MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 32, Number 4, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2013.0002
A Novel Monoclonal Antibody Specific for Cocaine Hiroshi Nakayama, Noriko Kenjyou, and Nobuyuki Shigetoh
Detection systems for the illegal drug cocaine need to have a high sensitivity and specificity for cocaine and to be relatively easy to use. In the current study, a monoclonal antibody (MAb) with a high specificity for cocaine was produced. Enzyme-linked immunosorbent assay and fluorescence quenching immunoassay were used to screen the hybridomas. The MAb S27Y (IgG1) was shown to be sensitive and specific for cocaine and quenched fluorescence. Thus, S27Y has the potential to be used in screening assays for the rapid and sensitive detection of cocaine.
Materials and Methods
rug abuse has become a serious social problem during the past few decades and cocaine is one of the most dangerous illegal drugs.(1) As such, it has been necessary to develop sensitive and useful methods for the detection of cocaine. Several methods often used for the detection of cocaine include thin layer chromatography (TLC), liquid chromatography (LC),(2,3) gas chromatography/mass spectrometry (GC/MS),(4,5) and capillary zone electrophoresis.(6,7) These assays are generally time consuming to perform. In addition, there are various types of immunological assays(8–10) used as qualitative and quantitative methods for the detection of cocaine. A number of immunological biosensors have been developed for the detection of cocaine including the direct potentiometric method.(11,12) A variety of antibodies with highly specific binding properties are useful for screening the presence of cocaine in urine.(13) A rapid and sensitive method for monitoring drug levels is the noncompetitive immunoenzymometric assay using enzyme labels.(14) This method, however, requires the synthesis of labeled immunoconjugates. Currently, all homogeneous immunoassays used for cocaine detection require both labeling and bound/free (B/F) separation. We therefore considered it necessary to produce a monoclonal antibody (MAb) specific for cocaine (anti[a]-cocaine) that was able to reduce fluorescence intensity upon the formation of affinity complexes (cocaine:a-cocaine) without the need for labeling. Here we describe the production of a novel monoclonal antibody specific for cocaine. This MAb was not only able to recognize cocaine but was also used to measure the concentration of cocaine in the absence of any labeling or B/F separation.
Synthesis of immunogen We dissolved 500 mg cocaine (Shionogi & Co., Osaka, Japan) in 21 mL of mixture consisting of acetonitrile and water (1:2). The solution was adjusted with acetic acid to a pH of *5.0. Then 21 mL potassium permanganate solution (534 mg) was added dropwise to the solution. The solution was stirred overnight at room temperature. The liquid was purified by silica-gel thin layer chromatography (TLC, Merck, Darmstadt, Germany) using an eluent of ammonia-saturated chloroform to produce 269 mg norcocaine; the norcocaine, with 314 mg bromobutyl phthalimide and 147 mg anhydrous sodium carbonate, was dissolved in 10 mL benzene, and the mixture was refluxed under a nitrogen atmosphere for 72 h. The reaction mixture was purified by TLC (1:3 ammonia-saturated chloroform-benzene) to produce 270 mg butyl phthalimide derivative. 100 mg butyl phthalimide derivative was dissolved in 3 mL of 95% ethanol, to which hydrazine hydrate (0.20 mmol) was added, and refluxed for 2 h. The reaction mixture was purified by TLC (10:1 ammonia-saturated chloroform-methanol) to produce 57 mg amino derivative. The above 84 mg amino derivative and 73 mg o-succinimidyl-3-(2-pyridyl-dithio)-1-propionate (Dojindo Molecular Technologies, Kumamoto, Japan) in 1 mL ethanol were stirred at room temperature for 1 h, and the solution was purified by TLC (ammonia-saturated chloroform) to produce 133 mg of 2-pyridyl dithio derivative. The cocaine-KLH/PBS solution was produced by combining 48 mL of 1.73 · 10 - 5 M keyhole limpet hemocyanin (KLH)-SPDP (SH-free)/phosphate-buffered saline (PBS) solution with 4.63 mg (8.3 · 10 - 3 mol) of the pyridyl dithio derivative; the solution was stirred and kept at 4C overnight.
Bioscience Technology Development Office, R&D Division, Panasonic Co., Soraku-gun, Japan. All authors contributed equally to this work.
