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An enzyme immunoassay (EIA) for progesterone in horse plasma
Journal of Immunological Methods, 28 (1979) 211--217
211
© Elsevier/North-Holland Biomedical Press
AN ENZYME PLASMA
IMMUNOASSAY
(EIA) F O R P R O G E S T E R O N E
IN H O R S E
K. SEEGER, H. THUROW, W. HAEDE and E. KNAPP Hoechst AG, D-6230 Frankfurt 80, G.F.R.
(Received 30 November 1978, accepted 30 January 1979)
A simple enzyme immunoassay (EIA) for the measurement of progesterone is described. Antibody against ll-OH-hemisuccinate-BSA is bound to polystyrene tubes. l l-OH-hemisuccinyl-~-D-galactosidase is used as enzyme-coupled antigen and methylumbelliferyl-~-D-galactoside as substrate. Concentrations down to 0.156 ng/ml plasm or amounts of 93 pg/tube are detectable. Probit analysis gave a linear relationship between log concentration and percentage of binding. A comparison of EIA and radioimmunoassay gave a correlation coefficient of 0.81. The assay is sufficiently sensitive to estimate progesterone levels in plasma.
INTRODUCTION F o r studies o f r e p r o d u c t i o n in animals, m e a s u r e m e n t o f p r o g e s t e r o n e levels is o f t e n essential. If t h e s e l e c t i o n o f a n i m a l s for trials d e p e n d s u p o n t h e stage o f t h e r e p r o d u c t i v e c y c l e p l a s m a levels m u s t be o b t a i n e d q u i c k l y . E n z y m e i m m u n o a s s a y ( E I A ) , as d e s c r i b e d b y D r a y et al. ( 1 9 7 5 ) p r o v i d e s an a l t e r n a t i v e m e t h o d to r a d i o i m m u n o a s s a y ( R I A ) , w h e n r a d i o a c t i v e c o m p o u n d s have t o be e x c l u d e d f r o m l a b o r a t o r i e s a d j a c e n t t o t h e a n i m a l houses. T h e m e t h o d was slightly m o d i f i e d t o suit t h e e q u i p m e n t available. MATERIALS AND METHODS Proges teron e
l l ~ - O H - p r o g e s t e r o n e - h e m i s u c c i n a t e c o u p l e d t o ~-D-galactosidase (22.5 U / m l ) (PDG). R a b b i t a n t i s e r u m was raised against l l - ~ - O H - p r o g e s t e r o n e - h e m i s u c c i n a t e c o u p l e d to b o v i n e s e r u m a l b u m i n ( k i n d l y s u p p l i e d b y t h e P a s t e u r I n s t i t u t , Paris a n d t h e K i m r o n V e t e r i n a r y I n s t i t u t e , Beit Dagan). H o r s e s e r u m (HS) a n d b o v i n e s e r u m a l b u m i n (BSA) w e r e b o t h free f r o m p r o g e s t e r o n e . B u f f e r A was p r e p a r e d a c c o r d i n g to D r a y et al. ( 1 9 7 5 ) , a n d b u f f e r B a c c o r d i n g to C a t t ( 1 9 6 9 ) . 4-Methyl-umbelliferyl-/3-D-galactosid (MUG). f l - D - g a l a c t o p y r a n o s i d a s e f r o m E s c h e r i c h i a coil (EC 3 . 2 . 1 . 2 3 ) (Boehringer, Mannheim).
