Jeongeun Choi

Immunological analysis of methamphetamine antibody and its use for the detection of methamphetamine by capillary electrophoresis with laser-induced fluorescence

An accurate, simple and rapid immunoassay is demonstrated for the detection of methamphetamine in urine by capillary electrophoresis (CE) with laser-induced fluorescence (LIF). An aminobutyl derivative of methamphetamine was conjugated with proteins, and used as an immunogen to produce antibodies for the assay. The methamphetamine derivative was also labeled with fluorescein isothiocyanate (FITC) to compete with free methamphetamine in the sample for the antibody binding site. Levels of free and antibody-bound FITC-labeled methamphetamine were monitored by performing CE-LIF using an untreated fused-silica column. This competitive immunoassay used antiserum instead of purified antibody or antibody fragment, yet was found to have good precision with a sensitivity of lower than 20 ng/ml. Various antibodies were also screened, and cross-reactivity of anti-MA antibody with methamphetamine analogues were also investigated. The results indicate that CE-LIF-based immunoassay is a powerful tool for the screening and characterization of antibody and may have possible applications in the detection of abused drugs in urine.

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.

Localization of the Epitope in Methamphetamine and Its Antibody Use for the Detection of Methamphetamine and Benzphetamine by Polarization Fluoroimmunoassay

An antibody was prepared, using a four carbon-bridged methamphetamine molecule as an immunogen in order to develop a polarization fluoroimmunoassay for urine screening of methamphetamine and benzphetamine. Also, its binding characteristics were investigated to locate epitope sites of methamphetamine. The study showed that the antibody was highly capable of eliciting a polarization fluoroimmunoassay response. However, the detection limit was much greater for benzphetamine (0.05 ppm) than for methamphetamine (0.2 ppm) and weakly antibody binding was found with methamphetamine. This difference in sensitivity may reflect the similarity of benzphetamine to the immunogen used to produce the antibody. Both benzphetamine and the immunogen have a tertiary amine attached to a carbon bridges whereas methamphetamine has only a secondary amine and amphetamine has a primary amine group. The difference of cross-reactivity data between phenylethylamine drugs and beta-hydroxyl phenylethylamine drugs indicates that the beta-carbon position have a major influence on the antibody interaction. Thus, the substitution of hydroxyl group on beta-carbon resulted in virtually no antibody affinity, even if a tertiary amine or secondary amine group was present in the molecule. This suggests that the beta-carbon chain plays a primary role as the epitope site with cooperative binding site of tertiary amine or secondary amine in alpha-carbon position. A hydroxyl group at the beta-carbon position plays an important inhibitory role to the antibody binding.

The optimization of ELISA for methamphetamine determination: the effect of immunogen, tracer and antibody purification method on the sensitivity

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.

A new visual enzyme immunoassay of methamphetamine using linear water-soluble polyelectrolytes

A new visual enzyme immunoassay (EIA) technique has been developed. Oppositely charged synthetic linear water-soluble polyelectrolytes (poly-N-ethyl-4-vinyl-pyridine as polycation and polymethacrylate as polyanion) were used as carriers for reagent immobilization. The ability of these molecules to form an insoluble complex was applied for the separation of bound and free components of the immunoassay reaction mixture. This approach was realized in methamphetamine visual EIA. In the first stage of the assay two specific reactions took place during incubation of the analytical reagents with the probe to be analyzed: (1) competition between methamphetamine and hapten conjugated with peroxidase for the interaction with specific antibodies and (2) interaction of these antibodies with the protein A-polymethacrylate conjugate. As a result of these reactions the (polyanion-protein A)-antibody-(hapten-peroxidase) complex was formed. Then the reaction mixture was filtered through an Ultrabind membrane (0.45 microns) with adsorbed poly-N-ethyl-4-vinylpyridine, and the immunological complexes were immobilized to the membrane by electrostatic interaction. The level of peroxidase binding on the membrane was measured by diaminobenzidine substrate. The system described was optimized to achieve both high rapidity (20 min) and an appropriate sensitivity (0.4 micrograms/ml) for methamphetamine assay.

Production and Characterization of Monoclonal Antibodies Specific to Atrazine Group Compounds: Effects of Coating Ligand Structure on the Variation of Sensitivity and Specificity

Hybridoma cells were prepared by immunizing mice with carboxylic derivatives of atrazine conjugate to bovine serum albumin. After the screening of culture supernatant of hybridomas, five cell lines producing monoclonal antibodies were established and 1.8-5.3 ml of ascitic fluid per mouse was obtained from each cell line. The protein A affinity purification yielded 0.35-0.65 mg per ml of ascitic fluid from each cell line. The characterization studies in terms of sensitivity and specificity indicate that MAb 2F9 and MAb 4B9 showed the best responses with atrazine and its group of ametryne and cyanazine, using microtiter plate coated with simazine derivative of 6-amino hexanoic acid; no cross-reactivity was shown with simazine and cyanuric chloride.

