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Preparation of specific antiserum to estriol 3-sulfate 16-glucuronide
0022-473I /84 $3.00+ 0.00 Copyright I(‘ 1984 Pergamon Press Ltd
J. steroid Eiochem. Vol. 21, No. 2. pp. 199-203, 1984 Printed in Great Britain. All rights reserved
PREPARATION OF SPECIFIC ANTISERUM 3-SULFATE 16-GLUCURONIDE*
TO ESTRIOL
TOSHIO NAMBARA, TOSHIFUMI NIWA
and KAZUTAKE SHIMADA Pharmaceutical Institute, Tohoku University, Sendai 980, Japan (Received 22 September 1983)
Summary-The preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier through an (0-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Coupling of 6-oxoestriol 3-sulfate 16-glucuronide acetate-methyl ester 6-(0-carboxymethyl)oxime with BSA by the activated ester method followed by removal of the protecting groups with alkali provided the desired conjugate. The antisera raised against the conjugate in rabbits were highly specific to the double conjugate, estriol 3-sulfate 16-glucuronide, discriminating from ring A or D monoconjugated and unconjugated estrogens. The specificity of antisera elicited has been discussed on the basis of stereochemistry of the hapten-[C-6]-BSA
conjugate.
INTRODUCTION
The physiological significance of estriol 3-sulfate 16-glucuronidet, which is one of important double conjugates in the feto-placental unit, appears to be an attractive subject to be investigated [l-3]. Development of a simple and reliable radioimmunoassay method is required for the direct determination of the conjugate without hydrolysis and/or solvolysis. In preceding papers [4-lo], we reported the preparation of specific antisera to estrone 3-sulfate, estradiol 17-glucuronide and estriol 16-glucuronide, and their use for direct radioimmunoassay without prior deconjugation. These antisera were raised in the rabbit with hapten-[C-6]-bovine serum albumin (BSA) conjugates. On the basis of these findings the use of estriol 3-sulfate 16-glucuronide-[C-6]-BSA conjugate appeared to be promising for obtaining specific antiserum to the double conjugate. The present paper deals with the preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-BSA conjugate in which the hapten is linked to the carrier through an (O-carboxymethyl)oxime bridge at C-6 on the steroid nucleus. MATERIALS AND METHODS Chemicals
and reagents
[6,9-3H]Estriol 16-glucuronide (22.4 Ci/mmol) was supplied by Amersham International plc (Amersham, U.K.), and the radiochemical purity was
*Part CLXXXXVII of Studies on Steroids by T. Nambara; Part CLXXXXVI: T. Nambara, T. Niwa and K. Shimada: Jap. J. clin. Cbem. 12 (1983) 227-233. tThe following trivial names are used in this paper: 16-glucuronide = 3,17/?-dihydroxy- 1,3,5(10)estriol estratrien-16a-yl-fi-D-glucopyranosiduronate; estriol = 1,3,5(10)-estratriene-3,16a,l7/?-trial.
checked by TLC prior to use. Conjugated steroids were prepared in these laboratories by known methods. All free steroids were kindly donated by Teikoku Hormone Mfg. Co. (Tokyo, Japan). BSA Fraction V and bovine serum gamma-globulin were purchased from Sigma Chemical Co. (St Louis, MO), Sep-pak C,* cartridge and PBondapak C,, column from Waters Assoc., Inc. (Milford, MA), Amberlite XAD4 resin from Rohm and Haas Co. (Philadelphia, PA), complete Freund’s adjuvant and other reagents from Nakarai Chemicals Ltd. (Kyoto, Japan), respectively. A Hartley strain male guinea pig weighing 610 g was used for the sulfotransferase preparation.
Synthesis
of hapten-BSA
conjugate
All melting points were taken on a micro hot-stage apparatus and are uncorrected. Optical rotations were measured with a JASCO mode1 DIP-4 automatic polarimeter. Ultraviolet (u.v.) and infrared (i.r.) spectra were obtained with a Hitachi mode1 124 spectrometer and a JASCO model IRA-IS spectrometer, respectively. Mass spectra (MS) were measured with a Hitachi model M-52 spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded using tetramethylsilane as an internal standard on a JEOL model FX-100 spectrometer at 100 MHz (‘H). Abbreviations used s = singlet, d = doublet, dd = doublet of doublets and m = multiplet. Silica gel 60 HFlx4 (E. Merck AG, Darmstadt) was used for preparative TLC and silica gel 60 (70-230 mesh) or silica gel G (Type 60) (E. Merck AG) for column chromatography. Methyl (I 78 -acetoxy -3-benzyloxy -60x0 - 1,3,5(10)estratrien- I&-yl-2’,3’,4’-tri-O-acetyl-b-o-glucopyranosid)aronate (2) 6-Oxoestriol 199
l6-glucuronide
acetate-methyl
ester
200
TOSHIO NAMBARA
et al.
Methyl (17fl-acetoxy-3-hydroxy-6-oxo-1.3,5(10)3-benzyl ether (1) [9] (973 mg) was dissolved in pyriestratrien-16c(-yl-2’,3’,4’-tri-O-a~etyl-l)-~~-glu~odine (6 ml)-acetic anhydride (6 ml), and the solution was warmed at 70°C for 1 h. The reaction mixture pyranosidjuronate 6-(O-carhoxymethyl)oxime (4) was poured into ice-water and extracted with ethyl To a solution of 3 (202mg) and carboxyacetate. The organic layer was washed successively methoxylamine l/2 HCl (482 mg) in methanol (32 ml) with 5% HCl, water, 5% NaHCO, and water, dried was added sodium acetate (485 mg) in water (4 ml), over anhydrous Na,SO, and evaporated in vacua. and the whole was stirred at 35°C overnight. After The residue was subjected to column chroremoval of the methanol by evaporation, the rematography on silica gel. Elution with hexane-ethyl sulting solution was diluted with water and extracted acetate (2:1, v/v) and recrystallization of the eluate with ethyl acetate. The organic layer was washed with from dichloromethane-methanol gave 2 (450 mg) as water, dried over anhydrous Na,SO, and evaporated colorless needles: m.p. 16@164”C, [xl’,”- 35.1” in uacuo. The residue was subjected to column chro(c =O.lO, CHCl,). NMR (CDCl,) 6 ppm 0.78 (3H, matography on silica gel. Elution with s, 18-CH,), 2.02, 2.03, 2.06, 2.07 (each 3H, each s, chloroform-methanol-water (80:20:2.5, by vol) 17/3-, 2’-, 3’-, 4’-OCOCHI), 3.78 (3H, s, -COOCH,), gave 4 (267 mg) as a colorless oily substance: NMR 3.99 (lH, m, 5/-H), 4.30 (lH, in, 16/?-H), 4.57 (lH, d, (CDCl,) 6 ppm 0.67 (3H, s, 18-CH,), 2.02, 2.05 and J = 8 Hz, 1’-H), 4.90-5.40 (3H, m, 2’-, 3’-, 4-H), 5.01 2.08 (6H, 3H and 3H, each s, 17fl-, 2’-, 3’-. (lH, d, J = 6 Hz, 17c(-H), 5.13 (2H, s, 3-OC&C,H,), 4’-OCOCH,), 3.74 (3H, s, -COOCH,), 3.97 (lH, d, 7.Oc7.70 (8H, m, aromatic H). Anal. calcd for J = 9 Hz, 5/-H), 4.60 (3H, m, I’-H, =NOCH>--), C40H46014: C, 63.99; H, 6.17. Found: C, 64.00; H, 6.73 (lH, d, J = 8 Hz, 2-H), 6.79 (lH, d, J = 8 Hz, 6.38. I-H), 7.23 (lH, s, 4-H).
