• Bulletin of the Korean Chemical Society
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• 제23권4호
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• pp.605-609
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• 2002

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of organophosphorus insecticide cyanophos. An analogue (hapten) of cyanophos was synthesized and was coupled to BSA to produce polyclonal antibodi es from rabbits. The antisera were screened against another hapten coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigen-coated ELISA was developed, which showed an I50 of 310 ng/mL with the detection limit of 20 ng/mL. The antibodies showed negligible cross-reactivities with other organophosphorus pesticides except for parathion-methyl, which makes the assay suitable for the selective detection of cyanophos.

• Bulletin of the Korean Chemical Society
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• 제23권8호
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• pp.1116-1119
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• 2002

Monoclonal antibodies have been generated against a generic hapten, ο,ο-diethyl ο-(5-carboxy-2-fluorophenyl) phosphorothioate, for the determination of organophosphorus (OP) pesticides in a class-specific manner. In an indirect competitive enzyme-linked immunosorbent assay (ELISA) format, employing a heterologous coating antigen, these monoclonal antibodies showed desirable properties for use in the class-specific determination, i.e., broad specificity and high sensitivity. The IC50 values of four commonly used ο,ο-diethyl OP pesticides were fairly uniform ranging from 0.1 to 0.3 ㎛/mL. The IC50 values of three ο,ο-dimethyl derivatives were between 0.3 and 1.4 ㎛/mL. These values, together with the limits of detection (LOD), were better, in terms of the specificity and sensitivity, compared with the values obtained previously with polyclonal antibodies.

• Asian-Australasian Journal of Animal Sciences
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• 제15권3호
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• pp.410-415
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• 2002

Adrenal glucocorticoids, secreted by the stimulus of adrenocorticotropic hormone (ACTH), are catabolic hormones in the pig. The present study was conducted to find whether active immunization against ACTH would suppress cortisol secretion accompanied by an increased growth rate in growing-finishing barrows. ACTH was conjugated to keyhole limpet hemocyanin or human histone using glutaraldehyde or 3-maleimidobenzoic acid N-hydroxysuccinimide, under a 2 (ACTH vs no hapten)${\times}$2 (carrier)${\times}$2 (crosslinker) factorial arrangement of treatments. Cross-bred barrows weighing approximately 25 kg were injected with an ACTHcarrier or carrier only conjugate every 4th wk and slaughtered at approximately 110 kg body weight. Antibodies against ACTH were detected in serum, as determined by $[^{125}I]$ACTH-binding activity, in most animals immunized against the ACTH conjugate, but not in carrier only-injected animals, except for the animals which had received the hapten conjugated to histone via glutaraldehyde. The $[^{125}I]$ACTH-binding activity of serum increased after the second booster injection, but overall ACTH antibody titer was very low. Main effect was not detected not only for the carrier and crosslinker but for the hapten in serum cortisol concentration, ADG, loin muscle area, backfat thickness and longissimus muscle composition including fat and protein. In addition, bound $[^{125}I]$ACTH percentage had no relation to cortisol concentration or to any of the above growth-related variables. Results suggest that ACTH or its conjugates used in the present study were not immunogenically potent enough to affect the glucocorticoid secretion and thus the growth of the immunized pigs.

• 한국환경농학회지
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• 제12권3호
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• pp.298-308
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• 1993

Immunochemical assay, ELISA for small molecules such as pesticides are rapid, sensitive, cost effective and can easily analyze with large samples. ELISA is one of several powerful biotechnologies immediately applicable to pesticide analysis. This review lists the advantages and disadvantages of the ELISA and elucidate the steps in assay development using examples from this laboratory. The focus is primarily on hapten synthesis strategies, protein conjugation, Immunization, assay format, and assay validation.

• Bulletin of the Korean Chemical Society
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• 제24권11호
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• pp.1605-1608
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• 2003

To development an immunodetection method for DDT, 1,1,1-trichloro-2,2-bis(4-chorophenyl)ethane (p,p'-DDT) and its metabolites (p,p'-DDA, p,p'-DDE, p,p'-DDD), five derivatives of DDT haptens have been synthesised and characterized as coating ligands for antibody evaluation. The appropriate lengths of linkers were introduced to investigate a matching pair of coating ligand and antibody. Among these hapten derivatives, 2,2-bis(4-chlorophenyl)acetic acid (DDA), 5,5-bis(4-chlorophenyl)-5-hydroxypentanoic acid (DDHP) and 5,5-bis(4-chlorophenyl)-5-chloropentanoic acid (DDCP) were conjugated with keyhole limpet hemocyanin (KLH) for its use as an immunogen. The bovine serum albumin (BSA) conjugates of these derivatives were prepared as a coating ligand for monoclonal antibody screening. Fifteen monoclonal antibody clones were screened using these probes. 6,6-Bis(4-chlorophenyl)-6-hydroxyhexanoic acid (DDHH) and 3-[6,6-Bis(4-chlorophenyl)-6-hydroxyhexanoylamino]propanoic acid (DDHHAP), in addition to the above hapten derivatives, were conjugated to ovualbumin (OVA) and bovine serum albumin (BSA) for their use as coating ligands to measure the titration level of the antibody and the displacement of free analytes. The indirect competitive ELISA results indicate that the titration level and free analyte displacement were greatly influenced by the DDT derivatives and carrier proteins used. Three matching pairs of monoclonal antibodies and coating ligands were selected for the DDT immunoassay: antibody clone 1A3 and coating ligand DDA-OVA, 1A1 and DDHHAP-BSA, and 1A4 and DDHP-OVA.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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• pp.273-273
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• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.

