• Journal of Microbiology
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• 제41권2호
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• pp.144-147
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• 2003

Listeria monocytogenes is a pathogen of major concern to the food industry and the potential cause of severe infections such as listeriosis. Early detection of this foodborne pathogen is important in order to eliminate its potential hazards. So, immunomagnetic separation (IMS) has been suggested as a means of reducing the total analysis time and for improving the sensitivity of detection. Atomic force microscopy (AFM) has been used for measuring the topographic properties of sample surfaces at nanometer scale. In this study, we used AFM to confirm both the sensitivity and the specificity of IMS. Regarding AFM analysis, the length and the width of the bacteria, which were in agreement with literature values, were found to be 2.993 $\mu\textrm{m}$ and 0.837 $\mu\textrm{m}$, respectively. As a result, AFM helped us both characterize and measure the bacterial and bead structures.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.509-516
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• 2003

A model system for the immnunochemical detection of Escherichia coli O157:H7 using a combined immunomagnetic separation (IMS) and test-strip liposome immunoassay (LIA) procedure was developed. Immunomagnetic beads coated with anti-E. coli O157 IgG antibodies were used to separate the E. coli O157 (including the H7 serotype) from culture. Immunoliposomes, whose surface was conjugated to goat anti-E. coli O157:H7 IgG and which encapsulated the marker dye, sulforhodamine B, were used as a detection label. The test strip, onto which antibodies to goat IgG were immobilized, was the immunosensor capturing immunoliposomes that did not bind to E. coli O157:H7 on the immunomagnetic bead-E. coli O157:H7 complexes. In experiments, pure cell culture suspensions of $10^5 E.$ coli O157:H7 organisms per ml produced a measurable signal inhibition, whereas a weak yet detectable signal inhibition occurred with $10^3CFU/ml$. The inhibition signals increased, when the incubation time for IMS was extended to 90 min and higher IgG-tag density (0.4mol%) was used on the liposomes. With 0.2 and 0.4mol% IgG-tagged liposomes, the IMS-LIA procedure showed more improved signal inhibitions than those of a direct (no IMS) LIA. The combined assay, which measures the instantaneous signal from immunoliposomes, can be completed within 90 min, making it significantly faster than conventional plating methods and enzyme-linked immunosorbent assay (ELISA). Accordingly, it is quite feasible to use the combined immunoassay format of IMS and dye-loaded immunoliposomes for the detection of E. coli O157:H7.

• 한국수산과학회지
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• 제30권6호
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• pp.948-955
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• 1997

Immunomagnetic separation of virus coupled with .reverse transcription-polymerase chain reaction (IMS-PCR) was performed with infectious hematopoietic necrosis virus (IHNV). A DNA fragment of expected size was synthesized in the RT-PCR with total RNA extracted from IHNV inoculated CHSE-214. In a SDS-PAGE analysis, a protein band of over 70kDa was detected from non-infected cells and cells inoculated with IHNV and infectious pancreatic necrosis virus (IPNV). This protein was detected in the Western blot analysis probably because of non-specific reaction to monoclonal antibody against IHNV nucleocapsid protein. In the immunomagnetic separation, magnetic beads coated with monoclonal antibody against the IHNV nucleocapsid protein was incubated with supernatant from IHNV inoculated CHSE-214 cells. During this process, the non-specifically reacting protein could be removed by washing the magnetic bead with PBS in the presence of an external magnetic field, and viral proteins were detected from the remaining, cleaned magnetic beads. It was necessary to extract viral RNA from the captured virus particles before RT-PCR, and no DNA product was detected when the captured virus was only heated 5 min at $95^{\circ}C$. A PCR-product of expected size was synthesized from IMS-PCR with magnetic beads double coated either by goat anti-mouse IgG antibody -monoclonal antibody or streptavidin - biotin conjugated monoclonal antibody.

• Journal of Microbiology and Biotechnology
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• 제18권10호
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• pp.1689-1694
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• 2008

The ability to detect Salmonella spp. is essential in the prevention of foodborne illness. This study examined a Salmonella spp. detection method involving the application of immunomagnetic separation and immunoliposomes (IMS/IL) encapsulating sulforhodamine B (SRB), a fluorescent dye. A quantitative assay was conducted by measuring the fluorescence intensity of SRB that was produced from an immunomagnetic bead-Salmonella spp.-immunoliposome complex. The results indicated detection limits of $2.7{\times}10^{5}$ and $5.2{\times}10^{3}$ CFU/ml for Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterka subsp. enterka serovar Typhimurium (S. Typhimurium), respectivley. The signal/noise ratio was improved by using 4% skim milk as a wash solution rather than 2% BSA. In addition, higher fluorescence intensity was obtained by increasing the liposome size. Compared with the conventional plating method, which takes 3-4 days for the isolation and identification of Salmonella spp., the total assay time of to h only including 6 h of culture enrichment was necessary for the Salmonella detection by IMS/IL. These results indicate that the IMS/ IL has great potential as an alternative rapid method for Salmonella detection.

• Parasites, Hosts and Diseases
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• 제47권4호
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• pp.353-357
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• 2009

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, $10^1$, $10^2$, and $10^3$ per $10{\mu}l$ were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < $10^2$ per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

• Journal of Magnetics
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• 제21권1호
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• pp.125-132
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• 2016

High gradient magnetic separation (HGMS) is the most commonly used magnetic cell separation technique in biomedical science. However, parameters determining target cell capture efficiencies in HGMS are still not well understood. This limitation leads to loss of information and resources. The present study develops a bead-capture theory to predict capture efficiencies in HGMS. The theory is tested with CD3- and CD14-positive cells in combination with paramagnetic beads of different sizes and a generic immunomagnetic separation system. Data depict a linear relationship between normalized capture efficiency and the bead concentration. In addition, it is shown that key biological functions of target cells are not affected for all bead sizes and concentrations used. In summary, linear bead-capture theory predicts capture efficiency ($E_t$) in a highly significant manner.