Methyl (I 7fl-acetoxy-3-hydroxy-6-oxo-1,3,5(10)estratrien-16c(-yl-2’,3’,4’-tri-O-acetyl-P_D-glucopyranosid)uronate (3)
A solution of 2 (437 mg) in methanol (200 ml) was shaken with 5% PdjC (527 mg) under a Hz gas stream at room temperature for 3.5 h. After removal of the catalyst by filtration through Celite, the filtrate was evaporated in vacua. The residue was subjected to column chromatography on silica gel using hexaneethyl acetate (1: 1, v/v). Elution of the adsorbent corresponding to the spot (R, 0.34) on TLC from eluate recrystallization of the and dichloromethane-methanol gave 3 (185 mg) as colorless needles: m.p. 207-210°C [u.]‘,”- 160.9” (c = 0.09, CHCl,). NMR (CDCI,) 6 ppm 0.78 (3H, s, 18-CH,), 2.02, 2.03, 2.06, 2.07 (each 3H, each s, 17p-, 2’-, 3’-, 4’-OCOCH,), 3.77 (3H, s, -COOCH,), 3.97 (lH, d, J = 10 Hz, 5/-H), 4.55 (lH, d, J = 7 Hz, l/-H), 7.03(1H,dd,.I=8,3Hz,2-H),7.18(1H,d,J=8Hz, I-H), 7.46 (lH, d, J = 3 Hz, 4-H). MSm/z 660 (M+). EtOH calcd for nm: 257, 317. Anal. U.V. Amax Cj3H400,4.H20: C, 58.41; H, 6.24. Found: C, 58.49; H, 6.20. Elution of the adsorbent corresponding to another spot (R, 0.15) on TLC and recrystallization of the gave dichloromethane-methanol from eluate 65 -hydroxy compound (40 mg) as colorless needles: m.p. 230_235”C, [a]‘,”- 106.3” (c = 0.09, CHCl,). NMR (CDCl,) 6 ppm 0.78 (3H, s, 18-CH,), 2.02, 2.03 and 2.06 (3H, 3H and 6H, each s, 17/3-, 2’-, 3’-, 4’-OCOCH,), 3.77 (3H, s, -COOCH3), 3.97 (lH, d, J = 10 Hz, 5/-H), 4.55 (lH, d, J = 7 Hz, I’-H), 6.72 (lH, dd, J = 9, 3 Hz, 2-H), 7.Oc7.55 (2H, m, 1-,4-H). MS m/z 644 (M - H,O)+. U.V. I.FgH nm: 283, 290 (shoulder). Anal. calcd for Cj3H,,0,,.HzO: C, 58.23; H, 6.51. Found: C, 58.61; H, 6.41.
Methyl (17b’-acetoxy-3-hydroxy-6-oxo-1,3,5(10)estratrien- 16z-yl-2’,3’,4’-tri-O-acetyl-B_u-glucopyranosid)uronate 3-sulfate 6-(O-carboxymethyl)oxime (5)
To a solution of 4 (242 mg) in pyridine (3 ml) was added pyridine (1 .Omltchlorosulfonic acid (0.5 ml) complex under ice-cooling, and the whole was stirred at room temperature overnight. The reaction mixture was poured into 5% NaHCO, (50 ml) and stirred for 30min. The solution was percolated through an Amberlite XAD-4 column (11 x 1 cm i.d.). After washing with water, the sulfate fraction was eluted with methanol containing one drop of concn NH,OH. The eluate was then subjected to column chromatography on silica gel. Elution with chloroform-methanol-water (80:20:2.5, by vol.) gave 5 (87 mg) as a colorless amorphous substance: NMR (CDCl,) 6 ppm 0.78 (3H, s, 18-CH,), 1.97, 1.98 and 2.04 (3H, 3H and 6H, each s, 17/I-, 2’-, 3’-, 4’-OCOCHj), 3.70 (3H, s, -COOCH,), 4.22 (lH, d, J = 9 Hz, I’-H), 4.77 (2H, s, =NOCH,-), 7.1c7.80 (3H, m, aromatic H). i.r. v~~;crn~‘: 1735 (C=O), 1240 (-OCOCH,), 1040 (-OSO,H). Methyl (17/I-acetoxy-3-hydroxy-6-oxo-1,3,_5(10)estratrien- 16u-yl-2’,3’,4’-tri-O-acetyl-t!3-o-gIucopyranosid)uronate 3-sulfate 6-[0-(p-nitrophenox_y) carboxylmethyljoxime (6)
To a solution of 5 (42 mg) in acetonitrile (4 ml) were added dicyclohexylcarbodiimide (65 mg) and p-nitrophenol (100 mg), and the whole was stirred at room temperature for a day in the dark. After removal of the acetonitriie by evaporation under an N, gas stream, the residue was subjected to preparative TLC using ethyl acetate as a developing solvent. Elution of the adsorbent corresponding to the spot
Specific antiserum to estriol 3-sulfate 16-glucuronide
(R, 0.4) with acetonitrile gave 6 (23 mg) as a colorless oily substance: NMR (CD,OD) 6 ppm 0.80 (3H, s, 1%CH,), 1.99, 2.06 (each 6H, each s, 17/?-, 2’-, 3’-, 4’-OCOCH,), 3.71 (3H, s, -COOCH,), 6.86 (2H, d,
J=9Hz,
NO,), 7.2c7.80
m, I-, 2-, 4-H), 8.09
(3H,
NW.
