PRELIMINARY COMMUNICATION
/ . Biochem., 80, 191-194 (1976)
Enzyme Immunoassay of Testosterone Using the Testosterone-Glucoamylase Complex Kayoko TATEISHI,* Hiroshi YAMAMOTO,** Toshio OGIWARA,* Chozo HAYASHI,* and Masayasu KITAGAWA* *Central Laboratory for Clinical Investigation, Osaka University Hospital, and "Institute for Cancer Research, Osaka University Medical School, Fukushima-ku, Osaka, Osaka 553 Received for publication, April 7, 1976
A highly sensitive, reproducible method was established for enzyme-coupled immunoassay of testosterone, involving coupling of testosterone to glucoamylase [EC 3.2.1.3] with a water-soluble coupling reagent carbodiimide. No enzyme activity was lost during this coupling procedure. The sensitivity of the method was comparable to that of competitive protein binding assay.
sterone involving its coupling through oxime bridges to glucoamylase without any loss of its enzyme activity. Carbodiimide in aqueous solution was used as coupling agent. After formation of the immune complex, activity could be measured satisfactorily. Preparation of testosterone-protein complex: Testosterone 3-(O-carboxymethyl) oxime was prepared by the method of Erlanger etal. (7). A solution of 50 mg of glucoamylase from Rhizopus niveus (fine grade, Seikagaku Kogyo Co., Ltd., Tokyo) in 25 ml of water was adjusted to pH 5.5 and mixed with 30 mg of 1-cyclohexy 1- 3- (2- morphol iny 1 - 4 - ethy 1) carbodiimide methyl-/>-toluenesulfonate. Then a solution of 20 mg of testosterone-3-(0-carboxymethyl) oxime in 2 ml of dimethylformamide was added dropwise with stirring, the pH was readjusted to 5.5 and stirring was continued for 3 hr at room temperature (#). The reaction mixture was passed through a Sephadex G-25 column and fractions containing the glucoamylase-testosterone complex were collected
Recently enzyme-coupled immunoassay has been developed as an alternative to radioimmunoassay to avoid the problems of exposure to radioisotopes and their disposal {1—6). The most crucial point in establishing a sensitive and reproducible enzyme immunoassay system is to couple the antigen to the enzyme easily without loss of antigenicity and enzymic activity. Van Weemen and Schuurs (5,6) reported an enzyme immunoassay of estrogen involving coupling of estrogen to peroxidase [EC 1.11.1.7] with sec-butyl chloroformate in the organic solvent following the method of Erlanger etal.(7). However, when we tried to apply this method to coupling of testosterone with glucoamylase [EC 3.2.1.3], we found that most of the activity was lost during the coupling procedure. This paper reports a method for enzyme immunoassay of the steroid hormone, testoAbbreviations: PB-BSA, 0.02 M sodium phosphate buffer (pH 7.8)containing0.1% bovine serum albumin. Vol. 80, No. 1, 1976
191
PRELIMINARY COMMUNICATION
192
and lyophilized. The Iyophili2ed material was dissolved in 0.02 M sodium phosphate buffer (pH 7.8) just before use. Antisera: Antiserum against testosterone produced in rabbits immunized with testosterone 3-oxime-bovine serum albumin ( 9 ) was kindly supplied by Dr. C.A. Nugent, University of Arizona. The serum was diluted with phosphate buffer (pH 7.8) containing 0.1% bovine serum albumin (PB-BSA). Goat anti-rabbit IgG antiserum was purchased from Eiken Kagaku Co., Ltd., Tokyo. Goat antiserum and normal rabbit serum were diluted 1 : 20 and 1 : 200, respectively with PB-BSA. Competitive binding of testosterone and the testostercne-giucoamylase complex: For the enzyme immunoassay, 0.1 ml of