212
Coupling of 11-a-OH-progesterone to ~-D-galactosidase P r o g e s t e r o n e was c o u p l e d to /3-galactosidase ( f r o m E. coli) in a two-step reaction. In the first step 1 1 - h y d r o x y p r o g e s t e r o n e - h e m i s u c c i n a t e was activ a t e d by l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - c a r b o d i i m i d e (EDC, Merck). T h e activated c a r b o x y l groups were reacted with the amine groups of the e n z y m e in the second step. A solution of 11.0 /~mol Na l l - h y d r o x y p r o g e s t e r o n e - h e m i s u e c i n a t e and 1 0 0 0 # m o l EDC dissolved in 10 ml o f a m i x t u r e of d i m e t h y l s u l f o x i d e (DMSO) and w a t e r 2 : 1 (v/v) was stirred at r o o m t e m p e r a t u r e for 30 min. A pH of 4.7 was m a i n t a i n e d by t i t r a t i o n with 0.1 N HC1. 1 / 1 0 of the activated material was m i x e d at 4 ° C with 1 ml of a solution o f 5 mg ~-galactosidase in 0.1 M s o d i u m citrate, 2 mM MgC12, pH 5.5. A f t e r i n c u b a t i o n for 15 h at 4°C the m i x t u r e was dialysed against 0.1 M s o d i u m p h o s p h a t e , 2 m M MgC12, pH 7. T h e c r u d e material was c h r o m a t o g r a p h e d on a c o l u m n of S e p h a d e x G-200 which had p r e v i o u s l y been equilibrated with the same buffer. The fractions c o n t a i n i n g the e n z y m e activity were collected.
Principle o f the method C o n s t a n t a m o u n t s of a n t i b o d y against 1 1 - O H - p r o g e s t e r o n e - B S A were b o u n d to the inner surface o f p o l y s t y r e n e tubes (Catt and Tregear 1967). U n c o u p l e d p r o g e s t e r o n e c o m p e t e s for these p o l y s t y r e n e - b o u n d r e c e p t o r s with the p r o g e s t e r o n e - e n z y m e c o m p l e x which is present in k n o w n c o n s t a n t quantities. The a m o u n t of p r o g e s t e r o n e ~ e n z y m e c o m p l e x binding to the r e c e p t o r s is thus d e t e r m i n e d by the a m o u n t o f u n c o u p l e d p r o g e s t e r o n e present. The b o u n d p r o g e s t e r o n e - - e n z y m e c o m p l e x is evaluated by measuring the e n z y m e activity in the tube. A standard curve with k n o w n c o n c e n t r a t i o n s o f u n c o u p l e d p r o g e s t e r o n e is established. The c o n c e n t r a t i o n s in the test samples are calculated f r o m the s t a n d a r d curve.
Procedure 0.1 ml a n t i s e r u m (diluted 1 : 5000 in b u f f e r B) was a d d e d to each tube. Tubes t o c o n t r o l n o n s p e c i f i c binding were t r e a t e d with n o n - i m m u n e r a b b i t serum instead of antiserum. The tubes were r o t a t e d for 2 h at 37°C and 400 r e v . / m i n on a shaker, and their c o n t e n t s were t h e n p o u r e d off. The tubes were first washed with 1 ml 0.15 M NaC1 solution c o n t a i n i n g 0.5% progest e r o n e - f r e e BSA. A s e c o n d washing with 1 ml b u f f e r A per t u b e was perf o r m e d . T u b e s thus p r e p a r e d were k e p t at 4°C and used within u t m o s t 4 days. P r o g e s t e r o n e solutions o f k n o w n c o n c e n t r a t i o n s or test solutions were e x t r a c t e d a c c o r d i n g t o H o f f m a n n (1977). Standards and samples were e x t r a c t e d in triplicate. E x t r a c t s were redissolved in 0.02 ml PDG and 0.12 ml b u f f e r A (containing 20% p r o g e s t e r o n e - f r e e horse serum). T o t u b e s c o a t e d with a n t i b o d y a n d to t u b e s c o a t e d with n o r m a l rabbit serum, on PDG was added. These served as c o n t r o l s for 100% binding (Eo) and for n o n s p e c i f i c binding (Eu).
