• Mycobiology
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• 제42권4호
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• pp.339-345
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• 2014

During 2011~2013, a total of 729 samples for 19 types of medicinal plant were collected from Seoulyekryungsi in Seoul, Korea, and investigated for the presence of aflatoxins. The samples were analyzed using immunoaffinity column cleanup and high-performance liquid chromatography coupled to a fluorescence detector after post-column derivatization. Aflatoxins were found in 124 out of the 729 analyzed samples: 65 containing aflatoxin B1 (AFB1), 24 with aflatoxin B2 (AFB2), 15 with aflatoxin G1 (AFG1), and 20 samples with aflatoxin G2 (AFG2). The ranges for positive samples were $0.1{\sim}404.7{\mu}g/kg$ for AFB1, $0.1{\sim}10.0{\mu}g/kg$ for AFB2, $0.1{\sim}635.3{\mu}g/kg$ for AFG1, $0.1{\sim}182.5{\mu}g/kg$ for AFG2, and $0.1{\sim}1,043.9{\mu}g/kg$ for total aflatoxins. Most of the medicinal plant samples (721, 98.9%) were below legal limits, but 8 samples exceeded the legal limits of 10 and $15{\mu}g/kg$ established by the Korean standard for AFB1 and total aflatoxins (the sum of AFB1, AFB2, AFG1 and AFG2), respectively.

• 생명과학회지
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• 제13권1호
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• pp.67-72
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• 2003

위염, 위궤양 및 위암의 원인 균인 Helicobacter pylori 가균체 표면에 다량 함유하며 주된 생존 인자이며 병원성 인자인 urease를 대장균에서 발현시키고 이 효소에 대한 항체 또는 기질과의 특이 상호 작용을 이용하여 두 단계의 간편한 방법에 의하여 정제하였다. 우선 anti-H. pylori Urease IgG-Sepharose column과 urea-Sepharose column을 각각 제조하고 DEAE-Sepharose 음이온 교환수지를 통하여 1차 정제한 시료를 각각 적용하고 제반 조건에서 용출시켰다. Anti-H. pylori urease IgG-Sepharose column의 경우에는 urease 시료가 너무 강력하게 결합함으로써 극단적인 pH조건에서만 용출이 가능함이 관찰되었으므로, 100 mM 탄산 완충액(pH 10.5)으로 최종 용출하였을 때 비교적 순수한 효소를 얻었으나, 비활성이 다소 감소된 것으로 나타났다. 한편, urea-Sepharose에 적용시킨 시료는 100 mM urea-HEB 완충액(pH 7.5)으로 비교적 용이하게 용출되어 비교적 높은 순도와 비활성의 urease 효소를 얻을 수 있었으나 이 경우에는 urease의 smaller subunit인 UreA peptide band의 강도가 다소 감소한 것이 관찰되었다.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.89-97
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• 1994

These studies were carried out to investigate the properties of H-Y antigen purified by immunoaffinity chromatography using monoclonal H-Y antibody. Immunoaffinity column was prepared by the coupling of monoclonal antibody to the Aminolink Coupling Gel. Murine testis supernatant was applied onto the column and eluted by O.lM glycine-HCl buffer and 31${\mu}g$ of H-Y Ag was eluted from one testis. Purified H-Y Ag strongly reacted with Con A and lentil from 6 different kinds of lectins tested, which may indicate that sugar moiety of H-Y Ag is composed of glucose, mannose and their derivatives. Con A-sepharose affinity column was used to purified H-Y Ag based on that H-Y Ag is glycoprotein. The fraction eluted by 0.2M Me-${\alpha}$-D-mannoside from the column loaded with murine testis supernatant was identified to be H-Y Ag by dot blot test. Molecular weight of the purified H-Y Ag was estimated by Sepharose G-75 gel filtration and SDS-PAGE, and showing that it was about 67,000 dalton. In fluorescence test, the ratio of XY embryos and XX embryos was 1:1.

• 한국식품위생안전성학회지
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• 제8권4호
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• pp.251-254
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• 1993

Extraction and Clean-up procedures coupled with quantitation by high performance liquid chromatography(HPLC) was evaluated for the detection of 4 aflatoxins, B1, B2, G1 and G2, in peanut butter. The Sep-pak clean-up method showed poorer separation and repeatability than did the modified DeVries' and an immunoaffinity column clean-up methods. No significant difference of detected aflatoxins between the affinity column clean-up and modified DeVries' method. The coefficients of variation for the 4 aflatoxins were ranging from 6.3∼32.3 by the modified DeVries' method and 5.3∼9.8 by the affinity column clean-up.

