A comparative study on antibody immobilization strategies onto solid surface

이지은,서정현,김창섭,권윤경,하정협,최석순,차형준 한국화학공학회 2013 Korean Journal of Chemical Engineering Vol.30 No.10

Antibody immobilization onto solid surface has been studied extensively for a number of applications including immunoassays, biosensors, and affinity chromatography. For most applications, a critical consideration regarding immobilization of antibody is orientation of its antigen-binding site with respect to the surface. We compared two oriented antibody immobilization strategies which utilize thiolated-protein A/G and thiolated-secondary antibody as linker molecules with the case of direct surface immobilization of thiol-conjugated target antibody. Antibody immobilization degree and surface topography were evaluated by surface plasmon resonance and atomic force microscope, respectively. Protein A/G-mediated immobilization strategy showed the best result and secondary antibody-mediated immobilization was the worst for the total immobilization levels of target antibodies. However, when considering realto-ideal ratio for antigen binding, total target antigen binding levels (oriented target antibody immobilization levels)had the following order: secondary antibody-mediated immobilization>protein A/G-mediated immobilization>direct thiol-conjugated immobilization. Thus, we confirmed that protein A/G- and secondary antibody-mediated strategies,which consider orientation of target antibody immobilization, showed significantly high antigen binding efficiencies compared to direct random immobilization method. Collectively, the oriented antibody immobilization methods using linker materials could be useful in diverse antibody-antigen interaction-involved application fields.

調經種玉湯이 Immobilization Stress를 負荷한 C57BL/6J 생쥐의 免疫系에 미치는 影響

민경헌,김주연,김송백,조한백 대한한방부인과학회 2004 大韓韓方婦人科學會誌 Vol.17 No.4

The purpose of this research was to investigate the effects of Chokyungjongoktang water extract(CJT) on immune system by immobilization stress in C57BL/6J mice. CJT decreased the serum level of histamine and corticosterone increased by immobilization stress. In addition, CJT did not affect the viability of thymocytes and enhanced the viability of splenocytes decreased by immobilization. Also, CJT increased the viability of thymocytes and decreased the viability of splenocytes in vitro. CJT did not affect DNA fragmentation of thymocytes and splenocytes increased by immobilization stress. CJT increased the population of thymic CD4+ cells increased by immobolization stress, and decreased the population of Thy1+ cells increased by immobilization stress and increased the population of splenic CD8+ cells increased by immobilization stress. CJT enhenced the production of -interferon and IL-2 decreased by immobilization stress. Also, CJT decreased the phagocytic activity decreased by immobilization stress and decreased the level of nitric oxide decreased by immobilization stress. These results indicate that CJT may be useful for the prevention and treatment of stress via suppression of serum histamine and corticosterone level, and enhancement of viability of splenocytes and cytokines production from Th1 cells in splenocytes.

Optimized Conditions for In Situ Immobilization of Lipase in Aldehyde-silica Packed Columns

Seo Woo Yong,Lee Kisay The Korean Society for Biotechnology and Bioengine 2004 Biotechnology and Bioprocess Engineering Vol.9 No.6

Optimal conditions for the in situ immobilization of lipase in aldehyde-silica packed columns, via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding of the lipase, via amination between the amine group of the enzyme and the aldehyde terminal of the silica, and the reduction of the resulting imine group by NaCBH, could occur inside the bed, in situ. Mobile phase conditions in the ranges of pH $7.0{\~}7.8$, temperatures between $22{\~}28^{circ}C$ and flow rates from $0.8{\~}1.5\;BV/min$ were found to be optimal for the in situ immobilization, which routinely resulted in an immobilization of more than 70 mg­lipase/g-silica. Also, the optimal ratio and concentration for feed reactants in the in situ immobilization: mass ratio [NaCBH]/[lipase] of 0.3, at NaCBH and lipase concentrations of 0.75 and 2.5 g/L, respectively, were found to display the best immobilization characteristics for concentrations of up to 80 mg-lipase/g-silica, which was more than a 2-fold increase in immobilization compared to that obtained by batch immobilization. For tributyrin hydrolysis, the in situ immobilized lipase displayed lower activity per unit mass of enzyme than the batch-immobilized or free lipase, while allowing more than a $45\%$ increase in lipase activity per unit mass of silica compared to batch immobilization, because the quantity of the immobilization on silica was aug­mented by the in situ immobilization methodology used in this study.