213 All tubes were kept for 4 h at 20°C at 400 rev./min on the shaker. The liquid was then discarded and the tubes washed three times with buffer A. 1 ml o f a solution of 0.3% ~-mercaptoethanol and 25 t~g MUG/ml in buffer A were then added to each tube. The tubes were incubated at 45°C for 2 h, and the reaction was stopped with 1 ml 1 M Na2CO~ solution. The intensity of fluorescence was measured (ha : 360 nm; XF: 455 nm). The a m o u n t of binding (B) was calculated from relative fluorescence intensities as follows: B-
Ex --EL, × 1001%1 Eo -- Eu
Eo: Binding o f PDG to a n t i b o d y (specific binding). E~,: Binding of PDG to normal rabbit serum (nonspeeific binding) Ex: Binding of PDG in c o m p e t i t i o n with non-enzyme-coupled progesterone in test material. The co n cen tr ati ons were read from a graph of probits of ( E x - - E u / Eo - - E u ) × 100 pl ot t e d against log c. RESULTS
Specificity Specific binding to ant i body coated tubes as com pared with nonspecific binding to normal serum-coated tubes was 100% versus 27.8-+ 4.72%. Binding of steroids o t h e r than progesterone in c o m p e t i t i o n for the a n t i b o d y was n o t tested. Dray et al. (1975) r e p o r t selective binding of progesterone by a n t ib o d y in radioimmunoassay.
Sensitivity The relative fluorescence intensity for 0.156 ng/ml progesterone and 0 ng/ ml were significantly different (P < 0.05). The lowest measurable concentration of progesterone was thus taken to be 0.156 ng/ml or 93 pg/tube.
Precision Figure 1 shows the regression for c o n c e n t r a t i o n of progesterone and e n zy matic activity. The probability for linearity is greater than 95%. The 95% confidence limits are sufficiently small. The coefficient of variation (s/m) varied between 0.02 and 0.185 (Table 1).
Reproducibility The regression of values obtained from 5 i n d e p e n d e n t trials p e r f o r m e d on different days (Fig. 2), was calculated and showed linearity. Table 2 gives the correlation coefficients of regression lines for different tests.
Comparison with RIA Figure 3 shows results obtained with RIA and EIA for progesterone con-
214 Standard -- curve (single assay)
B (Probits)
Correlation coefficient
R=~0,9926
I Confidence limits (95:;) T
X measured values
~'~"~'~--T
-)t calculated regressionline
t
ng Prog. ml
3 Y 0,156
0,313
0,625
1,25
2,5
5
10
20
Fig. 1. S t a n d a r d curve o f a single assay, s i m u l t a n e o u s l y run with the samples w h o s e values are d e m o n s t r a t e d in Fig. 3. Y = A + B ' l o g X ; (Y in p r o b i t s ) ; A = 5.110; B = - - 0 . 7 0 0 ; r = --0.9928.
TABLE 1 D I F F E R E N T C O N C E N T R A T I O N S OF P R O G E S T E R O N E A N D T H E I R R E S P E C T I V E M E A N S (m) AND S T A N D A R D D E V I A T I O N S (s) OF T R A N S M I S S I O N Conc. (ng/ml)
0 0 0 0.313 0.625 1.25 2.5 5.0 10.0 20.0
n (3)
Eo EU Ex
% Transmission
Coeff. var. (s)
(m)
(s)
102.3 24.7 81.3 76.3 74.0 67.0 59.3 50.7 47.0 39.6
4.9 1.2 15 5.7 3.5 3.6 1.2 2.9 7.6 5.5
0.048 0.049 0.185 0.075 0.047 0.054 0.02 0.057 0.162 0.139
215 TABLE 2 C O R R E L A T I O N C O E F F I C I E N T S O F SOME S E R I A L M E A S U R E M E N T S F o r all regression lines linearity was d e m o n s t r a t e d . Trial
Day
r
1 2 3 4 5 pooled
07.07 05.07 01.07 29.06 27,06 l to 5
-0.9870 -0.9928 -0.9882 -0.9942 -0,9612 --0.9214
centrations
during
the reproductive
cycle of a mare. The absolute
values are
not equal but similar. Linearity of the correlation of RIA results with results was shown. The correlation coefficient between the methods r = 0.8119.
