Enzyme Immunoassay for the Quantification of Mitomycin C Using ÃŸ-Galactosidase As a Label1

[CANCER RESEARCH 42, 1487-1491, April 1982] 0008-5472/82/0042-OOOOS02.00 Enzyme Immunoassay for the Quantification of Mitomycin C Using ÃŸ-Galactosidase as a Label1

Kunio Fujiwara,2 Hitoshi Saikusa, Motomi Yasuno, and Tsunehiro Kitagawa

Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852, Japan

ABSTRACT were done with a structurally different cross-linker, N-(m-ma\- eimidobenzoyloxy)succinimide (9, 10). The success of this A mitomycin C (MMC) antibody was produced following procedure prompted us to develop an EIA for the quantification immunization of rabbits with a MMC-bovine serum albumin of MMC. conjugate, which was newly synthesized by coupling MMC to In this report, we detail our results in developing an EIA mercaptosuccinylated bovine serum albumin via a cross-linker, method for the direct determination of MMC in serum samples, W-maleoyl aminobutyric acid. Enzyme labeling of MMC was using rabbit anti-MMC antiserum and MMC-/?-Gal conjugate as performed using /S-o-galactosidase (EC 3.2.1.23) via m-ma- label. leoyl benzoic acid. An enzyme immunoassay for MMC was developed utilizing these reagents by a double-antibody tech MATERIALS AND METHODS nique. The standard curve of the assay was linear on a logit- log plot, and the lower limit of detection was 12 nM (0.2 ng/ Materials. MMC was purchased from Kyowa Hakko Kogyo Co., Ltd., tube) so the enzyme immunoassay was found to be approxi Tokyo, Japan. /3-Gal (EC 3.2.1.23; Lot 127818) from Escherichia coli mately 25 times more sensitive than a microbiological assay. was obtained from Boehringer Mannheim, Mannheim, Germany. MABA Further, the enzyme immunoassay is practically free from in and MBA were synthesized by the methods of Rich et al. (22) and terference by any other anticancer drugs. No significant de Kitagawa and Aikawa (14), respectively. crease in MMC immunoreactivity was observed following 24 hr Preparation of Immunogen to MMC. The preparative process was essentially the same as our method of synthesizing the immunogen to of incubation of the drug in normal human serum or urine at 37Â°. Using this assay, serum or urine levels of MMC can be pepleomycin (10) (Chart 1). MMC was acylated with MABA through an amide bond using N.N'-dicyclohexylcarbodiimide (1). To a solution determined accurately after administration of the drug to rats containing 1.65 mg (9.01 jumol) of MABA and 6.28 mg (18.8 /imol) of at a single dose of 600 jug/kg. The sensitivity and specificity of MMC in 1.0 ml of dry tetrahydrofuran, 1.90 mg (9.22 /rniol) of the the enzyme immunoassay for MMC should provide a valuable carbodumide was added and incubated at 25Â° for 1 hr, followed by new tool for use in pharmacokinetic and toxicity studies of further incubation at 4Â°for 48 hr with stirring. The reaction mixture MMC. was concentrated to about 0.1 ml by flashing dry nitrogen to remove the solvent. At the same time, acetylmercaptosuccinyl bovine serum albumin [10 mg; from the same batch as that used previously (10)] was INTRODUCTION incubated in 0.2 ml of 0.1 M hydroxylamine, pH 7.2, at 25Â°for 30 min The anticancer antibiotic MMC3 isolated from Streptomyces to remove the protecting acetyl group. The resulting MS-BSA, esti caespitosus (23, 27) exhibits broad activity against trans mated to contain 17 thiol groups/BSA molecule (6), was diluted with planted tumors (24) and has recently been used effectively for 3 ml of 0.1 M phosphate buffer, pH 7.0, and added immediately to the the treatment of a variety of neoplastic diseases by the special MABA-acylated MMC solution and incubated for 30 min with vigorous stirring. Purification of the conjugate was achieved by column chro- chemotherapy of restricting dosage regimens to afford maxi matography on Sephadex G-100. Then, the purified conjugate was mum chemotherapeutic benefit combined with closer monitor examined spectrophotometrically and estimated to contain about 4 ing of the hematological toxicity of the drug (2,7,11,15). The molecules of MMC per BSA molecule assuming the molar extinction development of a simple and sensitive assay method for the coefficients of MMC and BSA to be 23,000 at 363 nm and 46,000 at quantification of MMC will greatly facilitate the design of dose 280 nm, respectively. treatment schedules for use with cancer patients and in phar Immunization. Three randomly bred rabbits were immunized s.c. macokinetic studies in human and animal systems. The deter and i.m. with 1 mg of MMC-BSA conjugate emulsified in Freund's mination of MMC in biological materials has previously been complete adjuvant, and thereafter booster injections totaling 1.5 mg possible only by a laborious microbiological assay technique were given 3 times at biweekly intervals. The rabbits were bled from an ear vein 2 weeks after each injection. The samples of serum were that lacks sensitivity and specificity (8). separated by centrifugation, and heated at 55Â°for 30 min, and stored Quite recently, we reported the development of ElAs for the at -30Â°. anticancer drugs pepleomycin and daunomycin, in which anti Labeling of MMC. MMC and /8-Gal were coupled by what is essen bodies were elicited in rabbits with each hapten drug conju tially a repetition of the same 2-step procedure as in the preparation of gated with MS-BSA, with A/-(y-maleimidobutyryloxy)succini- the immunogen for MMC, using MBA as a heterobifunctional cross- mide as a cross-linker, and /8-Gal labelings of the hapten drugs linker. 0.5 mg (1.5 ^mol) of MMC and 1.5 ng (6.9 nmol) of MBA were incubated with 1.59 Â¿ig(7.7 nmol) of N,N'-dicyclohexylcarbodiimide in 1 This work was partially supported by Cancer Research Grants from the 0.5 ml of tetrahydrofuran at 4Â°for 48 hr with stirring. The reaction Ministry of Education and Ministry of Health and Welfare of Japan. 2 To whom requests for reprints should be addressed. mixture was concentrated to about 50 Â»Iand added directly to /(-Gal 3 The abbreviations used are: MMC, mitomycin C; EIA, enzyme immunoassay; MS-BSA, mercaptosuccinyl bovine serum albumin; />'-Gal, /<-D-galactosidase; solution containing 78 fig (0.14 nmol) of the enzyme (3) in 1.0 ml of MABA. W-maleoyl aminobutyric acid; MBA, m-maleoyl benzoic acid; BSA, bovine 0.1 M phosphate buffer, pH 6.0. The mixture was stirred for 30 min at 25Â° and then applied to a Sepharose 6B column (2 x 38 cm) equili serum albumin. Received August 4, 1981 ; accepted January 8, 1982. brated with 0.02 M phosphate buffer, pH 7.0, containing 0.1 M NaCI,

