Journal oflmmunological Methods, 28 (1979) 51--57 © Elsevier/North-Holland Biomedical Press
51
D E T E R M I N A T I O N O F P H E N O B A R B I T A L BY R A D I O I M M U N O A S S A Y
A. YAMAOKA and T. TAKATORI 1
Department of Legal Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan (Received 13 December 1978, accepted 26 December 1978) A radioimmunoassay for phenobarbital has been studied. Antiphenobarbital antisera were obtained by repeated immunization of rabbits with p-succinamidophenobarbital conjugated to bovine serum albumin. Less than 0.2 pmol of phenobarbital could be measured by this procedure. The specificity of the antibodies was directed to substituents on the nitrogen atoms of the barbituric ring as well as to substituents at the carbon 5-position of the ring. INTRODUCTION P h e n o b a r b i t a l has b e e n w i d e l y u s e d as a sedative a n d h y p n o t i c . F o r clinical a n d forensic p u r p o s e s , a m e t h o d is f r e q u e n t l y r e q u i r e d t o d e t e r m i n e t r a c e a m o u n t s o f p h e n o b a r b i t a l in biological materials. A l t h o u g h a f e w c h r o m a t o graphic d e t e r m i n a t i o n s o f p h e n o b a r b i t a l h a v e b e e n r e c e n t l y d e s c r i b e d ( K u m p s and Mardens, 1 9 7 5 ; T o u c h s t o n e et al., 1 9 7 7 ; B o w m a n et al., 1 9 7 8 ) , r a d i o i m m u n o a s s a y is a m o r e sensitive and reliable m e t h o d f o r the d e t e r m i n a tion of drug haptens. T h e r a d i o i m m u n o a s s a y f o r b a r b i t u r a t e s has b e e n r e p o r t e d b y S p e c t o r a n d F l y n n ( 1 9 7 1 ) and F l y n n a n d S p e c t o r ( 1 9 7 2 ) , a n d C h u n g et al. ( 1 9 7 3 ) a n d S a t o h et al. ( 1 9 7 4 ) d e s c r i b e d t h e r a d i o i m m u n o a s s a y f o r p h e n o b a r b i t a l . In t h e p r e s e n t p a p e r we d e s c r i b e t h e use o f p - s u c c i n a m i d o p h e n o b a r b i t a l conjug a t e d to b o v i n e s e r u m a l b u m i n (BSA), a d i f f e r e n t derivative f r o m t h e i m m u n o g e n i c c o n j u g a t e s d e s c r i b e d p r e v i o u s l y , ip t h e p r o d u c t i o n o f p h e n o b a r b i t a l specific a n t i b o d i e s a n d t h e r a d i o i m m u n o a s s a y p r o c e d u r e . MATERIALS AND METHODS
Preparation o f the imrnunogen Fig. 1 illustrates t h e m e t h o d o f s y n t h e s i s o f p - s u c c i n a m i d o p h e n o b a r b i t a l conjugated to bovine serum albumin, p-Nitrophenobarbital, m.p. 216-220°C, was p r e p a r e d b y t h e m e t h o d s o f B o u s q u e t a n d A d a m s ( 1 9 3 0 ) a n d Butler ( 1 9 5 6 ) , s u s p e n d e d in 50% e t h a n o l s o l u t i o n a n d r e d u c e d t o p - a m i n o p h e n o b a r b i t a l , m . p . 1 9 8 . 5 - - 1 9 8 . 8 ° C , in t h e p r e s e n c e o f iron p o w d e r in 1 To whom reprint requests should be addressed.
52 H
0 CzHs
l + HNO3
H ~ O ~CzHs
~l Reduction . H . ~ "O /C2H5
4- ~H2"COXO CH2--COp H
~ -I.,.BSA- NH2 H~-~O ,C2H; O~N ~ H C O C H 2 C H 2 C O N H - B S A Fig. 1. Synthesis of the p-succinamidophenobarbital-conjugated bovine serum albumin.