EIA was
B
(Probits) Standard-curve
(five assays)
Correlation coefficient
R=-0,9214
T confidence limits (95 .) X measured values ~"
~ calculated regression-
~
line
l
:
X
X
x
t
Ix ng Prog. ml
,f/ 0,156
0,313
0,625
1.25
2.5
5
10
20
Fig. 2. Values o f 5 s t a n d a r d curves f r o m 5 d i f f e r e n t assays a n d days. Y = A + B • log X ; ( Y in p r o b i t s ) ; A = 5 . 4 2 ; B = - - 0 . 9 2 0 ; r = - - 0 . 9 2 1 4 .
216 ncJ. ml Proge ste rone
PROGESTERONE Mare
20
b.
23. Aug. - 6.0kt.
76 • RIA - values
j~a
to
s
• EIA -values
.:\
...-\ ...." -\ ,...\ -\ -\
///.;"'...
........
..
..,,,
t.
"~.
Estrus
~
//
.,: I 2
~~
//
;i":
Est rus
/.:
O
\ 4
i" ::
i
I:
1
"
,
] / .;"
'"~,
,. " ,
24
i ............/
30 ~2
3"e ~
Day of observation
40
43 45
Fig. 3. P r o g e s t e r o n e c o n c e n t r a t i o n s in mare plasma. C o m p a r i s o n o f RIA and EIA values. C o r r e l a t i o n is linear (P ~ 0.05); r = - - 0 . 8 1 1 9 .
DISCUSSION
One o f the m a j o r advantages o f the m e t h o d is t h a t no radioactive material is necessary. T h e e n z y m e - - p r o g e s t e r o n e c o m p l e x is stable unlike radioactive c o m p o u n d s which have half-lives. T h e use o f p o l y s t y r e n e as a solid carrier o f a n t i b o d y facilitates the separation of b o u n d and u n b o u n d p r o g e s t e r o n e e n z y m e , saving 5 p r o c e d u r a l steps c o m p a r e d with the second a n t i b o d y m e t h o d for separation. M o r e o v e r the use o f a refrigerated c e n t r i f u g e is n o t necessary. Sensitivity is less t h a n with the m e t h o d o f Dray et al. ( 1 9 7 5 ) b u t n o t p r o h i b i t i v e l y so. This is p r o b a b l y due to the use o f l l a - O H - p r o g e s t e r o n e - h e m i s u c c i n a t e - B S A as i m m u n i z i n g antigen and to the use of 11c~-OHprogesterone-hemisuccinate-~-D-galactosidase as c o m p e t i t o r to p r o g e s t e r o n e . A c c o r d i n g to Van W e e m e n and Schuurs ( 1 9 7 6 ) such a system shows a lack o f sensitivity c o m p a r e d with a s y s t e m where the coupling to BSA and the c o u p l i n g to the e n z y m e have been with d i f f e r e n t c o u p l i n g links owing to the high affinity o f the h e m i s u c c i n a t e b o n d in e n z y m e c o u p l e d antigen. Comp a r e d with the m e t h o d o f J o y c e et al. (1976), the sensitivity is a l m o s t identical. We c o n c l u d e t h a t the present m e t h o d is simpler and faster b u t s o m e w h a t less sensitive than the m e t h o d o f Dray et al. (1975).
217 ACKNOWLEDGEMENTS The help o f Dr. F, Dray and Dr. Claude Gros is gratefully a c k n o w l e d g e d . We also t h a n k Dr. S h e m e s h and Professor A y a l o n for the gift o f p r o g e s t e r o n e a n t i s e r u m . Dr. Seidel, H o e c h s t AG, k i n d l y m e a s u r e d p r o g e s t e r o n e by R I A . REFERENCES Catt, K., 1969, Acta Endocr. 63 (Suppl. 142), 222. Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Dray, F., J.-M. Andrieu and F. Renaud, 1975, Biochim. Biophys. Acta 403, 131. Hoffmann, B., 1977, Entwicklung yon Messverfahren und physiologische Daten (Springer, Berlin). Joyce, B.G., G. Read and D.R. Fahmi, 1976, J. Endoer. 71, 53P. Van Weemen, B.K. and A.H.W.M. Sehuurs, 1976, in: Immunoenzymatie Techniques, eds. Feldmann et al. (North-Holland, Amsterdam).
