A radioimmunoassay (RIA) of estradiol was performed with the use of antiserum produced by estradiol-6-oxim-BSA. The precision of the assay was such as to result in a within-assay variance of 10.1% and a between-assay variance of 31.8%. The concentrations of estradiol thus determined in the serum of women with normal menstrual cycles were 69.0±40.6 pg/ml inthe menstrual phase, 127.8±70.3 pg/ml in the follicular phase, 281.9±113.5 pg/ml in the ovulatory phase, and 165.4±79.5 pg/ml in the luteal phase. The blood estradiol level durinh pregnancy ranged from 0.28 to 13.17 ng/ml in the first trimester, 2.6 to 48.1 ng/ml in the second trimester, and 13.5 to 130.0 nh/ml in the third trimester

Iseimaru Tadayuki

Kase Yasuaki

Miura Seiran

Mori Atsumi

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Acta med. Nagasaki. 21 :45-53Radioimmunoassay of Estradiolusing Estradiol-6-oxim-BSA                   Anti SerumAtsumi MORI, Tadayuki ISEIMARU, Yasuaki KASE              and Seiran MIURA*Department of Obstetrics & Gynecology, Nagasaki University School of Medicine           nagasaki, JapanReceived for publication, November 18, 1976     A radioimmunoassay (RIA) of estradiol was performed with the use of antiserum produced by estradiol-6-oxim-BSA. The precision of the assay was such as to result in a within-assay variance of 10.1% and a between-assay variance of 31.8%.      The concentrations of estradiol thus determined in the serum of women with normal menstrual cycles were 69.0±40.6 pg/ml inthe menstrual phase, 127.8±70.3 pg/ml in the follicular phase, 281.9±113.5 pg/ml in the ovulatory phase, and 165.4±79.5 pg/ml in the luteal phase.      The blood estradiol level durinh pregnancy ranged from 0.28 to 13.17 ng/ml in the first trimester, 2.6 to 48.1 ng/ml in the second trimester, and 13.5 to 130.0 nh/ml in the third trimester.                   INTRODUCTION      There have been a number of reports"~" on the radioimmunassay of serum estradiol since 1969 when this assay method was first revealed by ABRAHAM'). The study reported on here involved the RIA of estradiol using antiserum produced by estradiol-6-oxim BSA.*　 　森 　 淳 躬,石 丸 忠 之　 　 加 瀬 泰 昭,三 浦 清 轡                     METHODS 1) Reagents and Appliances      Radioactive estradiol : Estradiol-17i9-6.7-3H (New England Nuclear Corporation, Boston, U. S. A., specific activity 46.6 Ci/mM, E2-3H) was stored at 4°C after puri-fication in a Sephadex LH-20 microcolumn and dilution to a concentration of 1 x 105 dpm/ ml with methanol.       Non-radioactive estradiol : A pure substance (Teikokuzoki Co., Tokyo) was used, and a series of dilutions ranging in concentration from 5 ng/ml to 125 pg/ml were prepared and stored at 4°C. Bovine plasma r-globulin (ICN Pharmaceuticals Inc. , Ohio, U.S.A.) and bovine serum albumin (BSA) were used as serums to be added to the buffer.      Dextran charcoal solution : One gram of purified charcoal and 0.1 g of Dextran T-70 (Pharmacia Fine Chemicals Co., Sweden) were dissolved in 100 ml of 0.05M borate buffer and then stored at 4°C.      Scintillation solution : A dioxane scintillation solution was prepared by dissolving 0.5 g of 1.4 - Bis (5-Phenyl-oxazolyl) - benzene ' (POPOP) , 20 g of 2. 5-D phenyloxazole (PPO), and 200 g of naphthalene in 2,000 ml of dioxane.      Sephadex LH-20 column chromatography : The chromatographic procedure was in accordance with the method of WU et all). Sephadex LH-20 was suspended in a 85 :15 mixture of benzene and methanol and packed to a height of 60 mm in a 7 x 100 mm microcolumn.     Liquid scintillation counter : The ALOKA LSC-651 (Tokyo) liquid scintillation counter was utilized. Each sample was subjected to a scintillation count for five minutes. Counting vials were of the low background type.Table 1. Cross reaction with various steroidsof estradiol-6-oxim         BSA antibody (From Yoshida et all).)     Steroids Cross rreaction Steroids Cross reaction                    (/o) (%) estradiol-17(3 100 androst-4-ene, 3-17-dione 0.44 16 oxoestradiol 0.80 dehpdroepiandrosterone X0.08 estradion-17(3 0.80 pregnenolone <0.08 estrone 3.20 progesterone <0.08 2-methoxy-estrone <0.08 17 3-hydroxyprogesterone <0.08 estrone-3-sulfate 8.00 cortisol <0.08 estriol 1.77 corticosterone <0. 08 16-epiestriol 5.33 cortisone X0.08 estriol-16-glucuronide <0.08 cholesterol <0.08 testosterone 0.29     Estradiol antiserum : Estradiol was combined with BSA by introducing at OXO group at C-6, a position that has no bearing on the biologic activity of the substance, while the active OH groups at C-3 and C-17 were kept intact. The resulting estradiol-6-oxim-BSA was injected into rabbits to obtain an estradiol antiserum. The specificity of the antiserum is shown in Table 1. 2) Plasma Extraction, Separation and Purification      Blood samples were immediately centrifuged at 2,500-3,000 rpm for 10 minutes and then the serum was stored at -25'C. Al x 103 dpm volume of 3H-estradiol was added to the serum to produce an internal standard for recovery estimations. The substance under investigation was then extracted with 4ml of ethyl ether, following which the extract wasFig. 