(CANCER RESEARCH 45, 5835-5839, November 1985]

Measurement of Human Serum Immunoreactive 7-Glutamyl Transpeptidase in

Patients with Malignant Tumors Using Enzyme-linked ImmunosorbentAssay1

Naoyuki Taniguchi,2 Susumu lizuka, Zhang Nai Zhe, Stephanie House, Noriko Yokosawa, Minoru Ono,

Kenji Kinoshita, Akira Makita, and Chihiro Sekiya

Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060, Japan [N. T., S. l., Z. N. Z., S. H., N. Y., M. 0., K. K., A. M.], andDepartment of Internal Medicine, Asahikawa Medical College, Asahikawa 78-11, Japan [C. S.J

ABSTRACT

An enzyme-linked immunosorbent assay for human immuno-reactive 7-glutamyl transpeptidase (7-GTP) was developed. This

assay was found to be simple, reproducible, and sensitive to 10ng of the enzyme. Serum immunoreactive 7-GTP content was

significantly elevated in patients with various malignant tumorsincluding liver cell cancer, lung cancer, gastric cancer, esopha-geal cancer, and colorectal cancer. On the other hand, in sera ofpatients with nonneoplastic diseases, the immunoreactive 7-GTPcontent was not significantly elevated. No correlation was foundbetween the serum levels of 7-GTP determined by enzymaticassay and enzyme-linked immunosorbent assay, which indicates

that, due to the presence of endogenous inhibitors and/or activators in sera, the enzyme activity may not reflect the trueamount of enzyme protein. The measurement of immunoreactive7-GTP protein in sera appears to be useful for the detection andmonitoring of certain malignant tumors.

INTRODUCTION

7-GTP3 is widely distributed in mammalian tissues (see, forreviews, Refs. 8 and 17). A high 7-GTP activity has been reportedin patients with some malignant diseases such as metastaticliver cancer, mammary cancer, and colorectal cancer. However,it is well known that the enzyme activity also increases in somenonmalignant diseases including cholestatic liver disease andacute and chronic hepatitis. Therefore it is difficult to use theenzymatic assay of 7-GTP in serum as an indicator of cancer.

Previous studies on rat 7-GTP in our laboratory indicated thatthe immunoreactive level of 7-GTP in the hepatoma tissues does

not correlate well with the enzyme activity (16). Quite recentlywe also found that the manganese-containing Superoxide dis-

mutase exists in lung tumor tissues in an enzymatically inactivebut immunochemically active form (4). This indicates that, due tothe presence of endogenous inhibitors or activators, the enzymeactivity may not reflect the true amount of enzyme protein. Thesestudies prompted us to develop a method for the determinationof immunoreactive 7-GTP in sera of patients with malignanttumors. An immunochemical assay of 7-GTP, in which 7-GTPwas quantified as a protein entity, could provide a reliable meansof measuring 7-GTP overproduction or release into the blood

1This work was supported in part by grants-in-aid for scientific research from

the Ministry of Education, Science, and Culture, Japan, and for cancer researchfrom the Ministry of Health and Welfare, Japan. This work was done in part at theCentral Research Institute of the Hokkaido University School of Medicine.

2To whom requests for reprints should be addressed.3The abbreviations used are: >-GTP, -x-glutamyl transpeptidase; ELISA, en

zyme-linked immunosorbent assay.Received 11/6/84; revised 8/7/85; accepted 8/14/85.

stream in cancer patients. We found that the assay of immuno-reactive 7-GTP in sera using ELISA provides important infor

mation for diagnosis and monitoring of several malignant tumors.

MATERIALS AND METHODS

Chemicals. Alkaline phosphatase (calf intestine) was purchased fromSigma Chemical Co., St. Louis, MO. All other reagents used were ofanalytical grade.

Tissues. Normal human kidney and liver tissues were obtained atautopsy of a male patient who died of a heart attack. The tissuescontained no microscopically detectable pathological involvement. Autopsy was performed within a few hours of death. The patient did nothave carcinoma or hepatic diseases. Liver cell cancer tissue was obtainedat surgery. After removal, the tissues were stored at -80°C until use.

