Production and characterisation of antisera to diphenylhydantoin suitable for radioimmunoassay

Journal o f Immunological Methods, 10 (1976) 317--327 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

317

P R O D U C T I O N A N D C H A R A C T E R I S A T I O N O F A N T I S E R A TO DIPHENYLHYDANTOIN SUITABLE FOR RADIOIMMUNOASSAY

J.W. PAXTON *, F.J. ROWELL ** and J.G. RATCLIFFE *** Departments o f Materia Medica * and Chemistry **, University o f Glasgow, G12 8QQ, U.K. and Radioimmunoassay Unit ***, Stobhill General Hospital, Glasgow, G21 3UW, U.K.

(Received 13 September 1975, accepted 29 September 1975)

The effect of carrier protein, nature of hapten-carrier bridge, and density of hapten substitution on the immunogenicity of diphenylhydantoin (DPH) derivatives in rabbits is described. DPH-3-valerate-bovine serum albumin (BSA) with a hapten : protein ratio of 27 : 1 yields antisera of high titre and specificity for DPH. An antiserum to DPH-valerateBSA was employed to develop a rapid, sensitive, one stage double antibody radioimmunoassay suitable for clinical application to serum, saliva, and urine samples. The assay, using 14C-DPH as tracer, is accurate and precise over the range 0.5--50 /ag DPH per ml. Results by radioimmunoassay correlate closely with those obtained by gas-liquid chromatography (r = 0.97y:

INTRODUCTION D i p h e n y l h y d a n t o i n (DPH) has long been a drug o f first c h o i c e in the control o f grand mal and p s y e h o m o t e r epilepsy. R e c e n t studies suggest t h a t ser u m DPH levels s h o u l d be m o n i t o r e d regularly in patients o n long t e r m thera p y because o f individual differences in drug p h a r m a e o k i n e t i c s and the narr o w t h e r a p e u t i c range ( L u n d , 1 9 7 4 ; L a n c e t , 1 9 7 5 ; Richens and D u n l o p , 1 9 7 5 ) . H o w e v e r , n o n e o f the existing m e t h o d s o f assay is ideal for r o u t i n e clinical use. R a d i o i m m u n o a s s a y s for DPH o f f e r significant p o t e n t i a l advantages for this p u r p o s e ( C o o k et al., 1 9 7 3 ; Tigelaar et al., 1 9 7 3 ; R o b i n s o n et al., 1 9 7 5 ) b u t t h e r e is little i n f o r m a t i o n on reliable m e t h o d s o f d e v e l o p i n g antisera to DPH with suitable specificity and a f f i n i t y characteristics. We have c o m p a r e d the e f f e c t o f carrier p r o t e i n , n a t u r e o f h a p t e n - c a r r i e r bridge, and d e n s i t y o f h a p t e n s u b s t i t u t i o n o n t h e i m m u n o g e n i e i t y o f DPH derivatives in rabbits and describe the d e v e l o p m e n t o f a specific and sensitive r a d i o i m m u n o a s s a y for DPH w h i c h is suitable for clinical and e x p e r i m e n t a l studies. MATERIALS 5 , 5 - D i p h e n y l h y d a n t o i n , 5 - ( p - H y d r o x y p h e n y l ) - 5 - p h e n y l h y d a n t o i n (HPPH), 5 - ( p - m e t h y l p h e n y l ) - 5 - p h e n y l h y d a n t o i n (MPPH) were f r o m Aldrich Chemical

318 Company, Inc. (Milwaukee, Wisc.), and 5,5-Diphenyl (4--14C)hydantoin (S.A. 14.3 mCi/mMol) was from the Radiochemical Centre, Amersham. The drugs used in specificity studies were gifts from their respective manufacturers. The other reagents were obtained as follows: Freund's Complete Adjuvant, Difco Labs., Detroit, Michigan; Tween 20, Bovine Thyroglobulin (Tg), l-ethyl-3(3-dimethylaminopropyl)-carbodiimide, Sigma Chemical Co., St. Louis, Missouri; Bovine Serum Albumin (BSA), Armour Pharmaceutical Co., Eastbourne, England; Donkey anti-rabbit precipiiating serum, Wellcome Reagents Ltd., Beckenham, England; Visking 32/32 tubing, Scientific Instrument Centre Ltd., London; Scintillation fluid NE 260, Nuclear Enterprises Ltd., Edinburgh; Polyethylene scintillation vials, Koch-Light Laboratories Ltd., Colnbrook, Bucks, England. METHODS