• 한국식품과학회지
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• 제44권5호
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• pp.638-642
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• 2012

돼지축사에서 채집해온 6개의 육성돈의 분변에서 식중독 유발 바이러스인 HAV와 HEV를 검출하였으며, HAV는 88.3%의 검출율을 보였으며, HEV는 33.3%의 검출율을 보였다. 결과에는 제시하지 않았으나, 염기서열 분석결과 HEV는 사람에게 전염이 가능한 유전자형인 III형이었으며, 실험적으로 사람의 간세포인 PLC/PRF/5에 접종하였을 때 증식이 됨을 확인하였다. 식중독 유발 바이러스인 HAV와 HEV는 오염된 식품이나 물을 섭취하거나 교차 오염에 의해 전염이 가능하기 때문에 돼지축사에서 위생상태의 개선뿐만 아니라 육류를 섭취하기 전인 운송 및 가공과정까지 식중독 유발 바이러스에 의한 교차오염을 막는 노력이 필요하다. HAV와 HEV 모두 검출된 분변에서 HAV를 순수분리하고 빠르게 검출하기 위해 IMS-RT-PCR을 적용하였으며, 항원-항체 반응에 의해 순수하게 HAV만을 분리할 수 있었다. 또한 HAV만이 순수분리 되었는지 재확인하기위해 세포감염을 통해 증식된 바이러스를 확보한 후 nested RT-PCR을 수행한 결과, HAV만을 순수 분리할 수 있음을 확인하였다. 이는 IMS 활용기술이 단순히 항체를 교체함으로써 다른 특정 식중독 유발 바이러스의 다양한 시료에서 바이러스 순수분리 및 검출에 활용 가능성이 있음을 확인하였다.

• Journal of Dairy Science and Biotechnology
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• 제33권4호
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• pp.271-275
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• 2015

Colloidal semiconductor CdSe-ZnS core-shell nanocrystal quantum dot (Qdot) are luminescent inorganic fluorophores that show potential to overcome some of the functional limitations encountered with organic dyes in fluorescence labeling applications. Salmonella Enteritidis has emerged as a major cause of human salmonellosis worldwide since the 1980s. A rapid, specific, and sensitive method for the detection of Salmonella Enteritidis was developed using Qdot as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-Salmonella Enteritidis antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-Salmonella antibodies were added to form sandwich immune complexes. After magnetic separation, the immune complexes were labeled with Qdot via biotin-streptavidin conjugation, and fluorescence measurement was carried out using a fluorescence measurement system. The detection limit of the Qdot method was a Salmonella Enteritidis concentration of $10^3$ colony-forming units (CFU)/mL, whereas the conventional fluorescein isothiocyanate-based method required over $10^5CFU/mL$. The total detection time was within 2 h. In addition to the potential for general nanotechnology development, these results suggest a new rapid detection method of various pathogenic bacteria from a complex food matrix.

• 한국식품저장유통학회지
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• 제21권2호
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• pp.170-174
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• 2014

식중독 유발 바이러스인 HAV는 제 1군 감염병원으로 규정되면서 감염 시 원인식품을 빠르게 분석하게 되었으며 그로 인해 정확하면서 빠른 검출기술을 요구하게 되었다. 본 연구에서는 HAV에 오염된 상추에서 신속하게 바이러스를 검사하기 위해서 IMS를 통하여 신속하게 HAV를 순수 분리 및 농축하였고, 형광물질인 quantum dot을 활용하여 형광검출을 실시하였다. 또한 일반적으로 바이러스 농축에 사용되는 PEG 농축방법과 비교하였을 때 검출능은 유사한 결과를 얻었으나, 농축시간 면에서는 IMS를 통한 방법이 효과적이었다. 또한 IMS 방법으로 확보된 항원을 Quantum dot을 활용하여 10분 이내에 바이러스를 검출할 수 있었다. 본 연구에서 제시된 검출기반은 식품 유통 과정 중 다양한 식중독 바이러스로부터 소비자를 보호 할 수 있는 검사방법으로 활용될 수 있다고 기대된다.

• 한국축산식품학회지
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• 제23권3호
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• pp.278-283
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• 2003

식중독 세균 검출에 있어서 리포좀의 이용 가능성을 병원성 식중독 세균인 E. coli O157:H7을 대상으로 검토하였다. 리포좀의 표면에 E. coli O157:H7을 인식하는 항체를 공유결합시키고 리포좀의 내부 수용액상에는 형광 표지물질인 sulforhodamine B를 포집시켰다. 이렇게 합성된 면역리포좀이 진단시약으로써의 기능을 하는지 알아보기 위해 두 가지 분석 방법을 적용하였다. 첫번째 방법으로써, test-strip을 이용한 분석은 E. coli O157:H7의 존재 유무를 test-strip 상에 나타난 분석 신호을 육안으로 관찰하는 것으로써 E. coli O157:H7 존재시 분석 신호가 저하되는 것을 원리로하였다. 이 방법은 고가의 실험 장비가 필요하지 않아 현장적용성이 용이한 장점이 있다. 두번째 방법은, IMS 후 목표 세균에 결합되어 있는 리포좀을 파괴시켜 나오는 형광도의 세기를 측정하는 방법으로 이때는 형광도의 강도와 E. coli O157:H7 세균 수와 비례적 관계가 나타났다. 상기의 두 방법에서 얻은 결과는, 리포좀을 E. coli O157:H7 검출에 응용할 수 있는 가능성을 제시하며 추후에 식품 적용 실험이 요구된다.