1 Standard curves with different antserum dilutionsdried by spraying of N2 gas in a thermostat at 37-40°C. The dried residue was dissolved in 0. 2ml of an 85 : 15 mixture of benzene and methanol. 3) Antiserum Titer      Binding rate of 1 x 104 dpm of 3H-estradiol to the antiserum was 86.3-L3.8% at an antiserum dilution of 2 x 104, 83.1±1.0% at an antiserum dilution of 4 x 104, and 71.64-1.8% at an antiserum dilution of 8 x 104 (Fig. 1).      The binding rate of standard estradiol in a range of 0-1,000 pg varied in an almost linear fashion when the antiserum dilution rate was 4 x 104. Within this standard range, the binding values displayed a standard deviation of ±0.2-±1.8, with a coefficient of 0.5-6.0%. These findings led us to utilize an antiserum dilution of 4 x 104 in the present study. 4) Incubation Time and Temprature      A study, using a 4 x 104-fold dilution of antiserum, was made as to the relationship between incubation time and incubation temperature. It was found that the incubation time required for 3H-estradiol to be bound to antiserum at a rate exceeding 80% was 90-120 minutes at 4°C and 20-30 minutes at room temperature (15-28'C). 5) Chromatography      A chromatographic study was made with a Sephadex LH-20 microcolum in accordance with the method of Wu5) et al. The elution patterns thus obtained are shown in Fig. 2.Fig. 2 Fractionation of estrogen in Sephadex LH-20 column chromatography6) Precision      A coefficient of variation of 2.5-17.0% was obtained with estradiol added, in amounts of 0-500 pg, to 1ml of distilled water. The precision was such as to give (4) within-assay variance of 10.1% (n=10) and a between-assay variance of 31.8% (n::--9). 7) RIA      After considering all of the afore-mentioned results and the conditions involved in the present study, we decided on the estradiol radioimmunoassay procedure as outlined in Fig. 3.      Incidentally, the concentration in lml of serum of estradiol of the unconjugated type was calculated from the following formula :        Estradiol level (Pg/ml) = R %Pg R e - (M+B))                                 X 1                                              Volume of seru m under                                             examination (ml)Serum, 0.1-2.Oml           Addition of estradiol-3H (1000 dpm) for correction             of recovery rate Extraction, with 4ml of ethyl ether (X2 mal) Drying with N2 gas spray in thermostat at 37-40°C Sephadex LH-20 column chromatography            0.5ml for correction of recovery rate            1.Oml submitted to assay Radioimmunoassay           1 x 104dpm of estradiol-3H added           Dried by N2 gas spray            Incubated for 30 minuttes at room temperature with           0.3ml of antiserum (1: 4 x 104 dilution) added. Separation of free from bound from            0.3m1 of dextran-coated charcoal addedin ice            water bath. Allowed to stand for minutes.           Centrifuged at. 2,600-3,000 rpm for 1.0 minutes. Determination            Concentrationcalculated from the standard curve.            Fig. 3 Sequential steps in estradiol RIAwhere E denotes the total amount of eluate in chromatography ; Re, recovery rate (%) ; R, the amount of eluate used in RIA ; M, the concentration of 3H-estradiol for correction of the recovery rate (Pg), % Pg, the concentration of estradiol as obtained from the sta-ndard curve (Pg) ; and B, the concentration of estradiol for the blank (Pg).                     RESULTS      In women with a normal menstrual cycle, the serum level of estradiol was 69.0 ± 40.6 Pg/ml (n=57) for the menstrual phsae, 281.9±113.5 Pg/ml (n= 15) for the ovulatory phase, and 165.4±79.5 Pg/ml (n=63) for the luteal phase. Determinations of serum estradiol levels were made daily throughout each cycle of four women experiencing normal menstrual cycles. The results are shewn in Fig. 4.      The serum estradiol level during pregnancy ranged from 0.28 to 13.17 ng/ml (n= 50) for the first trimester (7th-16th weeks), from 2.6 to 48. 1 ng/ml (n = 17) for the second trimester (17th-28th weeks), and from 13.5 to 130.0 ng/ml (n=111) for the third trimester (29th-43rd weets). The measurements made every week after the 36th week in 18 cases of pregnancy are plotted in Fig. 5.Fig. 4 Variation in estradiol level in 4 women with normal menstrual cycle        (The thick line indicates mean values for -,,cases.)          