Synthesis of DPH derivatives Preparation of 5,5-diphenyl-hydantoin-3-acetic acid (DPH-AA ) The sodium salt of DPH (2.74 g) was refluxed with freshly distilled ethyl chloroacetate (2.45 g) in dimethyl formamide (5 ml) for 2 h. The solvent was distilled off under vacuum and the residue recrystallised from ethyl acetate/petroleum to yield ethyl-5,5-diphenyl-hydantoin-3-acetic acici (1.90 g) m.p. = 181--182°C (reported 178--180°C by Sandberg, 1951). The ester (1.5 g) was refluxed in 3 N hydrochloric acid-dioxane solution (50 ml) (1/1 v/v) for 1.5 h. The cold solution was added to ice-water and the resulting precipitate was filtered, washed with water, then recrystallised from methanol-ethyl acetate to yield 5,5-diphenylhydantoin-3-acetic acid (1.3 g) m.p. = 282--283°C (reported b y Sandberg (1951) as 282--284°C).

Preparation of 5,5-diphenylhydantoin-3-co-valeric acid (DPH-VA ) This was prepared using the reaction of sodium diphenylhydantoin and methyl-5-bromovalerate, with subsequent hydrolysis of the ester by the method of Cook et al. (1973).

Synthesis of protein conjugates Preparation of DPH-3-acetate-thyroglobulin (DPH-AA-Tg) DPH-3-acetic acid (18.3 mg) was dissolved in a water--pyridine mixture (4 ml of 4/1, water/pyridine). To the stirred solution was added 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (C.D.I.) (19.2 mg) followed by bovine Tg (325 mg). After mixing for 30 min at room temperature the solution was dialysed against 3 × 1 litre of distilled water at room temperature for three days, then lyophilised. The conjugate (189 mg) contained 12 DPH residues per Tg molecule. The incorporation of DPH into carrier was determined by including trace amounts of 14C_DP H initially.

319

Preparation of DPH-3-valerate-BSA (DPH-VA-BSA ) DPH-3-c0-valeric acid (20.8 mg) was dissolved in a water--pyridine mixture (4 ml of 4/1, water/pyridine). To the stirred solution was added C.D.I. (19.7 mg), then BSA (30 mg). After mixing for 30 min at room temperature the solution was dialysed against distilled water as described above. The conjugate (11.7 mg) precipitated out and contained 27 DPH residues per BSA molecule.

Preparation of DPH-3-valerate- Tg (DPH- VA- Tg) DPH-3-co-valeric acid (20.8 mg) was dissolved in a water--pyridine mixture (4 ml of 4/1, water/pyridine). To the stirred solution was added C.D.I. (19.7 mg) followed by bovine thyroglobulin (325 mg). The mixture was made up to 10 ml with distilled water and stirred at room temperature for 19 h. After dialysis, the precipitate which formed was removed by centrifugation. The conjugate (76 mg) contained 41 DPH residues per Tg molecule (DPH-VA-Tg (ppt)). The supernatant liquid from the centrifugation was also lyophilised yielding a conjugate (16.7 mg) containing 70 DPH residues per Tg molecule (DPH-VA-Tg (sol)).

Immunisation Groups of New Zealand White rabbits of both sexes were immunised with each of the immunogens (DPH-AA-Tg; DPH-VA-BSA; DPH-VA-Tg soluble precipitate) (table 1). Each rabbit received 2 ml of an emulsion (25% saline, 4% of 15% Tween 20, 71% Freunds complete adjuvant) containing the speci-. fied amount of immunogen distributed intradermally at 40--50 sites into its shaven back (Vaitukaitis et al., 1971). Boosters identical in composition and amount with the primary injections were given six weeks after the primary at approximately 2--4 weekly intervals for up to 32 weeks into subcutaneous, intramuscular and intraperitoneal sites. Blood was taken 10--14 days after each booster injection, and serum stored at --20 ° C.

Radioimmunoassay Phosphate buffer (0.05 M) pH 7.5 containing 0.1% BSA and 0.1% w/v sodium azide was used as the diluent.

