No abstract

Multiple sequence alignments can be used in the template-based modelling of protein structures to build fragment-based assembly models. Therefore, useful functional information on the 3D structure of the anti-MCF-7 scFv protein can be obtained using available bioinformatics tools. This paper utilises several commonly-used bioinformatics tools and databases, including BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Data Bank), KABAT numbering and SWISS-MODEL, to gain specific functional insights into the anti-MCF-7 scFv protein and the assembly of single-chain fragment variable (scFv) antibodies, which consist of a variable heavy chain (VH) and a variable light chain (VL) connected by the linker (Gly 4-Ser) 3. The linker has been built as a loop structure using the Insight II software. The accuracy of the loop structure has been evaluated using Root Mean Square Deviation (RMSD). The accuracies of the VL and VH template-based structures are enhanced by using the evaluation methods Verify3D, ERRAT and Ramchandran plotting, which measure the error in the residues. In the results, 100% of the light-chain residues scored above 0.2, whereas 88.5% of the heavychain residues' scored above 0.15 in the Verify3D evaluation method. Meanwhile, using ERRAT, the alignments of both chains scored more than 70% in space. Additionally, the Ramchandran plot evaluation method showed large numbers of residues in the favoured areas in both chains; these findings demonstrated that all of the chosen templates were the best candidates.

Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production.

BackgroundLung cancer consider as one of leading cause of death around the world, normally the disease detection is difficult in early stages, Anew methods for diagnostic using non‐invasive way are developed using Nano‐molecules called exosomes. Exosomes are cell derived vesicles carrying different kind of molecules (proteins, DNA, RNA, microRNA) and also, displaying many proteins on their membrane surfaces. Those exosomes molecules are identical of parental cancer cells that induce them.MethodsIn this study, exosomes were isolated and characterized using multiple centrifugations, ultrafiltration, and chemical precipitation techniques. The resulted products were captured by two electro‐Microscopes (EM) methods for characterizing the morphology of exosomes as to differentiate exosomes from other extracellular vesicles Besides, the resulted products also further characterized by blotting the proteins extracted from exosomes against different monoclonal antibodies (CD81, CD63, CD9) that confirmed the existence of ligands attached to exosomes surfaces in western blot test.Results and Discussionthe exosomes isolated products were screened under Transmission Electron Microscopy (TEM). The size distributions of extracellular vesicles were significantly different from exosomes 160 nm P < 0.001). The size distributions of apoptotic vesicles (APV) 450nm and necrotic bodies, (NCB) 280 nm were significantly different from exosomes (P < 0.001). The same size band was observed in PVDF membranes of western blot test in all three methods using each monoclonal antibody. However, the filtration devices give higher protein yield and greater purity.In ConclusionsThe current results confirmed the accessible use of exosomes as biomarker to detect lung cancer cells.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract

Chicken anaemia virus (CAV) causes a viral disease in chickens worldwide and thus has economic importance. The main aim of this study was to develop a rapid, sensitive and specific VP1-CAVI indirect ELISA for the detection of CAV infection. The CAV-VP1, was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV-VP1 protein was then coated as an antigen on an ELISA plates to evaluate its reactivity against chicken sera. The resulting indirect ELISA was then compared with a commercial ELISA. The specificity and sensitivity of the indirect ELISA were measured as 93.3% and 100%, respectively. A t-test produced a t-value of 15.805 for the indirect ELISA and revealed a significant difference between CAV-positive serum and CAV-negative serum (p-value of 0.001). For the second variable (i.e., a commercial ELISA), the t-test yielded a t-value of 5.063, which revealed a significant difference between CAV-positive serum and CAV-negative serum (p-value of 0.015). This intervention produces statistically significant improvements in both variables (p-values < 0.05). The correlation coefficient for the indirect ELISA was r = 0.93. Therefore, this work can be considered as a new achievement in diagnosis for Chicken anaemia virus in chicken flocks.

The recombinant antibody technology used in this study is a rapid and effective approach that will aid in the development of the next generation of immunodiagnostic reagents.

Lung cancer is considered one of the leading causes of death worldwide. Usually, disease detection is difficult in the early stages; new methods for diagnosis using non-invasive ways are developed using Nano-molecules called exosomes. Exosomes are cell-derived vesicles carrying different molecules (proteins, DNA, RNA, microRNA) and displaying many proteins on their membrane surfaces. In this study, NCI 1975 Cells line has been used as a source of exosomes. First, the isolation processes were carried out using different ultra-centrifugation devices. Then isolated exosomes were characterized using surface protein biomarkers. Finally, the Exosome isolated products were screened under Transmission Electron Microscopy (TEM) and compared with Scanning Electron Microscopy (SEM). The results showed that the size distributions of apoptotic vesicles (APV) 450nm and necrotic bodies (NCB) 280 nm (extracellular vesicles )were significantly different from exosomes(P < 0.001). This study confirmed the accessible use of exosomes in the diagnosis and prognosis of Non-Small Cell Lung Cancer (NSCLC).