Production and purification of anti-VEGFR2 single chain fragment variable antibody

Introduction: The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). VEGFR and its receptor (VEGFR2) play an important role in angiogenesis associated with tumor growth and metastasis. Methods and Results: To pursue production of scFv antibody fragments against human VEGFR2, we performed four rounds of biopanning using stepwise decreased amount of VEGFR2 peptide (1 to 0.1 μg), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR and Western blot analyses as well as fluorescence microscopy and Wound healing assay. Based upon binding affinity to VEGFR2, 5 clones were selected out of 30 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human VEGFR2. The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the HUVEC cell line. The effectiveness of the selected scFv fragments was further validated by Western blot analyses and Wound healing assay. Conclusions: Based on these findings, we propose the selected fully human anti- VEGFR2 scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications

Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance

Purpose: The single-chain variable fragment (scFv) domain of antibodies is now considered asone of the therapeutic tools that can be produced by phage display technology (PDT). Antibodypurification is one of the most important steps in antibodies production. The aim of study waspurification and characterization of anti-VEGFR2 scFv antibody fragments.Methods: After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptidein ELISA microplates, the phage display library of Tomlinson was used for antibody isolation.The targeted scFv was purified by chromatography using a zeolite-based column. The purity andfunctional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity bindingwas evaluated by surface plasmon resonance (SPR).Results: The desired scFv was selected after four stages of biopanning. SDS-PAGE analysisshowed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed thebinding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibodyanalyzed by SPR was about 60 μM.Conclusion: In this study, the novel scFv antibody against VEGFR2 peptide was purified bychromatography column containing zeolite. Based on our findings the produced antibody maybe applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies

INTESTINAL HELMINTHS FROM THE VIEWPOINT OF TRADITIONAL PERSIAN MEDICINE VERSUS MODERN MEDICINE

Background: Traditional Persian Medicine (TPM) has a history of almost 10,000 years with practice and experience aspects. The existing information and experiences of physicians such as Avicenna clearly show the vast amount of knowledge in the classification and treatment of pathogenic worms. The aim of this paper was the description of the various types of helminths along with their treatment in medieval Persia and comparing them with new medical findings. Materials and Methods: We searched main Traditional Persian Medical and pharmacological texts about etiology, manifestation, diagnosis and treatment of worms in the human digestive system and the out come was compared with the data extracted from modern medical sources. A list of medicinal plants was also extracted from traditional pharmacological books and the anthelmintic properties of these plants were checked in Google Scholar, Scopus, PubMed and Ulrich's databases. Results: The results show the existence of theories on pathogenicity, physiopathology, symptoms and the classification of worms in TPM. TPM philosophers have divided worms into four groups and there is not great difference between old Persian and Modern classification. The old Persian scholars have explained a treatment procedure using a list of 48 medicinal plants and the anthelmintic effect of 23 plants have been shown in modern medicine studies. Conclusion: This study shows a more in-depth and thorough classification of pathogenic worms, their pathogenicity, symptoms and treatments in Traditional Persian Medical compared to Greek Medicine so that old Persian classification may be the base of modern taxonomy. More clinical trials are suggested for the efficacy and safety of these plants