0021-972X/78/4705-0980$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 5 Printed in U.S.A.
Enzyme Immunoassay of Human Chorionic Gonadotropin Employing /?-Galactosidase as Label MOTOSUKE KIKUTANI, MASATSUNE ISHIGURO, TSUNEHIRO KITAGAWA, SADAOMI IMAMURA, AND SEIRAN MIURA Faculty of Pharmaceutical Sciences (M.K., M.I., T.K.) and the Department of Obstetrics and Gynecology (S.I., S.M.), Nagasaki University, Nagasaki 852, Japan Difficulty was experienced when this method was utilized for the determination of hCG in plasma samples from patients. Since the presence of the plasma may have affected this assay method, the following improvements were made: 1) the same volume of hormone-free plasma was added to the standard solutions of hCG, and 2) the volume of plasma sample was 10 /xl. The performance and validity of this assay were comparable to the RIA using [l2rTJhCG as tracer. The dose-response curves of both assays have the same slope and there was no significant difference between the values (correlation coefficient, Y = 0.96X + 1.53). (J Clin Endocrinol Mete&47:980,1978)
ABSTRACT. An enzyme-linked immunoassay was applied to the determination of hCG, a glycoprotein hormone usually assayed by RIA. For this purpose, an enzyme hormone conjugate was prepared by reacting hCG with /S-D-galactosidase (/?-Gal.) of E. coli in the presence of N-(m-maleimidobenzoyloxy) succinimide (MBS) as coupling reagent. The conjugate, after purification by affinity and gel chromatographies, was shown to exhibit sufficient enzyme activity and immunoactivity. The immunoassay of hCG was performed by the double antibody method and, using this assay, 0.4-250 mlU/ml hCG were detectable. This was about 10 times as sensitive as the RIA.
R
IA IS widely employed as a highly sensitive method for the determination of biologically active substances and is particularly useful for hormone research. This assay requires specific antisera of high affinity and labeled antigens of high specific activity. Labeling of antigens with radioactive isotopes (3H or 125I) is now extensively used for RIA, but it has some disadvantages, e.g. it is expensive and hazardous (1, 2). In order to eliminate the disadvantages of radiolabeling, several authors have modified the immunoassay using other kinds of labeling (3). Recent studies have shown that enzyme-linked immunoassay may overcome serious drawbacks inherent in the radiolabeling (4, 5). The present paper describes the preparation and purification of the /?-galactosidase-hCG conjugate and its application to the hCG assay. It is suggested that this method of coupling antigens to enzyme has general applicability. Received November 25, 1977. Address requests for reprints to: Motosuke Kikutani, Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki, 852, Japan.
Materials and Methods hCG (10,000 IU/mg) was obtained from Mochida Pharmaceutical Co., Japan. [l25I]hCG was prepared according to the procedure of Miura et al. (6) by the chloramine-T method. For immunization, rabbits were injected im five times on every 7 days with 10,000 IU hCG emulsified with Freund's complete adjuvant (1:1, vol/vol), followed by a similar booster injection on the 35th day after the first injection. The rabbit producing the highest amount of antibody was bled on the 40th day. y-Globulin fractions of antisera were prepared by 33% saturation with ammonium sulfate. Y-Globulin fractions were further treated with the 1:1 mixture of infantile urinary proteins and serum proteins from normal adult men to adsorb unwanted antibodies. After the precipitate formed was removed by centrifugation, the supernatant solution was used as the anti-hCG antibody and stored at a concentration of 50 mg/ml at —20 C. Assay of fi-galactosidase activity The /?-galactosidase preparation (EC 3.2.1.21) from E. coli obtained from Boehringer Mannheim Yamanouchi Co. contained 160,000 enzyme U/mg. The enzyme activity was determined by incubation at 30 C with 50 /d enzyme or sample solution and
980
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:03 For personal use only. No other uses without permission. . All rights reserved.