211
© Elsevier/North-Holland Biomedical Press
AN ENZYME PLASMA
IMMUNOASSAY
(EIA) F O R P R O G E S T E R O N E
IN H O R S E
K. SEEGER, H. THUROW, W. HAEDE and E. KNAPP Hoechst AG, D-6230 Frankfurt 80, G.F.R.
(Received 30 November 1978, accepted 30 January 1979)
A simple enzyme immunoassay (EIA) for the measurement of progesterone is described. Antibody against ll-OH-hemisuccinate-BSA is bound to polystyrene tubes. l l-OH-hemisuccinyl-~-D-galactosidase is used as enzyme-coupled antigen and methylumbelliferyl-~-D-galactoside as substrate. Concentrations down to 0.156 ng/ml plasm or amounts of 93 pg/tube are detectable. Probit analysis gave a linear relationship between log concentration and percentage of binding. A comparison of EIA and radioimmunoassay gave a correlation coefficient of 0.81. The assay is sufficiently sensitive to estimate progesterone levels in plasma.
INTRODUCTION F o r studies o f r e p r o d u c t i o n in animals, m e a s u r e m e n t o f p r o g e s t e r o n e levels is o f t e n essential. If t h e s e l e c t i o n o f a n i m a l s for trials d e p e n d s u p o n t h e stage o f t h e r e p r o d u c t i v e c y c l e p l a s m a levels m u s t be o b t a i n e d q u i c k l y . E n z y m e i m m u n o a s s a y ( E I A ) , as d e s c r i b e d b y D r a y et al. ( 1 9 7 5 ) p r o v i d e s an a l t e r n a t i v e m e t h o d to r a d i o i m m u n o a s s a y ( R I A ) , w h e n r a d i o a c t i v e c o m p o u n d s have t o be e x c l u d e d f r o m l a b o r a t o r i e s a d j a c e n t t o t h e a n i m a l houses. T h e m e t h o d was slightly m o d i f i e d t o suit t h e e q u i p m e n t available. MATERIALS AND METHODS Proges teron e
l l ~ - O H - p r o g e s t e r o n e - h e m i s u c c i n a t e c o u p l e d t o ~-D-galactosidase (22.5 U / m l ) (PDG). R a b b i t a n t i s e r u m was raised against l l - ~ - O H - p r o g e s t e r o n e - h e m i s u c c i n a t e c o u p l e d to b o v i n e s e r u m a l b u m i n ( k i n d l y s u p p l i e d b y t h e P a s t e u r I n s t i t u t , Paris a n d t h e K i m r o n V e t e r i n a r y I n s t i t u t e , Beit Dagan). H o r s e s e r u m (HS) a n d b o v i n e s e r u m a l b u m i n (BSA) w e r e b o t h free f r o m p r o g e s t e r o n e . B u f f e r A was p r e p a r e d a c c o r d i n g to D r a y et al. ( 1 9 7 5 ) , a n d b u f f e r B a c c o r d i n g to C a t t ( 1 9 6 9 ) . 4-Methyl-umbelliferyl-/3-D-galactosid (MUG). f l - D - g a l a c t o p y r a n o s i d a s e f r o m E s c h e r i c h i a coil (EC 3 . 2 . 1 . 2 3 ) (Boehringer, Mannheim).