(2H, d, J = 9 Hz,
GOxoestriol 3-sulfate 16-glucuronide oxymethyl)oxime-BSA conjugate (8)
6-(O-carb-
To a solution of BSA (27 mg) in 0.1 M phosphate buffer (pH 7.4) (1.2 ml) was added a solution of 6 (23 mg) in pyridine (0.5 ml), and the whole was stirred at room temperature for 16 h. The resulting solution was dialyzed against water for 7 h and then against 0.05 M NaHCO, at 4°C overnight. Lyophilization of 3-sulfate 6-oxoestriol gave solution the acetate-methyl ester 6-(O-carb16-glucuronide oxymethyl)oxime-BSA conjugate (7). A solution of 7 in water (50 ml) was adjusted to pH 12 with 5 M NaOH (0.08 ml) and stirred at room temperature for a day. The resulting solution was dialyzed against cold running water overnight. Lyophilization of the solution gave 8 (33 mg). The molar steroid: protein ratio of the conjugate was spectrophotometrically (at 600nm) determined to be 14 using the color reaction with concn H,S04. Immunization of rabbits
Three male albino rabbits were used for immunization. The antigen (0.5-l mg) was dissolved in sterile isotonic saline (0.5 ml) and emulsified with complete Freund’s adjuvant (0.5 ml). The emulsion was injected into rabbits subcutaneously at multiple sites over the back and foot pads. The procedure was repeated once a month. The rabbits were bled 10 days after the booster injection. The sera were separated by centrifugation at 3000 rpm for 15 min and stored at -20°C. The antisera were thawed and diluted with 0.05 M phosphate buffer (pH 7.4) containing 0.004% BSA for assay.
201
incubation mixture was deproteinized with heat (60°C) and centrifuged. The supernatant was acidified with dilute acetic acid, and passed through a Sep-pak C,, cartridge equilibrated with 0.5% NH4H2P04. After washing with 0.5% NH,H,P04 (10 ml), the conjugate fraction was eluted with methanol (10ml). The double conjugate fraction was separated from the starting material by highperformance liquid chromatography (HPLC) on using 0.5”/, column p Bondapak C,, a NH,H,PO,-methanol (2.5:1, v/v) as a solvent at a flow rate of 1 ml/min. The desired fraction (I?, 12.5-15.5 min) was passed through an Amberlite XAD-4 column (7.5 x 0.6cm i.d.). After thorough washing with water, the double conjugate was eluted with methanol (10 ml) containing one drop of concn NHdOH. The eluate was purified again by HPLC in the manner described above. The desired [3H]estrio1 3-sulfate 16-glucuronide fraction (4.3 PCi) was concentrated in vacua and diluted in an assay buffer. The radioactive fraction showed the same chromatographic behavior with cold standard estriol TLC using 16-glucuronide on 3-sulfate chloroform-methanol-14% NH,OH (35: 15:6, by vol.) (R, 0.16) and HPLC. Measurement of radioactivity
The samples were counted on a Beckman mode1 LS 7000 liquid scintillation spectrometer employing toluene-Triton X-100 (tT 21) scintillant [12]. Assay procedure
A standard curve was constructed by setting up duplicate centrifuge tubes (10 ml) containing 0, 50, 100, 250, 500, 700, 1000, 2000 and 5000 pg of nonlabelled estriol 3-sulfate 16-glucuronide in assay buffer (0.1 ml) and 3H-labelled estriol 3-sulfate 16-glucuronide (85 pg, 8000dpm) in assay buffer (0.1 ml). After addition of assay buffer to bring the whole volume to 0.3 ml, the diluted antiserum (1:2000) (0.1 ml) was added and the mixture was incubated at 4°C overnight. After addition of dextran (0.05x)-coated charcoal (0.5%) in assay buffer (0.4 ml), the mixture was centrifuged at 3000 rpm for 15 min. A 6/10 ml aliquot of each supernatant was transferred into a counting vial containing a scintillation cocktail (5 ml) and radioactivity was counted. Cross-reaction studies
Preparation of [6,9-‘H]estriol3-sulfate
16glucuronide
[6,9-‘H]Estriol 16-glucuronide (50 PCi) was incubated with 105,OOOg supernatant (0.5ml: 12mg protein) of guinea pig liver homogenate in 0.25 M sucrose, 0.28 mM 3’-phosphoadenosine-5’-phosphosulfate [I I] (0.5 ml) in 1 mM Tris-HCI buffer (pH 8.7) and D-glucaro-1,4-lactone (1 mg) in 0.1 M Tris-HCl buffer (pH 7.5) (1.0 ml) containing 0.1 mM dithiothreitol at 37°C for 2 h. The
The specificity of antiserum raised against the estriol 3-sulfate 16-glucuronide-BSA conjugate was tested by cross-reaction studies [13] with 18 kinds of related steroids. The relative amounts required to reduce the initial binding of 3H-labelled estriol 3-sulfate 16-glucuronide by half, where the mass of non-labelled estriol 3-sulfate 16-glucuronide was arbitrarily chosen as 100% were calculated by the standard curve.
202
T~SHIO
NAMBARA
OH
et
al.
OAC
OAc
0 1
3
2
OAc
OAc
OAc
I
HO,SO ijOCH2COOH 4
5
OAc
OH
,..,OG
/OG’
HO$O
HOj SO NOCH&O-NHBSA
B
7
Fig. 1. Structures of estriol 3-sulfate I6-glucuronide-BSA conjugate and its related compounds
RESULTS
AND DISCUSSION
Previously, we could obtain antisera highly specific to estrone sulfate [4] and estrogen ring D glucuronides [9] using hapten-[C-6]-BSA conjugates as immunogens. In the present study, therefore, the preparation of 6-oxoestriol 3-sulfate 16-glucuronide 6-(0-carboxymethyl)oxime-BSA conjugate possessing a bridge at the C-6 position has been undertaken. To ensure the unequivocal coupling to the hapten through the (0-carboxymethyl)oxime bridge the glucuronyl moiety was protected as the acetatemethyl ester until after the attachment to BSA and then was selectively hydrolyzed under mild condition to expose the glucuronide [14]. 6-Oxoestriol3-benzyl ether 16-glucuronide acetatemethyl ester (1) [9] obtainable from 6-oxoestriol in two steps, was taken as a pertinent starting compound. Treatment with acetic anhydride and pyridine in the usual manner provided the 17-acetate (2) which on hydrogenolysis over palladium-on-charcoal was led to 6-oxoestriol 17-acetate 16-glucuronide acetatemethyl ester (3). Condensation of 3 with carboxymethoxylamine proceeded readily to afford the oxime (4). Treated with chlorosulfonic acid in pyridine, 4 was converted into the 3-sulfate (5). For the purpose of coupling the hapten with BSA, the activated ester method was used. The p-nitrophenyl ester (6) was prepared from 5 and p-nitrophenol by the carbodiimide method. The activated ester was condensed with BSA, yielding the BSA conjugate (7). Simultaneous elimination of the protecting groups in both glucuronyl and steroid moieties with alkali under the mild condition furnished the desired 6-oxoestriol 3-sulfate 16-glucuronide 6-(O-carb-
oxymethyl)oxime-BSA conjugate (8). The molar ratio of hapten to BSA of the conjugate was spectrophotometrically determined to be 14. The hapten-BSA conjugate thus obtained was used for immunization of animals. Several months after an initial injection of the antigen, immunized rabbits yielded antibodies exhibiting remarkably increased binding activity to estriol 3-sulfate 16-glucuronide. The preliminary test indicated that there was no substantial difference in the affinity and specificity in three immunized rabbits. Evaluation of the titer was carried out by incubating various dilutions of antiserum with a constant 3-sulfate estriol 3H-labelled amount of 16-glucuronide. The dilution of antiserum which was able to bind 50% of the labelled antigen was defined
0
I
I 320 Bound
I 600
900
(PM)
Fig. 2. Scatchard plot for antiserum raised against estriol 3-sulfate I6-glucuronide-BSA conjugate.