Stabilization of Bovine carbonic anhydrase II through rational site-specific immobilization

Lee, Chang Hyun,Jang, Eui Kyoung,Yeon, Young Joo,Pack, Seung Pil Elsevier 2018 Biochemical engineering journal Vol.138 No.-

<P><B>Abstract</B></P> <P>Carbonic anhydrase (CA) is a prominent biocatalyst used for the enzymatic CO<SUB>2</SUB> capture process. For industrial applications, the immobilization of enzyme on a matrix support is a useful technique for stabilizing the enzyme and improving its reusability. Although there have been several trials for immobilization of CA, there is no systematic approach to investigate which site-specific immobilization is more effective for stabilization of CA. In this study, we investigated the effect of the immobilization position on the stability of CA using α-type Bovine CA (bCAII). Six candidate residues (K9, K36, T85, D151, E233, or N252) were selected and each Cys mutant was site-specifically immobilized on the magnetic beads. The thermal and long-term stabilities were compared. Interestingly, the immobilized K9C and K36C, in which the immobilized sites are located close to the N-terminus of bCAII, showed 4.0- and 9.8-fold enhanced thermostability, respectively, at 58 °C. They also maintained 60.6% and 55.5% of activity, respectively, at 45 °C after 20 days, when the wild-type bCAII (free and randomly immobilized) completely lost its activity. These results indicated that the site-specific immobilization of the flexible residues on the N-terminal region could be an effective strategy for the stabilization of bCAII, which would be useful guidelines for the immobilization of other CAs in a site-specific manner.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The effect of the immobilization position on bCAII stability was investigated newly. </LI> <LI> Six mutants, K9C, K36C, T85C, D151C, E233C, and N252C were designed for the site-specific immobilization. </LI> <LI> The immobilized K9C and K36C showed high thermostability and long-term stability. </LI> <LI> Flexible residues near N-terminal region was more proper targets for site-specific immobilization of bCAII. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

사전운동이 고정 후 골미세구조에 미치는 영향

석민화 ( Min-hwa Suk ),김창선 ( Chang-sun Kim ),오태웅 ( Tae-woong Oh ),신윤아 ( Yun-a Shin ) 한국운동생리학회(구 한국운동과학회) 2016 운동과학 Vol.25 No.3

PURPOSE: The purpose of this study was to analyze the differences of bone density and structure after immobilization prior aerobic exercise and resistance exercise. METHODS: Twenty four mice were randomly divided into 3 groups: CC: 4weeks control cage after 2weeks immobolization (n=7), RT: 4weeks resistance training after 2 weeks immobilization (n=7), AE: 4 weeks aerobic training after 2 weeks immobilization (n=7). The prior exercise was performed aerobic and resistance exercise. The body weight, muscle weight, and bone morphometric microstructure parameter including bone mineral density (BMD), bone specific surface (BS/BV), percent bone volume (BV/TV), trabecular thickness (Tb/Th), trabecular number (Tb/N), trabecular separation (Tb/Sp), structure model index (SMI), connectivity density, and cross-sectional area was evaluated using by microcomputed tomography (micro-CT). One-way repeated ANOVA was used to evaluate the differences between three groups and paired t-test analysis was applied to examine the different between immobilization leg and non-immobilization leg. RESULTS: The results of this study indicated that the body weight was increased after 4 weeks, and decreased after 2 weeks immobilization. The gastronemius and soleus muscle weight was significantly decreased in immobilization leg compared to non-immobilization leg. BMD, BV/TV, BS/BV, Tb/Th, Tb/N, Tb/Sp, and Cross-sectional area in immobilization leg of CC was lower than immobilization leg of RT and AE. CONCLUSIONS: These results suggest that 4 weeks prior exercise might improve the bone density and microstructure, even though 2 weeks immobilization. Especially, the prior resistance exercise has more beneficial effects on muscle weight, bone density and microstructure.