212
Coupling of 11-a-OH-progesterone to ~-D-galactosidase P r o g e s t e r o n e was c o u p l e d to /3-galactosidase ( f r o m E. coli) in a two-step reaction. In the first step 1 1 - h y d r o x y p r o g e s t e r o n e - h e m i s u c c i n a t e was activ a t e d by l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - c a r b o d i i m i d e (EDC, Merck). T h e activated c a r b o x y l groups were reacted with the amine groups of the e n z y m e in the second step. A solution of 11.0 /~mol Na l l - h y d r o x y p r o g e s t e r o n e - h e m i s u e c i n a t e and 1 0 0 0 # m o l EDC dissolved in 10 ml o f a m i x t u r e of d i m e t h y l s u l f o x i d e (DMSO) and w a t e r 2 : 1 (v/v) was stirred at r o o m t e m p e r a t u r e for 30 min. A pH of 4.7 was m a i n t a i n e d by t i t r a t i o n with 0.1 N HC1. 1 / 1 0 of the activated material was m i x e d at 4 ° C with 1 ml of a solution o f 5 mg ~-galactosidase in 0.1 M s o d i u m citrate, 2 mM MgC12, pH 5.5. A f t e r i n c u b a t i o n for 15 h at 4°C the m i x t u r e was dialysed against 0.1 M s o d i u m p h o s p h a t e , 2 m M MgC12, pH 7. T h e c r u d e material was c h r o m a t o g r a p h e d on a c o l u m n of S e p h a d e x G-200 which had p r e v i o u s l y been equilibrated with the same buffer. The fractions c o n t a i n i n g the e n z y m e activity were collected.
Principle o f the method C o n s t a n t a m o u n t s of a n t i b o d y against 1 1 - O H - p r o g e s t e r o n e - B S A were b o u n d to the inner surface o f p o l y s t y r e n e tubes (Catt and Tregear 1967). U n c o u p l e d p r o g e s t e r o n e c o m p e t e s for these p o l y s t y r e n e - b o u n d r e c e p t o r s with the p r o g e s t e r o n e - e n z y m e c o m p l e x which is present in k n o w n c o n s t a n t quantities. The a m o u n t of p r o g e s t e r o n e ~ e n z y m e c o m p l e x binding to the r e c e p t o r s is thus d e t e r m i n e d by the a m o u n t o f u n c o u p l e d p r o g e s t e r o n e present. The b o u n d p r o g e s t e r o n e - - e n z y m e c o m p l e x is evaluated by measuring the e n z y m e activity in the tube. A standard curve with k n o w n c o n c e n t r a t i o n s o f u n c o u p l e d p r o g e s t e r o n e is established. The c o n c e n t r a t i o n s in the test samples are calculated f r o m the s t a n d a r d curve.
Procedure 0.1 ml a n t i s e r u m (diluted 1 : 5000 in b u f f e r B) was a d d e d to each tube. Tubes t o c o n t r o l n o n s p e c i f i c binding were t r e a t e d with n o n - i m m u n e r a b b i t serum instead of antiserum. The tubes were r o t a t e d for 2 h at 37°C and 400 r e v . / m i n on a shaker, and their c o n t e n t s were t h e n p o u r e d off. The tubes were first washed with 1 ml 0.15 M NaC1 solution c o n t a i n i n g 0.5% progest e r o n e - f r e e BSA. A s e c o n d washing with 1 ml b u f f e r A per t u b e was perf o r m e d . T u b e s thus p r e p a r e d were k e p t at 4°C and used within u t m o s t 4 days. P r o g e s t e r o n e solutions o f k n o w n c o n c e n t r a t i o n s or test solutions were e x t r a c t e d a c c o r d i n g t o H o f f m a n n (1977). Standards and samples were e x t r a c t e d in triplicate. E x t r a c t s were redissolved in 0.02 ml PDG and 0.12 ml b u f f e r A (containing 20% p r o g e s t e r o n e - f r e e horse serum). T o t u b e s c o a t e d with a n t i b o d y a n d to t u b e s c o a t e d with n o r m a l rabbit serum, on PDG was added. These served as c o n t r o l s for 100% binding (Eo) and for n o n s p e c i f i c binding (Eu).
