Journal of Immunological Methods, 10 (1976) 161--170
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
AN I M M U N O - E N Z Y M A T I C A S S A Y O F C O R T I S O L U S I N G E. C O L I f l - G A L A C T O S I D A S E AS L A B E L
SERGIO COMOGLIO and FRANCO CELADA Laboratorio di Fisiologia Clinica C.N.R. Pisa and Laboratorio di Biologia Cellulare C.N.R. Roma, Italy
(Received 29 July 1975, accepted 4 September 1975)
An enzyme-assay of cortisol was established by using E. coli ~-galactosidase as a marker. The number of cortisol residues per molecule of enzyme and the method of separation of free from antibody-bound enzyme proved to be critical factors for the assessment of the assay. A sensitivity ranging from 100 to 150 pg of cortisol per tube has been achieved. A comparison with other methods, i.e. competitive protein binding assay, radioimmunoassay and fluorimetry, is reported.
INTRODUCTION I m m u n o a s s a y p r o c e d u r e s , e m p l o y i n g an e n z y m e as a m a r k e r f o r i m m u n o c h e m i c a l l y reactive c o m p o u n d s have b e e n r e p o r t e d b y several l a b o r a t o r i e s (van W e e m e n e t al., 1 9 7 1 , 1 9 7 2 ; Engvall et al., 1 9 7 1 , 1 9 7 2 ) . In particular, some e x p e r i m e n t a l evidence has b e e n given t h a t , if a c o n v e n i e n t e n z y m e is used and if t h e s u b s t a n c e t o be m e a s u r e d is o f low m o l e c u l a r w e i g h t (van W e e m e n , 1 9 7 4 ) , such m e t h o d s m a y possibly be as sensitive as r a d i o i m m u n o assay. In this p a p e r we describe a p r o c e d u r e , which is designed f o r t h e assay o f cortisol b y using as a m a r k e r E. coli fi-galactosidase. T h e reasons f o r c h o o s i n g 13-galactosidase are t h e high rate o f t u r n o v e r ( R o t m a n , 1 9 6 1 ) ; t h e relatively high n u m b e r o f a m i n o g r o u p s available f o r c o u p l i n g r e a c t i o n s and the stability b o t h o n storage ( a b o u t 1 y e a r at 4°C) and in various e x p e r i m e n t a l c o n d i t i o n s ( a b o u t 20 h at 37°C). O n the o t h e r h a n d , cortisol was c h o s e n as a m o d e l o f a small m o l e c u l e w h i c h can be q u a n t i t a t e d b y several m e t h o d s like f l u o r i m e t r y (Mattingly, 1 9 6 2 ) , c o m p e t i t i v e p r o t e i n binding assay ( K o l a n o w s k i et al., 1 9 6 9 ) , radioimm u n o a s s a y ( R u d e r e t al., 1 9 7 2 ; Rolleri et al., 1 9 7 6 ) , t h u s allowing a c o m p a r ison o f results t o be m a d e .
162 MATERIALS AND METHODS
Chemicals O-nitrophenyl-~-D-galactopyranoside (ONPG) and cortisol were purchased from Merck. 6-bromo-2-naphthyl-~-D-galactopyranoside was from Sigma. 1,2[ 3 H] cortisol (40 mCi/mmole) was supplied by CEA-IRE-SORIN. All other chemicals used were of analytical grade.
Diluents The assay buffer contained 10 mM Tris (Hydroxymethyl)-amino methane, 10 mM MgC12, 0.1 M NaC1 and its pH was adjusted to 7.2 with acetic acid (Tris buffer).
Elec troph oresis Polyacrylamide gel electrophoresis (5% gel) was for 2 h at 3.5 ma per tube. Gels were stained for enzymatic activity and protein with 6-bromo-2naphthyl-~-D-galactopyranoside and amido schwarz respectively (Steers et al., 1971).
Preparation o f [3 H] cortisol-21-hemisuccinate This derivative was obtained by reaction for 16 h at 40°C of succinic anhydride (414 mg) with cortisol (500 mg) in anhydrous pyridine (6 ml) after addition of 2 mCi of 1,2[ 3 H] cortisol. The product obtained after crystallization from ethyl acetate had a melting point 168.9°C and was homogeneous on silicagel thin layer chromatography.