Specific antiserum to estriol 3-sulfate 16-glucuronide Table
I.
Per cent cross-reactions of anti-estriol 3-sulfate 16-glucuronide
antiserum
with selected steroids % Cross-reactivity
Steroid
Estriol 3-sulfate 16-glucuronide Estradiol 3-sulfate 17-glucuronide Estrone sulfate Estradiol 3-sulfate Estriol 3-sulfate 2-Hydroxyestradiol 3-sulfate Dehydroepiandrosterone sulfate Androstenediol 3-sulfate Estrone glucuronide Estradiol 3-glucuronide Estradiol 17-glucuronide Estriol 3-glucuronide Estriol 16-glucuronide Estriol 17-glucuronide 2-Hydroxyestradiol 2-glucuronide Pregnanediol 3-glucuronide
Estrone Estradiol Estrioi
00%) 100 0.06
as a titer. The binding affinity was determined by incubating a constant amount of antiserum with an increasing amount of the non-labelled antigen. The ratio of the bound to free (B/F) observed with antiserum was plotted against the concentration of bound antigen according to Scatchard [ 1.51.The asso3-sulfate anti-estriol constant of ciation 16-glucuronide antiserum was thus estimated to be (2.48 f 0.24) x lO’M_‘* (Fig. 2). The dose-response curve was constructed with 1: 8000
203
against hapten-BSA conjugates having a linkage through the C-6 position. In the present work it has been demonstrated that the use of an immunologic complex having both sulfate and glucuronide moieties intact is responsible for production of specific antibody to the doubly conjugated estrogen. The design of hapten-carrier conjugate in a similar fashion may be widely applicable to the preparation of antisera specific to other double conjugates. Development of a simple and reliable radiofor estriol 3-sulfate system immunoassay 16-glucuronide in biological fluids using specific antiserum will be the subject of a future communication.
authors express their sincere thanks to Professor T. Ido, Cyclotron Radioisotope Center of this University, for his kind help. They are also indebted to all the staff of central analytical laboratory of this Institute for elemental analyses and spectral measurements. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.
Acknowledgements-The
REFERENCES 1. Troen P., Nilsson B., Wiqvist N. and Diczfalusy E.: Pattern of oestriol conjugates in human cord blood, amniotic fluid and urine. Acta endocr., Copenh. 38 (1961) 361-382.
2. Touchstone J. C., Greene, J. W. Jr, McElroy R. C. and Murawec T.: Blood estriol conjugation during pregnancy. Biochemistry 2 (1963) 653-657. 3. Goebelsmann U., Chen L.-C., Saga M., Nakamura R. M. and Jaffe R. B.: Plasma concentration and protein binding of estriol and its conjugates in pregnancy. Acta endocr., Copenh. 74 (1973) 592-604. 4. Nambara T., Shimada K. and Ohta H.: Preparation of specific antiserum to estrone sulfate. J. steroid Biochem.
final dilution of anti-estriol 3-sulfate 16-glucuronide antiserum. The per cent bound radioactivity vs logarithm of the amount of estriol 3-sulfate 16-glucuronide showed a linear relationship over the range of 50 to 2000 pg. The specificity of antiserum was assessed by testing the ability of the closely related compounds to com13 (1980) 1075-1079. pete for binding sites on the antibody. The per cent 5. Nambara T., Ohkubo T. and Shimada K.: Determination of estrone sulfate in plasma by radiocross-reactions determined by the method of Abraimmunoassay without deconjugation. C/in. chim. Acta ham [13] with one of the three antisera are collected I19 (1982) 81-87. in Table 1. It is evident from the data that anti-estriol 6. Nambara T., Ohkubo T. and Shimada K.: A simple 3-sulfate 16-glucuronide antiserum was highly radioimmunoassay for estrone sulfate in unextracted specific. There were no remarkable cross-reactions plasma without deconjugation. Japan. J. clin. Chem. 11 (1982) 330-334. with estradiol 3-sulfate 17-glucuronide (0.0699, es7. Nambara T., Numazawa M., Tanaka T. and Ohkubo trio1 3-sulfate ( < 0.0 1%) and estriol 16-glucuronide T.: Preparation of specific antisera to estriol (0.74%). Other conjugated steroids and unconjugated 16-glucuronide by immunization with steroids attached estrogens exhibited no significant cross-reactivities. to protein through positions in ring A. J. pharmac. Dyn. 1 (1978) 55-61. In recent years several attempts have been made to develop direct radioimmunoassay of steroid gluc- 8 Nambaia T., Numazawa M., Tanaka T. and Ohkubo T.: Preparation of specific antisera to estradiol uronides and sulfates without prior deconjugation. It 17-glucuronide. J. steroid Biochem. 9 (1978) 785-790. is sufficiently substantiated that the structure remote 9 Nambara T., Shimada K., Ohkubo T. and Niwa T.: from the attached position of a carrier is readily Preparation and antigenic properties of estriol 16-glucuronideand estradiol 17-glucuroniderecognized in animals with hapten-protein conjugate, [C-6l_bovine serum albumin conjugates. J. steroid Bioand the immunologic specificity is less dependent on them. 16 (1982) 533-538. the individual steroid than on the particular func- 10. _. Numazawa M., Tanaka T. and Nambara T.: Detertional groups occupied. The previous studies revealed mination of estrogen ring D glucuronides in pregnancy plasma by direct radioimmunoassay without hvdrolvsis. that highly specific antisera discriminating the strucClin. chim. Acta 91 (1979) 169-17>. . . ture around the conjugated position of estrogen monoglucuronides and monosulfates are raised Il. Singer S. S.: Enzymatic sulfation of steroids VI. A *Mean & SD (n = 5).
simple rapid method for routine enzymatic preparation of 3’-phosphoadenosine-5’-phosphosulfate. Analyt. Biochem.
96 (1979) 34-38.
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TOSHIO NAMBARA et al.
12. Patterson M. S. and Greene R. C.: Measurement of low energy /I-emitters in aqueous solution by liquid scintillation counting of emulsions. Analyf. Chem. 37 (1965) 854-857. 13. Abraham G. E.: Solid-phase radioimmunoassay of 17/J-estradiol. J. clin. Endocr. Metab. 29 (1969) 866-870.
14. Chaudhri R. and Coulson W. F.: An improved hapten design for the radioimmunoassay of steroid glucuronides. J. steroid Biochem. 13 (1980) 691-696. 15. Scatchard G.: The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51 (1949) 660-672.
