• Title/Summary/Keyword: Immunofluorescence assay

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Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

  • Koompapong, Khuanchai;Sutthikornchai, Chantira;Sukthana, Yowalark
    • Parasites, Hosts and Diseases
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    • v.47 no.4
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    • pp.353-357
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    • 2009
  • Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, $10^1$, $10^2$, and $10^3$ per $10{\mu}l$ were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < $10^2$ per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

Differential Expression of HSP90β in MDA-MB-231 and MCF-7 Cell Lines after Treatment with Doxorubicin

  • Jokar, Fereshte;Mahabadi, Javad Amini;Salimian, Morteza;Taherian, Aliakbar;Hayat, Seyyed Mohammad Gheibi;Sahebkar, Amirhossein;Atlasi, Mohammad Ali
    • Journal of Pharmacopuncture
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    • v.22 no.1
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    • pp.28-34
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    • 2019
  • Background: Breast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including $HSP90{\alpha}$ and $HSP90{\beta}$ in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed. Material and Methods: The cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. immunofluorescence and western blotting techniques were used to determine the expression of $HSP90{\beta}$ in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of $HSP90{\alpha}$. Results: The MTT assay results showed that the MDA-MB-231 cells ($IC_{50}=14.521{\mu}M$) were more sensitive than the MCF-7 cells ($IC_{50}=16.3315{\mu}M$) to DOX. The immunofluorescence results indicated that the expression of $HSP90{\alpha}$ in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that $HSP90{\beta}$ expression decreased in the MCF-7 cells but increased in the MDA-MB-231 cells after DOX treatment. Conclusion: The obtained results suggested that $HSP90{\alpha}$ and $HSP90{\beta}$ expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, $HSP90{\alpha}$ expression was reduced while $HSP90{\beta}$ was found to be overexpressed following DOX treatment.

Serological study on the cross-reactivity of bacteroides gingillalis, bacteroides intermedius and bacteroides asaccharolyticus by indirect immunofluorescence and enzym-linked immunsorbent assay (형광 현미경법 및 효소결합 면역협착법을 이용한 Bacteriodes gingivalis, Bacteriodes intermedius 및 Bacteriodes asaccharolyticus의 혈청학적 연구)

  • Chung, C.P.;Lee, J.Y.;Lee, Y.H.;Chung, H.W.;Chung, H.J.
    • Journal of Periodontal and Implant Science
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    • v.16 no.1
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    • pp.100.1-100.1
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    • 1986
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Isolation of bovine Coronavirus from calves with diarrhea (소 Coronavirus 분리에 관한 연구)

  • 안재문;유기조;이종인
    • Korean Journal of Veterinary Service
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    • v.20 no.2
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    • pp.195-203
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    • 1997
  • Fecal samples were collected from 47 calves with diarrhea and 12 clinically normal co-h-abitants, and tested for virus using MDBK cell cultures. Three cytopathic viruses were isolated from 8 fecal samples obtained from diarrheic calves. The isolated viruses were neutralized by bovine coronavirus hyperimmune serum In plaque reduction assay and were detected in the cytoplasm of MDBK cell by bovine coronavirus hyperimmune serum using immunofluorescence staining. The viruses agglutinated mouse erythrocytes only among the various animal erythrocytes tested and new isolates were identified as bovine coronavirus.

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Evaluation of a cell enzyme-linked immunosorbent assay for the detection of Borna disease virus antibodies in experimentally infected animals (보르나 바이러스를 실험감염시킨 동물에서 항체검출에 대한 세포효소면역반응법의 평가에 대한 연구)

  • Lee, Du-sik
    • Korean Journal of Veterinary Research
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    • v.32 no.3
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    • pp.377-380
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    • 1992
  • The value of the cell enzyme-linked immunosorbent assay as a possible replacement for the indirect immunofluorescence antibody test for the estimation of antibodies against BD virus was assessed in four animal models. The serum antibody response was measured by both assay systems;the variability of both tests was less than one diluent step, and correlation of the two tests was assessed using regression analysis. The study showed that the all four animal models gave satisfactory correlation of CELISA and IFA. There, CELISA is acceptable for use in mouse, rabbit, chicken and rat models.