Serum Samples. Serum samples were obtained from healthy volunteers whose liver function tests were normal and from hospitalizedpatients. Sera were stored at -80°C until used. All patients in this study

were hospitalized at Asahikawa Medical College Hospital and KuriyamaRed Cross Hospital, Hokkaido, Japan. Five ml of fresh blood werecollected by venipuncture, randomly numbered, and immediately sent tothe laboratory for these studies. Clinical diagnoses were not known priorto assay, and patients' diagnoses were not matched with test code

numbers until the assay was complete. The sera were obtained frompatients prior to any treatment or operation. The sera of three patientswith liver cell cancer and three patients with adenocarcinoma of the lungwere collected both before and after operation. The tumors of these sixpatients were all within 1 x 3 x 3 cm. Most of the cancer patients werein Stages III and IV. The origins of the metastatic liver cell cancers weregall bladder and stomach. Diagnoses of malignant tumors had beenconfirmed by X-ray, angiography, endoscopy, and computed tomography

findings, as well as by the biopsy or operation.Purification of Human Kidney and Liver Cell Cancer -,-GTPs. -, -

GTP was first purified from normal human kidney according to themethod of Tate and Ross (18). The purified enzyme had specific activityof 850 units/mg of protein. The kidney enzyme is composed of twononidentical subunits with molecular weights of 62,000 and 22,000 asjudged by polyacrylamide gel electrophoresis in the presence of sodiumdodecyl sulfate (Fig. 1). 7-GTPs from various human tissues wereimmunologically identical (9); therefore -y-GTPs from human liver cell

cancer and normal liver were purified with the use of an ¡mmunoaffinitycolumn in the final step, using the antibody against human kidney 7-GTP(13). The specific activities of -y-GTPs from human liver cancer and

normal liver were 280 and 230 units/mg of protein, respectively.Preparation of Polyclonal Antibody. Antiserum against the purified

enzyme from human kidney was raised in a goat. Purified vGTP (0.5mg) was injected s.c. with Freund's complete adjuvant, and a booster

injection of 0.3 mg of enzyme was given after 2 wk. Three wk after thebooster injection, the animal was bled, and antiserum was obtained. Thepurity and specificity of the antiserum were checked by the doubleimmunodiffusion technique and immunoelectrophoresis, and the anti-serum was found to be specific for 7-GTP. IgG fractions were obtainedby DEAE-cellulose chromatography as described (16). The antibodyprecipitated over 95% of the purified 7-GTP activity.

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HUMAN SERUM IMMUNOREACTIVE 7-GTP AND CANCER

origin

was measured using an ImmunoReader NJ 2000 (InterMed, Japan) at410 nm. 7-GTP content was defined as the amount of immunochemically

reactive protein determined by ELISA.Standard Curve for ELISA. All the standard samples (7-GTP purified

from human kidney) were applied to the microtiter plate in triplicate, andELISA was carried out as described above. Using this method, 7-GTP

was detectable between 10 ng and 1000 ng. Chart 1 shows a typicalstandard curve for the assay.

Reproducibility. The reproducibility of the assay was examined byestimating interassay and intraassay variances. For interassay precision,the coefficient of variance among 5 individual measurements was 10%,while for intraassay precision, the coefficient was 9.5% when 30 individual measurements were done.

Accuracy. The percentage of recovery of exogenously added kidney7-GTP was measured by successive ELISAs. Recoveries were 95%when 500 ng of kidney 7-GTP were added to each of 100-^1 samples of

serum at concentrations of 5 pg/m\. When serum was diluted over awide range, the measured 7-GTP content decreased proportionally. This

indicates the validity of the assay procedure.Activity Assay of 7-GTP. 7-GTP was assayed by the method de

scribed previously (12,14) using a Gilford Model 250 spectrophotometer.One unit of the enzyme is that amount which catalyzes the formationsof 1 fimo\ of p-nitroaniline per min at 30°C.

Statistical Analysis. The statistical evaluations were made by Student's f test.

