Enzyme immunoassay for captopril
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Enzyme lmmunoassay for Captopril
H. KINOSHITA,' R. NAKAMARU, s. TANAKA, Y. TOHIRA, AND M. SAWADA Received January 14, 1986, from Central Research Laboratories, Chugai Pharmaceutical Co., Tokyo, Japan. 14, 1986.
Accepted for publication April
Abrtract 0 A simple enzyme immunoassay for the determination of captopril was developed. A specific antibody for captopril was produced in rabbits that were immunized with a hapten-bovine immunoglobulin G conjugate, which was prepared by using 4-(maleimidomethyl)cyclohex- ane carboxylic acid as a spacer group. The limit of detection in plasma is 0.5 ng/mL. The assay has an adequate specificity, so isolation of captopril is unnecessary.
Captopril (l-[2(S)-3-mercapto-2-methylpropionyl]-~pro- line) is a sulfhydryl compound that is a potent and selective inhibitor of angiotensin I converting enzyme both in vitro and in vivo.1 Although captopril is being increasingly used as an antihypertensive drug in humans, such side effects as membranous glomerulopathy, neutropenia, proteinuria, and taste loss have been reported.2.3 Thus, it has become impor- tant to monitor the plasma concentration of captopril. Sever- al methods for determining captopril in biological fluids have been reported, including GC-MS,' G C , 5 and HPLC.M How- ever, these methods are complicated and a rapid and simple method is needed for measuring the plasma concentration. This report describes a sensitive and specific enzyme immu- noassay.
Experimental Section Apparatus and Reagents-Fluorometric measurements were per-
formed on a spectrofluorometer (model MPF-4, Hitachi Co. Ltd., Tokyo). Radioactivity was measured by a liquid scintillation counter (model LSC-900, Aloka Co. Ltd., Tokyo). A centrifuge (model KHAK 232505, Hitachi Co. Ltd., Tokyo), incubator (model personal H, Taiyo Scientific Co. Ltd., Tokyo), and 13 x 100-mm glass tubes (Corning Glass Works, NY) were used. Tritiated captopril acetate ester [ l - [~2S~-3-mercapto-2-methylpropionyll-~-pro~ine~,4-~~, acetate (es- ter)], N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochlo- ride (DEC), bovine immunoglobulin G (BIgG), bovine serum albumin (BSA), 02-~-galactosidase, immunobead second antibody goat anti- rabbit immunoglobulin (ISA), and keund's complete adjuvant were procured from commercially available sources (New England Nucle- ar, Boston, MA, E. Merck Co., Ltd., Darmstadt, FRG, Sigma Chemi- cal Co., St. Louis, MO; Armour Pharmaceutical Co., Chicago, IL; Boehringer Mannheim Biochemicals, Mannheim- Woldhof, FRG Bio-Rad Laboratories, Richmond, CA, and Iatoron Laboratories, Tokyo; respectively). 7-(~~-Galactosyloxy)-4-methylcoum~n and mercaptoethanol were purchased from Nakarai Chemicals Ltd., Kyoto, Japan. 4-(Maleimidomethyl)cyclohexane carboxylic acid (MCC) was synthesized in our laboratories from N-cr44-carboxycy- clohexyl)methylmaleamide~ according to the procedure of Yamada et aL;9 mp 158-160 "C (lit.9 mp 144-146 "C); TLC (silica gel): R, 0.83 (chloroform:methanol, 5:2); 'H NMR (CDCl,): 6 1.8-2.5 (m, 10, CHz), 3.83 (d, 2, CH,N), 6.71 (8, 2, CH=CH), and 11.07 ppm (br 8, 1, COOH); MS: mlz 237(M+', 72%), 191(47), 111(97), 94(55), and 8l(base peak); IR(KBr): 3040-2400 (COOH) and 1700 (C=O) cm-l. Buffer A consisted of 0.1 mM MgC12, 0.1% BSA, and 0.1% NaN3 in 20 mM sodium phosphate buffer (pH 7.0). Buffer B consisted of 1 mM NazEDTA, 0.1% NaN3, and 0.1% BSA in 20 mM sodium phosphate buffer (pH 7.4).