A n tisera Antiserum to cortisol-21-hemisuccinate bovine serum albumin conjugate was raised in rabbits according to the method proposed by Ruder (1972). Anti rabbit 7-globulin serum (a gift of Dr. A.M. Isetta, Farmitalia, Milano) was obtained b y injecting a sheep twice with 40 mg purified rabbit IgG in Freund's complete adjuvant, intramuscularly. The m e t h o d used to isolate rabbit IgG was DEAE-cellulose chromatography after precipitation by Na2 SO4. The antiserum obtained 30 days after the second injection was used to separate b o u n d from free antigen.
Immunosorbent ~/-globulin fractions of sheep anti rabbit serum, obtained by precipitation with solid Na2 SO4, were coupled to microcrystalline cellulose activated
163 with CNBr (Axen, 1967; Wide, 1970). T w e n t y mg of 7-globulins in 20 ml of 0.1 M NaHCO3 were incubated for 16 h at room temperature under stirring with 3 g activated cellulose.
[J-galactosidase The enzyme was extracted and purified from E. coli, strain 3300, as described elsewhere (Celada et al., 1971). The specific activity of the preparation used was a b o u t 400,000 EU/mg. Hydrolysis of ONPG was used to measure enzymatic activity. One international unit is defined as the a m o u n t of enzyme that digests 10 -9 moles of substrate per minute. The EU of a given sample was calculated by utilizing the molar extinction coefficient of O-nitrophenate (4700) at 420 mp and the dilution factor. Usually the reaction was carried o u t at 37°C in a volume of 2 ml of 3 mM ONPG and stopped by rapid addition of 0.5 ml of 1.2 M Na2 CO3. Since no significant loss of activity occurs when the enzyme is in solid phase, measurements could be performed in immunoprecipitates.
Preparation of cortisol-[J-galactosidase conjugates The cortisol derivative was coupled to the amino groups of the enzyme with a mixed anhydride reaction, according to Erlanger (1959). A representative procedure is as follows: 9.24 mg of [3 H] cortisol-21hemisuccinate and 3.6 mg tri-n-butyl amine were dissolved in 0.4 ml of dioxan and cooled to 10°C. Iso-butyl chloroformate (2.8 mg) was added and the mixture was stirred for 30 min at 10°C. The organic solution was then added to 5.8 mg fl-galactosidase, dissolved in 2 ml distilled water. The pH of the latter solution had been previously brought to 9.4 with 1 M NaOH. The reaction was allowed to proceed for 4 h at 10°C and 30 min at 23°C while keeping the pH at 9 with 1 M NaOH. After dialysis against Tris buffer with several changes of buffer, the conjugate was adsorbed to a column of DEAE-Sephadex A-50 equilibrated with Tris buffer and eluted with a linear NaC1 gradient (Craven et al., 1965). The fractions showing specific activity greater than 300,000 EU/mg were pooled, dialyzed against Tris buffer and stored at 4°C in the presence of 2%o NaN3. The other conjugates were obtained following the same procedure by varying the a m o u n t of the cortisol derivative with respect to the enzyme. The number of cortisol residues per molecule of enzyme was estimated by measuring the radioactivity of aliquots of conjugate solution, dissolved in a scintillation liquid.
Assay conditions Two m e t h o d s of separation of free from antibody-bound tracer were used. A) Double antibody (DA method): to 2.5 ml plastic tubes containing 0.1
164
ml Tris buffer or 0.1 ml of free cortisol in Tris buffer was added 0.1 ml of a dilution of cortisol antiserum in normal rabbit serum 1 : 40 in Tris buffer (NRS). After 30 min at room temperature, 0.1 ml of cortisol-fi-galactosidase conjugate in NRS was added and allowed to react at 4°C for 4 h. At the end of the incubation, 0.2 ml of sheep anti-rabbit serum diluted 1 : 5 in Tris buffer was added and the reaction mixture was left overnight at 4°C. After centrifugation at 4000 rpm for 10 min the pellets were washed 3 times and assayed for enzymatic activity in 2 ml ONPG at 37°C as described above. B) Double antibody solid phase (DASP method): the procedure is similar to that described in (A), except that the Tris buffer contained 2% bovine serum albumin instead of NRS. To the reaction medium (total volume 0.5 ml) was added, after the described incubation, 0.5 ml of a suspension of the immunosorbent (about 16 mg/ml cellulose). After stirring overnight at 4°C and centrifuging at 4000 rpm for 5 min, 0.1 ml of the supernatant was assayed for enzymatic activity. For the measurements of plasma cortisol, standard plasma volumes were extracted with dichloromethane (e.g. 0.1 ml of plasma was extracted with 2.5 ml of solvent by shaking for 60 sec on a Vortex mixer and 0.5 aliquots of the extracts were evaporated to dryness with a nitrogen stream.
