• 대한수의학회지
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• 제29권4호
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• pp.541-548
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• 1989

오제스키병 바이러스를 배양세포에다 감염시켜, 냉동 및 araldite포매 초박절편에서 protein A-gold labeling을 통해 바이러스항원 검출을 시도하였다. 오제스키병 바이러스항원은 10nm gold probes로 표지되었으며, 전자밀도가 높은 gold 입자들은 바이러스의 nucleocapsid와 envelope에서 주로 관찰되었고, 초냉동박절표본에서의 immunogold labeling은 조직구조물들과 극히 미미한 비특이결합만을 보였다. 초냉동박절표본에서의 immunogold labeling은 오제스키병 바이러스항원을 검출하는데 있어 효과적이었으며, 이는 또한 여러가지 바이러스들과 속주세포들간의 상호작용에 관한 면역세포화학적 연구에 크게 이용될 수 있을 것으로 생각된다.

• Applied Science and Convergence Technology
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• 제24권1호
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• pp.1-8
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• 2015

Protein immobilization on a gold surface plays an important role in the usefulness of biosensors that utilize gold-coated surfaces such as surface plasmon resonance (SPR), quartz crystal microbalance (QCM), etc. For developing high performance biosensors, it is necessarily required that immobilized proteins must remain biologically active. Loss of protein activity and maintenance of its stability on transducer surfaces is directly associated with the choice of immobilization methods, affecting protein-protein interactions. During the past decade, a variety of strategies have been extensively developed for the effective immobilization of proteins in terms of the orientation, density, and stability of immobilized proteins on analytical devices operating on different principles. In this review, recent advances and novel strategies in protein immobilization technologies developed for biosensors are briefly discussed, thereby providing an useful information for the selection of appropriate immobilization approach.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권2호
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• pp.143-146
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• 2004

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His$\sub$6/-Ub-hPTHF(1-34)) expressed in Escherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker$\^$TM/ B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilizaton of capture proteins on solid matrices. The soluble and insoluble fractions of an E. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His$\sub$6/-Ub-hPTHF(1-34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.

• Applied Microscopy
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• 제19권2호
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• pp.74-84
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• 1989

인삼 종자의 배유조직에서 Tris 완충액 가용성 저장단백질을 추출한 후 전기 영동적 분석으로 분리하여 $SP_{1}$(MW=160,000)과 $SP_2$(MW=70,000)의 두가지 저장단백질을 정제하였다. 이 두가지 저장단백질을 항원으로 사용하여 토끼에 피하주사하여 항체를 얻었으며, 이 항체를 이용하여 면역 세포화학적 금입자표지법을 실시한 결과, $SP_1$과 $SP_2$ 모두 구형의 protein body내에 산재하여 있음을 확인되었으며, globoid에는 이러한 두가지 단백질중 어느 것도 함유되어 있지 않는 것으로 나타났다. 또한 각각의 protein body에 함유된 $SP_1$과 $SP_2$의 상대적 함량에는 서로 차이가 있음이 확인되었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.112-116
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• 2003

An immunosensor based on surface plasmon resonance (SPR) onto a protein G layer by Self-assembly technique was developed for detection of Legionella pneumophila. The protein G layer by self-assembly technique was fabricated on a gold (Au) surface by adsorbing the 11-mercaptoundecanoic acid (MUA) and an activation process for the chemical binding of the free amino (-NH$_2$) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of the protein G layer by self-assembly technique on the Au Substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The Surface topographies of the fabricated thin films on an Au substrate were also analyzed by using an atomic force microscope (AFM). Consequently, an immunosensor for the detection of L. pneumophila using SPR was developed with a detection limit of up to 10$^2$CFU per mL.

• 대한수의학회지
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• 제33권4호
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• pp.573-577
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• 1993

청설모(Sciurus vulgaris corea)의 췌장에 분포하는 glucagon 및 somatostatin 세포의 분포와 그들 분비과립의 특징을 밝히기 위해 면역조직화학적 및 면역전자현미경적 방법으로 관찰하였다. Glucagon 면역반응세포는 주로 췌도의 주변부에 분포하였고, 때로는 외분비부에서 관찰되었다. 이 세포의 분비과립은 직경이 240~320nm였으며, 전자밀도가 높은 core를 전자밀도가 낮은 halo가 둘러싸고 특히 gold particle들은 전자밀도가 높은 core에 강한 반응을 보였다. Somatostatin면역반응세포는 췌도의 주변부를 따를 면역반응이 아주 약한 세포들로 산재하였으며 또한 외분비에서 단독 혹은 소집단으로 관찰되었다. 이 세포의 분비과립은 전자밀도가 낮고 직경이 250~275nm였으며, gold particle는 분비과립에 중등도로 고른 반응을 보였다.

