• BMB Reports
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• 제38권6호
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• 한국가금학회지
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• 제38권4호
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• pp.323-330
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• 2011

닭의 단일 클론 항체인 13C8 항체는 조류의 콕시듐증을 유발하는 것으로 알려진 Eimeria acervulina의 포자충(sporozoites) 항원에 결합하는 닭 항체이다. 그러나 닭 하이브리도마 유전자의 불안정성 때문에 분비되는 항체의 생산량이 낮은 단점이 있다. 이러한 단점을 보완하기 위해 hybridoma로 부터 항체의 중사슬 가변 부위(VH)유전자와 경사슬 가변 부위(VL) 유전자를 증폭하여 linker peptide로 연결해준 재조합 ScFv 항체 유전자를 구축하고, E. coli를 형질 전환시켜 재조합 단백질로 발현 정제하였다. ELISA 분석 결과 재조합 13C8 ScFv 항체는 세 종류의 Eimeria spp.에 대해 모두 항원 결합력을 나타내었으며, 염기서열 분석을 수행하여 germline sequence와 비교한 결과 닭 항체유전자의 다양성(diversity)은 pseudogene들의 gene conversion 기작에 의해 이루어짐을 제시해 주었다.

• Radiation Oncology Journal
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• 제22권3호
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• pp.217-224
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• 2004

목적: 저산소증은 방사선 감수성을 현저히 감소시키며, 이에 대한 적응 반응에서 hypoxia-inducible factor 1 $\alpha$(HIF-1 u$\alpha$가 중요한 역할을 하고 있다. HIF-1 $\alpha$의 발현과 방사선 감수성과의 상관 관계를 알아보고자 하였다. 대상 및 방법: 쥐의 간암 세포주인 hepalclc7 세포와 HIF-1 $\beta$가 결손되어 HIF-1 $\alpha$의 기능이 억제된 hepaIC4 세포를 사용했다 저산소 유사 물질인 desferrioxamine (DFX)을 전처치하고 6시간 뒤에 방사선조사를 하여 western blot으로 HIF-1 $\alpha$ 발현을 조사하였다. Apoptosis는 DNA 분절화, propidium iodide 핵염색, 그리고 apoptotic cell death detection ELISA kit를 이용하였다. MTT assay법으로 방사선 감수성을 측정하고 SF2$_{2}$ SF$_{8}$, 그리고 mean inactivation dose (MID)를 산출하여 통계적 분석을 하였다. 결과: Hepalclc7 세포에서는 DFX 전처치를 한 경우 방사선에 의해 HIF-1 $\alpha$의 발현이 증가했으나, hepalC4 세포 주에서는 변화가 없었다 Hepa1C4 세포의 방사선 감수성은 DFX처리에 따른 영향이 없었으나 hepalclc7 세포의 방사선 감수성은 DFX를 전처치했을 때 유의하게 감소하였다. 결론: 저산소 유사 물질인 DFX에 의해 유도된 HIF-1 $\alpha$가 쥐의 간암 세포주에서 apoptosis와 방사선 감수성을 감소시켰다 이러한 결과는 종괴내의 저산소 세포에서 방사선에 의해 HIF-1 $\alpha$가 유도되고 이로 인해 저산소 세포에서 방사선 감수성을 저하시키는 것으로 생각되었다.

• Bulletin of the Korean Chemical Society
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• 제30권5호
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• pp.1101-1106
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• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• Journal of Plant Biotechnology
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• 제7권4호
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• pp.259-265
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• 2005

Indian citrus ringspot virus (ICRSV)-free plants of Kinnow mandarin (Citrus nobilis Lour x C. deliciosa Tenora) were raised from virus-infected plants using unfertilised ovules as explants. Plants were tested by indirect ELISA and RT-PCR before using their explant. An amplified product of 539 bp was obtained by RT- PCR in ICRSV infected plants. Unfertilized ovules were excised from unopened flower buds of plants tested postive for virus and were cultured on Murashige and Skoog's (MS) basal medium supplemented with various concentrations of kinetin (KN) or malt extract (ME). Maximum induction (31.94%) of embryogenic callus was observed on MS medium supplemented with KN ($9.29\;{\mu}M$). Transfer of embryogenic calli to similar media composition resulted in somatic embryogenesis in all cultures, with an average number of 60.36 globular, 17.39 heart and 7.71 cotyledonary-shaped somatic embryos per culture. All cotyledonary shaped embryos developed into complete plantlets within 60 days on transfer to similar medium. Embryogenic callus induction, somatic embryo formation, maturation, germination and plantlet formation were achieved on MS medium supplemented with KN ($9.29\;{\mu}M$) alone. The plantlets derived from somatic embryos were transferred to sterilized soil, sand and vermiculite (3:1:1) mixture. After acclimatization, the plantlets were transferred to screen house and were indexed for ICRSV employing indirect ELISA and RT-PCR and found free of virus. A distinct feature of this study is the induction of somatic embryogenesis from unfertilised ovules to produce virus-free plants.