APRIL 1982 1487

conjugate. Typical binding curves are shown in Chart 3 for BSA-NH2 serum from bleedings of one of the 3 rabbits. The antibody titer in the serum increased gradually and reached a maximum 2 weeks after the final injection. There was no significant degree MITOMYCIN C of binding when comparable dilutions of prebled serum were v tested. EIA of MMC. The standard dose-response curve of MMC in BSA-NHCOCH2Ã•HCOOH a buffer system was linear on a logit-log plot (Chart 4). The 0 MABA AMS BSA sensitivity of the assay, defined as the smallest amount of MMC that could be detected, was 12 nw (0.2 ng/tube). For practical cARBODIIMIDE H2NOH purposes, the working range was from 60 to 600 nM (1 to 10 o ng/tube). Assay precision, defined as the ratio between the 2OCONH2 SH BSA-NHCOCH2CHCOOH standard deviation of Y and the slope of the regression line A between mean values of logit Y and the corresponding log X MS BSA was A = 0.055 at 3.2 ng. MABA-ocyloted MMC Detection of the drug in serum samples (50 pi) was also performed by adding known concentrations. The standard R" curve yielded was essentially linear and steeper than that in (CH,)foNfMMC| the buffer system (Chart 4). The sensitivity of the assay was 12 , nw (0.2 ng/tube). For practical purposes, the working range | BSAJNHCOCHyCHCOOH

MMC-BSA 40 Chart 1. Scheme for synthesizing immunogen for MMC. A, not identified; AMS BSA, acetylmercaptosuccinyl bovine serum albumin.