h y d r o ch lo r ic acid as catalyst. In order to prepare p-succinamidophenobarbital, 200 mg o f p -a m i nophenobar bi t a l and 115 mg of succinic anhydride in 3 ml o f dry pyridine were refluxed for 3 h in an oil bath. After the solvent was removed by evaporation in vacuo in a water bath, the reactants were separated by thin-layer c h r o m a t o g r a p h y on plates coated with silica gel H using a solvent system of benzene--acetone--acetic acid (50 : 50 : 1, by vol.). The Rf value o f p-succinamidophenobarbital purified was 0.41 and its melting p o in t was 107--110°C. Anal. calculated for C16H1706N3: C, 55.3; H, 4.9; N, 12.1 -- found: C, 53.1; H, 5.1; N, 11.8%. The phenobarbital hapten, p-succinamidophenobarbital (SAPh), was coupled to bovine serum albumin (BSA) according to the m e t h o d of Erlanger et al. (1958) with a slight modification. 45 mg of SAPh and 55 pl of tri-n-butylamine were dissolved in 1.9 ml of dioxane. After the solution was cooled to 10°C, 14 pl o f i s o b u t y l c h l o r o c a r b o n a t e was added to it to form the mixed anhydride and the m i xt ur e was then stirred for 30 min at 4°C. This mixed solution was added dropwise to a cooled solution of BSA (145
53 mg) dissolved in 3.8 ml of distilled water, 2.6 ml of dioxane and 0.145 ml of 1 N NaOH. The pH was kept between 7.5--9 with 1 N NaOH during the addition. The reaction mixture was further stirred for 3 h at 4°C, and dialyzed against distilled water for 3 days. Some precipitates in the dialysate were dissolved by the addition of 8% aqueous NaHCO3 solution. For the determination of the amounts of phenobarbital coupled to the carrier protein, 1.0 pCi of [3H]phenobarbital was added to the unlabelled phenobarbital, and the above procedure was followed. By measuring the radioactivity of the dialysate as the antigen, the degree of the conjugation was calculated to be 42.1 mol of the hapten per one mol of BSA (mol. wt. 68,000). The dialysate was kept at --20°C until used as antigen. The protein c o n t e n t was determined by the m e t h o d of Lowry et al. (1951).
Immunization A 0.5 ml volume of SAPh-BSA solution in saline, which contains 0.9 mg protein, was emulsified with an equal volume of complete Freund's adjuvant. Male albino rabbits, weighing 2.5--3.0 kg, were injected subcutaneously in six different sites at the back and in two thighs. Three rabbits received 1.2 ml of the emulsion twice the first m o n t h and then once 3 weeks for 9 weeks as a booster injection. Blood samples were collected from the vein of the rabbit ear 7--10 days after each booster injection and clotted at 4°C. The serum was separated by centrifugation and served as the source of antibodies.
Radioimmunoassay procedure The radioimmunoassay is based on competitive binding of unlabelled and labelled [3H]phenobarbital to the specific antiphenobarbital antibodies present in rabbit antisera. 0.01 M phosphate-buffered saline (pH 7.6) was used for the dilution of antisera, normal rabbit serum and for the preparation of labelled [3H]phenobarbital and cold ligands. Each assay tube contained the following components: 0.1 ml of [3H]phenobarbital (specific activity 5.0 Ci/. mmole, about 5,000 dpm, 0.45 pmole), 0.1 ml of diluted antiserum (1 : 2,000), 0.1 ml of normal rabbit serum which was added to obtain sufficient a m m o n i u m sulfate precipitates, 0.1 ml of either unlabelled phenobarbital or other ligands solution and 0.1 ml of phosphate-buffered saline. The incubation mixture was allowed to stand for 2 h at room temperature. After the addition of 0.5 ml of saturated a m m o n i u m sulfate solution to the mixture, the precipitate containing phenobarbital bound to antibody was collected by centrifugation for 15 min at 3,000 rpm. The precipitate was washed twice with 0.5 ml of 50% saturated a m m o n i u m sulfate solution and then dissolved in 1 ml of distilled water; 0.5 ml of its solution was transferred to 10 ml of dioxane scintillator containing 7 g of DPO, 0.3 g of POPOP and 100 g of naphthalene in 1,000 ml of dioxane, and its radioactivity was measured for 10 min in a liquid scintillation spectrometer (Aloka LSC-502, Japan).