J. steroid Eiochem. Vol. 21, No. 2. pp. 199-203, 1984 Printed in Great Britain. All rights reserved
PREPARATION OF SPECIFIC ANTISERUM 3-SULFATE 16-GLUCURONIDE*
TO ESTRIOL
TOSHIO NAMBARA, TOSHIFUMI NIWA
and KAZUTAKE SHIMADA Pharmaceutical Institute, Tohoku University, Sendai 980, Japan (Received 22 September 1983)
Summary-The preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier through an (0-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Coupling of 6-oxoestriol 3-sulfate 16-glucuronide acetate-methyl ester 6-(0-carboxymethyl)oxime with BSA by the activated ester method followed by removal of the protecting groups with alkali provided the desired conjugate. The antisera raised against the conjugate in rabbits were highly specific to the double conjugate, estriol 3-sulfate 16-glucuronide, discriminating from ring A or D monoconjugated and unconjugated estrogens. The specificity of antisera elicited has been discussed on the basis of stereochemistry of the hapten-[C-6]-BSA
conjugate.
INTRODUCTION
The physiological significance of estriol 3-sulfate 16-glucuronidet, which is one of important double conjugates in the feto-placental unit, appears to be an attractive subject to be investigated [l-3]. Development of a simple and reliable radioimmunoassay method is required for the direct determination of the conjugate without hydrolysis and/or solvolysis. In preceding papers [4-lo], we reported the preparation of specific antisera to estrone 3-sulfate, estradiol 17-glucuronide and estriol 16-glucuronide, and their use for direct radioimmunoassay without prior deconjugation. These antisera were raised in the rabbit with hapten-[C-6]-bovine serum albumin (BSA) conjugates. On the basis of these findings the use of estriol 3-sulfate 16-glucuronide-[C-6]-BSA conjugate appeared to be promising for obtaining specific antiserum to the double conjugate. The present paper deals with the preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-BSA conjugate in which the hapten is linked to the carrier through an (O-carboxymethyl)oxime bridge at C-6 on the steroid nucleus. MATERIALS AND METHODS Chemicals
and reagents
[6,9-3H]Estriol 16-glucuronide (22.4 Ci/mmol) was supplied by Amersham International plc (Amersham, U.K.), and the radiochemical purity was
*Part CLXXXXVII of Studies on Steroids by T. Nambara; Part CLXXXXVI: T. Nambara, T. Niwa and K. Shimada: Jap. J. clin. Cbem. 12 (1983) 227-233. tThe following trivial names are used in this paper: 16-glucuronide = 3,17/?-dihydroxy- 1,3,5(10)estriol estratrien-16a-yl-fi-D-glucopyranosiduronate; estriol = 1,3,5(10)-estratriene-3,16a,l7/?-trial.
checked by TLC prior to use. Conjugated steroids were prepared in these laboratories by known methods. All free steroids were kindly donated by Teikoku Hormone Mfg. Co. (Tokyo, Japan). BSA Fraction V and bovine serum gamma-globulin were purchased from Sigma Chemical Co. (St Louis, MO), Sep-pak C,* cartridge and PBondapak C,, column from Waters Assoc., Inc. (Milford, MA), Amberlite XAD4 resin from Rohm and Haas Co. (Philadelphia, PA), complete Freund’s adjuvant and other reagents from Nakarai Chemicals Ltd. (Kyoto, Japan), respectively. A Hartley strain male guinea pig weighing 610 g was used for the sulfotransferase preparation.
Synthesis
of hapten-BSA
conjugate
All melting points were taken on a micro hot-stage apparatus and are uncorrected. Optical rotations were measured with a JASCO mode1 DIP-4 automatic polarimeter. Ultraviolet (u.v.) and infrared (i.r.) spectra were obtained with a Hitachi mode1 124 spectrometer and a JASCO model IRA-IS spectrometer, respectively. Mass spectra (MS) were measured with a Hitachi model M-52 spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded using tetramethylsilane as an internal standard on a JEOL model FX-100 spectrometer at 100 MHz (‘H). Abbreviations used s = singlet, d = doublet, dd = doublet of doublets and m = multiplet. Silica gel 60 HFlx4 (E. Merck AG, Darmstadt) was used for preparative TLC and silica gel 60 (70-230 mesh) or silica gel G (Type 60) (E. Merck AG) for column chromatography. Methyl (I 78 -acetoxy -3-benzyloxy -60x0 - 1,3,5(10)estratrien- I&-yl-2’,3’,4’-tri-O-acetyl-b-o-glucopyranosid)aronate (2) 6-Oxoestriol 199
l6-glucuronide
acetate-methyl
ester
200
TOSHIO NAMBARA
et al.
Methyl (17fl-acetoxy-3-hydroxy-6-oxo-1.3,5(10)3-benzyl ether (1) [9] (973 mg) was dissolved in pyriestratrien-16c(-yl-2’,3’,4’-tri-O-a~etyl-l)-~~-glu~odine (6 ml)-acetic anhydride (6 ml), and the solution was warmed at 70°C for 1 h. The reaction mixture pyranosidjuronate 6-(O-carhoxymethyl)oxime (4) was poured into ice-water and extracted with ethyl To a solution of 3 (202mg) and carboxyacetate. The organic layer was washed successively methoxylamine l/2 HCl (482 mg) in methanol (32 ml) with 5% HCl, water, 5% NaHCO, and water, dried was added sodium acetate (485 mg) in water (4 ml), over anhydrous Na,SO, and evaporated in vacua. and the whole was stirred at 35°C overnight. After The residue was subjected to column chroremoval of the methanol by evaporation, the rematography on silica gel. Elution with hexane-ethyl sulting solution was diluted with water and extracted acetate (2:1, v/v) and recrystallization of the eluate with ethyl acetate. The organic layer was washed with from dichloromethane-methanol gave 2 (450 mg) as water, dried over anhydrous Na,SO, and evaporated colorless needles: m.p. 16@164”C, [xl’,”- 35.1” in uacuo. The residue was subjected to column chro(c =O.lO, CHCl,). NMR (CDCl,) 6 ppm 0.78 (3H, matography on silica gel. Elution with s, 18-CH,), 2.02, 2.03, 2.06, 2.07 (each 3H, each s, chloroform-methanol-water (80:20:2.5, by vol) 17/3-, 2’-, 3’-, 4’-OCOCHI), 3.78 (3H, s, -COOCH,), gave 4 (267 mg) as a colorless oily substance: NMR 3.99 (lH, m, 5/-H), 4.30 (lH, in, 16/?-H), 4.57 (lH, d, (CDCl,) 6 ppm 0.67 (3H, s, 18-CH,), 2.02, 2.05 and J = 8 Hz, 1’-H), 4.90-5.40 (3H, m, 2’-, 3’-, 4-H), 5.01 2.08 (6H, 3H and 3H, each s, 17fl-, 2’-, 3’-. (lH, d, J = 6 Hz, 17c(-H), 5.13 (2H, s, 3-OC&C,H,), 4’-OCOCH,), 3.74 (3H, s, -COOCH,), 3.97 (lH, d, 7.Oc7.70 (8H, m, aromatic H). Anal. calcd for J = 9 Hz, 5/-H), 4.60 (3H, m, I’-H, =NOCH>--), C40H46014: C, 63.99; H, 6.17. Found: C, 64.00; H, 6.73 (lH, d, J = 8 Hz, 2-H), 6.79 (lH, d, J = 8 Hz, 6.38. I-H), 7.23 (lH, s, 4-H).