1 rnw MgCI2, 0.1 % BSA, and 0.1 % NaN3 (Buffer A) and eluted with the ! 30 same buffer. The elution profiles of the enzyme and immunoreactive X "c enzyme activities are shown in Chart 2. These assays were done by the method described below, using 5 pi of a 1:30 solution of each > 20 fraction. The immunoreactive enzyme activity of the conjugate was almost parallel with the pure enzyme activity. The immune specificity of the conjugate was determined by an EIA procedure involving a competition for the anti-MMC antibody with 12.5 ng of MMC. This IO showed that the conjugate was more differentiated from MMC in the latter peak fractions than in the former peak fractions. Fractions 17 to 19 of the main peak were thus chosen for the EIA. Measurement of /.'-Gal Activity. Five pi of diluted enzyme solution were incubated with 0.15 ml of 0.1 mw 7-/?-D-galactopyranosyloxy-4- 5 io e to es so as methylcoumarin in Buffer A at 30Â°for 1 hr. The reaction was stopped FRACTION NUMBER (3.0 ml/tubÂ«) by the addition of 2.5 ml of glycine-NaOH buffer (0.2 M; pH 10.3), and Chart 2. Elution profiles of MMC-/5-Gal conjugate from a Sepharose 6B col the 7-hydroxy-4-methylcoumarin liberated was measured spectrofluo- umn. â€¢,enzyme activity of the conjugate in 5 pi of a 1:30 solution of each rometrically at wavelengths of 365 nm for excitation and 448 nm for fraction; O, immunoreactive enzyme activity, determined by the EIA described in "Materials and Methods" except that 5 pi of the conjugate and a 1/5000 solution emission. The amount of conjugate was expressed in units of /8-Gal of anti-MMC serum were used in the absence of MMC; D, competitive immuno activity, defining 1 unit of enzyme activity as the amount that hydrolyzes reactive enzyme activity determined in the presence of 12.5 ng of MMC by the 1 pmol of the substrate per min. same manner. Method of EIA. The assay was performed by the double-antibody method (26). Five pi of MMC-/?-Gal conjugate (125 microunits), 50 pi of MMC or sample, as appropriate, and 50 pi of a 1/10,000 solution of the antiserum in 0.06 M sodium phosphate, pH 7.4, containing 0.01 M EDTA and 0.1 % BSA (Buffer B) were mixed and incubated at 25Â°for 8 hr. Then, 50 pi of a 10% solution of goat anti-rabbit IgG and 50 pi of a 0.33% solution of normal rabbit serum were added. After further incubation for 3 hr, the immune precipitate was washed twice by the addition of 1.0 ml of Buffer A and centrifuged at 2000 rpm for 20 min. The supernatant was decanted, and the enzyme activity in the immune precipitate was measured.

RESULTS

Antibody Response. An antibody against MMC was pro 10 ICT duced in each of 3 rabbits immunized with a MMC-BSA con DILUTION (fold) jugate and was detected by the reaction of diluted antiserum with MMC-yS-Gal conjugate. The serum collected 2 weeks after Chart 3. Quantitative estimation of anti-MMC antibody. Samples 2, 3, 4, and 5 are the samples of antiserum collected 2 weeks after each injection. Sample 1 each injection was tested for its binding ability to MMC-/8-Gal was bled before immunization. The samples of serum were diluted with Buffer B.