Radioimmunoassay (RIA ), competitive protein binding assay (CPB) and fluorimetric assay of cortisol The RIA of cortisol was set up using [3 H] cortisol as tracer and following the m e t h o d of Rolleri et al. (1976). The CPB assay was performed with the same tracer as described by Kolanowski and Pizzarro (1969), and the fluorimetric test as indicated by Mattingly (1962). RESULTS
Enzymatic characteristics o f conjugates The properties of the enzymatic tracers after the coupling procedure described above were compared with those of native enzyme. The resulting data listed in table 1 indicate that the coupling reactions do not affect substantially the properties of fi-galactosidase. The superimposable electrophoretic patterns obtained with native and conjugated enzyme on polyacrylamide gel, stained for protein and enzymatic activity, further confirm the absence of structural modifications.
Substitution degrees and maximal binding ability As table 2 shows, different substitution degrees (s.d.) were obtained (moles cortisol/mole ~-galactosidase) by varying the molar ratios of cortisol21-hemisuccinate for conjugation.
165 TABLE 1 Effects o f c o n j u g a t i o n o n t h e e n z y m a t i c p r o p e r t i e s .
/3-galactosidase Cortisol-~galactosidase ***
Ks *
Vma x **
E n z y m a t i c activity r e c o v e r e d (%)
2 . 4 " 10 -4 2 . 2 ( + 0 . 1 5 ) 1 0 -4
44 • 10 -3 4 0 . 5 ( + 3 . 5 ) 1 0 -3
100 80(+10)
* K s in moles/1. ** Vmax in pm/ml/min/standard c o n c e n t r a t i o n o f e n z y m e (0.3 p g / t u b e ) a t 3 7 ° C in O N P G . *** R e s u l t s o b t a i n e d f o r 4 d i f f e r e n t p r e p a r a t i o n s w i t h s u b s t i t u t i o n degrees ranging f r o m 2 t o 10. In p a r e n t h e s e s t h e r a n g e o f v a r i a t i o n .
Fig. 1 shows the binding curves, obtained for a fixed a m o u n t of enzymeb o u n d cortisol (at 4 different degrees of substitution) b y diluting the antiserum. It is apparent that the binding ability depends on the substitution degree; e.g. only with the higher substituted conjugate it was possible to obtain quantitative binding in antibody excess. For setting up the EIA m e t h o d enzymatic tracers with a maximal binding ability greater than 40% were used.
Inhibition of binding by free cortisol Binding inhibition has been studied in relation to s.d. and to the method of separation of free from antibody-bound tracer (B/F). Three representative dose--response curves are shown in fig. 2, as obtained by using the conjugate at a s.d. of 10 and 4. F o r the upper curve the separation B/F has been performed with the DASP m e t h o d , while the lower one was obtained with the DA procedure. It can be seen that (lower curves) irrespective of the s.d. practically the same slope resulted, b u t a lower antiserum titer was required in the case of s.d. = 4 to maintain the initial binding ability of a b o u t 40%. TABLE 2 E f f e c t of t h e c o n c e n t r a t i o n o f cortisol derivative o n t h e c o u p l i n g yield. Mass r a t i o *
S u b s t i t u t i o n degree **
1 5 20 100
2 4 6.5 10
* Moles cortisol d e r i v a t i v e / m o l e a m i n o g r o u p s ( a b o u t 1 2 0 a m i n o g r o u p s are available per m o l e ~-galactosidase). ** Moles c o r t i s o l / m o l e ~-galactosidase.