testosteroneglucoamylase complex solution (equivalent to 3 ng of testosterone) was transferred to test tubes containing the reference and test samples of testosterone. Then, 0.2 ml of diluted antitestosterone antiserum was added and the mixtures were incubated for 2 hr at room temperature. After incubation, 0.2 ml each of diluted goat anti-rabbit IgG antiserum and normal rabbit serum were added. The mixtures were incubated for 24 hr at 4°, and then centrifuged at l,500Xff for 15 min. The supernatants were removed and the immune precipitates were washed twice with 1.5 ml of PB-BSA with centrifugation. Assay of glucoamylase activity in the immune precipitate: To the immune precipitates were added successively 0.4 ml of distilled water, 0.1 ml of sodium acetate buffer (pH 5.0) and 0.1 ml of 1% amylose A (M.W. approx. 4,000) solution. The mixtures were incubated for 2 hr at 37°, mixed with 10 fi\ of 0.5 N NaOH and centifuged for 15 min. The glucose produced was measured by the method of Greengard (10). Mixtures contained 0.5 ml of the above supernatant, 10 nmoles of triethanolamine-HCl buffer (pH 8.0), 1.5 ^moles of magnesium chloride, 50 nmoles of NADP + , 50 nmoles of ATP, and 22.5 fig of glucose-6phosphate dehydrogenase [EC 1.1.1.49]/hexokinase [EC 2. 7.1.1] from yeast (Boehringer, Mannheim) in a total volume of 1.06 ml. The mixtures were allowed to stand for 20 min at room temperature and then the intensity of
-50
at O40
O >-3O
0.1
1.0
10.0
Fig. 1. Determination of testosterone by fluorimetry. For competitive binding of testosterone and the testosterone-glucoamylase complex to anti-testosterone antiserum, 0.2 ml of antiserum was incubated with increasing amounts of testosterone and 0.1 ml of testosterone-glucoamylase complex (equivalent to 3 ng of testosterone) for 2 hr at room temperature. After further incubation of the mixture with goat anti-rabbit IgG anti-serum for 24 hr at 4°, the resulting immune precipitate was washed twice, and incubated to measure glucose production. Details of the procedure are described in the text. Means of duplicate determinations are plotted and the values of each determination are indicated by vertical bars.
fluorescence was measured in a fluorometer (Model 204, Hitachi Ltd., Tokyo). Preliminary experiments showed that the most suitable dilution of anti-testosterone antiserum for the immunoassay was 1 : 500 and this dilution was used throughout. Figure 1 shows the relation between the amount of testosterone added and the intensity of fluorescence of the reaction mixture, using 1 : 500 diluted anti-testosterone antiserum. This curve shows that testosterone can be determined within the range of 0.4 to 8 ng in this assay system. The sensitivity of the assay is comparable to that of competitive protein binding assay (77,72). The reactivity of anti-testosterone antiserum with dihydrotestosterone was 63% of that with testosterone. The accuracy of this assay was tested as follows. Two to 20 mg of testosterone in ethanol was added to tubes in duplicate, dried, mixed with 0.5 ml of water, and extracted with 3 ml of ether. One-third portion of extract / . Biochtm.
ENZYME IMMUNOASSAY OF TESTOSTERONE
5
ng 20
ji 10
10 20 ng Testosterone added
Fig. 2. Estimation of the accuracy of the enzyme immunoassay. Amounts of testosterone of 2 to 20 ng were added to 0.5 ml of water and extracted with 3 ml of ether. One-third portion of the extract was dried and subjected to enzyme immunoassay. Each point represents the mean of duplicate measurements.
