• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.256-261
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• 2004

In order to detect chitooligosaccharides (COS) in soybean paste, tandem immunoaffinity chromatography and enzyme-linked immunosorbent assay (ELISA) were developed. Polyclonal anti-chitooligosaccharides mixture (CaSM) antibody specific to COSM was attached to Sepharose gel for initial sample cleanup and concentration of COS in soybean paste. COS was eluted and quantified by competitive direct ELISA (cdELISA). Average ELISA recoveries from the column using binding buffer spiked with COSM at levels of 0.5, 2.0, 5.0, and $10.0\mu$g/ml were 79.8, 72.0, 77.7, and 60.6%, respectively, with a mean recovery of 72.5%. Mean inter-well and inter-assay coefficients of variation (CV) were 7.7% and 10.3%, respectively. Average recoveries from soybean paste spiked with COSM at levels of 2, 6, 20, and $60\mu$g/g were 115, 91.7, 91, and 73.3%, respectively, with a mean recovery of 92.8%. Mean inter-well and inter-assay CV were 12.9% and 16%, respectively. The COS was detected from 24 out of 25 homemade Korean soybean paste samples at an average of $14.0\mu$g/g (n, 25; range, $0-51.2 \mu$g/g) and from 13 out of 14 commercially made soybean paste samples at an average of $4.1\mu$g/g(n, 14; range, $0-18.4\mu$g/g). The tandem immunoaffinity chromatography-cdELISA that was developed in this study showed that the level of COS eluted from homemade soybean paste was higher than that of the commercially made ones. In addition, the level of COS eluted from commercially available soybean paste in Korea was higher than that of the ones in Japan.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.571-579
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• 2016

In this work, we prepared a panel of monoclonal anti-idiotypic antibodies to ovine prolactin (oPRL) by the hybridoma technique. Among these antibodies, one anti-idotypic antibody (designated B7) was chosen for further characterization by a series of experiments. We first demonstrated that B7 behaved as a typical $Ab2{\beta}$ based on a series of enzyme-linked immunosorbent assays. Subsequently, the results of a competitive receptor-binding assay confirmed that B7 could specifically bind to the prolactin receptor (PRLR) expressed on target cells. Finally, we examined its biological activities in CHO-PRLR and Nb2 cells and observed that B7 could activate Janus kinase 2-signal transducer and activator of transcription signalling in CHO-PRLR and Nb2 cells and induce BaF3 proliferation. The present study suggests that i) B7 can serve as a PRLR agonist or PRL mimic and has potential applications in regulating mammary gland development, milk production and maintenance of lactation in domestic animals and ii) B7 may be a biological reagent that can be used to explore the mechanism of PRLR-mediated intracellular signalling.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.69-69
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• 1997

In the screening of tropical medicinal plants using PAE receptor binding assay, the ether extract of Ardisia crispa showed the potent antagonistic activity. Ardisia crispa have been used to heal the scurf, earache, orchitis, fever and diarrhoea, cough and given to the mother after childbirth to ‘wash out dirty blood’ in Malaysia. By means of activity guided isolation, compound AC7-1 was isolated as the potent PAF antagonist. In this study, antiplatelet effects of compound AC7-1 were examined in vitro platelet aggregation assay using the chronolog aggregometer. Compound AC7-1 inhibited PAF-, collagen-, ADP-, thrombin-induced platelet aggregation in human, rabbit and rat platelet rich plasma. In vitro rabbit platelet aggregation, the IC$\_$50/ value of compound AC7-1 was 5 ${\times}$ 10$\^$-6/ M against PAF(5 ${\times}$ 10$\^$-7/M)-induced aggregation. The IC$\_$50/ values of AC7-1 on PAF-induced platelet aggregation increased with increase of the concentration of PAF used. This result suggested the competitive nature of the AC7-1 antagonism. In vitro rat platelet aggregation, the IC$\_$50/ values of AC7-1 on collagen-, ADP-induced platelet aggregation were 4 ${\times}$ 10$\^$-6/ M, 2 ${\times}$ 10$\^$-5/ M, respectively. Also in vitro human platelet aggregation, AC7-1 potently inhibited both the primary phase and secondary phase of thrombin-induced aggregation.

• 약학회지
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• 제45권6호
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• pp.694-700
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• 2001