Methyl (I 7fl-acetoxy-3-hydroxy-6-oxo-1,3,5(10)estratrien-16c(-yl-2’,3’,4’-tri-O-acetyl-P_D-glucopyranosid)uronate (3)
A solution of 2 (437 mg) in methanol (200 ml) was shaken with 5% PdjC (527 mg) under a Hz gas stream at room temperature for 3.5 h. After removal of the catalyst by filtration through Celite, the filtrate was evaporated in vacua. The residue was subjected to column chromatography on silica gel using hexaneethyl acetate (1: 1, v/v). Elution of the adsorbent corresponding to the spot (R, 0.34) on TLC from eluate recrystallization of the and dichloromethane-methanol gave 3 (185 mg) as colorless needles: m.p. 207-210°C [u.]‘,”- 160.9” (c = 0.09, CHCl,). NMR (CDCI,) 6 ppm 0.78 (3H, s, 18-CH,), 2.02, 2.03, 2.06, 2.07 (each 3H, each s, 17p-, 2’-, 3’-, 4’-OCOCH,), 3.77 (3H, s, -COOCH,), 3.97 (lH, d, J = 10 Hz, 5/-H), 4.55 (lH, d, J = 7 Hz, l/-H), 7.03(1H,dd,.I=8,3Hz,2-H),7.18(1H,d,J=8Hz, I-H), 7.46 (lH, d, J = 3 Hz, 4-H). MSm/z 660 (M+). EtOH calcd for nm: 257, 317. Anal. U.V. Amax Cj3H400,4.H20: C, 58.41; H, 6.24. Found: C, 58.49; H, 6.20. Elution of the adsorbent corresponding to another spot (R, 0.15) on TLC and recrystallization of the gave dichloromethane-methanol from eluate 65 -hydroxy compound (40 mg) as colorless needles: m.p. 230_235”C, [a]‘,”- 106.3” (c = 0.09, CHCl,). NMR (CDCl,) 6 ppm 0.78 (3H, s, 18-CH,), 2.02, 2.03 and 2.06 (3H, 3H and 6H, each s, 17/3-, 2’-, 3’-, 4’-OCOCH,), 3.77 (3H, s, -COOCH3), 3.97 (lH, d, J = 10 Hz, 5/-H), 4.55 (lH, d, J = 7 Hz, I’-H), 6.72 (lH, dd, J = 9, 3 Hz, 2-H), 7.Oc7.55 (2H, m, 1-,4-H). MS m/z 644 (M - H,O)+. U.V. I.FgH nm: 283, 290 (shoulder). Anal. calcd for Cj3H,,0,,.HzO: C, 58.23; H, 6.51. Found: C, 58.61; H, 6.41.
Methyl (17b’-acetoxy-3-hydroxy-6-oxo-1,3,5(10)estratrien- 16z-yl-2’,3’,4’-tri-O-acetyl-B_u-glucopyranosid)uronate 3-sulfate 6-(O-carboxymethyl)oxime (5)
To a solution of 4 (242 mg) in pyridine (3 ml) was added pyridine (1 .Omltchlorosulfonic acid (0.5 ml) complex under ice-cooling, and the whole was stirred at room temperature overnight. The reaction mixture was poured into 5% NaHCO, (50 ml) and stirred for 30min. The solution was percolated through an Amberlite XAD-4 column (11 x 1 cm i.d.). After washing with water, the sulfate fraction was eluted with methanol containing one drop of concn NH,OH. The eluate was then subjected to column chromatography on silica gel. Elution with chloroform-methanol-water (80:20:2.5, by vol.) gave 5 (87 mg) as a colorless amorphous substance: NMR (CDCl,) 6 ppm 0.78 (3H, s, 18-CH,), 1.97, 1.98 and 2.04 (3H, 3H and 6H, each s, 17/I-, 2’-, 3’-, 4’-OCOCHj), 3.70 (3H, s, -COOCH,), 4.22 (lH, d, J = 9 Hz, I’-H), 4.77 (2H, s, =NOCH,-), 7.1c7.80 (3H, m, aromatic H). i.r. v~~;crn~‘: 1735 (C=O), 1240 (-OCOCH,), 1040 (-OSO,H). Methyl (17/I-acetoxy-3-hydroxy-6-oxo-1,3,_5(10)estratrien- 16u-yl-2’,3’,4’-tri-O-acetyl-t!3-o-gIucopyranosid)uronate 3-sulfate 6-[0-(p-nitrophenox_y) carboxylmethyljoxime (6)
To a solution of 5 (42 mg) in acetonitrile (4 ml) were added dicyclohexylcarbodiimide (65 mg) and p-nitrophenol (100 mg), and the whole was stirred at room temperature for a day in the dark. After removal of the acetonitriie by evaporation under an N, gas stream, the residue was subjected to preparative TLC using ethyl acetate as a developing solvent. Elution of the adsorbent corresponding to the spot
Specific antiserum to estriol 3-sulfate 16-glucuronide
(R, 0.4) with acetonitrile gave 6 (23 mg) as a colorless oily substance: NMR (CD,OD) 6 ppm 0.80 (3H, s, 1%CH,), 1.99, 2.06 (each 6H, each s, 17/?-, 2’-, 3’-, 4’-OCOCH,), 3.71 (3H, s, -COOCH,), 6.86 (2H, d,
J=9Hz,
NO,), 7.2c7.80
m, I-, 2-, 4-H), 8.09
(3H,
NW.