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Glutamate dehydrogenase antigen detection in Plasmodium falciparum infections

  • Dominguez, Neira-De;Alexis, Rodriguez-Acosta
    • Parasites, Hosts and Diseases
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    • v.34 no.4
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    • pp.239-246
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    • 1996
  • The usefulness of malaria diagnosis by Plusmodium JaLcipawn-GDH (NADP+), obtained by affinity chromatography. is demonstrated in ELISA assays, testing IgG antibodies against GDH (NADP+) from patients with acute malaria, who have had two or more episodes of malaria. or from sera of hyperimmune patients. GDH (NADP+) thermal stability was demonstrated in a high heat resistance assay. The immunofluorescence assay demonstrated that anti-culture (P. falciporum) supernatant serum and anti-GDH (NADP+) of Proton app. recognized epitopes in Venezuelan isolates and Colombian and Brazilian malarial strains. The antigen is soluble, with high specificity is a potent imnlunogen and is thermoresistant. Key words: antigenic enzymes. glutamate dehydrogenase, malaria diagnosis, Plasmodium berghei, Plcswlodium ccthemelum, PlusmoniumJnlcipnmm, Plosmonium uiuox. soluble antigens.

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A Study on Serologic Diagnosis for Dengue Virus Infection

  • Sang-Wook Park;Je-Hoon Yang;Hyung-Joon Bae;Hi-Joo Moon;Young-Dae Woo
    • Biomedical Science Letters
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    • v.8 no.4
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    • pp.269-273
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    • 2002
  • Dengue fever (DF) is an acute febrile illness caused by dengue viruses in the family Flaviviridae, genus Flavivirus. DF has so far posed any problem in Korea, however it has been recently believed to be associated with oversea's traveler infected with dengue virus. Antibody titers of sera from DF patients against dengue virus were measured by indirect immunofluorescence assay (IFA) and plaque reduction neutralization test (PRNT), including the haematologic test. Three of patients with DF showed highly fluorescent and neutralizing antibody titers by IFA and PRNT assay. Two of them showed higher, remarkably. Meanwhile, one of them was tested and resulted in severe tirombocytopenia, elevated serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities as well as mild leucopenia, increased monocytes and basophils and depressed lymphocytes in haematological differential count.

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Selection of Peptides Binding to HCV E2 and Inhibiting Viral Infectivity

  • Hong, Hye-Won;Lee, Seong-Wook;Myung, Hee-Joon
    • Journal of Microbiology and Biotechnology
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    • v.20 no.12
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    • pp.1769-1771
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    • 2010
  • The envelope glycoprotein E2 of hepatitis C virus (HCV) binds to various cell surface receptors for viral infection. We performed biopanning against this protein and selected peptides from phage display peptide libraries. Two short peptides, pep7-1 and pep12-1, were selected and their ability to inhibit the infection process was investigated. When pep7-1 was present, the infectivity of HCV particles in cell culture was notably decreased. This decrease was demonstrated by Western blot analysis, immunofluorescence assay, and reverse transcription PCR assay. However, pep12-1 showed little inhibitory effect on HCV infection.

Antitumor effects of valdecoxib on hypopharyngeal squamous carcinoma cells

  • Trang, Nguyen Thi Kieu;Yoo, Hoon
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.6
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    • pp.439-446
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    • 2022
  • The antitumoral effects of valdecoxib (Val), an United States Food and Drug Administration-approved anti-inflammatory drug that was withdrawn due to the side effects of increased risk of cardiovascular adverse events, were investigated in hypopharyngeal squamous cell carcinoma cells by performing a cell viability assay, transwell assay, immunofluorescence imaging, and Western blotting. Val markedly inhibited cell viability with an IC50 of 67.3 µM after 48 h of treatment, and also downregulated cell cycle proteins such as Cdks and their regulatory cyclin units. Cell migration and invasion were severely suppressed by inhibiting integrin α4/FAK expression. In addition, Val activated the cell cycle checkpoint CHK2 in response to excessive DNA damage, which led to the activation of caspase-3/9 and induced caspase-dependent apoptosis. Furthermore, the signaling cascades of the PI3K/AKT/mTOR and mitogen-activated protein kinase pathways were significantly inhibited by Val treatment. Taken together, our results indicate that Val can be used for the treatment of hypopharyngeal squamous cell carcinoma.

Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

  • Yang, Zhaoshou;Ahn, Hye-Jin;Park, Young-Hoon;Nam, Ho-Woo
    • Parasites, Hosts and Diseases
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    • v.54 no.1
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    • pp.31-38
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    • 2016
  • Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.