Synthesis of the Immunogen-The pathway for the synthesis of immunogen is shown in Scheme I. A mixture of 23.4 mg of 4- (maleimidomethy1)cyclohexane carboxylic acid (MCC), 11.5 mg of N -
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Q Scheme 1-Pathway for synthesis of immunogen. Reagents: a, N- hydroxysuccinimide/N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (DEC)/THF to give 4-(maleimidoethyl)cyclohexane car- boxylic acid (MCC); b, captopril; c, bovine immunoglobulin G (BlgG)/ phosphate buffer (pH 7.0).
hydroxysuccinimide, and 19.2 mg of N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride in 2 mL of tetrahydrofuran was stirred at room temperature for 1 h. Fifty milligrams (10 mCi) of ['Hlcaptopril, which was prepared by hydrolyzing the tritiated captopril acetate ester in alkaline solution and extracting with ethyl acetate, was added to the mixture. This mixture was then added to 50 mL of 0.1 mM phosphate buffer (pH 7.0) containing 300 mg of bovine immunoglobulin G (BIgG) and the preparation was stirred for 1 h. Ammonium sulfate (3 g) was then added and the mixture was stirred gently a t room temperature for 1.5 h and centrifuged a t 10 000 rpm for 10 min. The precipitate was solubilized with 10 mM sodium phosphate buffer (pH 7.4) and then dialyzed against 10 mM sodium phosphate buffer (pH 7.4). The number of hapten residues conjugated per mole of the BIgG-conjugate was calculated to be 16 by measuring the radioactivity of ['Hlcaptopril.
Immunization-Three JW/CSK rabbits, weighing 2 kg, were in- jected intradermally with 1 mg of the immunogen which was emulsified with 0.75 mL of Freund's complete a4uvant and 0.25 mL of isotonic saline. The rabbits were immunized again at 2-week intervals with the same amount of immuno en. Three months after
ed in the sera of all rabbits. Ten days aRer the last booster injection, the rabbits were sacrificed and the antisera of each rabbit was frozen. The frozen antisera with the highest binding activity was used for the captopril assay.
Preparation of Captopril-4-(Maleimidomethyl)cyclohexane Carboxylic Acid-B-PGalactosidase Coqjugat-A mixture of 2.34 mg of MCC, 1.15 mg ofN-hydroxysuccinimide, and 1.92 mg of N-(3- dimethylaminopropy1)-N'-ethylcarbodiimide hydrochloride in 0.2 mL of tetrahydrofuran was stirred at room temperature for 1 h. Captopril(2.15 mg) was added to the mixture. Aliquots (2.5,5.0, 10, 20, and 40 pL) of this mixture were added ta 1 mL of phosphate buffer (20 mM, pH 7.0) containing p-Pgalactosidase (1 mg). The molar ratios of enzyme to hapten were 1:125, 1:250, 1:500, 1:1000, and 1:2500, respectively. The mixture was incubated at room tem- perature for 2 h. After ultrafiltration, the supernatant, which was adjusted to a concentration of 1 mmoUmL, was used in the experi- ment.
Enzyme Immunoassay Procedure for Captopril-To each 13 x 100-mm glass tube was added 100 pL of sample or standard, 100 KL of phosphate buffer B, 100 pL of normal plasma, and 50 of 4-
the first immunization, binding activity to [ Q Hlcaptopril was detect-
0022-3549/86/0700-0711$0 1 .OO/O 0 1966, American Pharmaceutical Association
Journal of Pharmaceutical Sciences / 71 1 Vol. 75, No. 7, July 1986
(maleimidomethy1)cyclohexane carboxylic acid (MCC) in buffer B (2.5 pg/mL). The mixture was incubated at room temperature for 30 min to afford adducts of captopril-MCC. Then, 50 pL of the mercap- toethanol solution (40 pg/mL of phosphate buffer B) was added and the solution was incubated at room temperature for 30 min. After- ward, 100 & of captopril-MCGpD-galactosidase conjugate solu- tion (1:80 000)) and 100 pL of antiserum solution (1:200 000) were added to each tube. The mixture was incubated at 4 "C for 16 h. Immunobead second antibody goat anti-rabbit immunoglobulin (ISA; 100 FL) was added to this solution. After incubating at 37 "C for 1 h, 3 mL of buffer A was added and the tube was centrifuged at 2500 rpm for 5 min. This procedure was repeated twice. The pellet was resuspended in 500 & of buffer A containing 7-(p~-galactosy- loxy)-4-methylcoumarin (0.1 mM) as the substrate. After incubation a t 37 "C for 1 h, 3 mL of 0.2 M g1ycine:NaOH buffer (pH 10.6) was added to the mixture which was then centrifuged at 2500 rpm for 5 min. The fluorescence intensity of the 7-hydroxy-4-methylcoumarin formed was measured at A (ex) 360 nm and A (em) 450 nm.
