• Journal of Food Hygiene and Safety
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• v.10 no.4
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• pp.225.2-262
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• 1995

Ochratoxin A was produced from Aspergillus ochraceus ATCC 18472 which was then orally administered into the experimental mice to study the toxic levels of ochratoxin A. AELISA (enzyme-linked immunosorbent assay) method which is more rapid and safe than conventional analytical method, was developed by using ochratoxin A antibody. This method was successfully used to measure the levels of ochratoxin Ain blood, liver and kidney of mice. In order to produce a large amount of ochratoxin A to study toxicity in the mice, Aspergillus ochraceus ATCC 18412 was incubated in the rice medium and as a result 0.5 g of ochratoxin A form rice medium (kg) was produced after extraction and purification Feed consumption and gain in body weight of mice with ochratoxin A at a level of $10 \mu\textrm{g}/g$ body weight was significantly (p<0.05) reduced as compared with control during period of 3 weeks. Ochratoxin A-BSA conjugate was made by putting 13 mole of ochratoxin A on I mole of BSA. This conjugate was used to develop ELISA method. The minmum detection level of ochratoxin A by established ELISA method was 0.5 ppb. After oral adminstraton of ochratoxin A dose of $10\;\mu\textrm{g}/g$ every two day for 3 weeks, concentration of ochratoxin A was measured in the blood, liver and kidney by ELISA method. The level of ochratoxin A was $11\;\mu\textrm{g}/dl,\;0.9 \mu\textrm{g}/g\;and\;3.7\;\mu\textrm{g}/g$ in the blood, liver and kidney, respectively.

• Preventive Nutrition and Food Science
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• v.19 no.4
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• pp.268-273
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• 2014

The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with $0.1{\sim}1.0{\mu}M$ BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; $0.5{\mu}M$ and $10{\mu}M$ NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with $0.1{\sim}2.0{\mu}M$ BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with $1{\mu}M$ and $2{\mu}M$ BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with $1{\mu}M$ and $2{\mu}M$ NAC-BITC caused 1.6-and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in $0.1{\sim}2{\mu}M$ BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in $0.1{\mu}M$ NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., $1{\sim}2{\mu}M$) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.7 no.1
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• pp.33-40
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• 2021

Recently, antibody-drug conjugates (ADCs) are used to deliver efficient cytotoxic payloads selectively in cancer cells. In the designing of an ADC, the antibody is connected to a toxic payload via a covalent linker, which helps to solubilizes the typical hydrophobic payload as well as stabilizes the linkage over circulation. The development of the linkers for the antibody drug conjugate is still in demand. Initially, the acid, disulfide, and cathepsin-sensitive ADCs attracted considerable attention for the delivery of a potent cytotoxic payload but suffer from instability in human and mouse plasma with a short half-life. In addition, It also suffer from a solubility issue that induces aggregation, which is the major problem in their development. ADCs associated with sulfatase and phosphatase cleavable linker are highly soluble due to the anionic nature of sulfate and phosphate groups. The ADCs also showed high stability in human and mouse plasma. Therefore, to overcome these limitations, sulfatase and phosphatase cleavable linkers were developed. This review focuses on the recently reported advantages of sulfatase and phosphatase cleavable linkers for ADCs.

• Biomedical Science Letters
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• v.2 no.1
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• pp.121-126
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• 1996

Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay).These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

• Korean Journal of Veterinary Research
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• v.31 no.4
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• pp.491-500
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• 1991