Synthetic pyrethroids are analysis of a natural chemical moiety, pyrethrin derived from the pyrethrum plant Chrysanthemum. The natural pyrethrin structure has been modified to be highly lipophilic and photostable, creating an effective pesticide and resulting in an increased presence in the environment. Worldwide, they are commonly used insecticides against ticks, mites, mosquitoes, and as treatment for human head lice and scabies. Therefore, human exposure to their compounds in extensive. Several studies on the effects of pyrethroids on thyroid hormone regulation, estrogen and androgen function have been reported and yet little has been done try assess their potential hormonal activities. Among humans, a pyrethroid compound was suggested to be the causal agent for gynecomastia in a group of Haitian men. The reports suggest that some pyrethroid compounds are capable of disrupting endocrine function. Therefore, we examined estrogenic/antiestrogenic potential of three pyrethroid insecticides, that is permethrin, allethrin and fenvalerate in human breast cancer cell and action mechanism mediated by the estrogen receptor. Fenvalerate showed weak estrogenic activity but aallethrin and permethrin showed no effect. In combination with high levels (10$^{-10}$ M, 10$^{-11}$ M) of 17$\beta$-estradiol and three synthetic pyrethroids inhibited cert proliferations in MCF7-BUS cell by 17$\beta$-estradiol. Whereas, fenvalerate increased cell proliferative activity at lower level of estradiol (10$^{-12}$ M, 10$^{-13}$ M). The relative affinities to the estrogen receptor were observed by allethrin and permethrin treatment, but not by fenvalerate. These results indicated that some of pyrethroid insecticides may modulate estrogen functions in human breast cancer cell. The action mechanisms of estrogen receptor mediated antiestrogenicity by allethrin and permethrin were postulated.

• 대한의생명과학회지
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• 제5권1호
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• pp.1-10
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• 1999

본 연구에서는 혈장이나 양수에 있는 $\alpha$-fetoprotein (AFP)을 인식 할 수 있는 모노클로날 항체를 제조하고, 모노클로날 항체를 이용한 효소면역분석법을 개발하고자 하였다. 양수로부터 얻은 AFP를 쥐에 주사한 후 비장을 분리하여 종양세포 (Sp2/O-Ag-14)와 융합하였고, 하이브리도마 기술을 이용하여 모노클로날 항체를 제조하였다. 모노클로날 항체를 클로닝하였으며, 생성된 항체를 MabF22로 명명하였고, IgG1 중사슬과 k 경사슬의 isotype을 나타냈다. 또한 immunoblotting 방법과 ELISA로 특이도를 조사한 결과 모노클로날 항체는 AFP와만 반응하였고, 결합 친화상수는 0.8$\times$$10^{-10}$M이었다. 두 종류의 효소면역분석법 -경쟁적 또는 비경쟁적 분석 -을 이용하여 항체의 효용성을 조사하였으며, 두 방법 모두 AFP와 농도에 비례하여 반응하였다. 따라서 본 연구에서 생산된 모노클로날 항체는 연구목적으로 뿐만 아니라 AFP 농도를 측정하기 위한 면역진단시약의 개발에도 유용할 것으로 생각된다.

• 한국가축번식학회지
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• 제9권2호
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• pp.133-139
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• 1985

An immunoassay may be defined as an analytical procedure involving the competitive reaction between a limiting concentration of specific antibody and two populations of antigen, one of which is labelled or immobillized. The advent of immunoassay has revolutionised our knowledge of reproductive physiology and the practice of veterinary and clinical medicine. Radioimmunoassay (RIA) was the first of these methods to be developed, which meausred the analyte with good sensitivity, accuracy and precision (1,2). The essential components of RIA are:-(i) a limited concentration of antibodies, (ii) a reference preparation, and (iii) an antigen labelled with a radioisotope (usually tritium or iodine-125). Most procedures invelove isolating the antibody-bound fraction and measuring the amount of labelled antigen. Good facilities are available for scintilltion counting, data reduction nd statistical analysis. RIA is undergoing refinement through:-(i) the introduction of new techniques to separate the antibody-bound and free fractions which minimize the misclassification of labelled antigen into these compartments, and the amount of non-specfic binding. (3), (ii) the development of non-extration for the measurement of haptens (4), (iii) the determination of a, pp.rent free (i.e. non-protein bound) analytes (5), and (iv) the use of monoclonal antibodies(6). In 1968, Miles and Hales introduced in important new type of immunoassay which they termed immunora-diometric assay (IRMA) based on t도 use of isotopically labelled specific antibodies(7) in a move from limited to excess reagent systems. The concept of two-site IRMAs (with a capture antibody on a solid-phase, and a second labelled antibody to a different antigenic determinant of the analyte) has enabled the development of more sensitive and less-time consuming methods for the measurement of protein hormones ovar wide concentration of analyte (8). The increasing use of isotopic methos for diverse a, pp.ications has exposed several problems. For example, the radioactive half-life and radiolysis of the labelled reagent limits assay sensitivity and imposes a time limit on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive cmpounds and the solvents and photofluors necessary for liquid scientillation counting are incompatable with the development of extra-laboratory tests. To date, the most practical alternative labels to radioisotopes, for the measurement of analytes in a concentration > 1 ng/ml, are erythrocytes, polystyrene particiles, gold sols, dyes and enzymes or cofactors with a visual or colorimetric end-point(9). Increased sensitivity to<1 pg/ml may be obtained with fluorescent and chemiluminescent labels, or enzymes with a fluorometric, chemiluminometric or bioluminometric end-point. The sensitivity of any immunoassay or immunometric assay depends on the affinity of the antibody-antigen reaction, the specific activity of the label, the precision with which the reagents are manipulated and the nonspecific background signal (10). The sensitivity of a limited reagent system for the measurement of haptens or proteins is mainly dependent upon the affinity of the antibodies and the smalleest amount of reagent that may be manipulated. Consequently, it is difficult in practice to improve on the sensitivity obtained with iodine-125 as the label. Conversely, with excess reagent systems for the measurement of proteins it is theoretically possible to increase assay sensitivity at least 1000 fold with alternative luminescent labels. To date, a 10-fold improvement has been achieved, and attempts are being made to reduce the influence of other variables on the specific signal from the immunoreaction.