166
A
0--"-0--"-0"-~ O~ 0
o
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
AN I M M U N O - E N Z Y M A T I C A S S A Y O F C O R T I S O L U S I N G E. C O L I f l - G A L A C T O S I D A S E AS L A B E L
SERGIO COMOGLIO and FRANCO CELADA Laboratorio di Fisiologia Clinica C.N.R. Pisa and Laboratorio di Biologia Cellulare C.N.R. Roma, Italy
(Received 29 July 1975, accepted 4 September 1975)
An enzyme-assay of cortisol was established by using E. coli ~-galactosidase as a marker. The number of cortisol residues per molecule of enzyme and the method of separation of free from antibody-bound enzyme proved to be critical factors for the assessment of the assay. A sensitivity ranging from 100 to 150 pg of cortisol per tube has been achieved. A comparison with other methods, i.e. competitive protein binding assay, radioimmunoassay and fluorimetry, is reported.
INTRODUCTION I m m u n o a s s a y p r o c e d u r e s , e m p l o y i n g an e n z y m e as a m a r k e r f o r i m m u n o c h e m i c a l l y reactive c o m p o u n d s have b e e n r e p o r t e d b y several l a b o r a t o r i e s (van W e e m e n e t al., 1 9 7 1 , 1 9 7 2 ; Engvall et al., 1 9 7 1 , 1 9 7 2 ) . In particular, some e x p e r i m e n t a l evidence has b e e n given t h a t , if a c o n v e n i e n t e n z y m e is used and if t h e s u b s t a n c e t o be m e a s u r e d is o f low m o l e c u l a r w e i g h t (van W e e m e n , 1 9 7 4 ) , such m e t h o d s m a y possibly be as sensitive as r a d i o i m m u n o assay. In this p a p e r we describe a p r o c e d u r e , which is designed f o r t h e assay o f cortisol b y using as a m a r k e r E. coli fi-galactosidase. T h e reasons f o r c h o o s i n g 13-galactosidase are t h e high rate o f t u r n o v e r ( R o t m a n , 1 9 6 1 ) ; t h e relatively high n u m b e r o f a m i n o g r o u p s available f o r c o u p l i n g r e a c t i o n s and the stability b o t h o n storage ( a b o u t 1 y e a r at 4°C) and in various e x p e r i m e n t a l c o n d i t i o n s ( a b o u t 20 h at 37°C). O n the o t h e r h a n d , cortisol was c h o s e n as a m o d e l o f a small m o l e c u l e w h i c h can be q u a n t i t a t e d b y several m e t h o d s like f l u o r i m e t r y (Mattingly, 1 9 6 2 ) , c o m p e t i t i v e p r o t e i n binding assay ( K o l a n o w s k i et al., 1 9 6 9 ) , radioimm u n o a s s a y ( R u d e r e t al., 1 9 7 2 ; Rolleri et al., 1 9 7 6 ) , t h u s allowing a c o m p a r ison o f results t o be m a d e .
162 MATERIALS AND METHODS
Chemicals O-nitrophenyl-~-D-galactopyranoside (ONPG) and cortisol were purchased from Merck. 6-bromo-2-naphthyl-~-D-galactopyranoside was from Sigma. 1,2[ 3 H] cortisol (40 mCi/mmole) was supplied by CEA-IRE-SORIN. All other chemicals used were of analytical grade.
Diluents The assay buffer contained 10 mM Tris (Hydroxymethyl)-amino methane, 10 mM MgC12, 0.1 M NaC1 and its pH was adjusted to 7.2 with acetic acid (Tris buffer).
Elec troph oresis Polyacrylamide gel electrophoresis (5% gel) was for 2 h at 3.5 ma per tube. Gels were stained for enzymatic activity and protein with 6-bromo-2naphthyl-~-D-galactopyranoside and amido schwarz respectively (Steers et al., 1971).
Preparation o f [3 H] cortisol-21-hemisuccinate This derivative was obtained by reaction for 16 h at 40°C of succinic anhydride (414 mg) with cortisol (500 mg) in anhydrous pyridine (6 ml) after addition of 2 mCi of 1,2[ 3 H] cortisol. The product obtained after crystallization from ethyl acetate had a melting point 168.9°C and was homogeneous on silicagel thin layer chromatography.