Fig. 1. Polyacrylamidegel electrophoresis in the presence of sodium dodecylSulfate.Purified normal kidney -y-GTPwas subjected to polyacrylamidegel electrophoresis in the presenceof sodium dodecyl sulfate. Ten /¿gof the purified enzymewere loaded on the gel and electrophoresed for 4 h at room temperature using acurrent of 20 mA/gel. Protein was stained with Coomassie brilliant blue.

Immunological Studies. The specificity of the antibody was examinedas follows. The 7-GTP purified from human kidney (0.005 units) waspreincubated with varying amounts of antibody in 50 rriM Tris-HCI, pH8.0, containing 0.15 M NaCI at 37 °Cfor 30 min and 4°Covernight. After

preincubation, the activities of the mixtures and the supernatants aftercentrifugation were assayed. Immunodiffusion in agar was conductedaccording to the method of Ouchterlony (10).

Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanolwas performed by the method previously described (6). Differential (2.5%and 7.0%) disc polyacrylamide gel electrophoresis was carried out asdescribed by Wright and Mallmann (20).

ELISA. ELISA was essentially done as described (16) following themethod of Engvall (2). Polystyrene microtiter plates (A/S Nunc, Roskilde,Denmark) were used as a solid phase. Alkaline phosphatase was usedfor labeling antibodies. All incubations were done in a moist chamber.Goat anti-human 7-GTP IgG solution was diluted 200-fold in 50 mw

sodium bicarbonate buffer, pH 9.6, and 100 ¡Aof the solution were addedto each well of the microtiter plates and incubated at 4°Covernight. The

solutions were removed by aspiration, and the wells were washed 3times with phosphate-buffered saline containing 0.05% Tween-20 and0.1% bovine serum albumin (washing buffer); the remaining protein-binding sites were blocked with the same buffer. Standard 7-GTP and

sample solutions were diluted in 50 mw potassium phosphate buffer, pH7.4, and 50 ¿ilof the solutions and the same volume of washing bufferwere added to each well. The plates were incubated overnight at 4°C.

After removal of this solution, the wells were washed thoroughly withthe washing buffer. Alkaline phosphatase coupled with goat anti-human7-GTP IgG was diluted 200-fold in the above phosphate buffer, and 100

¿ilof the diluted solution were added to each well. The plates were thenincubated for 4 h at room temperature and washed again with washingbuffer. To each well were added 100 n\ of a solution containing p-

nitrophenyl phosphate (2 mg/ml) in 50 mw sodium bicarbonate buffer,pH 9.8, containing 1 mM MgCI2, and the plates were incubated at roomtemperature for 1 to 4 h. The liberation of p-nitrophenol was stopped by

the addition of 100 ¡Aof 2 N NaOH, and the color intensity of each well

RESULTS

Specificity of Antisera and Immuno-Cross-Reactivity. Antibody against human kidney 7-GTP inhibited 7-GTP activity dosedependently up to approximately 50% in the incubation mixture.After centrifugation, the activity of the supernatants was almostzero. These data indicate that the antibody recognizes a site inthe vicinity of the active site; the antigen-antibody complexretains some activity (Chart 2). Ouchterlony tests (10) indicatedthat the kidney, liver, and liver cell cancer enzymes were anti-genically identical (Fig. 2). Reactivity of the antibody to humanserum was also tested as follows. Serum of a patient with livercell cancer was incubated with IgG from preimmune serum andanti-7-GTP IgG and subjected to differential (2.5% and 7.0%)

disc polyacrylamide gel electrophoresis. The gels were thenstained for enzyme activity according to the method of Sawabuef al. (11). As shown in Fig. 3, untreated serum and serumtreated with preimmune IgG exhibited several bands with 7-GTPactivity in the 7.0% (lower gel). The appearance of several bandsunder nondenaturing conditions indicates that isozymic forms of7-GTP are present, as reported by Sawabu ef a/. (11). In contrast,

0.8

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0.5 1.0 YGTP protein (ng) 100

Chart 1. A typical calibrationcurve for ELISA of 7-GTP. Eachpoint is the meanof triplicate determinations performed according to the method in "Materials andMethods."