Immobilization of Diatom Phaeodactylum tricornutum with Filamentous Fungi and Its Kinetics

Barzee Tyler J.,El-Mashad Hamed M.,Burch Andrew R.,Franz Annaliese K.,Zhang Ruihong 한국미생물·생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.2

Immobilizing microalgae cells in a hyphal matrix can simplify harvest while producing novel mycoalgae products with potential food, feed, biomaterial, and renewable energy applications; however, limited quantitative information to describe the process and its applicability under various conditions leads to difficulties in comparing across studies and scaling-up. Here, we demonstrate the immobilization of both active and heat-deactivated marine diatom Phaeodactylum tricornutum (UTEX 466) using different loadings of fungal pellets (Aspergillus sp.) and model the process through kinetics and equilibrium models. Active P. tricornutum cells were not required for the fungal-assisted immobilization process and the fungal isolate was able to immobilize more than its original mass of microalgae. The Freundlich isotherm model adequately described the equilibrium immobilization characteristics and indicated increased normalized algae immobilization (g algae removed/g fungi loaded) under low fungal pellet loadings. The kinetics of algae immobilization by the fungal pellets were found to be adequately modeled using both a pseudo-second order model and a model previously developed for fungal-assisted algae immobilization. These results provide new insights into the behavior and potential applications of fungal-assisted algae immobilization.

Immobilization of phenol-containing molecules on self-assembled monolayers on gold via surface chemistry

Hwang, Hye-Jeong,Choi, Inseong,Kim, Young-Jin,Kim, Young-Kwan,Yeo, Woon-Seok Elsevier 2019 Colloids and surfaces. B, Biointerfaces Vol.173 No.-

<P><B>Abstract</B></P> <P>Various phenol-containing molecules such as flavonoids have a wide range of biological effects including anticancer, antimicrobial, and anti-inflammatory properties, and, therefore, they have become subjects of active research for various medicinal and biological applications. To construct applicable materials incorporated with phenol-containing molecules, strategies for immobilization of phenol-containing molecules on solid substrates are required. Although several immobilization methods have been devised and reported, mostly harnessing phenol functionality, however, development of a general immobilization method has been hampered due to its complicated chemical reactions and low reaction yields on surfaces. Furthermore, the use of phenol as a reaction center may compromise the biological activity of phenol-containing molecules. Here, we describe a simple, fast, and reliable method for the surface immobilization of phenol-containing molecules by introducing chemical functional groups, carboxylic acid, thiol, and azide, while maintaining phenol functionality by way of the Mannich-type condensation reaction. We examined the chemical functionalization of naphthol, tyrosine, and flavanone and their immobilization to the self-assembled monolayers on gold via various surface chemistries: the carbodiimide coupling reaction, Michael addition, and the ‘click’ reaction. We strongly believe our method can be a general and practical platform for immobilization of various phenol-containing molecules on surfaces of various materials.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The Mannich reaction was used to functionalize phenolic molecules permitting their surface immobilization. </LI> <LI> The strategy did not compromise the phenol functionality and the biological activity of the immobilized molecules. </LI> <LI> The modification and the surface immobilization was confirmed by NMR, XPS, and MALDI-TOF MS. </LI> <LI> We showed the immobilized molecules can be functional in an enzyme activity assay. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Immobilization of laccase on carbon nanomaterials

Jung Hee Park,Keungarp Ryu,Huahua Xue,Jin Suk Jung 한국화학공학회 2012 Korean Journal of Chemical Engineering Vol.29 No.10

Laccase from Trametes versicolor was readily immobilized on carbon nanomaterials including multiwalled carbon nanotubes (MWNTs), carboxylated multiwalled carbon nanotubes (MWNT-COOHs), and graphene oxides (GOs),by physical adsorption without using coupling agents. The immobilized amount of laccase strongly depends on the pH of the aqueous buffers of the immobilization mixture. As the pH of the aqueous buffer for immobilization increases,the immobilized amount of laccase decreases. The activity of the immobilized laccase on the three carbon nanomaterials exhibits a bell-shaped dependence on the pH of the immobilization solution with maximum activity at pH 6 or 7. When the immobilization solution becomes acidic or basic, the activity of the immobilized laccase declines significantly. The amount and the activity of immobilized laccase were maximum for graphene oxides as substrate material for immobilization.

