• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.703-706
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• 2000

We constructed a biospecific affinity surface using hyper-branched dendrimers on gold for biospecific recognition, and characterized the resulting surfaces by using confocal fluorescence microscopy. The dendrimer monolayer was firstly constructed on the mercaptoundecanoic acid SAM/Au with pentafluorophenyl ester activation and further functionalized with sulfo-NHS-biotin, an activated ester of biotin. To confirm the formation of biospecific affinity surface, FITC(fluorescein isothiocyanate)-labeled avidin was loaded onto the biotinylated dendrimer monolayer, and fluorescence images of the bound avidins were investigated with a confocal microscope. The constructed biospecific affinity surface showed a much more dense and uniform fluorescence compared to those from poly-L-lysine- and cystamine SAM-based affinity surfaces. For the dependency on the concentration of added FITC-labeled avidin on the affinity surface, derived fluorescence could be detectable from as low as $1{\mu}g/ml$, and intensified up to $50{\mu}g/ml$. Further reaction of FITC-labeled avidin layer with TMR(tetramethylrhodamine)-biocytins resulted in the efficient FRET(fluorescence resonance energy transfer) phenomenon. As an extension of the study, we attempted a patterning of the affinity surfaces on gold by microcontact printing. Fluorescence of the patterned surface demonstrated that FITC-labeled avidin molecules were specifically bound to the biotinylated patches.

• Bulletin of the Korean Chemical Society
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• 제32권12호
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• pp.4171-4175
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• 2011

Bio-functionalized gold nanoparticles (AuNPs), which bio-specifically interact with biotin-(strept)avidin, were investigated in this study. AuNPs were functionalized with a synthetically-provided biotin-linked thiol (BLT), which was synthesized by amidation of the active ester of biotin with 2-mercaptoethylamine. The BLT-attached AuNP was bio-specific for streptavidin, making it potentially useful for biosensor applications. To test the bio-specific interactions, the colors, absorption spectra and TEM images were investigated for proteins such as streptavidin, cytochrome C, myoglobin and hemoglobin. The colors and absorption spectra changed when streptavidin was added to the BLT-attached AuNP solution. However, the color and spectra did not change when the other proteins were added to the same solution. These results show that the AuNPs provided a colloidal solution with excellent stability and highly selective absorption characteristics for streptavidin as a target molecule. Proteins were also screened in order to identify a general strategy for the use of optical biosensing proteins based on AuNPs. In addition, TEM images confirmed that streptavidin led the BLT-attached AuNPs to aggregate or precipitate.

• 대한수의학회지
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• 제33권1호
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• pp.119-123
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• 1993

The present study was intended to use the avidin-biotin-peroxidase-antiperoxidase complex (ABPAP) method for the identification of Mycobacterium bovis in the tissue sections of infected cattle. Antibodies and linksera for ABPAP procedure used in incubated order were rabbit anti-Mycobacterium polyvalent antibodies, goat anti-rabbit IgG, rabbit peroxidase-antiperoxidase complex, biotinyl-horse anti-rabbit IgG, and avidin-biotin-peroxidase complex. Where the bacterial antigen was localized by ABPAP, a dark brown deposit occurred in the cytoplasms of macrophages and Langerhans' giant cells of the granulomatous lesions. The method approved to be highly specific for the identification of the bacteria and allowed a precise localization of the bacterial antigen in infected cells.

• 대한수의학회지
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• 제33권2호
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• pp.255-261
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• 1993

For a immunohistochemical diagnosis of the frozen and paraffin-embedded tissues against rabies virus, mice were intracerebrally inoculated with challege virus standard(CVS) rabies virus and then were used to detect the rabies viral antigen by the immunoperoxidase(IP) and the avidin-biotin complex(ABC) method. In this study, the results confirmed that ABC and IP methods, although the former showed more specific and sensitive than the latter, were reliable and effective for the demonstration of rabies virus in both frozen and paraffin-embedded brain tissues prepared from rabies-infected mice. Additionally, IP technique using the monoclonal antibody against rabies virus could be recommended as a standard diagnostic tool instead of the present immunofluorescent method for the local veterinary services in Korea.

• Bulletin of the Korean Chemical Society
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• 제33권11호
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• pp.3681-3685
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• 2012

We developed a simple and effective method for the SERS-based detection of protein-small molecule complexes and label-free proteins using avidin-induced silver aggregation. Upon excitation with light of the appropriate wavelength (633 and 532 nm), the aggregated silver nanoparticles generate a strong electric field that couples with the resonance of the molecules (atto610 and cytochrome c), increasing the characteristic signals of these molecules and resulting in sensitive detection. The detection limit of biotin with the proposed method is as low as 48 ng/mL. The most important aspect of this method is the induction of silver aggregation by a protein (avidin), which makes the silver more biocompatible. This technique is very useful for the detection of protein-small molecule complexes.

• Molecules and Cells
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• 제24권2호
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• pp.268-275
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• 2007

Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.

• 대한의생명과학회지
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• 제2권2호
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• pp.275-282
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• 1996

저자들은 마우스를 실험모델로 하여 간흡충의 항원을 투여 했을 때 비장조직에 대한 CD3, CD4 및 CD8 모노클로날 항체의 반응 여부를 알아보고자 하였다. 즉, 간흡충에 대한 세포면역학적인 특성을 규명고자 하였으며 특히 비장 조직에 대한 phenotype을 관찰한 결과 다음과 같은 결과를 얻었다. 간흡충의 조항원을 면역증강제와 함께 복강 투여한 다음 일정 기간 후에 비장조직을 Avidin-biotin complex 면역조직염색을 실시한 결과 CD3에서 강한 양성 반응을 나타냈고 CD4와 CD8에서는 약한 반응을 나타냈다. 조직부위를 보면 피막, 혈관, 임파관, 백수부위와 림프구 및 대식 세포의 세포막에서 양성반응을 보였다.

