• Title/Summary/Keyword: Nitrocellulose membrane

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Development of Immunochromatography for the Rapid Detection of Listeria monocytogenes (Listeria monocytogenes 신속 검출을 위한 면역크로마토그래피법의 개발)

  • Choi, Jin-Gil;Shim, Won-Bo;Je, Jung-Hyun;Kim, Ji-Young;Lee, Kyu-Ho;Kim, Min-Gon;Ha, Sang-Do;Kim, Keun-Sung;Kim, Kwang-Yup;Kim, Cheol-Ho;Chung, Duck-Hwa
    • Korean Journal of Food Science and Technology
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    • v.39 no.3
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    • pp.299-303
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    • 2007
  • The objective of this study was the development of immunochromatography (ICG) for the rapid and accurate detection of Listeria monocytogenes. Here, monoclonal antibodies (MAb) were conjugated with 40 nm colloidal gold particles, where the conjugate was used as the detection reagent in the ICG. The ICG was composed of three pads (sample, conjugate, and absorbance pads) and one nitrocellulose membrane. The colloidal gold-MAb conjugate was applied to the conjugate pad, and the test line and control line on the membrane were treated with MAb (FKLM-3BI2-37) and anti-mouse IgG, respectively. The detection limit of the ICG was $10^{5}$ cell/mL and it showed no cross-reaction to food borne pathogens. We inoculated meat and lettuce samples with various counts of L. monocytogenes, and analyzed them by ICG. All the inoculated meat samples gave positive results after enrichment for 24 h in LEB. These results indicate that ICG was able to serve as a primary screening tool for L. monocytogenes in various foods and agricultural products within 20 min after enrichment.

Ultrafiltration for Quality Improvement of Wine (한외여과공정을 이용한 포도주의 품질개선)

  • Chung, Jae-Ho;Mok, Chul-Kyoon;Lim, Sang-Bin;Park, Young-Seo
    • Korean Journal of Food Science and Technology
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    • v.35 no.3
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    • pp.386-392
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    • 2003
  • Physicochemical and microbiological changes of grape wine fermented and aged at 25 and $15^{\circ}C$ for 2 and 14 weeks, respectively, were investigated. Viable bacterial cell number, $3.3{\times}10^2\;CFU/mL$ at the beginning of fermentation, increased to $2.3{\times}10^6\;CFU/mL$ after 2 weeks, then decreased to $1.9{\times}10^3\;CFU/mL$ after 14 weeks. Viable yeast cell number increased from $2.8{\times}10^2\;to\;2.2{\times}10^7\;CFU/mL$ during fermentation, then decreased to $1.6{\times}10^4\;CFU/mL$ after aging. Turbidity, pH, total sugar content, reducing sugar content, and solid content of grape wine decreased during fermentation, whereas acidity and alcohol content increased to 0.64 and 8.4%, respectively. Most physicochemical properties did not change significantly during aging. When grape wine was filtered through $0.45-{\mu}m$ nitrocellulose membrane, followed by various ultrafiltration membranes with different molecular weight cut-off values, Biomax 100K membrane with $100\;L/m^2/hr$ (LMH) of initial flux was chosen for ultrafiltration process. These membrane filtration treatments resulted in complete removal of microorganisms and decreases in turbidity, reducing sugar, and solid content. Physicochemical properties of wine did not change, and no microorganisms were found during storage at $30^{\circ}C$ for 12 weeks.

Fabrication and pH response characteristics of LAPS(Light addressable potentiometric sensor) with electrolyte/$Si_3N_4/SiO_2$/Si structure (Electrolyte/$Si_3N_4/SiO_2/Si$ 구조의 LAPS 제작 및 pH 응답특성)

  • Chang Su-Won;Koh Kwang-Nak;Kang Shin-Won
    • Journal of the Korean Electrochemical Society
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    • v.1 no.1
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    • pp.40-44
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    • 1998
  • The LAPS device of fast response and high sensitivity, based on electrochemical potential difference, and its system were fabricated for the precise measurement of pH changes and its characteristic were investigated. The electrostatic variation characteristics of LAPS according to the pH changes and parameters in the device were verified through a simulation using LAPS equivalent circuit model. The LAPS device and its system were fabricated on the basis of the result of simulation. The fabricated LAPS system showed linear sensitivity (about 56 mV/pH within the range of pH 2 to pH 11. In order to overcome the defect of general urea sensor (especially slow response time), urease immobilized nitrocellulose membrane was attached on the LAPS and resulted in the very fast response time, 0.29 mV/sec, 0.86 mV/sec at urea concentration of $50{\mu}g/ml,\; 500{\mu}g/ml$, respectively. And also in order to measure the uranyl ion, the uranyl ion selective sensing membrane with calix[6]arene derivative was used and its sensitivity was 25mV/concentration decade in the wide uranyl ion concentration range of $10^{-11}M\;to\;10^{-4}M$.

