FADD (Fas‐associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, ...measurement of FADD expression mainly depends on Western‐blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli‐expressed recombinant His6‐FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin‐conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His6–FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti‐FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.
The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichia coli. Recombinant FADD proteins were purified under the denatured condition. ...After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.
This paper describes the development of a Dig-dUTP based multiplex real time RT-PCR for the simultaneous detection of HCV viral amount in plasma samples. Viral genomes were identified in the same ...sample by Dig-dUTP PCR 216 bp region. Analysis of known scalar concentrations of reference plasma indicated that the multiplex procedure detects at least 500 copies/ml of HCV. In addition, we also assayed HCV viral load in eighty co-infected patients and in fifteen blood donors, confirming the sensitivity and specificity of the assay. This method may represent a useful alternative method for the detection of HCV co-infection, reliable for a rapid and relatively inexpensive screening of blood donors. The assay may be used to determine post-therapy viral clearance.Bangladesh Journal of Medical Science Vol.14(3) 2015 p.247-253
In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT (GenBank Accession No ...DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A (MT2A). There are in-framed multiple cloning sites (MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame (ORF) to achieve fusion expression.
Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the cDNA encoding the human ...MT2A protein was expressed as glutathione S‐transferase (GST) fusion protein in Escherichia coli. Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate‐polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST‐MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low‐cost antibody will be useful for detection in various immuno‐assays.
Edited by Ming‐Hua XU
This study examines students’ understanding of the normative connections between key concepts of nanotechnology in nanomedicine and underlying biological principles that are critical for an in-depth ...understanding of its therapeutic application in medical field. A structured questionnaire was distributed among randomly selected undergraduates at the Faculty of Medicine and Allied Sciences, University of Rajarata, Sri Lanka. A total of 80 students participated in this study and completed written questionnaire on nanomedicine. The outcome of this study shows that there is a strong positive response on basic knowledge on nanoscale, but the undergraduates had an average knowledge on therapeutic application related to nanomedicine. Almost all students had a good knowledge on nanoscale but they lack knowledge of the relationship between nano and nanomedicine. Specifically, students were challenged to demonstrate an integrated understanding of the nanomedicine therapeutic application. Almost 58% of them were unable to give an example of it. Also some students struggled to explain it. Furthermore, in this study it was observed that there is a positive correlation in risk benefit section related to nanomedicine. Although the outcome is preliminary in nature, the results provide cause for concern over the status of nanotechnology education in Sri Lanka which needed to be uplifted.
PDF