Chemicals [3H]Phenobarbital (specific activity 5.0 Ci/mmole) was purchased from
54 New England Nuclear Corp. Crystallized BSA was obtained from Sigma Chemical Co. Phenobarbital and allobarbital were generous gifts from Fujisawa Pharmaceutical Co., Osaka, Japan. Amobarbital was purchased from Nippon Shinyaku Co., Kyoto, and mephobarbital and hexobarbital were from Yoshitomi Pharmaceutical Co., Osaka. Nembutal and metharbital were obtained from Dainippon Pharmaceutical Co., Osaka, and thiopental was from Tanabe Pharmaceutical Co., Osaka. Isobutylchlorocarbonate was supplied by T o k y o Kasei Co., and all other reagents were purchased from Wako Pure Chemical Ind., Japan. All organic solvents were redistilled before use. RESULTS In the present radioimmunoassay procedure, the addition of increasing amounts of unlabelled phenobarbital to a constant a m o u n t of [3H]phenobarbital and antibody resulted in a linear-competitive inhibition of the formation of [3H]phenobarbital--antibody complex. Normal rabbit serum showed no binding activity to phenobarbital and in the absence of non-radioactive phenobarbital, the antibody at 1 : 2,000 dilution could bind 50% of the radioactivity by the procedure. As shown in Fig. 2, less than 0.2 pmol (46.4 pg) of phenobarbital could be detected by this assay and 1.8 pmol produced a 50% inhibition of binding. The intra-assay coefficient of variation in reciplicate (n = 8) was 3.4%. Several phenobarbital derivative ligands at various concentrations were tested to determine the specificity of the radioimmunoassay (Table 1). The antibody showed different binding affinities for each ligand, depending on 100
80
v
60
._ .Q .c c I
40
20
0
I
0.2
0.4
I
I
I
I
I
1
2
4
I
I
10
P h e n o b a r b i t a l ( pmol )
Fig. 2. Inhibition by non-radioactive phenobarbital of binding of [3H]phenobarbital by the antibody. Each point in the figure represents an average of three runs.
55 TABLE 1 INHIBITION BY PHENOBARBITAL LIGANDS OF BINDING OF [3H]PHENOBARBITAL BY THE ANTIPHENOBARBITAL ANTISERUM
1•3
RI\ /CO--N\
/ C\ /C=X R2 CO--NH Ligands
R1
R2
R3
X
50% Inhibition (pmol)
Phenobarbital
C2H 5
Q
H
O
1.8
p-Succinamidophenobarbital
C2Hs
HO2C(CH2 ) 2 C O N H - - ( " ~
H
O
2.5
p-Nitro phenobarbital
C2H s
NO 2 - - ( r ' ~
H
O
3.3
p-Amino phenobarbital
C2H5
NH2-~
H
O
3.7
Mephobarbital
C2H 5
(~
CH 3
O
45.5
Nembutal
C2H5
CH3CH2CH27H
H
O
86.7
H
O
221
/
Amobarbital
C2H5
CH3 CH3CHCH2CII2 /
Metharbital
C:Hs
CH3 C2Hs
CH3
O
> 2000
Allobarbital
CH2=CH--CH2
CH2=CH--CH2
H
O
>2000
Hexobarbital
CH3
O
CH 3
O
>2000
Barbituric acid
H
H
H
O
>2000
Thiopental
C2H5
CH3CH2CH2~H
H
S
>2000
CH3
56 the similarity of the chemical structure to that of phenobarbital. The barbituric ring seems to be important for the binding of ligands to the antibody. For example, the affinity of N-methylbarbiturate such as mephobarbital was shown to be approximately 25 times weaker than that of phenobarbital, indicating that the antibody appears to recognize the intact urea moiety of the ring. Nembutal and amobarbital, which have a single substituent at the carbon-5 position of the barbituric ring, were shown to be effective at 86.7 and 221 pmol, respectively, in the 50% inhibition of binding by the antibody (Table 1). However, the antibody failed to bind allobarbital and barbituric acid which have two substituents at the carbon-5 position. These results indicate that substitution on the carbon-5 position of the barbituric ring markedly contributes to the binding affinity. The antibody had no significant cross reaction with hexobarbital, metharbital and thiopental. The binding affinity of p-nitrophenobarbital, p-aminophenobarbital and p-succinamidophenobarbital for the antibody, the last c o m p o u n d being used as the immunogenic hapten, was almost equal to t h a t of phenobarbital. DISCUSSION For production of a suitable antibody against a hapten conjugated to BSA, the coupling