• 한국식품위생안전성학회지
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• 제23권2호
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• pp.108-112
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• 2008

경기지역 시중마트와 국내 온라인상에서 유통되고 있는 견과류 및 그 가공식품 79건, 장류 29건, 곡류가공품 등 가공식품 50건에 대해 immunoaffinity column 정제방법을 이용하여 HPLC-FLD로 아플라톡신 오염실태를 조사한 결과 총 158건 중 45건(28.5%)에서 아플라톡신 오염이 확인되었고, 오염수준은 아플라톡신 $B_1$으로서 $0.02{\sim}3.13\;{\mu}g/kg$, 총 아플라톡신으로서 $0.02{\sim}3.96\;{\mu}g/kg$ 범위로 국내 기준치인 $10\;{\mu}g/kg$을 초과하는 것은 없었다. 아플라톡신이 검출된 식품은 견과류 및 그 가공식품에서 34건(43.0%) 장류에서 8건(27.6%),그 외 가공식품에서 3건(6.0%)으로 견과류 및 그 가공식품에서 검출빈도가 가장 높았다. 식품을 개봉하여 1개월, 6개월 보관 후의 아플라톡신$B_1$ 함량은 저장기간이 길어질수록 땅콩, 옥수수스낵에서는 증가하는 경향을 보였으나 된장의 경우에는 다소 감소하였다.

• 한국식품위생안전성학회지
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• 제24권2호
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• pp.169-173
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• 2009

서울약령시장과 시중마트에서 유통되고 있는 생약 145건을 immunoaffinity column 정제 방법을 이용하여 HPLC-FLD로 아플라톡신 오염 실태를 조사한 결과 총 145건 중 10건(6.9%)에서 아플라톡신 오염이 확인되었고, 오염수준은 아플라톡신 $B_1$로서 $0.45{\sim}79.15\;{\mu}g/kg$, 총 아플라톡신으로 $0.05{\sim}97.77\;{\mu}g/kg$의 범위로 빈랑자 19건 중 5건(26%), 백자인 13건 중 4건(31%), 산조인 15건 중 1건(7%)으로 검출 되었고, 백자인 2건, 빈랑자 1건에서 국내 기준치인 $10\;{\mu}g/kg$을 초과 검출 되었다. 아플라톡신이 검출된 생약은 전통적인 방법으로 종이나 비닐팩에 보관하는 방법은 아플라톡신에 오염될 수 있는 가능성을 높임으로 진공 포장과 같이 미생물과의 접촉을 가능한 피할 수 있는 포장 등으로 변경되어야 한다. 또한 아플라톡신 생성균주가 잘 생장할 수 있는 환기가 잘 되지 않고, 고온 다습한 환경이 제공될 가능성이 높은 전통적인 저장 창고와 같은 시설을 항온 항습의 조절이 가능한 현대화 시설로 바꾸는 방안 또한 고려하여야 할 것이다. 곰팡이독소에 대하여 그 대상 품목 및 허용 한계가 다양하게 규제되고 있으나 규제를 하는 것은 아플라톡신이 오염된 식품이나 생약을 섭취 할 위험성을 줄이고자 하는 것이지 근본적인 해결책은 아니다. 서울약령시장 등 생약시장은 매우 영세하고 문제점이 있을 수 있으며 의료 목적으로 환자를 대상으로 사용한다는 점을 감안한다면 더욱 신중하고 신속한 저감화 방안의 마련이 필요할 것이다.

• Parasites, Hosts and Diseases
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• 제29권1호
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• pp.1-8
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• 1991

스파르가눔 생리식염수 추출액 내에 포함되어 있는 성분단백질 중 스파르가눔증 환자 혈청내 특이 IgG항체와 민감하고 특이하게 반응하는 항원단벼질인 36, 29 kDa단백질을 단세포군 항체를 이용한 면역친화성 크로마토그 래피로 순수분리할 수 있음은 이미 보고하였다. 이 연구에서는 스파르가눔 추출액 내에 포함된 이 36, 29 kDa단백질이 젤라틴을 고리로 한 친화성 크로마토그래피로 훨씬 쉽게 순수분리할 수 있음을 증명하고자 하였다. 젤라틴을 고리로 부착시킨 Sepharose 4B column에 스파르가눔 추출액을 통과시키고 젤라틴에 부착한 단백질은 4 M urea/0.1M NaCl 용액을 분리완충액으로 분리하였다. 이렇게 분리한 단백질은 SDS-PAGE에서 36, 29 kDa band로 구성되어 있었고, SDS-PAGE/immunoblot 결과 환자의 polyclonal 항체는 이들 band에만 반응하였다. 스파르가눔증, 기타 기생충증 환자 및 건강대조군 혈청내 스파르가눔 특이항체가(IgG)를 면역효소측정 법으로 측정 한 결과 순수분리한 이 단백질은 특히 특이도가 95.8%로 생리식염수 추출액의 89%보다 우수하였고 민감도는 차이가 없었다. 이상의 결과는 젤라틴을 고리로 이용한 친화성 크로마토그래피는 스파르가눔 생리식염수 추출액 내의 36 및 29 kDa 단백질을 간편하게 순수분리할 수 있고 단백질의 항원성도 유지할 수 있음을 보이고 있었다.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.497-503
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• 2001