was dried and submitted to the enzyme immunoassay and observed values were corrected for the recovery measured by adding ['Hitestosterone during the extraction procedure. Regression analysis of the data gave the equation: r=1.17X-0.03, as shown in Fig. 2, where Y is the concentration of testosterone estimated measured by the present enzyme immunoassay and X is the concentration of testosterone added. This indicates that the value determined by this procedure is slightly higher than the true value. Although in the present glucose assay, approximately 10~9 moles of glucose can be determined and correspond to 0.1 ng of testosterone, the range of 0.4 to 8.0 ng of testosterone, was used for the calibration curve because of practically available determination of serum level of testosterone. However, it should be possible to further increase the sensitivity of the method since theoretically 10~15 moles of glucose can be measured using the enzyme recycling system of Lowry et al. (13). This possibility is now under investigation. There are several crucial points in establishing a sensitive reproducible enzyme immunoassay system. In this communication, the authors dealt with following two points in establishing the enzyme immunoassay of Vol. 80, No. 1, 1976
193
steroids. First, sufficient enzyme activity should be retained inertly in the antigen-enzyme complex, and the activity should be measured easily with high sensitivity. In preliminary experiments we found that the glucoamylase activity decreased to one-thirtieth of the initial value during coupling by the procedure of Erlanger using sec-butyl chloroformate, whereas did not decrease using carbodiimide for coupling. The method using water-soluble carbodiimide is very simple and is obviously applicable to other steroids or biologically active substances of small molecular size. The procedure for immunoassay must include an easy procedure for removing free antigens or free enzymes contaminating the mixture. In the present method, free testosterone was removed by passing the testosterone-glucoamylase complex through a Sephadex G-25 gel column, but free glucoamylase was not. However, the free glucoamylase was easily removed by washing the immune precipitate twice with buffer as compared with the tedious washing out in the previous report of insulin ( 1 ) and a-fetoprotein (Yamamoto, H., Inada, T., & Kitagawa, M., unpublished data) using glutaraldehyde to from protein complex. It is considered that the amount of free or polymerized enzyme contaminating was very small and easily washed out. The values of testosterone obtained by the present enzyme immunoassay (Tateishi, K., Yamamoto, H., Hayashi, C , & Kitagawa, M., manuscript in preparation) are comparable with those reported by others (9,11,12). Thus, the present enzyme immunoassay can be applicable for routine assays. Very recently, during our preparation of this munuscript, F. Dray, et al. reported enzyme immunoassay of progesterone using progesterone-iS-galactosidase [EC 3.2.1.23] coupled with a water-soluble carbodiimide (14). The authors thank Miss R. Yamada for help in preparation of the manuscript. REFERENCES 1. Ishikawa, E. (1973) / . Biochem. 73, 1319-1321 2. Van Weemen, B.K. & Schuurs, A.H.W.M. (1971) FEBS Lett. 15, 232-236
194 3. Kato, K., Hamaguchi, Y., Fukui, H., & Ishikawa, E. (1975) FEBS Lett. 56, 370-373 4. Kitagawa, T. & Aikawa, T. (1976) / . Biochem. 79, 233-235 5. VanWeemen.B.K. & Schuurs, A.H.W.M. (1972) FEBS Lett. 24, 77-81 6. VanWeemen.B.K. & Schuurs, A.H.W.M. (1975) Immunochemistry 12, 667-670 7. Erlanger, B.F., Borek, F., Beiser, S.M., & Lieberman, S. (1959) / . Biol. Ckem. 234,1090-1094 8. Oliver, G.C., Parker, B.M., Bransfield, D.L., & Parker, C.W. (1968)/. Clin. Invest. 47, 1035-1042 9. Furuyama, S., Mayes, D.M., & Nugent, C.A. (1970) Steroids 16, 415-428
PRELIMINARY COMMUNICATION 10. Greengard, P. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.) pp.551-555, Academic Press, London/New York 11. Maeda, R., Okamoto, M., Wegienka, L.C., & Forsham, P.H. (1969) Steroids 13, 83-99 12. Mayes, D. & Nugent, C.A. (1968) / . Clin. Endocrinol. 28, 1169-1176 13. Lowry, O.H., Passonneau, J.V., Schltz, D.W., & Rock, M.K. (1961) / . Biol. Chem. 236, 2746-2755 14. Dray, F., Andrieu, J.M., & Renaud, F. (1975) Biochim. Biophys. Ada 403, 131-138
/ . Biochem.