Testing antisera The sera were screened for specific binding of ~4C-DPH by adding to each tube 200 pl buffer, 100 pl buffer containing 10 ng ~4 C-DPH (1000 cpm) and 100 pl of antiserum (final dilution 1 : 100 -- 1 : 6400). After incubation for 24 h at 4°C, antibody b o u n d and free ~4C-DPH were separated by addition of 100 pl of a d o n k e y anti-rabbit precipitating serum (1 : 50 final dilution}. This second antiserum gave complete precipitation of the ~4C-DPH b o u n d to rabbit antibody under the conditions employed. After further incubation for

320 24 h at 4°C the tubes were centrifuged (2500 g for 30 min at 4°C) and the supernatant counted (Packard Tri-carb liquid scintillation spectrometer Model 3320). Quench corrections were negligible. The titre employed in subsequent radioimmunoassays was the final dilution of antiserum required to bind 50% of 14C-DPH.

Radioimmunoassay procedure Standard curves were set up by incubating increasing amounts of unlabelled DPH (0.1--1000 ng) or test serum (20 pl of 1 : 10 dilution), with 10 ng 14C-DPH (100 pl), appropriate first and second antiserum dilutions (100 pl of each), and diluent (200 pl), to give a final incubation volume of 520 pl. Label blanks containing label and non-immune rabbit serum and zero standard controls were set up with all assays. Label blanks usually constituted 1-3% and zero standard tubes 50--60% of the total counts. Incubation was for 1 h at 37°C after which the tubes were centrifuged and the supernatant counted as described above. The specificity of the selected antiserum towards DPH, its metabolites and other related compounds and anticonvulsants was evaluated by determining the a m o u n t of each c o m p o u n d needed to produce 50% inhibition of the m a x i m u m binding in the assay system described above. Antibody specificity was expressed as a % cross-reaction and was calculated as described by Abraham (1969). Unlabelled DPH was taken as standard (100%). The percentage cross-reaction of the other compounds was expressed as mass standard (unlabelled DPH) X 100% mass investigated c o m p o u n d in which the amounts of standard DPH and c o m p o u n d being investigated were read from the displacement curves at 50% inhibition of the zero standard controls.

Gas chromatography DPH was determined in serum by a modification of the m e t h o d of MacGee (1970). 15 pg 5-(4-methylphenyl)-5-phenyl hydantoin (MPPH) in methanol was added to glass tubes as internal standard and blown dry under nitrogen. To each tube 1 ml serum, 0.5 ml 3 M sodium dihydrogen phosphate, and 5.5 ml toluene were added. After shaking and centrifugation for 5 min respectively, 5 ml of the toluene layer was transferred to a second tapered tube and 30 pl 24% t e t r a m e t h y l a m m o n i u m hydroxide in methanol (TMH) was added. After vigorous mixing for 30 sec and centrifugation for 5 min at 2,500 g the upper toluene layer was removed and 1 pl of the TMH phase was injected into the chromatographic unit. Standards were prepared likewise, by adding known amounts of DPH (0--50 pg) in methanol to the internal standard and after drying 1 ml of plasma to each. A standard curve was drawn in which the peak height ratio of the drug to the internal standard was plotted

321 against drug concentration. Using peak heights of samples, drug concentrations were read from the graph. A Pye series 104 Gas Chromatograph with flame ionisation detector was employed with a 1.5 m glass column, internal diameter 4 mm, packed with 3% OV-17 on Gas-Chrom Q 100/120 mesh. The injection temperature was 350°C with a flow rate of nitrogen of 60 ml/min. Column temperature was initially 177°C for 5 min, rising at a rate of 8°C per min to 243°C. RESULTS

Antibody production None of the rabbits immunised with DPH-AA-Tg and only one of those injected with DPH-VA-Tg (sol) showed significant specific binding of 14C-DPH after 3 booster injections. However, when three of these were tested with a labelled 3-N-acetamido derivative of DPH all showed significant specific binding. Both the DPH-VA-BSA and DPH-VA-Tg (ppt) immunogens produced positive responses in all animals when the sera were tested with ~4CDPH (table 1). In general, the titres were higher with the DPH-VA-BSA immunogen, with peak response occurring between 16 and 24 weeks after the primary immunisation (fig. 1).