of more than 20 mol of the hapten to 1 mol of BSA is generally required (Nisonoff, 1967}. In our experiments, approximately 42.1 tool of phenobarbital were f o u n d to be coupled to 1 mol of BSA on the basis of the radioactivity of [3H]SAPh-BSA compound. The antiphenobarbital antiserum obtained by multiple immunization showed a sufficient affinity for radioimmunoassay. In order to save the overall run-time for the assay, the incubation was tested under various conditions; the 2-h incubation at room temperature was established to be sufficient for the radioimmunoassay w i t h o u t reduction of the sensitivity. The specificity of the antibody was tested by determining the ability of several ligands to inhibit the formation of [3H]phenobarbital--antibody complex. It was found that the antibody recognized n o t only the intact urea m o i e t y of the barbituric ring but also alterations of phenyl and ethyl groups at the carbon-5 position of the ring. The antibody used in this study gave no cross-reaction with thiopental, differing from nembutal only by sulfur replacing oxygen of the urea portion of the barbituric ring, which suggests that the oxygen atom of the urea portion was also discriminated by the specific antibody. Accordingly, an alteration of any of the four substituents of the barbituric ring results in a far weaker binding affinity for the antibody, and more than two alterations nullify the binding ability to the antibody (Table 1). Chung et al. (1973) reported that their antibodies gave no significant cross-reaction with amobarbital, nembutal and hexobarbital, even though the antibodies used in our experiment showed detectable affinity for the first two compounds. Similar observations were reported by Satoh et al. (1974) who used p-azophenobarbital-carrier protein as the antigen. These differ-
57 ences in t h e specificities o f t h e a n t i p h e n o b a r b i t a l a n t i s e r a s e e m t o be d u e to t h e d i f f e r e n c e in c o n f o r m a t i o n as well as b i n d i n g site o f t h e h a p t e n t o the carrier p r o t e i n . T h e m u l t i p l e i m m u n i z a t i o n w i t h t h e h a p t e n c o n j u g a t e d to BSA p r o d u c e d a n t i b o d i e s o f higher t i t e r t h a n t h o s e o f S a t o h et al. {1974); this m a y be a t t r i b u t a b l e t o the e x t e n s i o n o f t h e bridge c h a i n at t h e p a r a posit i o n o f t h e p h e n y l g r o u p o f t h e b a r b i t u r i c ring as well as a g r e a t e r degree o f c o n j u g a t i o n o f t h e h a p t e n t o t h e carrier p r o t e i n . T h e p r e s e n c e o f a bridge c h a i n as a h a p t e n - c a r r i e r s p a c e r was r e p o r t e d to e n h a n c e t h e a f f i n i t y a n d spec i f i c i t y o f t h e a n t i b o d y t o a m p h e t a m i n e ( C h e n g et al., 1 9 7 3 ) . ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y Scientific R e s e a r c h G r a n t s f r o m the Japanese Ministry of Education. REFERENCES Bousquet, E.W. and R. Adams, 1930, J. Am. Chem. Soc. 52,224. Bowman, M.C. and L.G. Rushing, 1978, J. Chromatogr. Sci. 16, 23. Butler, T.C., 1956, J. Pharmacol. Exp. Ther. 116,326. Cheng, L.T., S.Y. Kim, A. Chung and A. Castro, 1973, FEBS Lett. 36, 1973. Chung, A., S.Y. Kim, L.T. Cheng and A. Castro, 1973, Experientia 29,820. Erlanger, B.F., F. Borek, S.M. Beiser and S. Lieberman, 1957, J. Biol. Chem. 228,713. Flynn, E.J. and S. Spector, 1972, J. Pharmacol. Exp. Ther. 181,547. Kumps, A. and Y. Mardens, 1975, Clin. Chim. Acta 62,371. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Nisonoff, A., 1967, in: Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York and London) p. 120. Satoh, H., Y. Kuroiwa, A. Hamada and T. Uematsu, 1974, J. Biochem. 75, 1301. Spector, S. and E.J. Flynn, 1971, Science 174, 1036. Touchstone, J.C., M.F. Schwarz and S.S. Levin, 1977, J. Chromatogr. Sci. 15,528.