/ . Biochem., 80, 191-194 (1976)
Enzyme Immunoassay of Testosterone Using the Testosterone-Glucoamylase Complex Kayoko TATEISHI,* Hiroshi YAMAMOTO,** Toshio OGIWARA,* Chozo HAYASHI,* and Masayasu KITAGAWA* *Central Laboratory for Clinical Investigation, Osaka University Hospital, and "Institute for Cancer Research, Osaka University Medical School, Fukushima-ku, Osaka, Osaka 553 Received for publication, April 7, 1976
A highly sensitive, reproducible method was established for enzyme-coupled immunoassay of testosterone, involving coupling of testosterone to glucoamylase [EC 3.2.1.3] with a water-soluble coupling reagent carbodiimide. No enzyme activity was lost during this coupling procedure. The sensitivity of the method was comparable to that of competitive protein binding assay.
sterone involving its coupling through oxime bridges to glucoamylase without any loss of its enzyme activity. Carbodiimide in aqueous solution was used as coupling agent. After formation of the immune complex, activity could be measured satisfactorily. Preparation of testosterone-protein complex: Testosterone 3-(O-carboxymethyl) oxime was prepared by the method of Erlanger etal. (7). A solution of 50 mg of glucoamylase from Rhizopus niveus (fine grade, Seikagaku Kogyo Co., Ltd., Tokyo) in 25 ml of water was adjusted to pH 5.5 and mixed with 30 mg of 1-cyclohexy 1- 3- (2- morphol iny 1 - 4 - ethy 1) carbodiimide methyl-/>-toluenesulfonate. Then a solution of 20 mg of testosterone-3-(0-carboxymethyl) oxime in 2 ml of dimethylformamide was added dropwise with stirring, the pH was readjusted to 5.5 and stirring was continued for 3 hr at room temperature (#). The reaction mixture was passed through a Sephadex G-25 column and fractions containing the glucoamylase-testosterone complex were collected
Recently enzyme-coupled immunoassay has been developed as an alternative to radioimmunoassay to avoid the problems of exposure to radioisotopes and their disposal {1—6). The most crucial point in establishing a sensitive and reproducible enzyme immunoassay system is to couple the antigen to the enzyme easily without loss of antigenicity and enzymic activity. Van Weemen and Schuurs (5,6) reported an enzyme immunoassay of estrogen involving coupling of estrogen to peroxidase [EC 1.11.1.7] with sec-butyl chloroformate in the organic solvent following the method of Erlanger etal.(7). However, when we tried to apply this method to coupling of testosterone with glucoamylase [EC 3.2.1.3], we found that most of the activity was lost during the coupling procedure. This paper reports a method for enzyme immunoassay of the steroid hormone, testoAbbreviations: PB-BSA, 0.02 M sodium phosphate buffer (pH 7.8)containing0.1% bovine serum albumin. Vol. 80, No. 1, 1976
191
PRELIMINARY COMMUNICATION
192
and lyophilized. The Iyophili2ed material was dissolved in 0.02 M sodium phosphate buffer (pH 7.8) just before use. Antisera: Antiserum against testosterone produced in rabbits immunized with testosterone 3-oxime-bovine serum albumin ( 9 ) was kindly supplied by Dr. C.A. Nugent, University of Arizona. The serum was diluted with phosphate buffer (pH 7.8) containing 0.1% bovine serum albumin (PB-BSA). Goat anti-rabbit IgG antiserum was purchased from Eiken Kagaku Co., Ltd., Tokyo. Goat antiserum and normal rabbit serum were diluted 1 : 20 and 1 : 200, respectively with PB-BSA. Competitive binding of testosterone and the testostercne-giucoamylase complex: For the enzyme immunoassay, 0.1 ml of