213 All tubes were kept for 4 h at 20°C at 400 rev./min on the shaker. The liquid was then discarded and the tubes washed three times with buffer A. 1 ml o f a solution of 0.3% ~-mercaptoethanol and 25 t~g MUG/ml in buffer A were then added to each tube. The tubes were incubated at 45°C for 2 h, and the reaction was stopped with 1 ml 1 M Na2CO~ solution. The intensity of fluorescence was measured (ha : 360 nm; XF: 455 nm). The a m o u n t of binding (B) was calculated from relative fluorescence intensities as follows: B-
Ex --EL, × 1001%1 Eo -- Eu
Eo: Binding o f PDG to a n t i b o d y (specific binding). E~,: Binding of PDG to normal rabbit serum (nonspeeific binding) Ex: Binding of PDG in c o m p e t i t i o n with non-enzyme-coupled progesterone in test material. The co n cen tr ati ons were read from a graph of probits of ( E x - - E u / Eo - - E u ) × 100 pl ot t e d against log c. RESULTS
Specificity Specific binding to ant i body coated tubes as com pared with nonspecific binding to normal serum-coated tubes was 100% versus 27.8-+ 4.72%. Binding of steroids o t h e r than progesterone in c o m p e t i t i o n for the a n t i b o d y was n o t tested. Dray et al. (1975) r e p o r t selective binding of progesterone by a n t ib o d y in radioimmunoassay.
Sensitivity The relative fluorescence intensity for 0.156 ng/ml progesterone and 0 ng/ ml were significantly different (P < 0.05). The lowest measurable concentration of progesterone was thus taken to be 0.156 ng/ml or 93 pg/tube.
Precision Figure 1 shows the regression for c o n c e n t r a t i o n of progesterone and e n zy matic activity. The probability for linearity is greater than 95%. The 95% confidence limits are sufficiently small. The coefficient of variation (s/m) varied between 0.02 and 0.185 (Table 1).
Reproducibility The regression of values obtained from 5 i n d e p e n d e n t trials p e r f o r m e d on different days (Fig. 2), was calculated and showed linearity. Table 2 gives the correlation coefficients of regression lines for different tests.
Comparison with RIA Figure 3 shows results obtained with RIA and EIA for progesterone con-
214 Standard -- curve (single assay)
B (Probits)
Correlation coefficient
R=~0,9926
I Confidence limits (95:;) T
X measured values
~'~"~'~--T
-)t calculated regressionline
t
ng Prog. ml
3 Y 0,156
0,313
0,625
1,25
2,5
5
10
20
Fig. 1. S t a n d a r d curve o f a single assay, s i m u l t a n e o u s l y run with the samples w h o s e values are d e m o n s t r a t e d in Fig. 3. Y = A + B ' l o g X ; (Y in p r o b i t s ) ; A = 5.110; B = - - 0 . 7 0 0 ; r = --0.9928.
TABLE 1 D I F F E R E N T C O N C E N T R A T I O N S OF P R O G E S T E R O N E A N D T H E I R R E S P E C T I V E M E A N S (m) AND S T A N D A R D D E V I A T I O N S (s) OF T R A N S M I S S I O N Conc. (ng/ml)
0 0 0 0.313 0.625 1.25 2.5 5.0 10.0 20.0
n (3)
Eo EU Ex
% Transmission
Coeff. var. (s)
(m)
(s)
102.3 24.7 81.3 76.3 74.0 67.0 59.3 50.7 47.0 39.6
4.9 1.2 15 5.7 3.5 3.6 1.2 2.9 7.6 5.5
0.048 0.049 0.185 0.075 0.047 0.054 0.02 0.057 0.162 0.139
215 TABLE 2 C O R R E L A T I O N C O E F F I C I E N T S O F SOME S E R I A L M E A S U R E M E N T S F o r all regression lines linearity was d e m o n s t r a t e d . Trial
Day
r
1 2 3 4 5 pooled
07.07 05.07 01.07 29.06 27,06 l to 5
-0.9870 -0.9928 -0.9882 -0.9942 -0,9612 --0.9214
centrations
during
the reproductive
cycle of a mare. The absolute
values are
not equal but similar. Linearity of the correlation of RIA results with results was shown. The correlation coefficient between the methods r = 0.8119.