51
D E T E R M I N A T I O N O F P H E N O B A R B I T A L BY R A D I O I M M U N O A S S A Y
A. YAMAOKA and T. TAKATORI 1
Department of Legal Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan (Received 13 December 1978, accepted 26 December 1978) A radioimmunoassay for phenobarbital has been studied. Antiphenobarbital antisera were obtained by repeated immunization of rabbits with p-succinamidophenobarbital conjugated to bovine serum albumin. Less than 0.2 pmol of phenobarbital could be measured by this procedure. The specificity of the antibodies was directed to substituents on the nitrogen atoms of the barbituric ring as well as to substituents at the carbon 5-position of the ring. INTRODUCTION P h e n o b a r b i t a l has b e e n w i d e l y u s e d as a sedative a n d h y p n o t i c . F o r clinical a n d forensic p u r p o s e s , a m e t h o d is f r e q u e n t l y r e q u i r e d t o d e t e r m i n e t r a c e a m o u n t s o f p h e n o b a r b i t a l in biological materials. A l t h o u g h a f e w c h r o m a t o graphic d e t e r m i n a t i o n s o f p h e n o b a r b i t a l h a v e b e e n r e c e n t l y d e s c r i b e d ( K u m p s and Mardens, 1 9 7 5 ; T o u c h s t o n e et al., 1 9 7 7 ; B o w m a n et al., 1 9 7 8 ) , r a d i o i m m u n o a s s a y is a m o r e sensitive and reliable m e t h o d f o r the d e t e r m i n a tion of drug haptens. T h e r a d i o i m m u n o a s s a y f o r b a r b i t u r a t e s has b e e n r e p o r t e d b y S p e c t o r a n d F l y n n ( 1 9 7 1 ) and F l y n n a n d S p e c t o r ( 1 9 7 2 ) , a n d C h u n g et al. ( 1 9 7 3 ) a n d S a t o h et al. ( 1 9 7 4 ) d e s c r i b e d t h e r a d i o i m m u n o a s s a y f o r p h e n o b a r b i t a l . In t h e p r e s e n t p a p e r we d e s c r i b e t h e use o f p - s u c c i n a m i d o p h e n o b a r b i t a l conjug a t e d to b o v i n e s e r u m a l b u m i n (BSA), a d i f f e r e n t derivative f r o m t h e i m m u n o g e n i c c o n j u g a t e s d e s c r i b e d p r e v i o u s l y , ip t h e p r o d u c t i o n o f p h e n o b a r b i t a l specific a n t i b o d i e s a n d t h e r a d i o i m m u n o a s s a y p r o c e d u r e . MATERIALS AND METHODS
Preparation o f the imrnunogen Fig. 1 illustrates t h e m e t h o d o f s y n t h e s i s o f p - s u c c i n a m i d o p h e n o b a r b i t a l conjugated to bovine serum albumin, p-Nitrophenobarbital, m.p. 216-220°C, was p r e p a r e d b y t h e m e t h o d s o f B o u s q u e t a n d A d a m s ( 1 9 3 0 ) a n d Butler ( 1 9 5 6 ) , s u s p e n d e d in 50% e t h a n o l s o l u t i o n a n d r e d u c e d t o p - a m i n o p h e n o b a r b i t a l , m . p . 1 9 8 . 5 - - 1 9 8 . 8 ° C , in t h e p r e s e n c e o f iron p o w d e r in 1 To whom reprint requests should be addressed.
52 H
0 CzHs
l + HNO3
H ~ O ~CzHs
~l Reduction . H . ~ "O /C2H5
4- ~H2"COXO CH2--COp H
~ -I.,.BSA- NH2 H~-~O ,C2H; O~N ~ H C O C H 2 C H 2 C O N H - B S A Fig. 1. Synthesis of the p-succinamidophenobarbital-conjugated bovine serum albumin.