Calculations-The standard curve was constructed on the day of analysis by plotting the values of E/Eo on the y-axis, and the log values of captopril concentrations on the x-axis, where E and Eo are the antibody-bound enzyme activities in the presence and absence of captopril, respectively. Assay Characteristicdross-reactivity (CR) was estimated by
measuring the 'concentration of a compound that would displace captopril-MCC-PD-galactosidase conjugate from the antibody. Per- cent CR is calculated from: %CR = ( IC~~Mso)lOO; where ICso is the concentration of a compound that causes a displacement of 50% of the captopril-MCC-PD-galactosidase to Eo, and IMso is the concen- tration of captopril that displaces 50% of the Captopril-MCC-pD- galactosidase conjugate ta Eo.
Results Extent of Conjugation of MCC-Captopril With pa-
galactosidas-For hapten-p-D-galactosidase conjugation, MCC-captopril was synthesized and linked to the amino groups of Pbgalactosidase under various molar ratios of PP galactosidase to MCC-captopril (1:125, 1:250, 1500, 1:1000, and 1:2500). With respect to the immunoreactivities of these conjugates, shown in Fig. 1, a standard curve showed a steep slope in the case where the molar ratio of enzyme to MCC- captopril for the conjugate was 1:500. For assay purposes, labeled enzyme, with a ratio of PD-galactosidase to MCC- captopril of 1:500, was used.
Sensitivity and Specificity of the Captopril Enzyme Immunoassay-When 100 pL of the plasma samples was used, as shown in Fig. 1, the enzyme immunoassay (EIA) measured a8 little as 0.5 ng/mL of captopril in plasma. Measurement of lower concentrations was achieved when larger plasma samples (200 pL) were used. The specificity of the antisera was assessed according to the cross-reactions. The percentage of cross-reaction to the captopril disulfide was 3.75%; captopril- and mercaptoethanol-MCC did not cross-react to the antibody (Table I).
Flgure 1-Standard curve of captopril. E and Eo are the antibody-bound enzyme activifies in the presence and absence of captopnl, respecfively.
Table &Cross-Reactlon of the CaptoprlC4~Malelrnldornethyl)- cyclohexane Carboxyllc Acid (MCC) Antlsera
CaptopriCMCC' 100 Captopril Disulfide 3.75 MercaptoethanoCMCC - b Captopril without MCCC - d
'lmmunoas~ay was performed with MCC. bNot detected when 10 pg/mL present. clmmunoassay was performed without MCC. dNot detected when 256 ng/mL present.
I 0. I I 10 I b O
ns I m L
Figure 2-Efecf of human plasma on captopril enzyme immunoassay. Key: (0-0) with human plasma; (A-A) withouf human plasma. E and Eo are the antibody-bound enzyme activities in the presence and absence of captopril, respecfively.
The possible interference due to plasma matrix difference was also examined. As shown in Fig. 2, endogeneous materi- als did not interfere with the assay in human plasma.
Aesay Variations and Recovery-Both intra- and interas- say variations were estimated by using plasma standards prepared in accordance with the procedure described. The intra-assay variance was calculated from assay values ob- tained from a single day, whereas the interassay variance was assessed on the basis of assay values from four consecu- tive days. The recovery of captopril was near 100% when the drug was added at different concentrations (0.5, 2,8, and 64 nglmL) to plasma. The results are summarized in Table 11.
Table ll-Assay Varlann and Recovery of the Captoprll Enzyme lmrnunoassay
Mean Captopril Intra-assay Variance' lnterassa Variance I Concentration. Recoverv. * %
0.125 0.25 0.50 1 .oo 2.00 4.00 8.00
0.006 4.8 0.003 2.4 0.01 4.0 0.01 4.0 0.01 1.5 0.01 2.4 99.6 0.02 1.9 0.04 3.6 0.04 1.9 0.11 5.2 102 0.06 1.6 0.07 1.8 0.44 6.0 0.14 1.8 97.3 1.63 4.8 1.56 5.3 2.52 4.0 4.26 7.4 89.7
'Intra-assay variance was calculated from assay values of plasma standards in a single day of analysis; n = 4. Interassay variance was calculated from assay values of plasma standards in four consecutive days of analysis; n = 4. CCoefficient of variation. dRecovery from a set of independently prepared spiked samples.
71 2 / Journal of Pharmaceutical Sciences Vol. 75, No. 7, July 1986
- The assay variances were