(2H, d, J = 9 Hz,
GOxoestriol 3-sulfate 16-glucuronide oxymethyl)oxime-BSA conjugate (8)
6-(O-carb-
To a solution of BSA (27 mg) in 0.1 M phosphate buffer (pH 7.4) (1.2 ml) was added a solution of 6 (23 mg) in pyridine (0.5 ml), and the whole was stirred at room temperature for 16 h. The resulting solution was dialyzed against water for 7 h and then against 0.05 M NaHCO, at 4°C overnight. Lyophilization of 3-sulfate 6-oxoestriol gave solution the acetate-methyl ester 6-(O-carb16-glucuronide oxymethyl)oxime-BSA conjugate (7). A solution of 7 in water (50 ml) was adjusted to pH 12 with 5 M NaOH (0.08 ml) and stirred at room temperature for a day. The resulting solution was dialyzed against cold running water overnight. Lyophilization of the solution gave 8 (33 mg). The molar steroid: protein ratio of the conjugate was spectrophotometrically (at 600nm) determined to be 14 using the color reaction with concn H,S04. Immunization of rabbits
Three male albino rabbits were used for immunization. The antigen (0.5-l mg) was dissolved in sterile isotonic saline (0.5 ml) and emulsified with complete Freund’s adjuvant (0.5 ml). The emulsion was injected into rabbits subcutaneously at multiple sites over the back and foot pads. The procedure was repeated once a month. The rabbits were bled 10 days after the booster injection. The sera were separated by centrifugation at 3000 rpm for 15 min and stored at -20°C. The antisera were thawed and diluted with 0.05 M phosphate buffer (pH 7.4) containing 0.004% BSA for assay.
201
incubation mixture was deproteinized with heat (60°C) and centrifuged. The supernatant was acidified with dilute acetic acid, and passed through a Sep-pak C,, cartridge equilibrated with 0.5% NH4H2P04. After washing with 0.5% NH,H,P04 (10 ml), the conjugate fraction was eluted with methanol (10ml). The double conjugate fraction was separated from the starting material by highperformance liquid chromatography (HPLC) on using 0.5”/, column p Bondapak C,, a NH,H,PO,-methanol (2.5:1, v/v) as a solvent at a flow rate of 1 ml/min. The desired fraction (I?, 12.5-15.5 min) was passed through an Amberlite XAD-4 column (7.5 x 0.6cm i.d.). After thorough washing with water, the double conjugate was eluted with methanol (10 ml) containing one drop of concn NHdOH. The eluate was purified again by HPLC in the manner described above. The desired [3H]estrio1 3-sulfate 16-glucuronide fraction (4.3 PCi) was concentrated in vacua and diluted in an assay buffer. The radioactive fraction showed the same chromatographic behavior with cold standard estriol TLC using 16-glucuronide on 3-sulfate chloroform-methanol-14% NH,OH (35: 15:6, by vol.) (R, 0.16) and HPLC. Measurement of radioactivity
The samples were counted on a Beckman mode1 LS 7000 liquid scintillation spectrometer employing toluene-Triton X-100 (tT 21) scintillant [12]. Assay procedure
A standard curve was constructed by setting up duplicate centrifuge tubes (10 ml) containing 0, 50, 100, 250, 500, 700, 1000, 2000 and 5000 pg of nonlabelled estriol 3-sulfate 16-glucuronide in assay buffer (0.1 ml) and 3H-labelled estriol 3-sulfate 16-glucuronide (85 pg, 8000dpm) in assay buffer (0.1 ml). After addition of assay buffer to bring the whole volume to 0.3 ml, the diluted antiserum (1:2000) (0.1 ml) was added and the mixture was incubated at 4°C overnight. After addition of dextran (0.05x)-coated charcoal (0.5%) in assay buffer (0.4 ml), the mixture was centrifuged at 3000 rpm for 15 min. A 6/10 ml aliquot of each supernatant was transferred into a counting vial containing a scintillation cocktail (5 ml) and radioactivity was counted. Cross-reaction studies
Preparation of [6,9-‘H]estriol3-sulfate
16glucuronide
[6,9-‘H]Estriol 16-glucuronide (50 PCi) was incubated with 105,OOOg supernatant (0.5ml: 12mg protein) of guinea pig liver homogenate in 0.25 M sucrose, 0.28 mM 3’-phosphoadenosine-5’-phosphosulfate [I I] (0.5 ml) in 1 mM Tris-HCI buffer (pH 8.7) and D-glucaro-1,4-lactone (1 mg) in 0.1 M Tris-HCl buffer (pH 7.5) (1.0 ml) containing 0.1 mM dithiothreitol at 37°C for 2 h. The
The specificity of antiserum raised against the estriol 3-sulfate 16-glucuronide-BSA conjugate was tested by cross-reaction studies [13] with 18 kinds of related steroids. The relative amounts required to reduce the initial binding of 3H-labelled estriol 3-sulfate 16-glucuronide by half, where the mass of non-labelled estriol 3-sulfate 16-glucuronide was arbitrarily chosen as 100% were calculated by the standard curve.
202
T~SHIO
NAMBARA
OH
et
al.
OAC
OAc
0 1
3
2
OAc
OAc
OAc
I
HO,SO ijOCH2COOH 4
5
OAc
OH
,..,OG
/OG’
HO$O
HOj SO NOCH&O-NHBSA
B
7
Fig. 1. Structures of estriol 3-sulfate I6-glucuronide-BSA conjugate and its related compounds
RESULTS
AND DISCUSSION
Previously, we could obtain antisera highly specific to estrone sulfate [4] and estrogen ring D glucuronides [9] using hapten-[C-6]-BSA conjugates as immunogens. In the present study, therefore, the preparation of 6-oxoestriol 3-sulfate 16-glucuronide 6-(0-carboxymethyl)oxime-BSA conjugate possessing a bridge at the C-6 position has been undertaken. To ensure the unequivocal coupling to the hapten through the (0-carboxymethyl)oxime bridge the glucuronyl moiety was protected as the acetatemethyl ester until after the attachment to BSA and then was selectively hydrolyzed under mild condition to expose the glucuronide [14]. 6-Oxoestriol3-benzyl ether 16-glucuronide acetatemethyl ester (1) [9] obtainable from 6-oxoestriol in two steps, was taken as a pertinent starting compound. Treatment with acetic anhydride and pyridine in the usual manner provided the 17-acetate (2) which on hydrogenolysis over palladium-on-charcoal was led to 6-oxoestriol 17-acetate 16-glucuronide acetatemethyl ester (3). Condensation of 3 with carboxymethoxylamine proceeded readily to afford the oxime (4). Treated with chlorosulfonic acid in pyridine, 4 was converted into the 3-sulfate (5). For the purpose of coupling the hapten with BSA, the activated ester method was used. The p-nitrophenyl ester (6) was prepared from 5 and p-nitrophenol by the carbodiimide method. The activated ester was condensed with BSA, yielding the BSA conjugate (7). Simultaneous elimination of the protecting groups in both glucuronyl and steroid moieties with alkali under the mild condition furnished the desired 6-oxoestriol 3-sulfate 16-glucuronide 6-(O-carb-
oxymethyl)oxime-BSA conjugate (8). The molar ratio of hapten to BSA of the conjugate was spectrophotometrically determined to be 14. The hapten-BSA conjugate thus obtained was used for immunization of animals. Several months after an initial injection of the antigen, immunized rabbits yielded antibodies exhibiting remarkably increased binding activity to estriol 3-sulfate 16-glucuronide. The preliminary test indicated that there was no substantial difference in the affinity and specificity in three immunized rabbits. Evaluation of the titer was carried out by incubating various dilutions of antiserum with a constant 3-sulfate estriol 3H-labelled amount of 16-glucuronide. The dilution of antiserum which was able to bind 50% of the labelled antigen was defined
0
I
I 320 Bound
I 600
900
(PM)
Fig. 2. Scatchard plot for antiserum raised against estriol 3-sulfate I6-glucuronide-BSA conjugate.