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HUMAN SERUM IMMUNOREACTIVE 7-GTP AND CANCER

04 8 16 20 24Antr-YGTP IgG ((ig)

Chart 2. Inhibition and precipitation of 7-GTP activity by antiserum. Diluted anti-7-GTP IgG solution was added to 0.005 unit of 7-GTP as described in "Materialsand Methods." Each point represents the average of three determinations. The

enzyme activity was determined in the mixture (O) and in the supernatant aftercentrifugation (•).

Fig. 2. Specificity and cross-reactivity of polyclonal antibody to human 7-GTP.Center well contains antiserum against human kidney 7-GTP. Wells A and 0, Band E, and C and F contain purified 7-GTP from human liver cell cancer, humannormal liver, and human normal kidney, respectively.

most of the 7-GTP activity of the serum treated with anti-7-GTPIgG was retained in the 2.5% gel (upper gel). These data indicatethat the anti-7-GTP IgG cross-reacted with serum 7-GTP to forma high-molecular-weight antigen-antibody complex which did not

enter the 7.0% gel.Correlation between the Concentration of 7-GTP in Normal

and Patients' Sera as Determined by Immunoreactivity andEnzymatic Activity. The concentration of 7-GTP in 43 normalserum samples measured by the ELISA did not correlate withthe enzyme activity determined using 7-glutamyl p-nitroanilideas substrate (Chart 3A). Similarly the concentration of 7-GTP insera of 34 patients with hepatic diseases including 9 liver cellcancers and 4 metastatic liver cancers did not correlate with theenzyme activity (Chart 3B). This indicates that, in the humansera, immunoreactive but enzymatically inactive or enzymaticallyactive but immunochemically inactive protein was present.

7-GTP Content in Serum of Healthy Adults. The mean 7-GTP content in sera from 128 normal individuals was 8.5 ±3.5Mg/ml. The measured normal range was 2-15.5 ng/m\. Thecontents in male (n = 68; 8.5 ±4.3 ng) and female (n = 60; 8.5±3.4) did not differ significantly.

Serum 7-GTP Content in Patients with Malignant Tumorsand Various Diseases as Measured by ELISA. Chart 4 showsthe serum 7-GTP contents of preoperative patients with variousneoplastic (Chart 4/4) and nonneoplastic (Chart 48) diseases.Significantly elevated 7-GTP content was observed in the seraof patients with liver cell cancer, lung cancer, gastric and esoph-

Fig. 3. Differential (2.5% and 7.0%) disc polyacrylamide gel electrophoresis ofhuman serum 7-GTP after treatment with preimmune IgG or anti-7-GTP IgG. Serum(100 ft\) of a patient with liver cell cancer was treated with 20 n\ of preimmune IgG(2 mg) or anti-7-GTP IgG (1 and 2 mg) and incubated at 37°Cfor 2 h. An aliquot

(60 (il) of each reaction was electrophoresed at 2 mA/tube for 20 min and then 4mA/tube for 2 h at 4°C.After electrophoresis activity, staining of the gels was

carried out as described (10). Lane 1, untreated sample; Lanes 2 and 3, sampletreated with 1 and 2 mg of anti-7-GTP IgG, respectively: Lane 4, sample treated

with 2 mg of preimmune IgG. Arrow indicates the marker dye (bromophenol blue)front. Note that the broad band near the buffer front in Lanes 2-4 is due to the

presence of bilirubin rather than to enzyme activity. In Lane 7 most of the bilirubinis probably bound to the bromophenol blue.

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Charts. Relationships between immunochemically measured 7-GTP and enzymatically measured 7-GTP levels. Samples tested included those of normalcontrols (A) and individuals with nonneoplastic liver diseases (O), liver cell cancer(A), and metastatic liver cancer (A) (B).

ageal cancers, and colorectal cancer. Assuming the upper limitof normal 7-GTP content in serum to be 15.5 tig/ml (the value

corresponds to the mean value plus 2 SD of 128 sera of normalcontrol subjects), abnormally high serum levels of 7-GTP werefound for 8 of 17 patients with liver cell carcinoma, 10 of 13patients with lung cancer, 7 of 30 patients with colorectal cancer,and 5 of 25 patients with gastric or esophageal cancer. Incontrast, 5 patients with gall bladder cancer and 3 with mammarycancer exhibited normal levels of 7-GTP. Twenty-nine patientswith chronic hepatitis, 13 patients with alcoholic liver injury, 18patients with cirrhosis of the liver, and 18 patients with duoden-ogastric ulcer also exhibited normal levels of 7-GTP. Of 9 patientswith acute hepatitis, 8 showed normal 7-GTP levels. Sera ofpatients with other diseases including hypertension, diabetes,