Fluorescein labeling of estrogen for application of fluorescence polarization binding assays for antibody and receptor

Abstract The fluorescence polarization binding assay (FPBA) using fluorescein-labeled estrogen tracer is a homogeneous assay applicable to both estrogen antibody and estrogen receptor-binding assays. Two estrogen-ethylendiamine fluoresceinthiobamyl (E-EDF) tracers were synthesized; estrogen-6-EDF (E-6-F) derived from 6-ketoestradiol 6-(o-carboxymethyl) oxime and estrogen-17-EDF (E-17-F) was from 17β-estradiol 17-hemisuccinate. In both FPBAs using antibody and receptor, E-6-F tracer (Rf365nm=0.58) showed a better binding response than E-17-F (Rf365nm=0.70) indicating that the 17-position of estrogen seems to play an essential role as a binding site for antibody or receptor. In the optimized conditions of FPBA for E2 using E-6-F tracer, antibody binding (Kd=9.4×10−9 M) is 50 times sensitive than receptor binding (Kd=4.6×10−8 M). Binding responses of estrogen and its related chemicals by FPBA indicate that antibody binding assay is able to screen the structural similarity of estrogen showing some response with methyltestosterone (Ki=2.1×10−5 M). On the other hand, the receptor assay is able to screen for estrogenic chemicals such as tamoxifen (Ki=4.5×10−9 M) and diethylstilbesterol (Ki=8.1×10−7 M). Therefore, E-6-F tracer is useful as a tracer for FPBA that is able to screen for chemicals structurally similar to estrogen using antibody, and that is able to screen for chemicals functionally similar to estrogen using receptor binding assay.

EFFECTS OF THE COMPETITOR ON ANTIBODY-HAPTEN BINDING IN IMMUNOASSAYS

ABSTRACT The effects of competitors on antibody (Ab)-hapten binding in an immunoassay were investigated using a goat anti-methamphetamine (MA) antibody (Ab). An N-4-aminobutyl derivative of methamphetamine (4-ABMA) was conjugated with keyhole limpet hemocyanine (KLH) and used as an immunogen. The antiserum was purified by affinity chromatography with various ligands, including 4-ABMA-protein conjugates, free haptens, and protein G. Direct and indirect competitive enzyme-linked immunosorbent assays (ELISA) were conducted with a competitor of 4-ABMA-fluorescein isothiocyanate (4-ABMA-FITC). The results were compared to those of ELISA with a different competing antigen, 4-ABMA-ovalbumin (4-ABMA-OVA), in terms of sensitivity and specificity. In both direct and indirect assay formats, the sensitivity was much improved with 4-ABMA-FITC, compared to that with 4-ABMA-OVA, suggesting that different labels on the same haptenic moiety for competitors considerably influence the assay performance. All the purified Abs also showed a distinct feature of strong affinity for benzphetamine with 4-ABMA-FITC, whereas they had their respective binding specificities with 4-ABMA-OVA. Comparing the results to those from other assay systems, we determined that the assay sensitivity was dependent on both the system and the competitor employed, and that the specificity was primarily dependent on the competitor used.

A simple device of the dry tetrabromophenolphthalein ethyl ester reagent strip for the detection of methamphetamine

A new device to detect methamphetamine (MA), amphetamine(A) and its metabolites in urine was developed using the paper strip method and the test tube method of dry chemical reagents. The reagent containing tetrabromophenolphthalein ethyl ester (TBPE) and borax. For the TBPE paper strip method, a device was prepared with a window at each end of the reagent paper strip; one window is for the sample application, and the other window is for the methylene chloride. The diffused sample from one window reacts with reagent in the paper and produces color at the point where it meets with methylene chloride which has diffused form the other side. A positive sample produces as red-purple color and the negative sample a greenish color, with a detection limit of 5–10 ppm. The result can be obtained within one minute. For the TBPE test tube method which contains dry reagents, the detection limit is 5 ppm and the result can be obtained within 30 seconds, however the carry-on is not as convenient as the paper strip method. The performance of both methods were evlauated by comparing with the results of gas chromatography (GC) and fluorescence polarization immunoassay (FPIA). The results were proven that both methods were useful as primary screening reagents to detect MA in urine and in dry powder.