Antibody specificity The specificities of the two antisera with the highest titre (rabbits 8, 10 of DPH-VA-BSA group) were further investigated by testing the effect of several hydantoin derivatives and metabolites and structurally similar drugs in inhibiting the binding of 14C-DPH to the antibody (fig. 2). The specificities of both antisera are summarised in table 2. Both antisera showed negligible cross-reactivity with other structurally similar anticonvulsant hydantoins and 3 3x10

-

<

10 3

II

j

~

1~ ;~

2'0 2'4 2~

33

WEEKS AFTER PRIMARY IMMUNISATION

Fig. 1. A n t i b o d y titres in 6 r a b b i t s ( N u m b e r s 7 - - 1 2 ) i m m u n i z e d w i t h D P H - V A - B S A conjugate. T h e t i t r e was t a k e n as t h e final d i l u t i o n of first a n t i b o d y w h i c h b o u n d 50% o f t h e labelled DPH.

D P H - 3 N - a c e t i c acid DPH-3N-Valeric acid DPH-3N-Valeric acid DPH-3N-Valeric acid

1 2 3 4

Tg BSA Tg ( p p t ) Tg (sol)

Carrier protein

2.5 0.5 3.5 2.0

Amount immunogen (mg) 12.5 50 50 50

Amount DPH administered (#g) 12 27 41 70

: : : :

1 1 1 1

Ratio hapten/ protein

0 / 6 ** (0%) 6/6 (100%) 5/5 (100%) 1/4 (25%)

Response *

DPH HPPH MPPH Methoin Ethotoin Phenobarbitone

Compound

100% 7.9% 7.5% 0.4% 0.05% 0.01%

Antiserum 8

% Cross-reactivity

Specificity o f t w o a n t i s e r a to D P H - V A - B S A .

TABLE 2

100% 1.1% 0.3% 0.1% 0.01% ~0.002%

A n t i s e r u m 10

* Sera b i n d i n g 40% o f trace ~~ C-DPH. ** T h r e e sera t e s t e d w i t h a labelled 3 - N - a c e t a m i d o D P H derivative s h o w e d s i g n i f i c a n t b i n d i n g .

Hapten

Group

I m m u n e r e s p o n s e s to DPH derivatives.

TABLE 1

l : 2000--1 : 3000 I :400- ] :]200 1 : 20 I : 8 0

Titre

t~ b~

323

70"

--------oTroxidone " ~ ' ~ " " Ethosuximide

60-

~ " ~ " ' ~ Primidone

50=

"~Pheno

t~

barbitone

Z

40-

X*Ethotoin

< 20-

in

HPpH

10-

DPH

0

'2 10

'-I 10

' I0

~ 102

' 103

' 104

" 105

AMOUNT (NANOGRAMS) Fig. 2. Standard curve for DPH using DPH-3-valerate BSA antibody showing cross-reactions with HPPH, MPPH, Methoin, Ethotoin, Phenobarbitone, Primidone, Ethosuximide and Troxidone.

with other anti-epileptic drugs such as phenobarbitone, primidone, ethosuximide and troxidone. From these results it is apparent that antiserum 10 is slightly more specific than antisera 8 for DPH. Antiserum 10 was further tested for cross reactivity with a variety of c o m p o u n d s including endogenous steroids (cholesterol, dehydroepiandrosterone sulphate, aetiocholanolone sulphate, epiandrosterone sulphate, pregnanetriol), folic acid and drugs (pheneturide, diazepam, nitrazepam, carbamazepine, beclamide, ethosuximide, haloperidol, orphenadrine citrate, amitryptiline HC1, methyl phenidate HC1, trifluoperazine HC1, chlorpromazine HC1, chlordiazepoxide HC1, sulthiame, amylobarbitone, barbitone, cyclobarbitone, phenobarbitone, butobarbitone, salicyclic acid and acetylsalicyclic acid) at 10 or 100 gg amounts b u t no significant cross reactivity was observed.