• Tuberculosis and Respiratory Diseases
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• 제60권4호
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• pp.412-418
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• 2006

연구배경 : 클라미디아 감염의 진단은 혈청검사로 이루어진다. 현재 표준 방법은 MIF(microimmunofluorescence)이나 이 방법은 주관적이고 시간이 많이 걸리는 단점이 있다. 최근을 SPR(surface plasmon resonance) 센서를 이용한 단백질 칩이 감염의 새로운 진단 방법으로 제시되고 있다. 클라미디아 감염의 진단을 위한 단백질 칩 개발을 위하여 금 칩 표면에 세균을 고정하고 클라미디아 균에 대한 항체와 표면 위 세균과의 반응을 SPR 센서를 이용하여 측정하고자 하였다. 방법 : 표면 항원으로 배양한 Chlamydophila pneumoniae LKK1의 EB를 정제하였다. 양전하를 띤 PDDA (polydiallyldimethylammonium chloride)를 이용하여 전하를 이용한 단백질 칩을 제작하였다. 클라미디아 균을 고정시킨 후에 atomic force microscopy를 이용하여 표면을 관찰하였다. 클라미디아 균에 대한 항체를 투여하고 나서 자체 제작한 SPR 센서를 이용하여 항원 항체 반응을 SPR 파장 변화로 측정하였다. 결과 : 양전하를 띤 PDDA 표면위에서 클라미디아 균이 고정되었음을 확인 하였다. 그리고, 항체를 투여한 후에 SPR 파장의 증가를 확인하였다. 파장 변화는 항원의 농도와 관련이 있었다. 결론 : 전하를 이용하여 클라미디아 폐렴균의 EB를 단백질 칩에 고정하였고, 단백질 칩 위에서의 항원 항체 반응을 확인하였다. 비정형 폐렴의 진단에 SPR 센서가 기여할 수 있을 것으로 사료되나, 실제 임상 시료에의 적용을 위해서는 좀더 연구가 필요할 것으로 사료된다.

• 센서학회지
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• 제20권6호
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• pp.434-438
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• 2011

A portable surface plasmon resonance(SPR) based immunosensor using thiolated protein G and protein G was developed for the detection of immunoglobulin G(IgG). The protein G has specific affinity with Fc fragment of IgG and was thiolated by 2-Iminothiolane for introduction of thiol groups. Anti-IgG, bovine serum albumin(BSA), and IgG have been sequently injected after surface modification of gold sensor chip with protein G and thiolated protein G. The output signal was increased with the injection of each protein and the actual signal was measured by subtracting signal of reference channel from signal of sample injected channel. The experimental results showed the higher detection capability of IgG using thiolated protein G compared with protein G. From these results, we can conclude that the current surface modification technique and the portable SPR sensor system can be applied to various immunosensors for diagnosis.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.141-144
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• 2006

An antibody layer was fabricated to detect Escherichia coli O157:H7. The micropattern of 16-mercaptohexadecanoic acid (16-MHDA) as alkylthiolate was formed on the gold surface by using the PDMS stamp with microcontact printing $({\mu}CP)$ techniques. In order to form antibody patterns on the template, protein G was chemically bound to the 16-MHDA patterns, and antibody was adsorbed on a self-assembled protein G layer. The formation of the 16-MHDA micropattern, self-assembled protein G layer and antibody pattern on Au substrate was confirmed by surface plasmon resonance (SPR) spectroscopy. Finally, the micropatterning method was applied to fabricate the antibody probe for detection of E. coli O157:H7, and monitoring of antigen by using this probe was successfully achieved.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권4호
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• pp.227-232
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• 2003

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibodybased biosensor through immobilizing the antibody molecules (lgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detecting E. coli O157:H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10$^2$ CFU/mL.