h y d r o ch lo r ic acid as catalyst. In order to prepare p-succinamidophenobarbital, 200 mg o f p -a m i nophenobar bi t a l and 115 mg of succinic anhydride in 3 ml o f dry pyridine were refluxed for 3 h in an oil bath. After the solvent was removed by evaporation in vacuo in a water bath, the reactants were separated by thin-layer c h r o m a t o g r a p h y on plates coated with silica gel H using a solvent system of benzene--acetone--acetic acid (50 : 50 : 1, by vol.). The Rf value o f p-succinamidophenobarbital purified was 0.41 and its melting p o in t was 107--110°C. Anal. calculated for C16H1706N3: C, 55.3; H, 4.9; N, 12.1 -- found: C, 53.1; H, 5.1; N, 11.8%. The phenobarbital hapten, p-succinamidophenobarbital (SAPh), was coupled to bovine serum albumin (BSA) according to the m e t h o d of Erlanger et al. (1958) with a slight modification. 45 mg of SAPh and 55 pl of tri-n-butylamine were dissolved in 1.9 ml of dioxane. After the solution was cooled to 10°C, 14 pl o f i s o b u t y l c h l o r o c a r b o n a t e was added to it to form the mixed anhydride and the m i xt ur e was then stirred for 30 min at 4°C. This mixed solution was added dropwise to a cooled solution of BSA (145
53 mg) dissolved in 3.8 ml of distilled water, 2.6 ml of dioxane and 0.145 ml of 1 N NaOH. The pH was kept between 7.5--9 with 1 N NaOH during the addition. The reaction mixture was further stirred for 3 h at 4°C, and dialyzed against distilled water for 3 days. Some precipitates in the dialysate were dissolved by the addition of 8% aqueous NaHCO3 solution. For the determination of the amounts of phenobarbital coupled to the carrier protein, 1.0 pCi of [3H]phenobarbital was added to the unlabelled phenobarbital, and the above procedure was followed. By measuring the radioactivity of the dialysate as the antigen, the degree of the conjugation was calculated to be 42.1 mol of the hapten per one mol of BSA (mol. wt. 68,000). The dialysate was kept at --20°C until used as antigen. The protein c o n t e n t was determined by the m e t h o d of Lowry et al. (1951).
Immunization A 0.5 ml volume of SAPh-BSA solution in saline, which contains 0.9 mg protein, was emulsified with an equal volume of complete Freund's adjuvant. Male albino rabbits, weighing 2.5--3.0 kg, were injected subcutaneously in six different sites at the back and in two thighs. Three rabbits received 1.2 ml of the emulsion twice the first m o n t h and then once 3 weeks for 9 weeks as a booster injection. Blood samples were collected from the vein of the rabbit ear 7--10 days after each booster injection and clotted at 4°C. The serum was separated by centrifugation and served as the source of antibodies.
Radioimmunoassay procedure The radioimmunoassay is based on competitive binding of unlabelled and labelled [3H]phenobarbital to the specific antiphenobarbital antibodies present in rabbit antisera. 0.01 M phosphate-buffered saline (pH 7.6) was used for the dilution of antisera, normal rabbit serum and for the preparation of labelled [3H]phenobarbital and cold ligands. Each assay tube contained the following components: 0.1 ml of [3H]phenobarbital (specific activity 5.0 Ci/. mmole, about 5,000 dpm, 0.45 pmole), 0.1 ml of diluted antiserum (1 : 2,000), 0.1 ml of normal rabbit serum which was added to obtain sufficient a m m o n i u m sulfate precipitates, 0.1 ml of either unlabelled phenobarbital or other ligands solution and 0.1 ml of phosphate-buffered saline. The incubation mixture was allowed to stand for 2 h at room temperature. After the addition of 0.5 ml of saturated a m m o n i u m sulfate solution to the mixture, the precipitate containing phenobarbital bound to antibody was collected by centrifugation for 15 min at 3,000 rpm. The precipitate was washed twice with 0.5 ml of 50% saturated a m m o n i u m sulfate solution and then dissolved in 1 ml of distilled water; 0.5 ml of its solution was transferred to 10 ml of dioxane scintillator containing 7 g of DPO, 0.3 g of POPOP and 100 g of naphthalene in 1,000 ml of dioxane, and its radioactivity was measured for 10 min in a liquid scintillation spectrometer (Aloka LSC-502, Japan).