Immobilization Studies and Biochemical Properties of Free and Immobilized Candida Rugosa Lipase onto Hydrophobic Group Carrying Polymeric Support

Aysegül Ü. Metin 한국고분자학회 2013 Macromolecular Research Vol.21 No.2

In this study, the hydrophobic group carrying support was prepared for the immobilization of Candida rugosa lipase through the reversible adsorption method. Epoxy groups containing poly(glycidyl methacrylate-methyl methacrylate-ethylene glycol dimethacrylate) (poly(GMA-MMA-EGDMA)) beads were synthesized by suspension polymerization, and the reactive epoxy groups of poly(GMA-MMA-EGDMA) beads were treated with ammonia while glutaraldehyde was used as a coupling agent. After the L-hydrophobic ligand phenylalanine amino acid was covalently attached on the activated polymeric matrix, the lipase enzyme was immobilized. The immobilization efficiency as a function of adsorption parameters was characterized. The maximum lipase immobilization capacity of the poly(GMA-MMA-EGDMA) beads was 68.25 mg/g. The activities of the free and immobilized enzymes were investigated in the hydrolysis reaction of olive oil. The activities of the free and the immobilized lipases were measured at different pH values and temperatures, and their thermal and storage stabilities were also determined. The optimum pH for immobilized lipase was 6.0, which was the same for free enzymes. In this pH value, the retained activity of immobilized lipase was 91.84%. The optimum activity temperatures of free and immobilized lipases were determined as 35 and 45 oC, respectively. The optimum temperature for the lipase immobilized on the hydrophobic group carrying matrix was 10 oC higher and it demonstrated activity in a broader temperature range than that of the free enzyme. The immobilized lipase had better resistance to temperature inactivation than the free form. The free enzyme lost all of its activity during the 28 days storage period, while immobilized lipase lost 34% of its activity during the 56 days storage period.

Anti-Stress Effects of Ginseng in Immobilization-Stressed Rats

Choi, Eun-Ha,Lee, Hyun-Jung,Kim, Cheol-Jin,Kim, Jong-Tae,Kwun, In-Sook,Kim, Yang-Ha The Korean Society of Food Science and Nutrition 2004 Preventive Nutrition and Food Science Vol.9 No.3

Stress is a global menace exacerbated by the advancement of industrialization. Failure of stress management is to a breakdown of the psychological and physiological protection mechanisms against stress. The aim of present study was to investigate the anti-stress potential of ginseng against immobilization stress. Male Sprague-Dawley rats (n=24) were divided into three groups; (i) control, (ii) immobilization stress (2hr daily, for 2 weeks), and (iii) immobilization stress (2 hr daily, for 2 weeks) plus oral administration of ginseng (200 mg/kg BW Id). Immobilization stress resulted in a significant inhibition of body weight gain by 45 % and a significant decrease in the tissue weights of thymus and spleen (p < 0.05). The concentrations of blood GOT and GPT were significantly increased in the immobilization-stressed group compared to the control group (p < 0.05). There were no differences in the blood cholesterol levels among groups. Ginseng administration in the immobilization-stressed group tended to reverse the lack of body weight gain and food intake, though not significantly. The ginseng-administered group showed a significant reversal in the stress-induced effect on spleen and thymus weight, increasing the tissue weights by 16% and 20%, respectively, compared to immobilization-stressed group (p<0.05). The plasma corticosterone level was significantly increased in the stressed group by 39 % compared to the control group (p<0.05), but ginseng administration significantly reversed the stress-induced increase in plasma corticosterone by 15 % compared to the immobilization-stressed group. The present study suggests that the anti-stress effect of ginseng is mediated by normalization of stress-induced changes in the circulating hormones and a reversal of tissue weight loss, thereby returning the body to normal homeostasis.