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

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Enzyme Immunoassay for the Quantification of Mitomycin C Using ß-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 ß-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 37°. 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 25° for 1 hr, followed by new tool for use in pharmacokinetic and toxicity studies of further incubation at 4°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 25°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 55°for 30 min, and stored Quite recently, we reported the development of ElAs for the at -30°. 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 ¿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 4°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 »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 25° 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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1982 American Association for Cancer Research. K. Fujiwara et al. 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-NHCOCH2ÕHCOOH 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 30°for 1 hr. The reaction was stopped FRACTION NUMBER (3.0 ml/tub«) 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.
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