Radioimmunoassay (RIA ) An antiserum pool for RIA was formed from bleeds 3, 4 and 5 from rabbit 10. The affinity constant estimated from a Scatchard plot was 5.5 X 109 1/mol which is similar to that reported by Cook et al. (1973) for their antiserum to DPH (6.8 X 109 1/mol). Addition of urine, horse or human serum at concentrations less than 10% v/v had no effect on the standard curve and therefore the DPH standard curve for assaying patients' sera could be made up in horse serum. For the assay of patients' sera, a 20 pl sample of a 1 : 10 dilution of serum was used. The inter and intra assay precision was determined over the clinically relevant range (0.5--50 pg/ml) b y measurement of 3 quality control samples

324 made up in horse serum. The coefficients of variation ranged between 4.3 and 7.5% and 8.3 and 9.2% for intra and inter assay precision respectively. The accuracy of the m e t h o d was assessed by the addition of known a mo u n ts o f unlabelled DPH t o 1 ml aliquots of normal serum and estimating the serum concentrations by RIA. Over the range of added DPH concentrations between 0.5 and 50 pg/ml the recovery was 97.6 -+ 9.28% {mean + S.D.). The DPH concentrations of 25 serum samples from epileptic patients were assayed by RIA and gas liquid c h r o m a t o g r a p h y . The regression equation was: y (RIA) = 0.98 x (GLC) + 1.10 : r = 0.97. This suggests t hat values by RIA are slightly higher than by GLC though these differences are negligible within the therapeutic range (10--20 pg/ml). DISCUSSION

Factors affecting immunogenicity Carrier protein Although it has been suggested that thyroglobulin is a particularly effective carrier for the p r o d u c t i o n of antisera to small molecules in rabbits (Skowsky and Fisher, 1972) our results indicate t hat BSA may be more efficient for DPH. The group of rabbits immunised with the DPH-VA-BSA i m mu n o g en gave significantly higher titres than both groups immunised with DPH-VA-Tg. However, the effect of small differences in the a m o u n t of injected carrier or density of hapten groups c a n n o t be completely excluded.

Density of hapten substitution There is no general agreement on the optimal density of hapten substit u tio n in a carrier molecule for generation of antisera for radioimmunoassay. James and J ef fcoat e (1974) considered that there is no evidence t hat titre or affinity o f antisera to steroid conjugates depends upon the degree o f substitution. However the classic work of Landsteiner (1945) had suggested t hat 10 haptenic groups per molecule was optimal with serum albumin as carrier, and Midgley et al. (1971) and Kuss et al. (1973) f o u n d molar ratios of 15-30 : 1 were most immunogenic for their steroid conjugates. We obtained consistently high a n t i b o d y titres with a DPH : BSA ratio of 27 : 1. Better responses were also obtained with DPH-VA-Tg conjugates with molar ratios o f 41 : 1 than with 70 : 1. This agrees with the data of Skowsky and Fisher (1972) who f o u n d thyroglobulin conjugates with ratios of approxi m at el y 50 : 1 were optimally immunogenic. I nt e r pret at i on of the immunogenicity of the DPH-VA-Tg conjugates is complicated however, in our study by the insolubility o f the less substituted material since insoluble complexes may be mo r e immunogenic (Chase, 1967). It is of interest in this c o n t e x t t hat the insoluble p h e n y t o i n conjugate e m p l o y e d by Tigelaar et al. (1973) appeared to p ro d u ce better antisera than the soluble conjugate.