testosteroneglucoamylase complex solution (equivalent to 3 ng of testosterone) was transferred to test tubes containing the reference and test samples of testosterone. Then, 0.2 ml of diluted antitestosterone antiserum was added and the mixtures were incubated for 2 hr at room temperature. After incubation, 0.2 ml each of diluted goat anti-rabbit IgG antiserum and normal rabbit serum were added. The mixtures were incubated for 24 hr at 4°, and then centrifuged at l,500Xff for 15 min. The supernatants were removed and the immune precipitates were washed twice with 1.5 ml of PB-BSA with centrifugation. Assay of glucoamylase activity in the immune precipitate: To the immune precipitates were added successively 0.4 ml of distilled water, 0.1 ml of sodium acetate buffer (pH 5.0) and 0.1 ml of 1% amylose A (M.W. approx. 4,000) solution. The mixtures were incubated for 2 hr at 37°, mixed with 10 fi\ of 0.5 N NaOH and centifuged for 15 min. The glucose produced was measured by the method of Greengard (10). Mixtures contained 0.5 ml of the above supernatant, 10 nmoles of triethanolamine-HCl buffer (pH 8.0), 1.5 ^moles of magnesium chloride, 50 nmoles of NADP + , 50 nmoles of ATP, and 22.5 fig of glucose-6phosphate dehydrogenase [EC 1.1.1.49]/hexokinase [EC 2. 7.1.1] from yeast (Boehringer, Mannheim) in a total volume of 1.06 ml. The mixtures were allowed to stand for 20 min at room temperature and then the intensity of
-50
at O40
O >-3O
0.1
1.0
10.0
Fig. 1. Determination of testosterone by fluorimetry. For competitive binding of testosterone and the testosterone-glucoamylase complex to anti-testosterone antiserum, 0.2 ml of antiserum was incubated with increasing amounts of testosterone and 0.1 ml of testosterone-glucoamylase complex (equivalent to 3 ng of testosterone) for 2 hr at room temperature. After further incubation of the mixture with goat anti-rabbit IgG anti-serum for 24 hr at 4°, the resulting immune precipitate was washed twice, and incubated to measure glucose production. Details of the procedure are described in the text. Means of duplicate determinations are plotted and the values of each determination are indicated by vertical bars.
fluorescence was measured in a fluorometer (Model 204, Hitachi Ltd., Tokyo). Preliminary experiments showed that the most suitable dilution of anti-testosterone antiserum for the immunoassay was 1 : 500 and this dilution was used throughout. Figure 1 shows the relation between the amount of testosterone added and the intensity of fluorescence of the reaction mixture, using 1 : 500 diluted anti-testosterone antiserum. This curve shows that testosterone can be determined within the range of 0.4 to 8 ng in this assay system. The sensitivity of the assay is comparable to that of competitive protein binding assay (77,72). The reactivity of anti-testosterone antiserum with dihydrotestosterone was 63% of that with testosterone. The accuracy of this assay was tested as follows. Two to 20 mg of testosterone in ethanol was added to tubes in duplicate, dried, mixed with 0.5 ml of water, and extracted with 3 ml of ether. One-third portion of extract / . Biochtm.
ENZYME IMMUNOASSAY OF TESTOSTERONE
5
ng 20
ji 10
10 20 ng Testosterone added
Fig. 2. Estimation of the accuracy of the enzyme immunoassay. Amounts of testosterone of 2 to 20 ng were added to 0.5 ml of water and extracted with 3 ml of ether. One-third portion of the extract was dried and subjected to enzyme immunoassay. Each point represents the mean of duplicate measurements.