Monoclonal Antibody CFC-6, which Binds to Helix II, Inhibits Erythropoietin-Induced Bioactivity

  • Ha, Byung-Jhip;Kim, Suk-Joon;Park, Ji-Sook;Yoo, Ree-Ann;Lee, Dong-Eok;Yoo, Ook-Joon;Woo, Koo;Kim, Hyun-Su;Oh, Myung-Suk
    • BMB Reports
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    • v.30 no.5
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    • pp.315-319
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    • 1997
  • It was discovered that monoclonal anti-erythropoietin (EPO) antibody CFC-6 can neutralize EPO-induced cell activation. To know the binding site of CFC-6, recombinant human erythropoietin (rhEPO) was digested with Glu-C, followed by a separation using high performance liquid chromato graphy (HPLC). Each HPLC fraction was blotted on the nitrocellulose membrane and the membrane was treated with anti-EPO antibody CFC-6 and anti-mouse antibody which is modified with peroxidase. Only one spot showed the color and the fraction was sequenced by Edman degradation. The results suggest that CFC-6 recognizes amino acid sequence V63-W-Q-G-L-A-L-L-S-E72 which is a part of helix II of the EPO molecule. Binding of CFC-6 to EPO may inhibit EPO binding to its receptor, which implies that the antibody binding site and the receptor binding site are close or overlapping.

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Early recognized antigen (p34) of Toxoplasma gondii after peroral ingestion of tissue cyst forming strain (Me49 strain) in mice

  • Park, Yun-Kyu;Nam, Ho-Woo
    • Parasites, Hosts and Diseases
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    • v.37 no.3
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    • pp.157-162
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    • 1999
  • Serum from mouse orally ingested with tissue cyst forming stain (Me49) of Toxoplasma gondii was assayed by Western blot and immunofluorescene assay (IFA) to establish early responses in antigenicity of the parasite in mouse model of foodborne toxoplasmosis. Sera were collected weekly to blot the RH antigen transferred onto nitrocellulose paper after being separated by 12% SDS-PAGE. With the second week serum, 34 kDa protein (p34) was detected uniquely, and all antigens of T.gondii were detected with the sera from 3 or 4 weeks. p34 was not a member of the major surface membrane proteins and confirmed to be localized in the rhoptry by IFA. It was secreted into parasitophorous vacuolar membrane (PVM) during the entry into host cells. 10.3% of sera detected p34, while all the ELISA positive sera detected the band. It has diagnostic usefulness of presumed T.gondii infection. We suggest the name of the p34 protein as ROP9.

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Improvement of the detection limit of rapid detection kit for Salmonella Typhimurium using image analysis system (이미지 분석을 이용한 살모넬라 신속 진단키트의 측정감도 향상)

  • Lee, Sangdae;Kim, Giyoung;Park, Saet-Byeol;Moon, Ji-Hea
    • Korean Journal of Agricultural Science
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    • v.39 no.3
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    • pp.421-425
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    • 2012
  • The objective of this study was to improve the detection limit of rapid detection kit for Salmonella Typhimurium by image analysis system. The rapid detection kit was comprised of four elements: sample pad, conjugate pad, nitrocellulose pad and absorbent pad. Gold nanoparticle and Salmonella antibody were used as a tag and a receptor. Salmonella antibody and goat rabbit IgG antibody were used as test and control lines on nitrocellulose membrane. The color intensity of test line began to increase from $10^5CFU/mL$ of Salmonella sample. A multiple linear regression analysis was employed to explain the relationship between predicted and measured number of Salmonella cells. The developed model could successfully predict the cell number of Salmonella with validation against extra-experimental result.

Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis

  • Lee, Jiyon;Choi, Myoung-Kwon;Kim, Jinju;Chun, SeChul;Kim, Hong-Gyum;Lee, HoSung;Kim, JinSoo;Lee, Dongwook;Han, Seung-Hyun;Yoon, Do-Young
    • Journal of Microbiology and Biotechnology
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    • v.31 no.5
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    • pp.705-709
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    • 2021
  • Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without cross-reactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

Simultaneous Quantitative Determination of Multiple Analytes with Fluorescence- Tagged Probes by Immunochromatogratphy

  • Jeong, Dong-Seok;Choi, Eui-Yul
    • Animal cells and systems
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    • v.7 no.1
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    • pp.89-92
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    • 2003
  • Immunoassays have become indispensable tools and achieved great importance in scientific and medical research. However, typical immunoassays are time-consuming and use complex, multi-step procedures. In this study, we introduce a new immunoassay system for the quantification of several analytes at a time without any washing steps. It is comprised of a detector solution with fluorescence-labeled antibodies and a test strip with immobilized capture antibodies. Using a micro-array scanner, the antigen-antibody complex was quantitatively determined by measuring the intensities of fluorescence on the capture lines or dots of nitrocellulose membrane. This method demonstrated its rapid quantitative determination of analytes without many processing steps as well as specific identification of multiple analytes in biological specimens.

Immune Response of Bacterial Proteins of Staphylococcus intermedius from Canine Atopic Dermatitis (개의 아토피성 피부염에서 분리한 Staphylococcus intermedius 균의 세균단백질의 면역반응)

  • Park, Hee-myung
    • Journal of Veterinary Clinics
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    • v.21 no.1
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    • pp.20-22
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    • 2004
  • Bacterial infection of canine atopic dermatitis is largely caused by Staphylococcus intermedius and may be a superficial or deep pyoderma. The Purpose of this study was to identify the major proteins of S. intermedius cell surface components in humoral immune response of atopic dermatitis dog. Sera samples were obtained from dogs with atopic dermatitis and superficial pyoderma referred to the Veterinary Medical Teaching Hospital, Konkuk University. An isolate of S. intermedius from a clinical case of canine atopic dermatitis was cultured in brain heart infusion broth overnight at $37^{\circ}C$ in aerobic conditions on an orbital shaker. Following culture, Staphylococci were harvested by centrifugation, washed in PBS, and resuspended in PBS containing lysostaphin. The soluble components were separated by centrifugation and were collected. The soluble extract of S. intermedius was separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred onto nitrocellulose membrane. Western blotting for the specificity of serum IgG antistaphylococcal antibody was performed with anti-dog-IgG and sera obtained from an atopic dermatitis case and a normal dog. The molecular masses of four major proteins of S. intermedius recognized by serum obtained from an atopic dermatitis case were 18, 31, 75, and 110 kDa as determined by Western blot analysis. The present study indicates that most dogs of S. intermedius infection with atopic dermatitis could have a significant humoral immune response to bacterial proteins of the causative organism.

The Distribution of Cytoplasm and Nuclei within the Extra-radical Mycelia in Glomus intraradices, a Species of Arbuscular Mycorrhizal Fungi

  • Lee, Jai-Koo
    • Mycobiology
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    • v.39 no.2
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    • pp.79-84
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    • 2011
  • Nuclear distribution within the extra-radical fungal structures and during spore production in the arbuscular mycorrhizae fungus Glomus intraradices was examined using an in vitro monoxenic culture system. A di-compartmental monoxenic culture system was modified using a nitrocellulose membrane and a coverglass slip for detailed observations. Nuclear distribution was observed using the fluorescent DNA binding probes SYBR Green I and DAPI. Both septate and non-septate mycelial regions were observed, but cytoplasmic contents were only found within non-septate mycelia. Nuclear fluorescent staining revealed that the non-septate hyphal region contained nuclei only with cytoplasm, and that nuclear distribution was limited by septa. Swollen hyphal bodies were often associated with septate and empty-looking hyphae. Cytoplasmic contents filled the swollen hyphal body from the non-septate hyphal region following removal of the septa. As a consequence, the swollen body developed into a new spore. These observations provide understanding about the distribution of AM fungal nuclei within extra-radical mycelia and during spore formation. The results suggest a mechanism by which the development of a cytoplasm-containing mycelium is controlled by the formation or removal of septa to efficiently maintain and proliferate essential contents. This mechanism may provide a survival strategy to the fungus.