325

Nature of the bridge The poor response obtained with the DPH-AA-Tg immunogen as tested with 14C-DPH suggests that a bridge with only three bond lengths does not yield adequate exposure of the hapten. Further experiments using a labelled 3-N-acetamido derivative of DPH showed significant binding by antisera raised to the DPH-AA-Tg immunogen which did not bind 14C-DPH. This suggests that antibody specificity is directed to the preferred conformation of immunogen and includes the fl-methylene group of a 3-N-n-alkyl substituted DPH molecule (Rowell and Paxton, 1975). With the DPH-AA-Tg immunogen, the acetate function would be a c c o m m o d a t e d within the recognition site and the preferred conformation of the acetate bridge would thus be recognised by the resulting antiserum in addition to the conformation of the DPH moiety. Interaction between the hydantoin carbonyl groups and the acetamido carbonyl would produce a preferred conformation in which the bridge carbonyl would be located above the hydantoin ring thus partially eclipsing one face of the ring and possibly altering the conformation of the phenyl groups at C-5. The poor binding of 14C-DPH compared with the Nacetamido derivative thus possibly reflects the formation of antibodies specific for the conformation of the DPH-AA-BSA immunogen. The presence of the N-3-acetamide substituent on DPH would therefore be an essential requirement for high avidity, as was found experimentally. In contrast, the valerate derivatives gave good antibody response. This could be explained by the fact that the hydrophobic valerate bridge in the DPH-VA-BSA immunogen orientates away from the polar hydantoin system, thus isolating the DPH moiety. Consequently, the specificity and binding of this antiserum would not be greatly influenced by the absence of an N-3 alkyl substituent on DPH. In practice, this antiserum showed a ten-fold greater binding of N-3 alkyl substituted derivatives and the specificity was directed towards both the phenyl groups at C-5 and the complete hydantoin ring (Rowell and Paxton, 1975). These results suggest the importance of considering possible interactions between hapten and bridge in the immunogen which may alter the conformation of hapten in the free molecule or produce a conformation of the immunogen in which hapten is masked by the bridge. Either possibility could produce antisera which show low avidity for free hapten in radioimmunoassay systems.

Specificity All the immunogens we employed were coupled through the N3 position to give maximum exposure of the two phenyl rings which are required for full biological activity and are also the main loci of metabolism. These predictions were realised in that the antiserum chosen for the assay showed only a b o u t 1% cross reactivity with 5-hydroxyphenyl-5-phenylhydantoin, the major metabolite and negligible reaction with phenobarbitone, which is usually

326 administered to epileptic patients together with DPH. None of the clinically used analogues, anti-epileptic drugs or endogenous compounds (eg. steroids) which are normally present in high concentrations showed significant cross reaction. Furthermore, the radioimmunoassay results correlated closely with those obtained by gas chromatography. The high degree of specificity for DPtt of the present assay and that of Cook et al. (1973) also using the N3 conjugates contrasts with the poor specificity obtained with an immunogen conjugated through the h y d r o x y p h e n y l group (5-(p-carboxymethoxypbenyl)-5-phenylhydantoin) to gamma globulin (Tigelaar et al., 1973). Conclusion

This radioimmunoassay offers several advantages over some previous methods for the measurement of DPH. First, it requires only 10 pl of serum for duplicate analysis. Second, the assay is quick and has a large sample capacity, and so can readily be applied to pharmacokinetic studies. Third, the assay is potentially much more sensitive than other currently available methods. The limit of detection can be reduced to 200 pg/ml by using a labelled ligand of higher specific activity than the 14C-DPH employed in this study (e.g. 3H or 12SI-labelled DPH, Paxton, Rowell and Ratcliffe, unpublished observations). It is therefore, applicable to the measurement of non protein bound and salivary DPH levels (Paxton, Rowell and Ratcliffe, unpublished observations). Furthermore, the radioimmunoassay is highly specific for DPH and has a precision and accuracy ecLual to that of the gas chromatographic method, which is currently the method of choice for clinical application. ACKNOWLEDGEMENTS This work was supported by a grant from the Radiochemical Centre, Amersham, England. We thank Professor A. Goldberg for his continued advice.

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327 Lund, L., 1974, Arch. Neurol. 3 1 , 2 8 9 . MacGee, S., 1970, Analyt. Chem. 4 2 , 4 2 1 . Midgley, A.R., G.D. Niswender, V.L. Gay and L.E. Reichert, 1971, Recent Prog. Hormone Res. 2 7 , 2 3 5 . Richens, A. and A. Dunlop, 1975, Lancet ii, 247. Robinson, J.D., B.A. Morris, G.W. Aherne and V. Marks, 1975, Brit. J. Clin. Pharmacol. 2, 345. Rowell, F.J. and J.W. Paxton, 1975, submitted for publication. Sandberg, A., 1951, Acta Physiol. Scand. 24, 149. Skowsky, W.R. and Fisher, D.A., 1972, J. Lab. Clin. Med. 8 0 , 1 3 4 . Tigelaar, R.E., R.L. Rapport, J.K. Inman and H.J. Kupferberg, 1973, Clin. Chim. Acta. 43,231. Vaitukaitis, J., J.B. Robbins, E. Nieschlag and G.T. Ross, 1971, J. Clin. Endocrinol. 33, 988.