• 대한본초학회지
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• 제23권2호
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• pp.99-109
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• 2008

Objectives : The present study was to investigate the effect of extract of Chelidonii herba (ECH) on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mouse by ovalbumin (OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dossiciated with collagenase (1 ${\mu}g$/ml). Eosinophils were activated by rIL-3/rmIL-5 co-treatments. The lung cells were treated with ECH, incubated for 48 hr at $37^{\circ}C$, and analyzed by flow cytometery, ELISA, RT-PCR, and immuno-histochemical analysis. Results : In FACS analysis, number of granulocyte/lymphocyte, $CD3e^-$/$CCR3^+$, $CD3e^+$/$CD69^+$, $CD4^+$ and $CD23^+$/$B220^+$ in asthma-induced lung cells were significantly decreased by ECH treatment compared to the control group. And mRNA expression for IL-4, IL-5, IL-13, CCR3 and eotaxin in asthma-induced lung cells, which was induced by rIL-3 plus rmIL-5 treatments, was significantly decreased by ECH treatment. In ELISA analysis, production levels of IL-3, IL-5, IL-13 and histamine in asthma-induced lung cells, which were induced by rIL-3 plus rmIL-5 co-treatment, were significantly decreased by ECH treatment. ECH treatments significantly inhibited the proliferation of eosinohils prepared from asthma-induced mouse lung tissues compared to the non-ECH treated control cells. Immunohistochemical analysis revealed that ECH treatment significantly decreased the levels of eosipnphil activation compared to non-treated cells. Conclusions : The present data suggested that Chelidonium majus L. may have an effect on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of Chelidonium majus L.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.133.1-133
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• 2003

Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.

• 대한임상검사과학회지
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• 제47권4호
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• pp.306-312
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• 2015

알러지 반응이 일어나면 histamine이 방출되기 때문에 histamine을 정량 측정함으로써 유발된 알러지의 정도를 확인 할 수 있다. 일반적으로는 항원-항체반응으로 microplate reader를 이용하여 흡광도를 측정하여 정량 한다. 본 연구에서는 histamine 방출량을 측정함에 있어서 일반적인 항원-항체반응과 분석 화학적인 방법으로 HPLC-MS를 이용한 방법을 비교하였다. 세포주는 RBL-2H3를 사용하였고, C48/80으로 자극시켜 알러지를 유발하였다. 유발된 알러지는 ${\beta}$-hexosaminidase의 측정으로 탈 과립을 확인하였으며 실험의 정당성을 위하여 세포독성 능을 확인하였다. Histamine 정량에서 항원-항체반응에 의한 측정의 정량한계는 10.257 ppm이었고, HPLC-MS에 의한 정량한계는 0.020 ppm으로 현저한 차이를 보였다. 알러지 활성 및 항 알러지 실험에 있어서 histamine의 측정은 HPLC-MS를 이용한 분석이 더 정밀하고 정확한 실험인 것을 확인하였다.

• Parasites, Hosts and Diseases
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• 제39권2호
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• pp.177-183
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• 2001

The objectives of the present study were to (1) determine the susceptibility of Anopheles sinensis to Korean isolates of Plasmodium vivax, (2) establish a method to collect large quantities of P. vivax sporozoites for use as antigen in seroepidemiological studies, and (3) investigate the characteristics of Korean isolates of P. vivax sporozoites. Females of Anopheles sinensis were collected at non-epidemic area, Seokwha-ri, Cheongwon-gun and Chungcheongbuk-do using tent-trap methods coupled with dry ice. The females were artificially infected with gameiocytes of P. vivax using blood obtained from P vivax malaria patients. Individual mosquitoes were infected using either a parafilm-covered glass feeding apparatus or were allowed to feed on naturally infected volunteers. Mosquitoes were sacrificed between 16 and 18 days post-feeding and an enzyme-linked immunosorbent assay (ELISA) was used to detect sporozoites. Four (33.4%) of 12 mosquitoes, which were fed on naturally infected volunteers directly, were positive for sporozoites. In cases, the mosquitoes allowed to feed on whole blood which were extract from three different patients with heparin treated vacuutainers using a parafilm-covered glass apparatus. Two of 55 (3.6%) were positive which blood sample was maintained at room temperature for 8 hours, 1 of 68 (1.5%) was positive which blood was maintained at $4^{\circ}C$ for 24 hours and 1 of 47 (2.3%) was positive at 4$^{\circ}C$ for 48 hours. The mean number of sporozoites was estimated about 818 (n=8; range of 648-1,056) based on optical density values of ELISA.

• 식물병연구
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• 제10권3호
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• pp.194-197
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• 2004

풋고추 수경재배에서 tobamovirus 병의 발병률은 생육 후기 단계에서 100%였다 바이러스 종류별 감염 빈도는 PMMOY 34%, TMGMY 41.5% 및 이들 2종의 바이러스 복합감염이 24.5%로 나타났다. 다클론 항체로 제작된 DAS-ELISA에서 각각의 항체에 대하여 특이적인 반응을 보였다. 수경재배하는 풋고추에서 77개의 tobamovirus를 순수분리하여 tobamovirus의 pathotype을 구분한 결과, $P_{0}$은 61% 및 $P_{1,2}$는 39% 빈도로 분리되었다. 모든 TMGMV 분리주는 pathotype $P_{0}$에 속하였다. PMMOY에서 구분된 pathotype $P_{0}$ 및 $P_{1,2}$의 외피단백질유전자에 상응하는 cDNA를 합성하여 제한효소에 대한 분석을 실시한 결과, $P_{1,2}$는 TaqI 위치가 두 곳에 존재하였으나 $P_{0}$은 단지 한 곳에만 존재하였다.존재하였다.