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Chart 4. -y-GTP content in sera of preoper

ative patients with various malignant tumors (A)and patients with nonmalignant diseases (8)determined by ELISA. The vertical dashed linedenotes the upper limit of normal serum immu-noreactive -y-GTP defined as 15.5 »ig/ml(mean

+ 2 SD of 128 normal subjects), ca, cancer.Each group included both male and female adultsubjects.

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arteriosclerosis, duodenogastric ulcer, epilepsy, neurological disease, lung tuberculosis, pneumonia, and chronic obstructive lungdisease were also assayed and showed normal 7-GTP levels.Sera from 6 patients whose liver cell cancers and lung cancerswere very small and could be surgically removed were collectedbefore and after the operation. In 2 cases, the level of immuno-reactive 7-GTP determined by ELISA was within normal rangeand did not change significantly after the operation. In 1 casewith liver cell cancer and 3 cases with lung cancer, however, thelevel decreased markedly following the operation (Chart 5). Thisresult suggests that the assay could be used to monitor livercancer and lung cancer patients during the course of treatment.In these studies, 7-GTP activity was also determined before andafter the operation. The activity did not change significantly.

DISCUSSION

7-GTP is one of the oncofetal proteins and is recognized as a

tumor marker for experimentally induced hepatocarcinogenesis(1,3,5,12-15, 19). In previous studies on the level of immuno-reactive 7-GTP in rat liver, no correlation was found between the

40

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6 8/1984Chart 5. Detection of immunoreactive i-GTP in sera of patients with liver cell

cancer (O, A, •)or lung cancer (D, •A) before and after operations using ELISA.Arrows indicate the day of operation. Abscissa indicates dates on which thepatients' sera were taken for analysis.

7-GTP levels determined by ELISA and enzymatic assay (16).Similarly enzyme activity does not seem to reflect the trueamount of manganese-containing Superoxide dismutase in lungcancer tissues (4). Therefore the question arose as to whetherenzymatic assay is a reliable means of assessing 7-GTP contentin human sera.

Polyclonal antibody against human kidney 7-GTP was foundto cross-react to human serum enzyme (9) as well as to human

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HUMAN SERUM IMMUNOREACTIVE y-GTP AND CANCER

liver and hepatoma enzymes, and it was used in developing asimple and sensitive ELISA. With this assay, it was possible tospecifically quantitate very low amounts of human 7-GTP protein.

The level of immunoreactive 7-GTP was significantly elevatedin sera of patients with liver cell cancer, lung cancer, gastric andesophageal cancers, and colorectal cancer. On the other hand,sera of patients with nonmalignant diseases including acute andchronic hepatitis and cirrhosis of the liver did not have increasedlevels of 7-GTP protein. The sera of 9 of the patients with livercell cancer were assayed for a-fetoprotein as well as for 7-GTP.

Little correlation was found between the levels of the 2 proteins(data not shown), which indicates that the expression of these 2proteins differs. In 1 patient with liver cell cancer and 3 patientswith lung cancer, surgical removal of the tumor brought about adecrease in the level of immunoreactive 7-GTP. This suggeststhat the immunoreactive 7-GTP level is a useful indicator for

monitoring treatment.In the present study, we also found discrepancies between

the 7-GTP levels determined by enzymatic assay and immuno-

chemical assay of sera of normal controls and patients withhepatic diseases including malignant tumors. The basis of thisdiscrepancy is still unknown. Quite recently, we observed that aprecursor form with a high molecular weight appears in culturedyolk sac tumor cells (23). This suggests that the sera of patientswith liver cell cancer or other malignant diseases may containlarge amounts of an excreted immunoreactive but enzymaticallyinactive precursor form of 7-GTP. However, the possibility thathuman sera contain an inhibitor(s) and/or activator(s) of 7-GTPor cross-reacting materials which would account for the discrepancy between the enzymatic and ¡mmunochemical levels of 7-