A n tisera Antiserum to cortisol-21-hemisuccinate bovine serum albumin conjugate was raised in rabbits according to the method proposed by Ruder (1972). Anti rabbit 7-globulin serum (a gift of Dr. A.M. Isetta, Farmitalia, Milano) was obtained b y injecting a sheep twice with 40 mg purified rabbit IgG in Freund's complete adjuvant, intramuscularly. The m e t h o d used to isolate rabbit IgG was DEAE-cellulose chromatography after precipitation by Na2 SO4. The antiserum obtained 30 days after the second injection was used to separate b o u n d from free antigen.
Immunosorbent ~/-globulin fractions of sheep anti rabbit serum, obtained by precipitation with solid Na2 SO4, were coupled to microcrystalline cellulose activated
163 with CNBr (Axen, 1967; Wide, 1970). T w e n t y mg of 7-globulins in 20 ml of 0.1 M NaHCO3 were incubated for 16 h at room temperature under stirring with 3 g activated cellulose.
[J-galactosidase The enzyme was extracted and purified from E. coli, strain 3300, as described elsewhere (Celada et al., 1971). The specific activity of the preparation used was a b o u t 400,000 EU/mg. Hydrolysis of ONPG was used to measure enzymatic activity. One international unit is defined as the a m o u n t of enzyme that digests 10 -9 moles of substrate per minute. The EU of a given sample was calculated by utilizing the molar extinction coefficient of O-nitrophenate (4700) at 420 mp and the dilution factor. Usually the reaction was carried o u t at 37°C in a volume of 2 ml of 3 mM ONPG and stopped by rapid addition of 0.5 ml of 1.2 M Na2 CO3. Since no significant loss of activity occurs when the enzyme is in solid phase, measurements could be performed in immunoprecipitates.
Preparation of cortisol-[J-galactosidase conjugates The cortisol derivative was coupled to the amino groups of the enzyme with a mixed anhydride reaction, according to Erlanger (1959). A representative procedure is as follows: 9.24 mg of [3 H] cortisol-21hemisuccinate and 3.6 mg tri-n-butyl amine were dissolved in 0.4 ml of dioxan and cooled to 10°C. Iso-butyl chloroformate (2.8 mg) was added and the mixture was stirred for 30 min at 10°C. The organic solution was then added to 5.8 mg fl-galactosidase, dissolved in 2 ml distilled water. The pH of the latter solution had been previously brought to 9.4 with 1 M NaOH. The reaction was allowed to proceed for 4 h at 10°C and 30 min at 23°C while keeping the pH at 9 with 1 M NaOH. After dialysis against Tris buffer with several changes of buffer, the conjugate was adsorbed to a column of DEAE-Sephadex A-50 equilibrated with Tris buffer and eluted with a linear NaC1 gradient (Craven et al., 1965). The fractions showing specific activity greater than 300,000 EU/mg were pooled, dialyzed against Tris buffer and stored at 4°C in the presence of 2%o NaN3. The other conjugates were obtained following the same procedure by varying the a m o u n t of the cortisol derivative with respect to the enzyme. The number of cortisol residues per molecule of enzyme was estimated by measuring the radioactivity of aliquots of conjugate solution, dissolved in a scintillation liquid.
Assay conditions Two m e t h o d s of separation of free from antibody-bound tracer were used. A) Double antibody (DA method): to 2.5 ml plastic tubes containing 0.1
164
ml Tris buffer or 0.1 ml of free cortisol in Tris buffer was added 0.1 ml of a dilution of cortisol antiserum in normal rabbit serum 1 : 40 in Tris buffer (NRS). After 30 min at room temperature, 0.1 ml of cortisol-fi-galactosidase conjugate in NRS was added and allowed to react at 4°C for 4 h. At the end of the incubation, 0.2 ml of sheep anti-rabbit serum diluted 1 : 5 in Tris buffer was added and the reaction mixture was left overnight at 4°C. After centrifugation at 4000 rpm for 10 min the pellets were washed 3 times and assayed for enzymatic activity in 2 ml ONPG at 37°C as described above. B) Double antibody solid phase (DASP method): the procedure is similar to that described in (A), except that the Tris buffer contained 2% bovine serum albumin instead of NRS. To the reaction medium (total volume 0.5 ml) was added, after the described incubation, 0.5 ml of a suspension of the immunosorbent (about 16 mg/ml cellulose). After stirring overnight at 4°C and centrifuging at 4000 rpm for 5 min, 0.1 ml of the supernatant was assayed for enzymatic activity. For the measurements of plasma cortisol, standard plasma volumes were extracted with dichloromethane (e.g. 0.1 ml of plasma was extracted with 2.5 ml of solvent by shaking for 60 sec on a Vortex mixer and 0.5 aliquots of the extracts were evaporated to dryness with a nitrogen stream.