Chemicals [3H]Phenobarbital (specific activity 5.0 Ci/mmole) was purchased from
54 New England Nuclear Corp. Crystallized BSA was obtained from Sigma Chemical Co. Phenobarbital and allobarbital were generous gifts from Fujisawa Pharmaceutical Co., Osaka, Japan. Amobarbital was purchased from Nippon Shinyaku Co., Kyoto, and mephobarbital and hexobarbital were from Yoshitomi Pharmaceutical Co., Osaka. Nembutal and metharbital were obtained from Dainippon Pharmaceutical Co., Osaka, and thiopental was from Tanabe Pharmaceutical Co., Osaka. Isobutylchlorocarbonate was supplied by T o k y o Kasei Co., and all other reagents were purchased from Wako Pure Chemical Ind., Japan. All organic solvents were redistilled before use. RESULTS In the present radioimmunoassay procedure, the addition of increasing amounts of unlabelled phenobarbital to a constant a m o u n t of [3H]phenobarbital and antibody resulted in a linear-competitive inhibition of the formation of [3H]phenobarbital--antibody complex. Normal rabbit serum showed no binding activity to phenobarbital and in the absence of non-radioactive phenobarbital, the antibody at 1 : 2,000 dilution could bind 50% of the radioactivity by the procedure. As shown in Fig. 2, less than 0.2 pmol (46.4 pg) of phenobarbital could be detected by this assay and 1.8 pmol produced a 50% inhibition of binding. The intra-assay coefficient of variation in reciplicate (n = 8) was 3.4%. Several phenobarbital derivative ligands at various concentrations were tested to determine the specificity of the radioimmunoassay (Table 1). The antibody showed different binding affinities for each ligand, depending on 100
80
v
60
._ .Q .c c I
40
20
0
I
0.2
0.4
I
I
I
I
I
1
2
4
I
I
10
P h e n o b a r b i t a l ( pmol )
Fig. 2. Inhibition by non-radioactive phenobarbital of binding of [3H]phenobarbital by the antibody. Each point in the figure represents an average of three runs.
55 TABLE 1 INHIBITION BY PHENOBARBITAL LIGANDS OF BINDING OF [3H]PHENOBARBITAL BY THE ANTIPHENOBARBITAL ANTISERUM
1•3
RI\ /CO--N\
/ C\ /C=X R2 CO--NH Ligands
R1
R2
R3
X
50% Inhibition (pmol)
Phenobarbital
C2H 5
Q
H
O
1.8
p-Succinamidophenobarbital
C2Hs
HO2C(CH2 ) 2 C O N H - - ( " ~
H
O
2.5
p-Nitro phenobarbital
C2H s
NO 2 - - ( r ' ~
H
O
3.3
p-Amino phenobarbital
C2H5
NH2-~
H
O
3.7
Mephobarbital
C2H 5
(~
CH 3
O
45.5
Nembutal
C2H5
CH3CH2CH27H
H
O
86.7
H
O
221
/
Amobarbital
C2H5
CH3 CH3CHCH2CII2 /
Metharbital
C:Hs
CH3 C2Hs
CH3
O
> 2000
Allobarbital
CH2=CH--CH2
CH2=CH--CH2
H
O
>2000
Hexobarbital
CH3
O
CH 3
O
>2000
Barbituric acid
H
H
H
O
>2000
Thiopental
C2H5
CH3CH2CH2~H
H
S
>2000
CH3
56 the similarity of the chemical structure to that of phenobarbital. The barbituric ring seems to be important for the binding of ligands to the antibody. For example, the affinity of N-methylbarbiturate such as mephobarbital was shown to be approximately 25 times weaker than that of phenobarbital, indicating that the antibody appears to recognize the intact urea moiety of the ring. Nembutal and amobarbital, which have a single substituent at the carbon-5 position of the barbituric ring, were shown to be effective at 86.7 and 221 pmol, respectively, in the 50% inhibition of binding by the antibody (Table 1). However, the antibody failed to bind allobarbital and barbituric acid which have two substituents at the carbon-5 position. These results indicate that substitution on the carbon-5 position of the barbituric ring markedly contributes to the binding affinity. The antibody had no significant cross reaction with hexobarbital, metharbital and thiopental. The binding affinity of p-nitrophenobarbital, p-aminophenobarbital and p-succinamidophenobarbital for the antibody, the last c o m p o u n d being used as the immunogenic hapten, was almost equal to t h a t of phenobarbital. DISCUSSION For production of a suitable antibody against a hapten conjugated to BSA, the coupling