• Food Science and Biotechnology
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• v.15 no.5
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• pp.694-697
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• 2006

The initiation of immunoglobulin E (IgE)-mediated allergic reactions requires binding of IgE antibody to its high-affinity receptor. Human basophilic KU812F cells express $Fc{\varepsilon}RI$ on the cell surface and act as effector cells in the allergic response. In this study, we investigated the effects of Scutellaria radix extract on the expression of the $Fc{\varepsilon}RI$ in human KU812F cells. Flow cytometric analysis showed that S. radix extract treatment caused a concentration-dependent decrease in $Fc{\varepsilon}RI$ expression on the cell surface. Furthermore, the level of $Fc{\varepsilon}RI$ ${\alpha}$, ${\beta}$, and ${\gamma}$ chain mRNA in KU812F cells was examined by RT-PCR. S. radix extract reduced total cellular $Fc{\varepsilon}RI$ $\alpha$ and ${\gamma}$ chain mRNA expression in a concentration-dependent manner. $Fc{\varepsilon}RI$-mediated histamine release was reduced from $21.75{\pm}1.34\;ng/10^6$ cells in non-treated cells to $16.46{\pm}1.98\;ng/10^6$ cells in S. radix extract treated cells. These results suggested that S. radix extract has the potential to down-regulate of FcRI expression and to inactivate basophils.

• Applied Chemistry for Engineering
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• v.25 no.5
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• pp.544-547
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• 2014

In this study, an enzyme-linked immunosorbent assay (ELISA) and immuno-chromatographic technique were combined to fabricate immuno-strip sensors for the detection of Legionella pneumophila. The immuno-strip sensor was manufactured with four different membranes. A nitrocellulose membrane was used to immobilize capture antibody and generate signals due to the high affinity to antibodies, and glass fiber membranes were used as a conjugate release pad and a sample application pad. A cellulose membrane was used as an absorption pad to induce sample flow by the capillarity. Colorimetric signals produced by sandwich immuno-reaction and enzyme reaction could be analyzed qualitatively and quantitatively within 30 min. Under the given experimental conditions, sensor signals with L. pneumophila samples were observed qualitatively by naked eyes and measured quantitatively in a range of $1.3{\times}10^3-1.3{\times}10^6CFU/mL$ with a digital camera and home-made image analysis software.

• Research in Plant Disease
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• v.20 no.1
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• pp.15-20
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• 2014

A rapid, user-friendly and simple immune-chromatographic dipstick kit named 'rapid immune-gold strip' (RIGS) kit was developed in a novel single strip format to detect on-site detection of Tomato spotted wilt virus (TSWV). Immunoglobulin G (IgG) from polyclonal antisera raised in rabbits against TSWV was purified through protein-A affinity chromatography and then the purified TSWV-IgG was conjugated to colloidal gold nano-particles which served as a test line on nitrocellulose membrane. Protein A that non-specifically binds to TSWV antibody was used as a control line on the same strip. The diagnosis process with the TSWV-RIGS involves simply grinding the suspect plant sample in a bag that contains the extraction buffer and inserting the strip the bag. Results can be seen in 2-5 minutes. The flow of the complexes of gold particles coated with TSWV-IgG and a crude sap from TSWV-infected pepper, tobacco and tomato plants resulted in intensive color formed on the test lines proportional to the concentrations of TSWV. The RIGS-TSWV kit did not show any cross-reactions against other tomato-infecting viruses unrelated to TSWV. These results indicate that the TSWV-RIGS kit is highly sensitive and is not required for laboratory training and experience prior to testing. The TSWV-RIGS kit is suitable for on-site detection of suspect TSWV-infected plants as well as for laboratory diagnosis.

• IMMUNE NETWORK
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• v.8 no.1
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• pp.7-12
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• 2008

Background: Members belonging to the interferon-lambda (IFN-${\lambda}$) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-${\lambda}$ biology, such as endocytosis of IFN-${\lambda}$, we produced monoclonal antibodies (Abs) against human IFN-${\lambda}$ and examined their usefulness. Methods: We purified recombinant human IFN-${\lambda}$1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-${\lambda}$1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-${\lambda}$1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-${\lambda}$ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-${\lambda}$1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-${\lambda}$1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-${\lambda}$1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-${\lambda}$ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-${\lambda}$1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-${\lambda}$.

