Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

In low prevalent countries of Human Immunodeficiency Virus (HIV) like Bangladesh, it is recommended that all HIVpositive sera tested by Enzyme Linked Immunosorbent Assay (ELISA) and/or rapid tests should be confirmed by Line Immune Assay (LIA) or Western Blot (WB) method. As these two tests are quite expensive, Indirect Immunofluorescence Assay (IFA) was evaluated as a confirmatory test and as an alternative to these two methods in the present study. A total of 92 subjects consisting of 46 HIV-antibody-positive patients and 46 controls were included in the study. All samples of sera were tested by ELISA and IFA methods, and some 34 of 46 HIV-antibody-positive samples were tested by LIA. One ELISA positive serum was found negative by both IFA and LIA. This result indicates that ELISA was 100% sensitive and 98.7% specific for detection of HIV antibody. Comparison between LIA with IFA on 34 samples, and WB with IFA on 26 samples showed 100% correlation between these methods. The study concluded that the IFA method is equal in performance as LIA and WB methods for the detection of antibody to HIV and can be used as a confirmatory test.

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Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

10.1101/2020.03.11.987586 ◽

2020 ◽

Author(s):

Yukiko Yasuoka ◽

Takashi Fukuyama ◽

Yuichiro Izumi ◽

Yushi Nakayama ◽

Hideki Inoue ◽

...

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

Western Blot ◽

Western Blotting ◽

Fold Increase ◽

Interstitial Cells ◽

Severe Hypoxia ◽

Liquid Chromatography Mass Spectrometry ◽

Western Blot Method ◽

Erythropoietin Production

AbstractThe detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin β pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules.These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.

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Anti-inflammatory activity of pectic enzyme-treated pectin on lipopolysaccharide-induced RAW 264.7 cells

Journal of Functional Food and Nutraceutical ◽

10.33555/jffn.v1i1.14 ◽

2019 ◽

Vol 1 (1) ◽

pp. 23-30 ◽

Cited By ~ 1

Author(s):

Cian-Song Huang ◽

Qiao-Lin Li ◽

Diana Lo ◽

Yuh-Tai Wang ◽

Ming-Chang Wu

Keyword(s):

Western Blot ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Pectic Enzyme ◽

Western Blot Method ◽

Tnf Α ◽

Cox 2 ◽

Necrosis Factor ◽

Normal Macrophage

The purpose of this study was to investigate the ability and pathway of the pectic enzyme-treated (PET) pectin to inhibit the inflammation of macrophage RAW 264.7 induced by lipopolysaccharide. Results showed that PET-pectin produced from 1% substrate and 48 h reaction time had the highest antioxidative activity, thus these parameters were used to produce PET-pectin used in this study. PET-pectin showed no cell cytotoxicity to normal macrophage RAW 264.7 and reduce the nitrite secretion from LPS-induced RAW 264.7 by 20%. Finally, the expression of cytokines, including NO synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and tumor necrosis factor (TNF-α) were analyzed by western blot. In the western blot method, it was found that iNOS, COX-2, NF-κB, TNF-α and other proteins that activated NO production had a downtrend. It was found that PET-pectin possess promising activity to mitigate the inflammatory response.

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Practical diagnostic testing for human immunodeficiency virus.

Clinical Microbiology Reviews ◽

10.1128/cmr.1.1.124 ◽

1988 ◽

Vol 1 (1) ◽

pp. 124-138 ◽

Cited By ~ 33

Author(s):

J B Jackson ◽

H H Balfour

Keyword(s):

Human Immunodeficiency Virus ◽

Western Blot ◽

Reverse Transcriptase ◽

Indirect Immunofluorescence ◽

Diagnostic Testing ◽

Enzyme Linked Immunosorbent Assay ◽

Hiv Reverse Transcriptase ◽

Reverse Transcriptase Enzyme ◽

Immunodeficiency Virus ◽

Hiv Antigen

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures.

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Effect of PGK1 on radiosensitivity through regulation of CIN and CFL1 in human glioma cells.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14631 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14631-e14631

Author(s):

Hongyi Liu ◽

Yuchi Liu ◽

Hong Xiao ◽

Mengjie Zhao ◽

Yong Xiao

Keyword(s):

