Myung Ja 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.

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.

Characterization of s-triazine antibodies and comparison of enzyme immunoassay and biotin-avidin enzyme immunoassay for the determination of s-triazine

Abstract Triazines have been used widely as herbicides and known to cause environmental contamination. For developing ELISA of s -triazines, six kinds of s -triazine derivatives (one from simazine, one from atrazine and four from cyanuric chloride) which contained a C3- or C6-carboxylic acid group bridge were prepared. These derivatives were conjugated to bovine serum albumin (BSA) for the use of immunogens and to KLH for the use of coating ligands on the microtiter plate wells. Polyclonal antibodies produced from rabbit or sheep using atrazine-BSA (1b-BSA) and simazine-BSA (1a-BSA) immunogens. These antibodies were characterized and biotinylated for the use of enzyme immunoassay (EIA). We evaluated EIA and biotin-streptavidin mediated EIA in terms of the sensitivities and specificities with these antibodies. The results in both assay systems showed that coating ligand synthesized from atrazine derivative was better than that from simazine derivative. Comparing binding ability between C3-carboxylic acid and C6-carboxylic acid spacers of N -alkyl group in s -triazine ring, C3-carboxylate-KLH (2c-KLH) derived from atrazine showed better sensitivity for the both assay systems. The detection limit was found to be 0.01 ppb in biotin-streptavidin mediated EIA (B-Av EIA). Comparing IC 90 values of EIA (0.5 ppb) and B-Av EIA (0.05 ppb), B-Av EIA was able to detect one order lower concentration range of atrazine than EIA.

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.

Solid-phase immunoassay using a flow cytometer: quantitative and qualitative determination of protein antigens and a hapten

A fluoroimmunoassay employing a flow cytometer as the fluorescence-detecting device is described. Three kinds of antigens, murine immunoglobulin (Ig), human chorionic gonadotropin (hCG) and progesterone were chosen as examples of the assay using fluorescein-labeled antibodies. Cyanogen bromide-activated agarose beads were used as solid-phase supporters. The flow cytometric immunoassay was applied to both qualitative and quantitative analyses; determination of murine Ig isotypes, quantitative determination of Ig, hCG and a hapten, progesterone. This assay produced very reproducible and less-fluctuating data since thousands of particles in the assay were collected and processed to produce a single value for fluorescence intensities. Furthermore, the working range of the assay in terms of antigen concentration was much broader than that of enzyme immunoassay. Therefore, we believe that microparticles like agarose beads could be useful solid-phase supporters in immunoassay, and the flow cytometer could provide a reliable alternative to the fluorescence-detecting device in immunoassay.

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.

Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell.

By the fusion of lymphocytes from hyperimmunized people with heteromyeloma cells, 600 human hybridoma cell lines were generated. Even though seven cell lines produced antibodies against tetanus toxoid, only two antibodies from hybrid CH8 and CH5 only neutralized the tetanus toxin and completely protected the mice that had been challenged with the toxin even at the level of 90 mean lethal dose. The cDNA of light (L) chain and heavy (H) chain variable region was isolated, and then inserted into expression vectors containing human IgG constant regions. After transfection of the recombinant human IgG gene into Chinese Hamster Ovary (CHO) cells, transformants secreting the complete human antibody were selected. The recombinant human antibodies produced from CHO cells possessed neutralizing activity against tetanus toxin just like the original human antibodies produced from human hybridoma cell lines. Western blot analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin and did not bind to its L chain. The neutralizing test showed that HmAb rCH5 had 4.55IU and HmAb rCH8 had 1.09IU/100 micro g of IgG, respectively. Mixing of the two HmAbs resulted in synergistic effects. On a weight basis (IU/100 micro g IgG), the highest potency values were obtained when the two HmAbs were combined in equal quantity. The neutralizing activity of rCH8 and rCH5 mixture was 6.94IU/100 micro g IgG.

High throughput determination of BTEX by a one-step fluorescence polarization immunoassay

Environmental Context.Benzene, toluene, ethylbenzene, and xylenes (BTEX) are used as solvents in paints and coatings and are constituents of petroleum products. BTEX can contaminate air, water or soil and is toxic; benzene, in particular, is a recognized human carcinogen. Most existing methods for detecting BTEX are time-consuming, complicated and very expensive for routine screening. A rapid immunoassay of BTEX is presented that greatly simplifies environmental monitoring of water contamination. Abstract.For the rapid screening of BTEX (benzene, toluene, ethylbenzene, xylenes), a fluorescence polarization immunoassay (FPIA) was developed using the fluorescence polarization analyzer Abbott TDx. Several fluorescence-labelled tracers were synthesized by binding ethylenediamine fluorescein thiocarbamyl (EDF) to various substituted phenylcarboxylic acids. The binding of 27 tracers with two antisera that can be considered as class-specific for BTEX was investigated to select optimal tracer–antibody pairs. Significant differences were found in binding, titer, sensitivity, and assay kinetics. A best pair of anti-BTEX antiserum and EDF-labelled p-tolylacetic acid tracer was selected for FPIA. To simplify the method, an immunocomplex single reagent was prepared to detect BTEX by a one-step FPIA. One-step FPIA is a rapid homogeneous type of immunoassay that has only one pipetting step, does not need separation of free and bound analyte and can be performed at room temperature. The within-run coefficient of variation was ranged between 3.4% and 5.7%. Furthermore, if the measurement can be done at constant temperature, standards for every assay run are unnecessary. Cross-reactivity studies of petroleum compounds and a BTEX mixture indicated that p-xylene was most reactive with a limit of detection (LOD) of 0.22 µg mL−1 in 50 µL of sample. The LOD for toluene and benzene was 2.1 and 11 µg mL−1 respectively. The immunocomplex single reagent has proven to be significantly more stable than the corresponding solutions of antibody and tracer.

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.