981
ENZYME IMMUNOASSAY OF hCG
150 /xl 10 4 M 4-methylumbelliferyl-/?-D-galactopy- the reaction and for stabilizing the MBS-resiranoside in 0.02 M phosphate buffer (pH 7.2) for 30 dues introduced, 0.5 ml 1 M citrate-phosphate min. The amount of 4-methylumbelliferone formed buffer (pH 5.0) was added to the mixture. The was estimated spectrofluorometrically (5). modified protein was separated from the reagents by Sephadex G-25 gel filtration (1.2 x hCG assay 45 cm, equilibrated with 0.1 M citrate buffer, The hCG assay employing /?-galactosidase-hCG pH 5.0). The eluate was monitored at 280 nm conjugate was carried out by measuring the com- for protein and was also assayed for maleimide petitive binding of hCG and the conjugate to anti- residues. hCG antibody. A mixture of 10 jul 40-fold diluted solution of the peak fraction from an Ultrogel AcA Coupling of MBS-acylated hCG with (3-galac44 column, 10 jul appropriate amount of hCG (0-64 tosidase. A mixture of MBS-acylated hCG ng) or serum sample solution, 100 /il antiserum to solution from the Sephadex G-25 column (3.6 hCG (200,000-fold diluted) in 0.02 M sodium phos- nmol as maleimide residues) and /?-galactosidphate buffer (pH 7.0) containing 0.1% bovine serum ase from E. coli (0.5 mg, 0.93 nmol) dissolved albumin (BSA), 1 mM magnesium chloride, 0.1 M in 1 ml 0.05 M phosphate buffer (pH 7.0) was sodium chloride, and 0.1% sodium azide (buffer A) incubated for 2 h at room temperature. in a final volume of 0.2 ml was incubated at 4 C for 72 h. Then, 100 ju.1 goat antirabbit 7S y-globulin Purification of the conjugate. The reaction antibody (Hyland, Division of Travenol Laborato- mixture was then passed through a Concanaries, Inc.) and 1% normal rabbit serum were added. valin A (Con A) Sepharose 4B column (1 X 20 After further incubation at 4 C for 24 h, the mixture cm) equilibrated with 0.05 M phosphate buffer was centrifuged at 800 X g for 30 min and /?-galaccontaining 0.15 M sodium chloride (pH 7.5) to tosidase activity of the precipitate was measured. remove unreacted /?-galactosidase. As Fig. 1 In the case of the RIA, all of the procedures were the same as described above, except that /?-galactosidase-hCG conjugate was replaced by l25I-la.05 M phosphate buffer,pH 7.5 0.2 M o
Vol. 47, No. 5 Printed in U.S.A.
Enzyme Immunoassay of Human Chorionic Gonadotropin Employing /?-Galactosidase as Label MOTOSUKE KIKUTANI, MASATSUNE ISHIGURO, TSUNEHIRO KITAGAWA, SADAOMI IMAMURA, AND SEIRAN MIURA Faculty of Pharmaceutical Sciences (M.K., M.I., T.K.) and the Department of Obstetrics and Gynecology (S.I., S.M.), Nagasaki University, Nagasaki 852, Japan Difficulty was experienced when this method was utilized for the determination of hCG in plasma samples from patients. Since the presence of the plasma may have affected this assay method, the following improvements were made: 1) the same volume of hormone-free plasma was added to the standard solutions of hCG, and 2) the volume of plasma sample was 10 /xl. The performance and validity of this assay were comparable to the RIA using [l2rTJhCG as tracer. The dose-response curves of both assays have the same slope and there was no significant difference between the values (correlation coefficient, Y = 0.96X + 1.53). (J Clin Endocrinol Mete&47:980,1978)
ABSTRACT. An enzyme-linked immunoassay was applied to the determination of hCG, a glycoprotein hormone usually assayed by RIA. For this purpose, an enzyme hormone conjugate was prepared by reacting hCG with /S-D-galactosidase (/?-Gal.) of E. coli in the presence of N-(m-maleimidobenzoyloxy) succinimide (MBS) as coupling reagent. The conjugate, after purification by affinity and gel chromatographies, was shown to exhibit sufficient enzyme activity and immunoactivity. The immunoassay of hCG was performed by the double antibody method and, using this assay, 0.4-250 mlU/ml hCG were detectable. This was about 10 times as sensitive as the RIA.