This study was conducted to detect the serum antibodies in the experimentally toxoplasma infected dogs and street dogs by use the of an enzyme-linked immunosorbent assay (ELISA). And this test was performed on the polystylene microplate by coating with the tachyzoites soluble antigen of T gondii (RH strain), incubated with sera diluted then, added with HPO-conjugated rabbit anti-dog IgG and o-phenylenediamine used as a substrate. Tachyzoites of T gondii harvested from mouse peritoneal cavity were purified by 30, 40 and 50% Percoll density gradient centrifugation and used as the source of antigen. The results obtained were summarized as follows; 1. The highest ratio of positive to negative (P/N ratio) was obtained at the level of $l{\mu}g/ml$ protein concentration of antigen with the 1/4000 dilution of the conjugate measured by checker-board titration. It was regarded as the optimum concentration of the antigen and conjugate. 2. Cut-off value in this IgG ELISA was 0.375 that was determined by mean absorbance (at 492nm) of IFA negative serum added with the dauble value of the standard deviation $(mean{\pm}2S.D.)$. 3. Serum ELISA IgG antibodies to T gondii in the exyerimentally infected dogs were detected firstly at the Week 3 after inoculation and the highest titer was recognized at the Week 4, 5 and 6 after inoculation. 4. Stability of the antigen absorbed in the microplates that were preserved at $4^{\circ}C$ and $-25^{\circ}C$ separately were prolonged up to 3 weeks and 10 weeks at $4^{\circ}C$ and $-25^{\circ}C$, respectively. However the reproducibility was not reliable after the preservation of 4 weeks and longer. 5. Positive rate of the specific antibodies in 312 test sera was 28.5% and there was no significant differences between the male (27.8%) and female (29.5%), respectively. 6. The IgG ELISA was proved to be a specific procedure for the detection of antibodies to canine toxoplasma infection and also evaluated as a screening test for the large scale of test samples in laboratory.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.1
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• pp.71-80
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• 1990

New immunoassay systems for the detection of anti-sperm antibodies were developed. For this, sperm surface protein was purified by the immunoaffinity column prepared by the coupling of rabbit anti-human IgG antibodies to Sepharose-4B. Fraction eluted by tris-HCI buffer containing SDS showed a single band having molecular weight of about 60KD on electrophoresis. Enzyme HRP labelled goat anti-human IgG and chemiluminescence aminobutylethyl-isoluminol(ABEI) labelled rabbit anti-human IgG were used for ELISA and CIA, respectively. These two labelled conjugate bound well with human IgG. When serum dilution curves were made to titrate positive serums, two kinds of curves with steep and sluggish slopes were obtained Serum samples were categorized into 3 groups: positive, weak positive and negative based on slope of curve and O.D. values at 1:160 dilution of serum. When ELISA and CIA were compared to conventional method Kibrick test by the determinations of 62 male serums with different diagnosis, the results of ELISA and CIA agreed well, but both disagreed with that of Kibrick test. This study showed that purified sperm surface antigen can be used to develope solid-phase immunoassay systems such as ELISA and CIA which may eliminate the problems encounted the immobilization of living sperm in other tests.

• KSBB Journal
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• v.14 no.1
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• pp.82-90
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• 1999

In order to eventually fabricate an analytical system for infectious microorganisms, we synthesized major immunochemical components, utilized them for the construction of model system, and investigated an assay concept for bacterial whole cells. For the preparation of system components, a polyclonal antibody, against Salmonella thompson as model analyte, purified by immuno-affinity chromatography was used to chemically link to streptavidin or an enzyme, horseradish peroxidase(HRP). The antibody and streptavidin was modified with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate and N-succinimidyl-3-[2-pyridyldithio]propionate(subsequently activated by dithiotheritol), respectively. The modified components were reacted to synthesize antibody-streptavidin conjugates which were then purified on a two-layer chromatography column of diaminobiotin gel and Sephadex G-100. For antibody-HRP conjugates, HRP molecules were activated by $NalO_4$ oxidation and then coupled to immunoglobulin. After stabilizing with ($NaCNBH_3$, the conjugates were purified by size exclusion chromatography on Biogel A5M column. To devise a model system, such produced components were combined with a dot-blotter in which a nitrocellulose membrane($12{\mu}m$ pre size) with immobilized biotin was already located. The analyte (S. thompson cells) was reacted with the both antibody conjugates in a liquid phase, and the complexes formed were captured on the membrane surfaces by applying vacuum in the bottom compartment of the blotter to invoke biotin-streptavidin reaction. Under optimal conditions, the system enabled to identify the analytical concept for bacterial whole cells, and the lower limit of detection was approximately $1{\mu}g/m{\ell}$($10^5-10^6$ cells/m$m{\ell}$). The controlling factors were the concentrations of each antibody conjugate that caused agglutination in the presence of analyte as they increased.