• Molecules and Cells
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• v.39 no.11
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• pp.807-813
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• 2016

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of $1.3{\times}10^6CFU/ml$. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate ($5{\times}10^4CFU/ml$). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of $2{\times}10^4CFU/mL$ of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

• Korean Journal of Food Science and Technology
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• v.43 no.2
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• pp.220-223
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• 2011

Human basophilic KU812F cells express a high affinity for IgE receptor, $Fc{\varepsilon}RI$, which plays an important role in immunoglobulin E (IgE)-mediated allergic reactions. Using the human basophilic KU812F cells, we assessed the inhibitory effects of the ethanolic extract from Chrysanthemum zawadsaki(CZ) on $Fc{\varepsilon}RI$ ${\alpha}$ chain expression. Flow cytometric analysis showed that cell surface $Fc{\varepsilon}RI$ ${\alpha}$ chain expression was suppressed when the cells were cultured with CZ extract. RT-PCR analysis showed that the mRNAs for $Fc{\varepsilon}RI$ ${\alpha}$ chain decreased by the treatment of CZ extract. Moreover, CZ extract was also observed to result in a reduction in the release of histamine from anti-$Fc{\varepsilon}RI$ antibody (CRA-1)-stimulated cells. These results suggest that CZ extract may exert anti-allergic activity through down-regulation of $Fc{\varepsilon}RI$ ${\alpha}$ chain expression and a subsequent decrease in histamine release.

• International Journal of Industrial Entomology and Biomaterials
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• v.11 no.1
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• pp.63-69
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• 2005

While observing silkworm larval samples received from field, microsporidian spores formed within the haemocytes of silkworm haemolymph were observed. The spores of microsporidian sp. were purified and characterized for morphological characters viz., size, shape as well as serological affinity with different Nosema spp. (M$_{11}$ and M$_{12}$). The infectivity of the isolated spores to silkworm was also studied. The microsporidian sp. was found to be highly pathogenic to silkworm, B. mori. The isolated microsporidian sp. was designated as NIK-5hm, which formed ovocylindrical spore in the haemocytes of silkworm and differed in spore size (length, 4.55 $\mu$m & width, 2.10 $\mu$m) and shape from Nosema bombycis (NIK-ls), NIK-2r (Nosema sp. Mysore [3.6 & 2.8 $\mu$m]), NIK-3h (Nosema sp. M$_{11}$ [3.8 & 1.8 $\mu$m]), NIK-4m (Nosema sp. M$_{12}$ [5.0 & 2.1 $\mu$m]) and Lb$_{ms}$ (Nosema sp. in Lamerine breed of silkworm [4.36 & 2.14]). In immonological test (Latex agglutination test), the isolated microsporidian spores did not react with antibody sensitized latex particles of N. bombycis, M$_{11}$, M$_{12}$ and Lb$_{ms}$ and thus are different type of microsporidian sp., parasitic to silkworm, Bombyx mori L.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.248-254
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• 2008

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and two-dimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.965-974
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• 2017

The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, $55.2{\pm}0.3^{\circ}C$, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, $52.5{\pm}0.3^{\circ}C$, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

• Preventive Nutrition and Food Science
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• v.22 no.1
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• pp.9-15
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• 2017

Vaccinium angustifolium, commonly known as the lowbush blueberry, is a rich source of flavonoids, with which various human physiological activities have been associated. The present study focuses on the investigation of the effect of the methanolic extract of V. angustifolium root extract (VAE) on high affinity immunoglobulin E receptor ($Fc{\varepsilon}RI$) ${\alpha}$ chain antibody (CRA-1)-induced allergic reaction in human basophilic KU812F cells. The total phenolic content of VAE was found to be $170{\pm}1.9mg$ gallic acid equivalents/g. Flow cytometry analysis revealed that the cell surface expression of $Fc{\varepsilon}RI$ was suppressed in a concentration-dependent manner upon culture with VAE. Reverse-transcriptase polymerase chain reaction analysis showed that the mRNA level of the $Fc{\varepsilon}RI$ ${\alpha}$ chain was reduced in a concentration-dependent manner as a result of VAE treatment. Western blot analysis revealed that the protein expression of $Fc{\varepsilon}RI$ and the phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 were concentration-dependently inhibited by VAE. We determined that VAE inhibited anti-CRA-1-induced histamine release, in addition to the elevation of intracellular calcium concentration ([$Ca^{2+}$]i), in a concentration-dependent manner. These results indicate that VAE may exert an anti-allergic effect via the inhibition of calcium influx and histamine release, which occurs as a result of the down-regulation of $Fc{\varepsilon}RI$ expression through inhibition of ERK 1/2 activation.