Hollow circles indicate values at onset of paids. Fig. 5 Variation in estradiol level at the 36th and later weeks of pregnancy                   DISCUSSION      Antiserum produced by estradiol-6-oxim BSA is more specific for estradiol and also of better quality respect to sensitivity and precision that conventional antiserum prod-uced by estradiol - 17(3 - succinyl BSA. On the other hand, it must be mentioned that estradiol is contained in the lipid extracted from serum, giving rise to a variation in the results of RIA, as pointed out by MAKINO47 and HASHIMOTO3). In the present study, a microcolumn was used with a view to removing lipid and purifying estradiol. As a result, a mean recovery rate of 68.7±10.2% was attained for the entire process of extraction, the rate being as high as 94.3±8.0% in as much as the microcolumn alone was concerned. The use of a Sephadex LH-20 column as a means of removing impurities in the blood involves a somewhat complicated procedure, whereas the microcolumn is capable of dealing with a number of specimens in a short period of time. The latter also is economical, permitting repeated use.      A review of actual measurements disclosed that the serum concentration of estradiol in women with a normal menstrual cycle roughly corresponded with the figures reported by other investigators (Table 2). Daily determination of blood estradiol level revealed that the level was at its lowest during the menstrual phase, then rose gradually during thefollicular phase, and sharply for4 to 5 days before the LH peak until a peak was reached during the ovulatory phase. It then showed a rapid decline, followed around 7 to 8 days after ovulation by a rise up to a peak for the luteal phase, and then finally declined.      The serum level of estradiol at different weeks of pregnancy varied markedly in different individuals. There was a prominent individual difference observed in the maximal value-ranging from 24.5 to 130 ng/ml-obtained in 18 cases from the 36th week until the onset of labor. The estradiol level at onset of pains was lower than that at 1-2 weeks before delivery in 14, but higher in 3, of 18 cases. No difference was noted in the one remaining case. Blood estradiol level rises sharply around 40 days after fertilizations), and the luteoplacental shift occurs around 50-60 days after fertilization) with the subsequent                    Table 2. Blood estradiol levels in women with normal                               menstrual cycles     Method of Time of Value obtained (pg/ml) Person in change     determina - determinatino (number of cases) of determination     tion                  Follicular phase 29-85 ( 9)      DIDD Ovulatory phase 318±98 (13) Baird (1969)                 Luteal phase 191±49 ( 6)                   Follicular phase 65.6±14.3 (15) Korenma n      CPBA Ov ulatory phase 124.5±49.6 (12) (1969,1970)                   Luteal phase 137.2±35.6 ( 6)                   Follicular phase 10^-100 (12)      CPB Ovulatory phase 140^-770 (12) Dufan (1970)                                     (350±145)                Luteal phase 190-340 (12)       RIA Follicular phase 108±13 Abraham (1969)                  (pooling)      RIA Women (pooling) 145±24 ( 6) Mikhail (1970)                 Ovulatory phase 513 42 8                  3rd day of menstrual 21 .4±2.62 ( 4)                   cycle (1971)       RIA 27th day of menstrual 68 .5±4.70 ( 4) Wu cycle                  Follicular phase 87± 34.5 (12)      RIA Ovulatory phase 352±128.8 (12) Yamaji (1972)                 Luteal pphase 110± 89.2 (16)                  Follicular phase 102.5±28.2 (10)       RIA Ovulatory phase 284.4±99.2 ( 5) Hashimoto(1974)                  Luteal phase 150.4±60.2 (17)                  Follicular phase 62.8±22.9       RIA Intermediate phase 204.9±84.7 Kawagoe (1974)                   Luteal phase 119.4±46.7                  Menstrual phase 69.0± 40.6 (29)                  Follicular phase 127.8± 70.3 (57)       RIA Ovulatory phase 281.9±113.5 (15) Miura (1976)                  Luteal phase 165.4- 79.5 (63)                  Peak of ovulation 384.7±127.7 ( 5)biosynthesis of estrogens in large quantities in the chorionic villi. It is evident that DHAS from the fetal adrenal is the precursor of estrogens, particularly estriol synthesized in the placenta. Hence, the urine or blood level of estriol is a reflection of the functional status of the fetus or placenta. At the same time, the variation in the level of estriol, whose activity is much less potent than that of estrogens, is also of importance in analyzing the physiologi-cal phenomena that occur during pregnancy. Further investigation is needed in this res-pect.                    REFERENCES 1) ABRAHAN, G. E. : J. Clin. Endocr., 29: 866 (1969). 2) CSAPO, A. I., et al. : Am. J. Obst. & Gynecol., 112: 1061 (1972). 3) HASHIMOTO, K. : Medical Postgraduates, 12 : 83 (1974) (Japanese). 4) MAKINO, T. : Folia Endocrinologica Japonica, 49: 629 (1972) (Japanese). 5) Wu, C. H. & LUDY, L. E. : Steroids, 18: 19 (1971). 6) YAMACHI, M. : Nippon Sanka-Fujinka Gakkai Zasshi, 24: 1045 (1972) (Japanese). 7) YOSHIDA, T. & MAKino, T. : Clinical Endocrinology, 21: 651 (1973) (Japanese).

Radioimmunoassay of Estradiolusing Estradiol-6-oxim-BSA Anti Serum

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Radioimmunoassay of Estradiolusing Estradiol-6-oxim-BSA Anti Serum

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