Western Blot ◽

Phosphoglycerate Kinase ◽

Glioma Cells ◽

Underlying Mechanism ◽

Migration And Invasion ◽

Normal Cells ◽

Western Blot Method ◽

Successful Establishment ◽

U373 Cells

e14631 Background: Phosphoglycerate kinase 1 (PGK1) has been detected overexpressed in many malignancies and usually correlated with increased tumorigenesis and poor survival. Our previous study revealed that PGK1 significantly up-regulated and positively correlated with cofilin-1 (CFL1) expression in radioresistant astrocytomas samples. However, the further mechanism remains unknown. Methods: In the present study, functional interactions among PGK1, chronophin (CIN) and CFL1 were analyzed by co-immunoprecipitation and immunofluorescence experiments. Transfection of shRNA and pcDNA3.1-plasmid were respectively utilized to silence and overexpress PGK1 or CIN in both normal glioma cells (U251 and U373) and estabilshed radioresistant (RR-U251 and RR-U373) cells. The effect of PGK1 on CIN and CFL1 expression levels was investigated with Western-blot method; while the effect of PGK1 or CIN on radiosensitivity of glioma cells was evaluated using cell viability, migration and invasion assays after irradiation. Results: Cell proliferation, migration and invasion abilities significantly increased in RR-U251 and RR-U373 cells compared with those of normal cells, indicating the successful establishment of RR-glioma cell models. Laser confocal experiment in vitro showed the colocalization of PGK1 and CIN. Meanwhile, PGK1 specifically and directly combined with CIN, while did not interplay with CFL1. Elevated PGK1 expression level was observed in radioresistant cells compared with that of normal cells. The western blot analysis showed that CFL1 and CIN levels were positively correlated with the PGK1 expression in both normal and radioresistant cells. The proliferation, migration and invasion capabilities significantly decreased in PGK1-silenced and CIN-silenced glioma cells, indicating the improvement of the radiosensitivity of radioresistant cells. Conclusions: Our research implied the effect of CIN-mediated PGK1 regulation on CFL1 level, thereby altering the migration and invasion abilities in nomal and notably radioresistant glioma cells. These findings clarify a novel underlying mechanism of radioresistance in glioma cells and provide valid basis for further explorations concerning radiotherapy.

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Effects of Intermittent Cisplatin Intervention on 6PGD Levels and Tumor Growth and Migration in A549 Lung Cancer Cells

Integrative Journal of Medical Sciences ◽

10.15342/ijms.2021.442 ◽

2021 ◽

Vol 8 ◽

Author(s):

WU Qiang ◽

TANG Lin-dong ◽

WU Yan-hui ◽

LI Qiang ◽

YU Li

Keyword(s):

Lung Cancer ◽

Tumor Growth ◽

Western Blot ◽

Cancer Cells ◽

A549 Cells ◽

Lung Cancer Cells ◽

Migration Ability ◽

Western Blot Method ◽

And Migration ◽

A549 Lung

Aim: To investigate the changes of A549 protein 6PGD expression, tumor growth and migration, NADPH and ROS levels in lung cancer cells after intermittent intervention with DDP. Methods: The sensitivity of A549 cells to DDP was detected by cck-8 method, A549 cells were treated by DDP intermitten, 6PGD protein expression was detected by Western blot method, and the ROS level of A549 cells was determined by ROS kit. NADPH levels of A549 cells were determined by NADPH kit. Result: After DDP intermittent intervention, the expression of 6PGD protein in lung cancer cells A549 was increased, the growth and migration of A549 cells were significantly inhibited, and ROS and NADPH levels were significantly increased. Conclusion: 6PGD were upregulated in after DDP intermittent intervention and affected the migration ability in lung cancer cells.

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The Expression and Significance of Matrix Metalloproteinase, Wnt5a and β-Catenin in Placental Tissue of Preeclampsia

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2852 ◽

2021 ◽

Vol 11 (12) ◽

pp. 2375-2380

Author(s):

Hui Zhang ◽

Wei Chen ◽

Shenmiao Chen ◽

Junrong Wang ◽

Shunfen Mao

Keyword(s):

Matrix Metalloproteinase ◽

Western Blot ◽

Pregnant Women ◽

Placental Tissue ◽

Normal Pregnancy ◽

Expression Level ◽

Control Group ◽

Cytotrophoblast Cell ◽

Western Blot Method ◽

Low Expression

In order to investigate the expression and clinical significance of MMPs (Matrix Metalloproteinase) (MPP-2 and MPP-9), Wnt5a as well as β-catenin in placental tissues of preeclampsia (PE) patients, 36 PE patients (PE group) and 25 pregnant women (control group) with normal pregnancy were selected for cesarean delivery. The expression and localization of MPP-2, MPP-9, Wnt5a as well as β-catenin proteins in placental tissue were detected by Western blot method and immunohistochemistry method. The correlation between the poor prognosis of mother and child and the expression level of MMPs, Wnt5a as well as β-catenin is analyzed. The results showed that Western blot results showed that MPP-2, MPP-9, Wnt5a as well as β-catenin proteins were expressed in placental tissue, and the expression level of them of placental tissue in PE group was significantly lower than that in control group (P < 0.01). Immunohistochemistry showed that MMPs, Wnt5a, as well as β-catenin proteins were expressed in the cytotrophoblast cell (CTB) and syncytiotrophoblast cell (STB) of placental tissue, among which Wnt5a proteins as well as β-catenin proteins were mainly expressed in the cytoplasm of STB and CTB respectively. MMPs proteins were mainly expressed in the cytoplasm of these two types of cells. Low expression of MPPs, Wnt5a, β-catenin proteins may be related to blood pressure as well as 24 h urine protein of pregnant women at admission. In conclusion, Low expression of MPPs, Wnt5a, β-catenin proteins may be involved in the PE pathogenesis.

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