• Journal of Pharmaceutical Investigation
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• 제29권2호
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• pp.87-92
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• 1999

Drug delivery to the brain may be achieved by producing chimeric peptide, attaching the drug to protein 'vectors' which are transported into the brain from the blood by a receptor-mediated transcytosis through the blood-brain barrier (BBB). Since the BBB expresses high concentrations of transferrin receptor, and it was reported that anti-transferrin receptor mouse monoclonal antibody (OX26) undergoes transcytosis through the BBB, it is logical to assume that a drug delivery system via transferrin receptor-mediated transcytosis is a promising strategy. In the present study, therefore, we tested feasibility of several OX26 based vectors for the brain delivery of a model drug. Avidin-based delivery vectors such as OX26-streptavidin (OX26-SA), OX26-neutralite avidin (OX26-NLA) were chemically synthesized vectors and OX26 immunoglobulin G 3 type $C_{H}3$ fusion avidin $(OX26\;IgG3C_H3-AV)$ was genetically engineered. To improve the efficiency of producing chimeric peptide, we used avidin-biotin technology. Pharmacokinetics of $[^3H]biotin$ bound to OX26-SA, OX26-NLA and $OX26\;IgG3C_H3-AV$ was determined by intravenous injection technique, and their stabilities in plasma were analyzed using HPLC. The brain delivery of $[^3H]biotin$ bound to OX26-SA, OX26-NLA and OX26\;$IgG3C_{H}3-AV$ (expressed as %ID/g brain) was $0.22{\pm}0.01$, $0.18{\pm}0.01$ and $0.25{\pm}0.09$, respectively. The areas under the plasma concentration versus time curve (AUC) for OX26-SA, OX26-NLA, $OX26\;IgG3C_H3-AV$ from time zero to 60 min were $209{\pm}10$, $195{\pm}9$, $134{\pm}29\;%ID\;min/ml$ respectively and their total clearances $(CL_{tot})$ were $1.00{\pm}0.09$, $1.08{\pm}0.07$ and $1.54{\pm}0.29\;ml/min/kg$, espectively. These results showed that these vectors possess preferable pharmaceutical (e.g., resonable stability) and pharmacokinetics (e.g., significant brain uptake and enhanced AUC) for brain delivery. Therefore, these vectors may be broadly useful in the brain delivery of drugs that are not transported into the brain to a significant extent.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권4호
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• pp.263-267
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• 2003

Magnetic nanoparticles prepared from an alkaline solution of divalent and trivalent iron ions could covalently bind protein via the activation of Nethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC). Trypsin and avidin were taken as the model proteins for the formation of protein-nanoparticle conjugates. The immobilized yield of protein increased with molar ratio of EDC/nanoparticie. Higher concentrations of added protein could yield higher immobilized protein densities on the particles. In contrast to EDC, the yields of protein immobilization via the a ctivation of cyanamide were relatively lower. Nanoparticles bound with avidin could attach a single-stranded DNA through the avidin-biotin interaction and hybridize with a DNA probe. The DNA hybridization was confirmed by fluorescence microscopy observations. Immobilized DNA on nanoparticles by this technique may have widespread applicability to the detection of specific nucleic acid sequence and targeting of DNA to particular cells.

• 한국수산과학회지
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• 제33권1호
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• pp.55-59
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• 2000

무지개 송어의 뇌하수체 및 배양액에 존재하는 GTH II 농도를 측정하기 위해 Avidin- Biotin complex를 이용한 sandwich EIA 계을 개발했다. Protein A sepharose affinity chromatography을 통해서 얻어진 연어 GTH II의 rabbit IgG에 biotinylation시킨 것 (Biotin-salmon GTH II rabbit IgG)을 제2 항체로 사용하였고, Non-Biotin salmon GTH II rabbit IgG는 단지 protein A sepharose affinity chromatography에서 얻어진 IgG를 제 1 항체로 사용하였다. EIA는 sandwich법에 의해서 이루어졌으며, 효소반응 기질로는 TMB(3,3'5,5-tetramethylbenzidine)를 이용했으며, 반응후 450 nm의 흡광도에서 automatic microplate reader로 측정하였다. 그 결과, $0.12\;{\~}\;125\;ng/ml$의 범위에서 용량반응곡선을 얻었으며, 측정감도 (최소 검출량)는 거의 0.58 ng/ml 정도 였다. 그리고 뇌하수체 추출물 및 배양액 각각의 희석곡선은 GTH II 표존곡선과 일치 하였다. 또한 이러한 GTH II의 표준곡선는 뇌하수체내 다른 peptide hormone와는 교차반응을 거의 나타내지 않았다. Testosterone을 처리한 미성숙 무지개 송어의 뇌하수체 세포배양계를 이용하여 sGnRH에 의한 GTH II 분비량을 본 sandwich EIA계와 RIA계를 비교 조사한 결과, 거의 같은 분비량을 나타냈을 뿐만아니라 같은 분비 pattern을 나타냈다. 이러한 결과로부터 본 sandwich법 EIA계에 의해서 연어과 어류의 뇌하수체 추출물 및 뇌하수체 배양액 중의 GTH II 함량 및 분비량을 측정하는데 있어서 안정된 assay계라고 생각되어진다.