NOVEL MAb SPECIFIC FOR COCAINE Cell line The mouse myeloma cells (P3X63-Ag8.653) were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum in a 37C humidified incubator. Hybridoma preparation For hybridoma preparation, 100 mL of the prepared adjuvant emulsion (1:1 cocaine-KLH conjugate [100 mg/mouse] and complete Freund’s adjuvant [Wako Pure Chemical Industries, Osaka, Japan]) was injected intraperitoneally into 8week-old, female A/J mice ( Japan SLC, Shizuoka, Japan). The mice received the same injections 2 weeks later. Following another 12-week interval, the mice were injected intraperitoneally with 100 mL booster solution containing 100 mg cocaineKLH conjugate. Immunity was assessed by enzyme-linked immunosorbent assay (ELISA). The fusion of myeloma cells was carried out using standard methodology. The hybridoma cells of positive wells were cloned by the limiting dilution method. The cloned hybridoma cells were injected into A/J mice to produce ascites. The mice were sacrificed 2 weeks post-injection and the ascites extracted and centrifuged at 5000 rpm for 30 min at room temperature. The supernatants were purified by affinity chromatography using protein A Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden). ELISA assays For ELISA assays, 96-well polystyrene plates (Costar, Corning, NY) were coated with 100 mg/well of cocaine-bovine serum albumin (BSA) conjugate in PBS (pH 7.4) overnight at room temperature. The plates were washed and the unbound sites were blocked with 1% (w/v) BSA in PBS (pH 7.4). After washing twice with PBS (pH 7.4), the wells were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (0.2 mg/mL) and the absorbance of each well determined at 492 nm. Fluorescence quenching measurement All fluorescence measurements were conducted at 30C using a fluorimeter (Nihon Bunko, Tokyo, Japan) and a quartz microcell (path length, 10 mm). The a-cocaine antibody was dissolved in PBS to a final concentration of 1.9 · 10 - 7 M, and 360 mL aliquots were used for fluorescence quenching measurements. Excitation was performed at a wavelength of 280 nm (band pass, 5 nm) and emission at 340 nm (band pass, 10 nm). Fluorescence intensity of the unquenched a-cocaine solution was *35. The degree of fluorescence quenching was measured upon application of 20 mL aliquots of cocaine buffer solutions containing different concentrations (10 - 13–10 - 5M) to the a-cocaine antibody solution. Results Synthesis of immunogen Cocaine-KLH conjugate was successfully synthesized for use as an immunogen in the generation of a cocaine-specific monoclonal antibody. Approximately seven molecules of cocaine were bound per 1 mol of KLH.
263 Table 1. Comparison of Relative Sensitivities for Detection of Cocaine Derivatives Name Norcocaine Ecgonine Methylecgonine Benzoylecgonine
Relative sensitivity 10 13 20 30
Relative sensitivity was the lowest detectable concentration ratio for every substance relative to cocaine. The greater the relative sensitivity, the lower the detectability.
Preparation of anti-cocaine MAb Mouse splenocytes produced a high-titer antibody immune response to the fusion protein and were fused with myeloma cells. The resulting hybridomas were screened for the secretion of a-cocaine-specific antibodies by ELISA. Seven hybridomas that were specific for cocaine were obtained. Relative sensitivities for cocaine and its derivatives were also determined by ELISA (Table 1). Characterization of anti-cocaine MAb One of the seven hybridomas (S27Y; IgG1) exhibited fluorescence quenching, whereby the addition of increasing concentrations of cocaine (10 - 13–10 - 5) to the antibody solution resulted in a decrease in fluorescence intensity. As shown in Figure 1, fluorescence quenching was detected when 10 - 8.5 M cocaine (final concentration) was added to the S27Y monoclonal antibody solution (Fig. 1). Discussion Cocaine is a highly addictive and illegal drug and its detection is important for a variety of reasons. Recent reports suggested that a-cocaine antibodies might play an important role in the development of immunoassays for the detection of cocaine. Carrera and colleagues(15) generated a murine monoclonal antibody (GNC92H2) using a cocaine derivativeKLH immunoconjugate, which was shown to bind free cocaine with excellent specificity and good affinity (kDa = 40 nM). In general, when an antibody is irradiated with ultraviolet radiation (UV) of an approximate wavelength of 280 nm, fluorescence is emitted at about 340 nm because of the actions of amino acids including tryptophan. Accordingly, in the case where an antigen has quenching capability and tryptophan resides near the antigen-binding site of the antibody, the fluorescence intensity of bound antibody compared with free antibody decreases proportionally with antigen content since the quencher moiety is associated with tryptophan. Thus, quantitative detection of the antigen is possible because of the decrease in fluorescence following irradiation with UV light. Since cocaine has a quencher moiety (phenyl residue), the fluorescence emitted from the antibody bound to cocaine is suppressed to a greater extent than that of the free antibody. Using this fluorescence quenching method, the distinction between bound antibody and free antibody is not necessary and consequently gives rise to a rapid and sensitive detection assay. In this study, we obtained a-cocaine antibodies with an excellent affinity (kDa = 1 nM) for cocaine, and which upon complex formation with cocaine reduced fluorescence
NAKAYAMA ET AL.
Fluorescence quenching due to anti-cocaine antibody-cocaine complex formation.
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Address correspondence to: Hiroshi Nakayama Bioscience Technology Development Office R&D Division Panasonic Company 3-4 Hikaridai, Seika-cho Soraku-gun 619-0237 Japan E-mail: [email protected]
Received: January 7, 2013 Accepted: March 26, 2013