of more than 20 mol of the hapten to 1 mol of BSA is generally required (Nisonoff, 1967}. In our experiments, approximately 42.1 tool of phenobarbital were f o u n d to be coupled to 1 mol of BSA on the basis of the radioactivity of [3H]SAPh-BSA compound. The antiphenobarbital antiserum obtained by multiple immunization showed a sufficient affinity for radioimmunoassay. In order to save the overall run-time for the assay, the incubation was tested under various conditions; the 2-h incubation at room temperature was established to be sufficient for the radioimmunoassay w i t h o u t reduction of the sensitivity. The specificity of the antibody was tested by determining the ability of several ligands to inhibit the formation of [3H]phenobarbital--antibody complex. It was found that the antibody recognized n o t only the intact urea m o i e t y of the barbituric ring but also alterations of phenyl and ethyl groups at the carbon-5 position of the ring. The antibody used in this study gave no cross-reaction with thiopental, differing from nembutal only by sulfur replacing oxygen of the urea portion of the barbituric ring, which suggests that the oxygen atom of the urea portion was also discriminated by the specific antibody. Accordingly, an alteration of any of the four substituents of the barbituric ring results in a far weaker binding affinity for the antibody, and more than two alterations nullify the binding ability to the antibody (Table 1). Chung et al. (1973) reported that their antibodies gave no significant cross-reaction with amobarbital, nembutal and hexobarbital, even though the antibodies used in our experiment showed detectable affinity for the first two compounds. Similar observations were reported by Satoh et al. (1974) who used p-azophenobarbital-carrier protein as the antigen. These differ-
57 ences in t h e specificities o f t h e a n t i p h e n o b a r b i t a l a n t i s e r a s e e m t o be d u e to t h e d i f f e r e n c e in c o n f o r m a t i o n as well as b i n d i n g site o f t h e h a p t e n t o the carrier p r o t e i n . T h e m u l t i p l e i m m u n i z a t i o n w i t h t h e h a p t e n c o n j u g a t e d to BSA p r o d u c e d a n t i b o d i e s o f higher t i t e r t h a n t h o s e o f S a t o h et al. {1974); this m a y be a t t r i b u t a b l e t o the e x t e n s i o n o f t h e bridge c h a i n at t h e p a r a posit i o n o f t h e p h e n y l g r o u p o f t h e b a r b i t u r i c ring as well as a g r e a t e r degree o f c o n j u g a t i o n o f t h e h a p t e n t o t h e carrier p r o t e i n . T h e p r e s e n c e o f a bridge c h a i n as a h a p t e n - c a r r i e r s p a c e r was r e p o r t e d to e n h a n c e t h e a f f i n i t y a n d spec i f i c i t y o f t h e a n t i b o d y t o a m p h e t a m i n e ( C h e n g et al., 1 9 7 3 ) . ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y Scientific R e s e a r c h G r a n t s f r o m the Japanese Ministry of Education. REFERENCES Bousquet, E.W. and R. Adams, 1930, J. Am. Chem. Soc. 52,224. Bowman, M.C. and L.G. Rushing, 1978, J. Chromatogr. Sci. 16, 23. Butler, T.C., 1956, J. Pharmacol. Exp. Ther. 116,326. Cheng, L.T., S.Y. Kim, A. Chung and A. Castro, 1973, FEBS Lett. 36, 1973. Chung, A., S.Y. Kim, L.T. Cheng and A. Castro, 1973, Experientia 29,820. Erlanger, B.F., F. Borek, S.M. Beiser and S. Lieberman, 1957, J. Biol. Chem. 228,713. Flynn, E.J. and S. Spector, 1972, J. Pharmacol. Exp. Ther. 181,547. Kumps, A. and Y. Mardens, 1975, Clin. Chim. Acta 62,371. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Nisonoff, A., 1967, in: Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York and London) p. 120. Satoh, H., Y. Kuroiwa, A. Hamada and T. Uematsu, 1974, J. Biochem. 75, 1301. Spector, S. and E.J. Flynn, 1971, Science 174, 1036. Touchstone, J.C., M.F. Schwarz and S.S. Levin, 1977, J. Chromatogr. Sci. 15,528.