1488 CANCER RESEARCH VOL. 42

3.0

2.0

o 1.0 o 0.0

- 1.0

- 2.0 0.2 0.5 I.O 2JO 5X> 10 20 50 100 200 500 COO UNLABELED DRUG (ng/tube) 0.2 0.5 I 2 5 IO 20 90 MITOMYCIN C /lube t S.D. (ng) Chart 5. Specificity of the EIA of MMC. The proportion of antibody bound to MMC-/6-Gal conjugate (B'B) is plotted as percentage bound; MMC <â€¢);5- Chart 4. The standard curves for the EIA of MMC in buffer system (A) or fluorouracil (A); fluorodeoxyuridine O: daunomycin (O); Adriamycin O); acti- serum system (B). The response, Y, to the dose, X, can be defined as a logit nomycin D (x); bleomycin (â€¢):neocarzinostatin (A). function of Y such that logit Y = ln(V/1 - /). Y is the logit of the ratio of antibody-bound enzyme activity (6) to enzyme activity at zero concentration of unlabeled MMC (8Â°).Point, mean for 6 replicates; bars, S.D. was from 60 DM to 3 Â¡IM(1 to 50 ng/tube). Assay precision was A = 0.044 at 6.2 ng. The presence of 50 n\ serum slightly reduced the inhibition of MMC. Thus, instead of obtaining 50% inhibition with 3.2 ng of MMC, 6.2 ng were required. This calibration curve (i.e., in the presence of 50 jtil of serum) is used to quantify MMC levels only in samples in which 50 /il of serum are present. The reduction is not seen with 50 n\ of undiluted urine. Therefore, to quantify MMC levels in such samples, a calibration curve obtained with the buffer system is used. Experiments were also designed to measure the change in 10 20 30 40 5O 60 TO immunoreactivity of MMC following incubation in normal human TIME AFTER INJECTION (min) serum. Known concentrations of MMC were incubated in serum for up to 24 hr at 37Â°,and the reactivity was measured at timed Chart 6. Serum MMC levels in rats after a single i.v. injection of the drug. Two rats, each weighing about 320 g, were given injections of 600 fig of MMC per kg. intervals. No significant decrease of MMC immunoreactivity At each time interval, blood was collected and the serum MMC content was caused by incubation in serum was observed, there being an estimated. overall mean recovery of 97.0 Â± 6.6% (S.E.) at 5 different tained from the common carotid artery at different time inter levels of MMC. Similar experiments with MMC incubated at 37Â° for 24 hr in normal human urine resulted in detection of vals, and MMC content in the serum was determined by this EIA. The results presented in Chart 6 demonstrate that a MMC 95.5 Â±4.0% of the added drug. dose of as little as 0.6 nmol/ml can be determined quantita The antibody specificity was determined by measuring the displacement of bound MMC-/8-Gal conjugate by other cancer tively in rat serum by the EIA. Furthermore, clearance of MMC from rat serum was rapid and most of the drug (about 80%) chemotherapeutic agents. As indicated in Chart 5, none of the was removed in the first 10 min, indicating a very short serum tested drugs competed significantly with MMC-/S-Gal conjugate half-life for the drug, and this was followed by a second mode in the EIA. Only Adriamycin caused a slight reduction in MMC- of removal, evidently characterized by a longer half-life. Rat ÃŸ-Galconjugate binding to the antibody, with a value of cross- urine was also collected and assayed for MMC by the EIA. The reactivity of less than 0.001 % at the amount of the drug for a 1-hr urine samples of 2 animals contained an average of 48.5 50% inhibition. Â±6.5 (S.D.) pg of MMC per ml, and the excretion accounted Experiments on a microbiological assay using Escherichia for 25.5 Â±3.2% of the dose administered. coli NIHJ were done in order to compare the sensitivity of MMC detection with the EIA. These showed that the minimal sensitiv DISCUSSION ity of the microbiological assay using a paper disc that ad sorbed a maximum of 80 /il of MMC solution was such that 0.1 Although MMC has been used for the treatment of various jug (300 nM) of MMC per ml could be detected. This is 25 times kinds of human cancer as one of the most effective chemo less sensitive than the EIA. It was also found that there was a therapeutic agents (2,11,15,19), little information concerning good correlation between results by the microbiological and the clinical pharmacology of MMC has been available because the EIA methods (r > 0.93; n = 25) (data not shown). The of the lack of a specific and sensitive quantitative assay. The microbiological assay, however, was less accurate when MMC development of an immunological assay which is very valuable was present in serum, owing to the effect of serum proteins. in the cases of numerous other drugs (20, 25) has been Quantification of MMC in Rat Serum by the EIA. Two rats, hampered by the difficulties of preparing an effective immu- each weighing 320 g, were given 600 p.g of MMC per kg by nogen, presumably due to the fact that MMC is of small rapid injection into the femoral vein. Blood samples were ob- molecular size (M, 334) and very labile (5).