The purpose of this study was to examine the efficacy and mechanism of the cryo-precipitation, solvent/detergent (S/D) treatment, monoclonal anti-FVIIIc antibody (mAb) column chromatography, Q-Sepharose column chromatography, and lyophilization involved in the manufacture of antithemophilic factor VII(GreenMono) from human plasma, in the removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including the bovine viral diarrhoea virus (BVDV), bovine herpes virus (BHV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. BHV and EMCV were effectively partitioned from a factor VII during the cryo-precipitation with a log reduction factor of 2.83 and 3.24, respectively. S/D treatment using the organic solvent, tri(n-butyl) phosphate (TNBP), and the detergent, Triton X-100, was a robust and effective step in inactivating enveloped viruses. The titers of BHV and BVDV were reduced from the initial titer of 8.85 and $7.89{log_10} {TCID_50}$, respectively, reaching undetectable levels within 1 min of the S/D treatment. The mAb chromatography was the most effective step for removing nonenveloped viruses, EMCV and PPV, with the log reduction factors of 4.86 and 3.72, respectively. Q-Sepharose chromatography showed a significant efficacy for partitioning BHV, BVDV, EMCV, and PPV with the log reduction the log reduction factors of 2.32, 2.49, 2.60, and 1.33 respectively. Lyophilization was an effective step in inactivating g nonenveloped viruses rather than enveloped viruses, where the log reduction factors of BHV, BVDV, DMCV, and PPV were 1.41, 1.79, 4.76, and 2.05, respectively. The cumulative log reduction factors of BHV, BVDV, EMCV, and PPV were ${\geqq}$11.12, ${\geqq}$7.88, 15.46, and 7.10, respectively. These results indicate that the production process for GreenMono has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• Archives of Pharmacal Research
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• 제20권5호
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• pp.396-403
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• 1997

In order to direct the form of the immune response in an antigen-specific manner, we constructed a fusion protein (OVA/IL12) that contained the T cell-dependent antigen, ovalbumin (OVA), covalently linked to murine interleukin-12 (IL-12). The OVA/IL12 protein was produced in a baculovirus expression system and was purified by anti-OVA immunoaffinity chromatography. The purified OVA/ILI2 protein displayed potent IL-12 bioactivity in an IL-12 proliferation assay. BALB/c mice immunized with the OVA/IL12 protein produced increased quantities of anti-OVA IgG2a antibody compared with mice immunized with recombinant OVA alone. Lymph node cells from the immunized mice with the OVA/IL12 protein produced large amounts of IFN-,Y when restimulated in vitro with OVA, while those from mice immunized with the OVA protein produced little or no IFN-.gamma.. In contrast, immunization with a mixture of OVA and free recombinant IL-12 also induced IFN-.gamma. production, which was not OVA-specific. These studies indicate that the OVA/IL12 fusion protein can induce OVA-specific, Th1-dominated immune responses, and that the covalent linkage of OVA and IL-12 confines the effect of IL-12 to OVA-specific cells.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.472-477
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• 1996

Natural human epidermal growth factor (nhEGF) was purified from pregnant human urine by benzoic acid adsorption, DEAE-Sepharose ion exchange, and immunoaffinity chromatography. The purified nhEGF was further separated into four fractions using Bondapak C$_{18}$ HPLC system. Following characterization by Western blot analysis and double immu- nodiffusion, we found that each fraction corresponds to four derivatives of the nhEGF. For biological analysis of nhEGF, we optimized the labeling time and serum concentration for the incorporatioin of 5-bromo-2'-deoxy uridine (BrdU), a non-radioactive alternative for [$^{3}$H]-thymidine uptake, into NIH 3T3 cells. The DNA synthesis of NIH 3T3 cells was gradually increased at the nhEGF concentrations between 0.1 - 10 ng/ml in the Dulbecco's Modified Eagles Medium (DMEM) containing 0.2% Fetal calf serum (FCS). When we assayed the biological activity of four fractions, the activity of the second fraction was superior to that of the others. Based on the results from the HPLC analysis spiked with recombinant human epidermal growth factor (rhEGF) and amino acid sequencing, we concluded that the second fraction was nhEGF and the other three fractions were the derivatives of nhEGF. In addition, the proportion of nhEGF was approximately 46% is compared with that of the other three derivatives.