Radioimmunoassay (RIA ), competitive protein binding assay (CPB) and fluorimetric assay of cortisol The RIA of cortisol was set up using [3 H] cortisol as tracer and following the m e t h o d of Rolleri et al. (1976). The CPB assay was performed with the same tracer as described by Kolanowski and Pizzarro (1969), and the fluorimetric test as indicated by Mattingly (1962). RESULTS
Enzymatic characteristics o f conjugates The properties of the enzymatic tracers after the coupling procedure described above were compared with those of native enzyme. The resulting data listed in table 1 indicate that the coupling reactions do not affect substantially the properties of fi-galactosidase. The superimposable electrophoretic patterns obtained with native and conjugated enzyme on polyacrylamide gel, stained for protein and enzymatic activity, further confirm the absence of structural modifications.
Substitution degrees and maximal binding ability As table 2 shows, different substitution degrees (s.d.) were obtained (moles cortisol/mole ~-galactosidase) by varying the molar ratios of cortisol21-hemisuccinate for conjugation.
165 TABLE 1 Effects o f c o n j u g a t i o n o n t h e e n z y m a t i c p r o p e r t i e s .
/3-galactosidase Cortisol-~galactosidase ***
Ks *
Vma x **
E n z y m a t i c activity r e c o v e r e d (%)
2 . 4 " 10 -4 2 . 2 ( + 0 . 1 5 ) 1 0 -4
44 • 10 -3 4 0 . 5 ( + 3 . 5 ) 1 0 -3
100 80(+10)
* K s in moles/1. ** Vmax in pm/ml/min/standard c o n c e n t r a t i o n o f e n z y m e (0.3 p g / t u b e ) a t 3 7 ° C in O N P G . *** R e s u l t s o b t a i n e d f o r 4 d i f f e r e n t p r e p a r a t i o n s w i t h s u b s t i t u t i o n degrees ranging f r o m 2 t o 10. In p a r e n t h e s e s t h e r a n g e o f v a r i a t i o n .
Fig. 1 shows the binding curves, obtained for a fixed a m o u n t of enzymeb o u n d cortisol (at 4 different degrees of substitution) b y diluting the antiserum. It is apparent that the binding ability depends on the substitution degree; e.g. only with the higher substituted conjugate it was possible to obtain quantitative binding in antibody excess. For setting up the EIA m e t h o d enzymatic tracers with a maximal binding ability greater than 40% were used.
Inhibition of binding by free cortisol Binding inhibition has been studied in relation to s.d. and to the method of separation of free from antibody-bound tracer (B/F). Three representative dose--response curves are shown in fig. 2, as obtained by using the conjugate at a s.d. of 10 and 4. F o r the upper curve the separation B/F has been performed with the DASP m e t h o d , while the lower one was obtained with the DA procedure. It can be seen that (lower curves) irrespective of the s.d. practically the same slope resulted, b u t a lower antiserum titer was required in the case of s.d. = 4 to maintain the initial binding ability of a b o u t 40%. TABLE 2 E f f e c t of t h e c o n c e n t r a t i o n o f cortisol derivative o n t h e c o u p l i n g yield. Mass r a t i o *
S u b s t i t u t i o n degree **
1 5 20 100
2 4 6.5 10
* Moles cortisol d e r i v a t i v e / m o l e a m i n o g r o u p s ( a b o u t 1 2 0 a m i n o g r o u p s are available per m o l e ~-galactosidase). ** Moles c o r t i s o l / m o l e ~-galactosidase.
166
A
0--"-0--"-0"-~ O~ 0
o