APRIL 1982 1489

We recently reported a potential new method for binding an subject in advance of treatment with MMC. It was also shown aliphatic amino group of haptens such as the anticancer drugs that the immunoreactivity of MMC is not affected by incubating daunomycin and pepleomycin to chemically modified MS-BSA the drug in serum or urine for 24 hr at 37Â°. as a carrier protein using the bifunctional reagent A/-(y-maleim- On the basis of the data reported above, we successfully idobutyryloxy)succinimide and reported results of using the used the EIA in determining MMC concentration in rat serum conjugate as an immunogen (9, 10). With the slightly modified or urine following a single i.v. administration of a limited amount method of using MABA, the intermediate product of the syn (600 /ig/kg) of MMC, comparable to the dose commonly used thesis of A/-(y-maleimidobutyryloxy)succinimide, as a cross- for the treatment of human cancers (4, 8, 11). linker, we successfully prepared the immunogen for MMC by The specific and sensitive EIA technique for MMC described conjugating the drug with MS-BSA. MMC was first acylated here will allow the quantification of MMC in physiological con with MABA by the carbodiimide-catalyzed reaction. [The acyl ditions hitherto impossible for clinical investigators, and thus ated position of MMC has not been strictly identified but is studies on the toxicity and the pharmacokinetics of MMC may supposedly at the imino group of the aziridine ring as shown be developed further. by the similar reaction of MMC with the carboxyl group of some other compounds using the carbodiimide as a catalyst (1 2, 13, ACKNOWLEDGMENTS 16)]. Thereafter, the maleimide group of MABA incorporated on a MMC molecule was directly coupled to the thiol group of We wish to thank Dr. T. Miura for her valuable suggestions for synthesizing MABA, H. Tanimori for his interest, and H. Asada, M. Isobe, and A. Ogawa for MS-BSA by thiolation under aqueous neutral conditions. This technical assistance. method is extremely mild and does not result in extensive (intra- or intermolecular) self-coupling of the carrier protein or REFERENCES hapten. The MMC-BSA conjugate thus synthesized, containing 1. Bezas, B., and Zervas, L. On the peptides of u-lysine. J. Am. Chem. Soc., about 4 molecules of MMC per BSA molecule, elicited suc 87. 719-723, 1961. cessfully the production of anti-MMC antibody in rabbits, the 2. Buzdar, A. U., Tashima, C. K., Blumenshein, G. R., Hortobagyi, G. N., Yap, highest titer of which was 2 weeks after the final booster H. Y., Krutchik. A. N., Bodey. G. P.. and Livingston, R. B. Mitomycin-C and megestrol acetate in the treatment of breast cancer refractory to hormonal injection (Chart 3). and combination chemotherapy. Cancer (Phila.), 41: 392-395, 1978. MMC-/?-Gal conjugate was also prepared, with a full retention 3. Craven, G. R., Steers, E., and Anfinsen, C. B. Purification, comparison, and of the enzyme activity by essentially the same 2-step procedure molecular weight of /8-D-galactosidase of Escherichia coli K 12. J. Biol. Chem.. 240. 2468-2477, 1965. as used in the preparation of the immunogen for MMC, using 4. Crooke, S. T., Johson, D. E., and Bracken, R. B. A phase l-ll study of MBA as a cross-linker. This cross-linker differs from MABA by mitomycin C (MMC) topical therapy in early transitional cell carcinoma of the substitution of a benzoic acid radical for an n-butyric acid the bladderâ€”a preliminary report. Proc. Am. Assoc. Cancer Res., 79: 321. 1978. radical, which avoids a possibility of cross-reaction of the anti- 5. Edward, D.. Selkirk, A. B., and Taylor, R. B. Determination of the stability of MMC antibody with MABA. The Chromatographie behavior of mitomycin C by high performance liquid chromatography. Int. J. Pharma ceutics, 4:21-26, 1979. the conjugate on a Sephadex G-1 00 column (Chart 2) shows 6. Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82. 70- that the conjugate in the latter peak fractions is more suitable 77, 1959. 7. Frank, W., and Osterberg, A. E. Mitomycin C (NRC-26980): an evaluation of for use in the EIA since it has a higher degree of immune Japanese reports. Cancer Chemother. Rep., 9: 114-119, 1960. binding activity and a greater tendency to be inhibited by 8. Fujita, H. Comparative studies on the blood level, tissue distribution, excre nonlabeled MMC than that in the former peak fractions. The tion and activation of anticancer drugs. Jpn. J. Clin. Oncol., 12: 151-162, MMC-jS-Gal conjugate thus purified was stable in Buffer A for 1971. 9. Fujiwara, K.,Yasuno, M., and Kitagawa, T. Novel preparation method of 3 months during which time no loss of the enzyme and immu- immunogen for hydrophobic hapten. Enzyme immunoassay for daunomycin noreactive enzyme activities was observed. and Adriamycin. J. Immunol. Methods, 45: 195-203, 1981. 10. Fujiwara, K., Yasuno, M., and Kitagawa, T. Enzyme immunoassay for pe Using both the reagents of anti-MMC serum and MMC-ÃŸ-Ga\ pleomycin, a new bleomycin analog. Cancer Res., 41: 4121-4126, 1981. conjugate, an EIA for MMC is described in this report. This 11. Godfrey. T. E., and Wilbur. D. W. Clinical experience with mitomycin C in assay can detect as little as 12 nM of MMC (0.2 ng/tube), 25 large infrequent dose. Cancer (Phila.), 29. 1647-1652. 1972. 12. Kinoshita, S., Uzu, K., Nakano, K., Shimizu, M., Takahashi, T., and Matsui, times more sensitive than the corresponding microbiological M. Mitomycin derivatives. I. Preparation of mitosane and mitosene com assay. Since the current clinical investigations of MMC are pounds and their biological activities. J. Med. Chem., 14: 103-109, 1971. 13. Kinoshita, S., Uzu, K., Nakano, K., and Takahashi, T. Mitomycin derivatives. often undertaken by MMC chemotherapy in combination with 2. Derivatives of decarbamoylmitosane and decarbamoylmitosene. J. Med. 5-fluorouracil or fluorodeoxyuridine in regional intrahepatic Chem., 14: 109-112, 1971. artery infusion (17, 18, 21), an EIA for MMC must be sufficiently 14. Kitagawa, T., and Aikawa, T. Enzyme coupled immunoassay of insulin using specific to avoid cross-reactivity with these drugs. Therefore, a novel coupling reagent. J. Biochem. (Tokyo), 79. 233-236, 1976. 15. Manheimer, L. H., and Vital, J. Mitomycin C in therapy of far advanced many commonly used antineoplastic agents were examined for malignant tumors. Cancer (Phila.), 79: 207-212, 1966. their interference with the EIA described here, and it was found 16. Matsui, M., Yamada, Y., Uzu, K., and Mirata, T. Studies on mitomycins. III. The synthesis and properties of mitomycin derivatives. J. Antibiotics (Tokyo), that no significant cross-reactivity occurred with any of the 21: 189-198, 1968. drugs tested. This means that this EIA for MMC may become 17. Misra, N., Jaiswal, M., Singh, R., and Das, B. Intrahepatic arterial infusion an important tool in clinical evaluation of combination chemo with combination of mitomycin C (MMC) and 5-fluorouracil (5-FU) for treat ment of primary and metastatic carcinoma of liver. Proc. Am. Assoc. Cancer therapy. Res., 75: 113, 1974. The presence of serum in the EIA systems interferes only 18. Miura. T., and Ishida, M. 5-Fluorouracil-mitomycin combination cancer chemotherapy by regional intra-arterial or Â¡ntra-aortic infusion with or without very slightly with MMC determination since standards prepared radiation. In: Tenth International Cancer Congress Abstracts. Houston, No. in either buffer or serum are essentially linear on a logit-log 827, p. 512. Austin: University of Texas Press. 1970. plot in almost the same range between 0.2 and 10 ng/tube 19. Moore, G. E., Brass, I. D. J., Ausman, R., Nadler, S., Jones, R., Jr., Stalk, N., and Rimm, A. D. Effects of mitomycin C (NSC-26980) in 346 patients (Chart 4). Nonetheless, accuracy could be slightly enhanced if with advanced cancer. Cancer Chemother. Rep. Part I, 52: 675-684, 1968. the serum system standard curve could be calculated for each 20. Pal, S. B. (ed.). Enzyme Labelled Immunoassay of Hormones and Drugs.