EIA was
B
(Probits) Standard-curve
(five assays)
Correlation coefficient
R=-0,9214
T confidence limits (95 .) X measured values ~"
~ calculated regression-
~
line
l
:
X
X
x
t
Ix ng Prog. ml
,f/ 0,156
0,313
0,625
1.25
2.5
5
10
20
Fig. 2. Values o f 5 s t a n d a r d curves f r o m 5 d i f f e r e n t assays a n d days. Y = A + B • log X ; ( Y in p r o b i t s ) ; A = 5 . 4 2 ; B = - - 0 . 9 2 0 ; r = - - 0 . 9 2 1 4 .
216 ncJ. ml Proge ste rone
PROGESTERONE Mare
20
b.
23. Aug. - 6.0kt.
76 • RIA - values
j~a
to
s
• EIA -values
.:\
...-\ ...." -\ ,...\ -\ -\
///.;"'...
........
..
..,,,
t.
"~.
Estrus
~
//
.,: I 2
~~
//
;i":
Est rus
/.:
O
\ 4
i" ::
i
I:
1
"
,
] / .;"
'"~,
,. " ,
24
i ............/
30 ~2
3"e ~
Day of observation
40
43 45
Fig. 3. P r o g e s t e r o n e c o n c e n t r a t i o n s in mare plasma. C o m p a r i s o n o f RIA and EIA values. C o r r e l a t i o n is linear (P ~ 0.05); r = - - 0 . 8 1 1 9 .
DISCUSSION
One o f the m a j o r advantages o f the m e t h o d is t h a t no radioactive material is necessary. T h e e n z y m e - - p r o g e s t e r o n e c o m p l e x is stable unlike radioactive c o m p o u n d s which have half-lives. T h e use o f p o l y s t y r e n e as a solid carrier o f a n t i b o d y facilitates the separation of b o u n d and u n b o u n d p r o g e s t e r o n e e n z y m e , saving 5 p r o c e d u r a l steps c o m p a r e d with the second a n t i b o d y m e t h o d for separation. M o r e o v e r the use o f a refrigerated c e n t r i f u g e is n o t necessary. Sensitivity is less t h a n with the m e t h o d o f Dray et al. ( 1 9 7 5 ) b u t n o t p r o h i b i t i v e l y so. This is p r o b a b l y due to the use o f l l a - O H - p r o g e s t e r o n e - h e m i s u c c i n a t e - B S A as i m m u n i z i n g antigen and to the use of 11c~-OHprogesterone-hemisuccinate-~-D-galactosidase as c o m p e t i t o r to p r o g e s t e r o n e . A c c o r d i n g to Van W e e m e n and Schuurs ( 1 9 7 6 ) such a system shows a lack o f sensitivity c o m p a r e d with a s y s t e m where the coupling to BSA and the c o u p l i n g to the e n z y m e have been with d i f f e r e n t c o u p l i n g links owing to the high affinity o f the h e m i s u c c i n a t e b o n d in e n z y m e c o u p l e d antigen. Comp a r e d with the m e t h o d o f J o y c e et al. (1976), the sensitivity is a l m o s t identical. We c o n c l u d e t h a t the present m e t h o d is simpler and faster b u t s o m e w h a t less sensitive than the m e t h o d o f Dray et al. (1975).
217 ACKNOWLEDGEMENTS The help o f Dr. F, Dray and Dr. Claude Gros is gratefully a c k n o w l e d g e d . We also t h a n k Dr. S h e m e s h and Professor A y a l o n for the gift o f p r o g e s t e r o n e a n t i s e r u m . Dr. Seidel, H o e c h s t AG, k i n d l y m e a s u r e d p r o g e s t e r o n e by R I A . REFERENCES Catt, K., 1969, Acta Endocr. 63 (Suppl. 142), 222. Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Dray, F., J.-M. Andrieu and F. Renaud, 1975, Biochim. Biophys. Acta 403, 131. Hoffmann, B., 1977, Entwicklung yon Messverfahren und physiologische Daten (Springer, Berlin). Joyce, B.G., G. Read and D.R. Fahmi, 1976, J. Endoer. 71, 53P. Van Weemen, B.K. and A.H.W.M. Sehuurs, 1976, in: Immunoenzymatie Techniques, eds. Feldmann et al. (North-Holland, Amsterdam).