GTP cannot be eliminated at present. Quite recently we foundthat a boiled extract of cancer tissues or sera of a patient withcancer inhibits 7-GTP activity.4 This finding suggests that cancer

tissues or sera of a patient with cancer contain an inhibitors) for7-GTP, and this inhibitor may bring about an enzymaticallyinactive but immunochemically active 7-GTP. We are now con

tinuing the work along these lines.In previous studies in our laboratory, we found that the 7-GTP

in experimental hepatoma cells underwent structural modificationof its carbohydrate moiety (21-23). Sawabu ef al. (11) found thatthe appearance of a novel type of 7-GTP in the sera of patientswith liver cell cancer is closely associated with modification of itscarbohydrate moiety. One serum sample which displayed a noveltype of 7-GTP according to the method of Sawabu (11) was

kindly supplied by Dr. Sawabu (Cancer Institute, KanazawaUniversity, Japan) and assayed using our ELISA method. Theserum was found to contain a large amount of immunoreactive7-GTP (45 iig/ml). This does not necessarily imply that ourantibody reacted specifically with the novel type of 7-GTP,because the novel 7-GTP cannot be distinguished from normal7-GTP using a conventional antibody (11).

Masuike ef al. (7) purified 7-GTP from human pancreas anddeveloped a radioimmunoassay for human sera. They reportedthat the mean concentration in normal sera was 3.43 /ug/ml, witha range of 2.03-5.52 ng/m\. In the present study, the 7-GTPcontent of normal sera was found to be 8.5 ±3.5 ng/m\. Theyalso reported that enzymatically inactive 7-GTP protein or fragments of the enzyme are present in normal sera. However, thelevel of the enzyme under pathological conditions has not been

•N.Taniguchi, S. lizuka, Z. N. Zhe, S. House, N. Yokosawa, M. Ono, K.

Kinoshita,A. Makita, and C. Sekiya. unpublisheddata.

reported.Quite recently we obtained a monoclonal antibody against 7-

GTP from hepatoma tissues (13). This antibody is highly reactiveto the enzyme from liver cell cancer but not to the normal liverenzyme. Consequently it may provide useful information aboutthe character of the abundant form(s) of 7-GTP in the sera ofcancer patients. At any event, the results of ELISA using poly-clonal antibody presented in this study suggest that immunoreactive 7-GTP content may be a marker for liver cell cancer,lung cancer, and several gastrointestinal cancers.

REFERENCES

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2. Engvall, E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol., 76:419-439,1980.

3. Fiala.S., Fiala,A. E., and Dixon, B. -y-Glutamyltranspeptidase in transplanta-ble, chemically induced rat hepatomas and spontaneous mouse hepatomas.J. Nati. Cancer Inst., 48:1393-1401,1972.

4. lizuka, S., Taniguichi,N., and Makita, A. Enzyme-linkedimmunosorbentassayfor manganese-containingSuperoxide dismutase. J. Nati. Cancer Inst., 72:1043-1049,1984.

5. Kalengayi, M. M. R., Ronchi, G., and Desmet, V. J. Histochemistry of 7-glutamyl transpeptidasein rat liverduring aflatoxin B, inducedcarcinogenesis.J. Nati. Cancer Inst., 55: 579-582, 1975.

6. Laemmli,U.K. Cleavageof structural proteins during the assemblyof the headof bacteriophageT4.Nature (Lond.),224: 149-154,1970.

7. Masuike, M., Ogawa, M., Kitahara, T., Murata, A., Matsuda, K., and Kosaki,G. Developmentof radioimmunoassayfor gamma-glutamyl transferase usingpancreaticenzyme.Ann. Clin. Biochem.,20: 247-250,1983.

8. Meister,A., Täte,S. S., and Griffith,O.W. i-Glutamyl transpeptidase.MethodsEnzymol.,92: 237-253, 1981.

9. Miura, T., Matuda, Y., Tsuji, A., and Katunuma, N. Immunological cross-reactivity of 7-glutamylteranspeptidasesfrom humanand rat kidney, liver, andbile. J. Biochem.,89: 217-222,1981.

