( 2 0 . 7 5 - 1 7 , 2 5 ) = 0 . 4 3 . Relative p o t e n c y , R, is given b y antflog M = a n t f l o g 0.43 = 2.7. T h e r e f o r e , r e l a t i v e p o t e n c y o f A: B is 1: 2.7. Epidermal growth factor preparation
Dosage (//g/g p e r d a y )
Log dosage
Eye opening r e s p o n s e s (days)
Mean response (days)
A
0 0.5 2.0 0 0.5 2.0
-1.6990 0.3010 _-1.6990 0.3010
13, 11, 9, 13, 9, 8,
13.0 1 1 . 0 (A1) 9 . 2 5 (A2) 13.0 9 . 7 5 (B1) 8.0 (B2)
B
13 11, 11, 11 9, 9, 10 13 10, 10, 10 8, 8, 8
60 given eye opening response on the ordinate, preferably the mean response value for all groups. The antilog of the log difference will give the relative potencies of the two samples. However, ideally this value is obtained by direct calculation, using the equation shown in Table I. In either situation, the calculation is invalid if significant departure from parallelism is present and this can be easily evaluated from the standard errors by conventional statistical analysis [10]. However, in suitably designed experiments, significant departure from parallelism is not normally obtained. In most assay procedures it is convenient to use one dose group for a reference preparation and this may be included in other assays to form an epidermal growth factor standard. In this way, meaningful comparisons can be obtained b e t w e e n epidermal growth factor preparations assayed on different test occasions, thereby minimising the apparent variations in activity obtained without a reference sample. If highly purified native epidermal growth factor or an equally active derivative is available, this may form an ideal standard for such assays in the absence of a recognised unit of epidermal growth factor activity, and the specific activity of other preparations such as lower molecular weight fragments could be meaningfully expressed in terms of the standard activity. The assay described offers a reliable and quantitative m e t h o d for the estimation of epidermal growth factor activity based on its biological effects in vivo. The assay is n o t as rapid or as sensitive as the radioimmunoassay but on the other hand it does not require the specialised materials such as radioactively labelled epidermal growth factor or anti-epidermal growth factor serum essential for the latter technique. Also, the fact that it relies upon an observation b y which epidermal growth factor activity was defined and not upon antigen recognition, makes it more reliable. It is considered therefore that this biological assay will be of value in the measurement of epidermal growth factor activity, especially in studies on the biological activity of lower molecular weight derivatives or analogues of epidermal growth factor. References 1 2 3 4 5 6 7 S 9
C o h e n , S. ( 1 9 6 2 ) J. Biol. C h e m . 2 3 7 , 1 5 5 5 - - 1 5 6 2 C o h e n , S. a n d E l l i o t t , G . A . ( 1 9 6 3 ) J. Invest. D e r m a t o l . 4 0 , 1 - - 5 S a v a g e , C . R . , I n a g a m i , T. a n d C o h e n , S. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 6 1 2 - - 7 6 2 1 Covelli, I., Rossi, R . , Mozzi, R. a n d F r a t i , L. ( 1 9 7 2 ) E u r . J. B i o c h e m . 2 7 , 2 2 5 - - 2 3 0 B y n n y , R . L . , O r t h , D . N . a n d C o h e n , S. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 1 2 6 1 - - 1 2 6 6 S a v a g e , C . R . a n d C o h e n , S. ( 1 9 7 2 ) J. Biol. C h e m . , 2 4 7 , 7 6 0 9 - - 7 6 1 1 Bliss, C.I. a n d M a r k s , H.P. ( 1 9 3 9 ) Q. J. P h a r m . P h a r m a c o l . 1 2 , 8 2 - - 1 1 0 Bliss, C.I. a n d M a r k s , H.P. ( 1 9 3 9 ) Q . J. P h a r m . P h a r m a c o l . 1 2 , 1 8 2 - - 2 1 1 E m m e n s , C.W. ( 1 9 5 0 ) in S t a t i s t i c a l M e t h o d s . H o r m o n e A s s a y ( E m m e n s , C.W., e d . ) , p p . 1 8 - - 1 9 , A c a d e m i c Press, N e w Y o r k 1 0 E m m e n s , C.W. ( 1 9 4 6 ) in P r i n c i p l e s o f B i o l o g i c a l A s s a y ( E m m e n s , C.W., e d . ) , p p . 8 8 - - 0 7 , C h a p m a n a n d Hall, L o n d o n