R
IA IS widely employed as a highly sensitive method for the determination of biologically active substances and is particularly useful for hormone research. This assay requires specific antisera of high affinity and labeled antigens of high specific activity. Labeling of antigens with radioactive isotopes (3H or 125I) is now extensively used for RIA, but it has some disadvantages, e.g. it is expensive and hazardous (1, 2). In order to eliminate the disadvantages of radiolabeling, several authors have modified the immunoassay using other kinds of labeling (3). Recent studies have shown that enzyme-linked immunoassay may overcome serious drawbacks inherent in the radiolabeling (4, 5). The present paper describes the preparation and purification of the /?-galactosidase-hCG conjugate and its application to the hCG assay. It is suggested that this method of coupling antigens to enzyme has general applicability. Received November 25, 1977. Address requests for reprints to: Motosuke Kikutani, Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki, 852, Japan.
Materials and Methods hCG (10,000 IU/mg) was obtained from Mochida Pharmaceutical Co., Japan. [l25I]hCG was prepared according to the procedure of Miura et al. (6) by the chloramine-T method. For immunization, rabbits were injected im five times on every 7 days with 10,000 IU hCG emulsified with Freund's complete adjuvant (1:1, vol/vol), followed by a similar booster injection on the 35th day after the first injection. The rabbit producing the highest amount of antibody was bled on the 40th day. y-Globulin fractions of antisera were prepared by 33% saturation with ammonium sulfate. Y-Globulin fractions were further treated with the 1:1 mixture of infantile urinary proteins and serum proteins from normal adult men to adsorb unwanted antibodies. After the precipitate formed was removed by centrifugation, the supernatant solution was used as the anti-hCG antibody and stored at a concentration of 50 mg/ml at —20 C. Assay of fi-galactosidase activity The /?-galactosidase preparation (EC 3.2.1.21) from E. coli obtained from Boehringer Mannheim Yamanouchi Co. contained 160,000 enzyme U/mg. The enzyme activity was determined by incubation at 30 C with 50 /d enzyme or sample solution and
980
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:03 For personal use only. No other uses without permission. . All rights reserved.
981
ENZYME IMMUNOASSAY OF hCG
150 /xl 10 4 M 4-methylumbelliferyl-/?-D-galactopy- the reaction and for stabilizing the MBS-resiranoside in 0.02 M phosphate buffer (pH 7.2) for 30 dues introduced, 0.5 ml 1 M citrate-phosphate min. The amount of 4-methylumbelliferone formed buffer (pH 5.0) was added to the mixture. The was estimated spectrofluorometrically (5). modified protein was separated from the reagents by Sephadex G-25 gel filtration (1.2 x hCG assay 45 cm, equilibrated with 0.1 M citrate buffer, The hCG assay employing /?-galactosidase-hCG pH 5.0). The eluate was monitored at 280 nm conjugate was carried out by measuring the com- for protein and was also assayed for maleimide petitive binding of hCG and the conjugate to anti- residues. hCG antibody. A mixture of 10 jul 40-fold diluted solution of the peak fraction from an Ultrogel AcA Coupling of MBS-acylated hCG with (3-galac44 column, 10 jul appropriate amount of hCG (0-64 tosidase. A mixture of MBS-acylated hCG ng) or serum sample solution, 100 /il antiserum to solution from the Sephadex G-25 column (3.6 hCG (200,000-fold diluted) in 0.02 M sodium phos- nmol as maleimide residues) and /?-galactosidphate buffer (pH 7.0) containing 0.1% bovine serum ase from E. coli (0.5 mg, 0.93 nmol) dissolved albumin (BSA), 1 mM magnesium chloride, 0.1 M in 1 ml 0.05 M phosphate buffer (pH 7.0) was sodium chloride, and 0.1% sodium azide (buffer A) incubated for 2 h at room temperature. in a final volume of 0.2 ml was incubated at 4 C for 72 h. Then, 100 ju.1 goat antirabbit 7S y-globulin Purification of the conjugate. The reaction antibody (Hyland, Division of Travenol Laborato- mixture was then passed through a Concanaries, Inc.) and 1% normal rabbit serum were added. valin A (Con A) Sepharose 4B column (1 X 20 After further incubation at 4 C for 24 h, the mixture cm) equilibrated with 0.05 M phosphate buffer was centrifuged at 800 X g for 30 min and /?-galaccontaining 0.15 M sodium chloride (pH 7.5) to tosidase activity of the precipitate was measured. remove unreacted /?-galactosidase. As Fig. 1 In the case of the RIA, all of the procedures were the same as described above, except that /?-galactosidase-hCG conjugate was replaced by l25I-la.05 M phosphate buffer,pH 7.5 0.2 M o