• Journal of Applied Biological Chemistry
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• 제48권1호
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• pp.16-25
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• 2005

To develop an enzyme-linked immunosorbent assay (ELISA) for the detection of the residues of the acaricide fenazaquin, five haptens were synthesized and assessed. A competitive indirect format was used with polyclonal antibodies. Under an optimized condition using the selected rabbit C antiserum, an $IC_{50}$ of $96.97\;ng{\cdot}ml^{-1}$, the detection range of $14.9{\sim}631\;ng{\cdot}ml^{-1}$, and the lowest detection limit of $8\;ng{\cdot}ml^{-1}$ were obtained. Some structurally related compounds of practical use showed low crossreactivities to the antibody. Highest cross-reactivity observed with hapten IV indicates that the antiserum C recognizes very well quinazoline ring, 4-tert-butylphenyl, and an adequate length of spacer arm. The length of spacer arm affected recognition of quinazoline ring and 4-tert-butylphenyl moieties. When applied to apple and pear, recoveries were within acceptable ranges of $93.18{\sim}104.77%$ (n = 4) and $79.40{\sim}111.95%$ (n = 4), respectively.

• Bulletin of the Korean Chemical Society
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• 제24권3호
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• pp.334-338
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• 2003

A simple synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (aminocarboxylic acid) attached at the pesticide thiophosphate group was developed and was applied to the synthesis of haptens for the OP fungicide tolclofos-methyl. Using the haptens, a selective enzyme-linked immunosorbent assay (ELISA) for tolclofos-methyl was developed. One of the haptens was coupled to BSA to use as an immunogen. Rabbits were immunized with this conjugate to obtain polyclonal antibodies to tolclofos-methyl. The antisera were screened against another hapten coupled to ovalbumin (OVA). Using the serum with highest specificity, an antigen-coated ELISA was developed, which showed an $IC_{50}$ of 160 ng/mL with the detection limit of 20 ng/mL. The antibodies showed negligible cross-reactivity with other OP pesticides. An antibody-coated ELISA was also developed, which showed an $IC_{50}$ of 410 ng/mL with a detection limit of 130 ng/mL.

• 생약학회지
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• 제30권2호
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• pp.115-122
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• 1999

In order to investigate the effects of Bupleuri Radix aqua-acupuncture solution (BRAS) on immuno suppression induced by glucocorticoid, ICR mice were administrated with glucocorticoid (80 mg/kg) for 7 days, and immunized with hapten, methamphetamine-horseradish peroxidase $(10\;{\mu}g/mouse)$. And then, BRAS (0.2ml/mouse) injected into $CV_4\;and\;BL_{23}$, which are the classical acupuncture points in traditional medicine, for 7 days. And then B and T cells proliferation and cytolytic activity of natural killer (NK) cells were measured. Intraperitoneal injection of glucocorticoid decreased lysozyme activity in macrophage and cytolytic activity of NK cell. B and T cell proliferation were significantly increased in aqua-acupuncture group compared to normal group. On the other hand, BRAS significantly increased the lysozyme activity in macrophage, and the cytolytic activity of purified NK cell on K562. These results suggest that BRAS at $CV_4\;and\;BL_{23}$ may proliferate B and T cells that are suppressed by glucocorticoid and activate NK cell activity.

• Parasites, Hosts and Diseases
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• 제37권2호
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• pp.93-99
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• 1999

Eosinohil and IgE responses of interleukin IL(-5 transgenic and normal C3H/HeN mice were studied after experimental infection with Nippostrongylus brasiliensis 9Nb). Intestinal worms were recovered at day 5 post-infection (PI), and numbers of total white blood cells (WBC) and eosinophils, and total serum IgE and anti-hapten (dinitrophenyl)(DNP) specific IgE titers, were measured at days 0,14 and PI. IL-5 mice appeared resistant to Nb infection showing a significantly ower worm recovery rate than normal mice (P<0.05). Total WBC and eosinophil counts (/mm3) were significantly increased in Nb infected normal mice (p<0.05), but unchanged (total WBC) or decreased (eosinophils) in IL-5 mice at day 21 PI. The total serum IgE level remarkably increased in normal mice, but only a little in IL-5 mice at days 14 and 21 PI. Priming with DNP brought about more remarkable increases of the total and anti-DNP specific IgE in normal mice than in IL-5 mice. The results show that IL-5 mice are resistant to Nb infection, and that eosinophil and IgE responses in these mice are not augmented by N infection.