• Analytical Science and Technology
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• v.23 no.6
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• pp.537-543
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• 2010

Dopamine (DA) is an important neurotransmitter molecule of catecholamines. Its deficiency could lead to brain disorder such as Parkinson's disease and schizophrenia. Therefore, it is necessary to establish a suitable analytical technique with sensitivity and simplicity. A competitive enzyme-linked immunosorbent assay for DA has been optimized and characterized. Assay sensitivity is controlled by two factors in competitive immunoassay. One is a nature and concentration of competitor, and the other is those of binder, antibody. Thus, optimization was performed: BSA-DA conjugate and antibody-avidin conjugate were prepared by dual heterobifunctional coupling method using SATA and SMCC. Assay condition was optimized with $6.66\;{\mu}gmL^{-1}$ of BSA-DA and $4.17{\times}10^{-10}\;M$ of antibody-avidin conjugate. A dose-response curve was constructed, and a limit of detection and a dynamic range for DA were accomplished to $2.3{\times}10^{-2}\;{\mu}g\;mL^{-1}$ and four orders of magnitude ($1.0{\times}10^{-7}\;M$ to $1.0{\times}10^{-3}\;M$), respectively. Calibration curve was constructed on dynamic range and least-squares regression of this data gave the following relationship: absorbance = -0.1098 log[DA]+0.0353 ($R^2$ = 0.9956).

• Korean Journal of Food Science and Technology
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• v.41 no.1
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• pp.111-115
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• 2009

In this study we analysed for peanuts in processed foods using an enzyme-linked fluorescent immunoassay (ELFA), and compared the results with labeled ingredients. Crude peanut protein (CPP) was immunized into rabbits to produce specific antibodies(Ab). A sandwich ELFA was established using anti-CPP Ab and Ab-horseradish peroxidase (HRP) conjugate. The cross-reactivities of the Ab toward CPP, peanuts, almonds, soybeans, and walnuts were 100, 9.8, $1.1{\times}10^{-2},\;4.4{\times}10^{-3}$, and 0%, respectively. The samples included 19 items consisting of biscuits, snacks, chocolates, and so on. The results from the sandwich ELFA showed that peanuts were contained in 7 of the processed food items, among which, 5 items were labeled as having peanuts present but 2 items were not. One of the 2 items that was peanut-detected but unlabeled was a biscuit labeled to contain almonds and assayed to contain $2.1{\times}10^{-3}%$ peanuts, which might have been due to the weak cross-reactivity of the Ab toward almonds. The other item was a snack labeled to contain soybeans and assayed to contain 0.098% peanuts, which might have been due to peanut cross-contamination during processing, since the crossreactivity of the Ab toward soybeans was very weak. These results suggest that ELFA is a good tool to detect peanuts in processed foods, and allergens in certain processed foods should be labeled correctly.

• Proceedings of the IEEK Conference
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• 2005.11a
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• pp.755-758
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• 2005

This paper describes the novel immunoassay sensing system for a portable clinical diagnosis system. It consists of a bead cage reactor and a CMOS integrated biosensor. It showed the simple and easy antibody coating method on beads by flow-through avidin biotin complex technology in a microfluidic device. It showed just 90 nL sample consumption and good result for the application of alpha feto protein. The bead cage reactor has the role of the antibody coating, antigen binding and enzyme linking for the electrochemical sensing method. The CMOS biosensor consists of ISFET (ion selective field effect transistor) biosensor and temperature sensor for detecting pH that is the byproduct of enzyme reaction. The sensitivity is 8 $kHz/^{\circ}C$ in a temperature sensor and 33 mV/pH in a pH sensor. After filling the 15 um polystyrene beads in bead cage, antibody flowed and reacted to beads. Subsequently, the biotinylated antigen flowed and bound to the antibody and GOD (glucose oxidase)-avidin conjugate flowed and reacted to the biotin of the biotinylated antigen. After this reaction process, glucose solution flowed and reacted to the GOD on beads. The hydrogen was generated by glucose-GOD reaction. And it was detected by the pH sensor.