10. Ouchteriony, 0. Antigen-antibody reaction in gels. Acta Pathol. Microbiol.Scand., 26: 507-515, 1949.

11. Sawabu, N., Nakagen, M., Osaki, K., Wakabayashi,T., Toya, D., Hattori, N.,and Ishii, N. Clinical evaluation of specific 7-GTP isoenzyme in patients withhepatocellularcarcinoma.Cancer (Phila.),57: 145-149,1983.

12. Taniguchi, N. Purification and some properties of 7-glutamyl transpeptidasefrom azo-dye-inducedhepatoma.J. Biochem., 75: 473-480, 1974.

13. Taniguchi, N., House, S., Kuzumaki, N., Yokosawa, N., Yamagiwa,S., lizuka,S., Makita, A., and Sekiya, C. A monoclonal antibody against 7-glutamyltranspeptidase from human primary hepatoma: its use in enzyme-linked immunosorbentassayof serumof cancer patients.J. Nati. Cancer Inst., in press,1985.

14. Taniguchi, N., Tsukada, Y., and Hirai, H. Acquirement of fetal properties inhepatomaon glutathione metabolism.Biochim. Biophys. Acta, 354:161-167,1974.

15. Taniguchi, N., Tsukada, Y., Mukuo, K., and Hirai, H. Effect of hepatocarcino-genic azo-dye on glutathione and relatedenzymes in rat liver. Gann,65: 381-387, 1974.

16. Taniguchi,N., Yokosawa, N., lizuka, S., Sako, F., Tsukada, Y., Satoh, M., andDempo. K. 7-Glutamyl transpeptidase of rat liver and hepatoma tissues: anenzyme immunoassayand immunostainingstudies. Ann. NY Acad. Sci., 477:203-212,1983.

17. Taniguchi,N., Yokosawa, N., lizuka, S., Tsukada, Y., Sako, F., and Miyazawa,N. 7-Glutamyl transpeptidase from tumor tissues—chemical,enzymatic, andbiologicalproperties. Gann Mongr. Cancer Res., 29: 263-272,1983.

18. Tate, S. S., and Ross,M. E. Humankidney7-glutamyltranspeptidase,catalyticproperties, subunit structure, and localizationof the 7-glutamyl bindingsite onthe light subunit. J. Biol. Chem., 252: 6042-6050, 1977.

19. Tsuchida, S., Hoshino, K., Sato, T., Ito, N., and Sato, K. Purification of 7-glutamyl transferase from rat hepatomas and hyperplastic hepatic nodules,and comparison with the enzyme from rat kidney. Cancer Res., 39: 4200-4205, 1979.

20. Wright, G. L, and Mallmann,W. L. Differentialdisc electrophoresisof serumproteins. Proc. Soc. Exp. Biol. Med., 723: 22-27,1966.

21. Yamashita,K., Hitoi,A., Taniguchi,N.,Yokosawa, N.,Tsukada,Y., and Kobata,A. Comparativestudy of the sugar chainsof 7-glutamyl transpeptidases fromrat liver and rat AH-66 hepatomacells. Cancer Res., 43: 5059-5063, 1983.

22. Yokosawa, N., Taniguchi, N., Tsukada, Y., and Makita, A. Physicochemicalcharacterizationof 7-glutamyl transpeptidasefrom yolk sac tumor and aserteshepatomaAH66. Oncodev. Biol. Med., 2: 165-177, 1981.

23. Yokosawa, N., Taniguchi, N., Tsukada, Y., and Makita, A. Evidence for thepresenceof a precursor form to 7-glutamyl transpeptidasein cultured yolk sactumor cells. Oncodev. Biol. Med., 4: C71-C78, 1983.

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1985;45:5835-5839. Cancer Res   Naoyuki Taniguchi, Susumu Iizuka, Zhang Nai Zhe, et al.   Enzyme-linked Immunosorbent AssayTranspeptidase in Patients with Malignant Tumors Using

-GlutamylγMeasurement of Human Serum Immunoreactive

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