• 한국식품영양과학회지
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• 제35권8호
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• pp.990-996
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• 2006

Ci-ELISA를 이용하여 여러 가지 물리적 처리(초고압, 가압가열, 초음파 및 microwave 처리)에 의한 새우 allergen 및 allergenicity의 변화를 살펴본 결과는 다음과 같다. 초고압 처리에 의해 새우의 allergenicity는 압력이 400 MPa까지 증가함에 따라 새우 allergen과 mAb, 환자혈청과의 결합력이 감소하였으며, 특히 400 MPa로 압력 처리했을 때 mAb와 새우 allergen과의 결합력이 50% 이하로 감소하였다. 새우 allergy의 allergenicity 변화를 알아보기 위해 5, 10, 30, 60분간 초음파 처리한 결과는 5, 10, 30분간 초음파 처리 시 처리시간이 길어짐에 따라 새우 allergen과 mAb와의 결합력이 감소하였으며 특히 60분간 처리한 경우에 있어서는 결합력이 60% 이하로 감소하였다. 가압 가열 처리$(121^{\circ}C,\;1.4\;kg/cm^2)$와 microwave 처리(2,450 MHz)에 의한 새우 allergy의 allgergenicity 변화를 살펴본 결과 처리시간이 증가하여도 새우 allergen과 mAb와의 결합력이 크게 감소하지 않았다. 즉 20분간 microwave 처리 시 85% 이상의 높은 결합력을 보였으며 60분간 가압 가열처리에 의해서는 70% 이상의 결합력을 보였다.

• 대한본초학회지
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• 제32권2호
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• pp.77-85
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• 2017

Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan(HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells. Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. $PGE_2$ were measured using EIA kit. Pro-inflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. $NF-{\kappa}B$/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays. Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, $TNF-{\alpha}$, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of $NF-{\kappa}B$ in response to competitive LPS. Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of $NF-{\kappa}B$ delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced $PGE_2$, NO, IL-6 and $TNF-{\alpha}$.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.693-699
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• 2016

Clostridium difficile toxin A is known to cause deacetylation of tubulin proteins, which blocks microtubule formation and triggers barrier dysfunction in the gut. Based on our previous finding that the Clostridium difficile toxin A-dependent activation of histone deacetylase 6 (HDAC-6) is responsible for tubulin deacetylation and subsequent microtubule disassembly, we herein examined the possible effect of potassium acetate (PA; whose acetyl group prevents the binding of tubulin to HDAC-6) as a competitive/false substrate. Our results revealed that PA inhibited toxin A-induced deacetylation of tubulin and recovered toxin A-induced microtubule disassembly. In addition, PA treatment significantly decreased the production of IL-6 (a marker of inflamed tissue) in the toxin A-induced mouse enteritis model. An in vitro HDAC assay revealed that PA directly inhibited HDAC-6-mediated tubulin deacetylation, indicating that PA acted as a false substrate for HDAC-6. These results collectively indicate that PA treatment inhibits HDAC-6, thereby reducing the cytotoxicity and inflammatory responses caused by C. difficile toxin A.

• Asian-Australasian Journal of Animal Sciences
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• 제28권4호
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• pp.573-583
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• 2015

B-32 is one of a panel of monoclonal anti-idiotypic antibodies to growth hormone (GH) that we developed. To characterize and identify its potential role as a novel growth hormone receptor (GHR) agonist, we determined that B-32 behaved as a typical $Ab2{\beta}$ based on a series of enzyme-linked immunosorbent assay assays. The results of fluorescence-activated cell sorting, indirect immunofluorescence and competitive receptor binding assays demonstrated that B-32 specifically binds to the GHR expressed on target cells. Next, we examined the resulting signal transduction pathways triggered by this antibody in primary porcine hepatocytes. We found that B-32 can activate the GHR and Janus kinase (2)/signal transducers and activators of transcription (JAK2/STAT5) signalling pathways. The phosphorylation kinetics of JAK2/STAT5 induced by either GH or B-32 were analysed in dose-response and time course experiments. In addition, B32 could also stimulate porcine hepatocytes to secrete insulin-like growth factors-1. Our work indicates that a monoclonal anti-idiotypic antibody to GH (B-32) can serve as a GHR agonist or GH mimic and has application potential in domestic animal (pig) production.