was dried and submitted to the enzyme immunoassay and observed values were corrected for the recovery measured by adding ['Hitestosterone during the extraction procedure. Regression analysis of the data gave the equation: r=1.17X-0.03, as shown in Fig. 2, where Y is the concentration of testosterone estimated measured by the present enzyme immunoassay and X is the concentration of testosterone added. This indicates that the value determined by this procedure is slightly higher than the true value. Although in the present glucose assay, approximately 10~9 moles of glucose can be determined and correspond to 0.1 ng of testosterone, the range of 0.4 to 8.0 ng of testosterone, was used for the calibration curve because of practically available determination of serum level of testosterone. However, it should be possible to further increase the sensitivity of the method since theoretically 10~15 moles of glucose can be measured using the enzyme recycling system of Lowry et al. (13). This possibility is now under investigation. There are several crucial points in establishing a sensitive reproducible enzyme immunoassay system. In this communication, the authors dealt with following two points in establishing the enzyme immunoassay of Vol. 80, No. 1, 1976
193
steroids. First, sufficient enzyme activity should be retained inertly in the antigen-enzyme complex, and the activity should be measured easily with high sensitivity. In preliminary experiments we found that the glucoamylase activity decreased to one-thirtieth of the initial value during coupling by the procedure of Erlanger using sec-butyl chloroformate, whereas did not decrease using carbodiimide for coupling. The method using water-soluble carbodiimide is very simple and is obviously applicable to other steroids or biologically active substances of small molecular size. The procedure for immunoassay must include an easy procedure for removing free antigens or free enzymes contaminating the mixture. In the present method, free testosterone was removed by passing the testosterone-glucoamylase complex through a Sephadex G-25 gel column, but free glucoamylase was not. However, the free glucoamylase was easily removed by washing the immune precipitate twice with buffer as compared with the tedious washing out in the previous report of insulin ( 1 ) and a-fetoprotein (Yamamoto, H., Inada, T., & Kitagawa, M., unpublished data) using glutaraldehyde to from protein complex. It is considered that the amount of free or polymerized enzyme contaminating was very small and easily washed out. The values of testosterone obtained by the present enzyme immunoassay (Tateishi, K., Yamamoto, H., Hayashi, C , & Kitagawa, M., manuscript in preparation) are comparable with those reported by others (9,11,12). Thus, the present enzyme immunoassay can be applicable for routine assays. Very recently, during our preparation of this munuscript, F. Dray, et al. reported enzyme immunoassay of progesterone using progesterone-iS-galactosidase [EC 3.2.1.23] coupled with a water-soluble carbodiimide (14). The authors thank Miss R. Yamada for help in preparation of the manuscript. REFERENCES 1. Ishikawa, E. (1973) / . Biochem. 73, 1319-1321 2. Van Weemen, B.K. & Schuurs, A.H.W.M. (1971) FEBS Lett. 15, 232-236
194 3. Kato, K., Hamaguchi, Y., Fukui, H., & Ishikawa, E. (1975) FEBS Lett. 56, 370-373 4. Kitagawa, T. & Aikawa, T. (1976) / . Biochem. 79, 233-235 5. VanWeemen.B.K. & Schuurs, A.H.W.M. (1972) FEBS Lett. 24, 77-81 6. VanWeemen.B.K. & Schuurs, A.H.W.M. (1975) Immunochemistry 12, 667-670 7. Erlanger, B.F., Borek, F., Beiser, S.M., & Lieberman, S. (1959) / . Biol. Ckem. 234,1090-1094 8. Oliver, G.C., Parker, B.M., Bransfield, D.L., & Parker, C.W. (1968)/. Clin. Invest. 47, 1035-1042 9. Furuyama, S., Mayes, D.M., & Nugent, C.A. (1970) Steroids 16, 415-428
PRELIMINARY COMMUNICATION 10. Greengard, P. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.) pp.551-555, Academic Press, London/New York 11. Maeda, R., Okamoto, M., Wegienka, L.C., & Forsham, P.H. (1969) Steroids 13, 83-99 12. Mayes, D. & Nugent, C.A. (1968) / . Clin. Endocrinol. 28, 1169-1176 13. Lowry, O.H., Passonneau, J.V., Schltz, D.W., & Rock, M.K. (1961) / . Biol. Chem. 236, 2746-2755 14. Dray, F., Andrieu, J.M., & Renaud, F. (1975) Biochim. Biophys. Ada 403, 131-138
/ . Biochem.