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Proceedings of the International Symposium on Enzyme Labelled Immu- 24. Sugiura, K. Antitumor activity of mitomycin C. Cancer Chemother. Rep., 13: noassay of Hormones and Drugs, Dim, West Germany. Berlin: Walter de 51-56, 1961. Gruyter, 1978. 25. Thorell, J. I., and Larson, S. M. Clinical applications. In: J. I. Thorell and S. 21. Part. Y. Z.. Chuang, V., Johnston, P. S.. Benjamin. R.. Valdivieso, M., Hersh, M. Larson (eds.), Radioimmunoassay and Related Techniques. Methodology E.. and Mavligit, G. M. Hepatic arterial infusion (HAI) of mitomycin C (MTC) and Clinical Applications, pp. 109-249. St. Louis: The C. V. Mosby Co.. and fluoroxyuridine (FUDR)â€”an effective treatment for metastatic colorectal 1978. carcinoma to the liver. Proc. Am. Assoc. Cancer Res., 20. 316. 1979. 26. Van Vunakis, H., and Levine, L. Use of the double antibody and nitro 22. Rich. D. H., Gesellchen, P. D.. Tong, A., Cheung, A., and Buckner, C. K. cellulose membrane filtration technique to separate free antigen from anti- Alkylating derivatives of amino acids and peptides. Synthesis of N-maleoyl body bound antigen in radioimmunoassays. In. S. J. Mule, I. Sunshine, M. amino acids, [1-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1982 American Association for Cancer Research. Enzyme Immunoassay for the Quantification of Mitomycin C Using β-Galactosidase as a Label

Kunio Fujiwara, Hitoshi Saikusa, Motomi Yasuno, et al.

Cancer Res 1982;42:1487-1491.

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