Specific antiserum to estriol 3-sulfate 16-glucuronide Table
I.
Per cent cross-reactions of anti-estriol 3-sulfate 16-glucuronide
antiserum
with selected steroids % Cross-reactivity
Steroid
Estriol 3-sulfate 16-glucuronide Estradiol 3-sulfate 17-glucuronide Estrone sulfate Estradiol 3-sulfate Estriol 3-sulfate 2-Hydroxyestradiol 3-sulfate Dehydroepiandrosterone sulfate Androstenediol 3-sulfate Estrone glucuronide Estradiol 3-glucuronide Estradiol 17-glucuronide Estriol 3-glucuronide Estriol 16-glucuronide Estriol 17-glucuronide 2-Hydroxyestradiol 2-glucuronide Pregnanediol 3-glucuronide
Estrone Estradiol Estrioi
00%) 100 0.06
as a titer. The binding affinity was determined by incubating a constant amount of antiserum with an increasing amount of the non-labelled antigen. The ratio of the bound to free (B/F) observed with antiserum was plotted against the concentration of bound antigen according to Scatchard [ 1.51.The asso3-sulfate anti-estriol constant of ciation 16-glucuronide antiserum was thus estimated to be (2.48 f 0.24) x lO’M_‘* (Fig. 2). The dose-response curve was constructed with 1: 8000
203
against hapten-BSA conjugates having a linkage through the C-6 position. In the present work it has been demonstrated that the use of an immunologic complex having both sulfate and glucuronide moieties intact is responsible for production of specific antibody to the doubly conjugated estrogen. The design of hapten-carrier conjugate in a similar fashion may be widely applicable to the preparation of antisera specific to other double conjugates. Development of a simple and reliable radiofor estriol 3-sulfate system immunoassay 16-glucuronide in biological fluids using specific antiserum will be the subject of a future communication.
authors express their sincere thanks to Professor T. Ido, Cyclotron Radioisotope Center of this University, for his kind help. They are also indebted to all the staff of central analytical laboratory of this Institute for elemental analyses and spectral measurements. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.
Acknowledgements-The
REFERENCES 1. Troen P., Nilsson B., Wiqvist N. and Diczfalusy E.: Pattern of oestriol conjugates in human cord blood, amniotic fluid and urine. Acta endocr., Copenh. 38 (1961) 361-382.
2. Touchstone J. C., Greene, J. W. Jr, McElroy R. C. and Murawec T.: Blood estriol conjugation during pregnancy. Biochemistry 2 (1963) 653-657. 3. Goebelsmann U., Chen L.-C., Saga M., Nakamura R. M. and Jaffe R. B.: Plasma concentration and protein binding of estriol and its conjugates in pregnancy. Acta endocr., Copenh. 74 (1973) 592-604. 4. Nambara T., Shimada K. and Ohta H.: Preparation of specific antiserum to estrone sulfate. J. steroid Biochem.
final dilution of anti-estriol 3-sulfate 16-glucuronide antiserum. The per cent bound radioactivity vs logarithm of the amount of estriol 3-sulfate 16-glucuronide showed a linear relationship over the range of 50 to 2000 pg. The specificity of antiserum was assessed by testing the ability of the closely related compounds to com13 (1980) 1075-1079. pete for binding sites on the antibody. The per cent 5. Nambara T., Ohkubo T. and Shimada K.: Determination of estrone sulfate in plasma by radiocross-reactions determined by the method of Abraimmunoassay without deconjugation. C/in. chim. Acta ham [13] with one of the three antisera are collected I19 (1982) 81-87. in Table 1. It is evident from the data that anti-estriol 6. Nambara T., Ohkubo T. and Shimada K.: A simple 3-sulfate 16-glucuronide antiserum was highly radioimmunoassay for estrone sulfate in unextracted specific. There were no remarkable cross-reactions plasma without deconjugation. Japan. J. clin. Chem. 11 (1982) 330-334. with estradiol 3-sulfate 17-glucuronide (0.0699, es7. Nambara T., Numazawa M., Tanaka T. and Ohkubo trio1 3-sulfate ( < 0.0 1%) and estriol 16-glucuronide T.: Preparation of specific antisera to estriol (0.74%). Other conjugated steroids and unconjugated 16-glucuronide by immunization with steroids attached estrogens exhibited no significant cross-reactivities. to protein through positions in ring A. J. pharmac. Dyn. 1 (1978) 55-61. In recent years several attempts have been made to develop direct radioimmunoassay of steroid gluc- 8 Nambaia T., Numazawa M., Tanaka T. and Ohkubo T.: Preparation of specific antisera to estradiol uronides and sulfates without prior deconjugation. It 17-glucuronide. J. steroid Biochem. 9 (1978) 785-790. is sufficiently substantiated that the structure remote 9 Nambara T., Shimada K., Ohkubo T. and Niwa T.: from the attached position of a carrier is readily Preparation and antigenic properties of estriol 16-glucuronideand estradiol 17-glucuroniderecognized in animals with hapten-protein conjugate, [C-6l_bovine serum albumin conjugates. J. steroid Bioand the immunologic specificity is less dependent on them. 16 (1982) 533-538. the individual steroid than on the particular func- 10. _. Numazawa M., Tanaka T. and Nambara T.: Detertional groups occupied. The previous studies revealed mination of estrogen ring D glucuronides in pregnancy plasma by direct radioimmunoassay without hvdrolvsis. that highly specific antisera discriminating the strucClin. chim. Acta 91 (1979) 169-17>. . . ture around the conjugated position of estrogen monoglucuronides and monosulfates are raised Il. Singer S. S.: Enzymatic sulfation of steroids VI. A *Mean & SD (n = 5).
simple rapid method for routine enzymatic preparation of 3’-phosphoadenosine-5’-phosphosulfate. Analyt. Biochem.
96 (1979) 34-38.
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TOSHIO NAMBARA et al.
12. Patterson M. S. and Greene R. C.: Measurement of low energy /I-emitters in aqueous solution by liquid scintillation counting of emulsions. Analyf. Chem. 37 (1965) 854-857. 13. Abraham G. E.: Solid-phase radioimmunoassay of 17/J-estradiol. J. clin. Endocr. Metab. 29 (1969) 866-870.
14. Chaudhri R. and Coulson W. F.: An improved hapten design for the radioimmunoassay of steroid glucuronides. J. steroid Biochem. 13 (1980) 691-696. 15. Scatchard G.: The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51 (1949) 660-672.