Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies

Abstract

The large molecular size of antibodies is considered one major factor preventing them from becoming more efficient therapeutically. It is well established that all camelids have unique antibodies circulating in their blood called heavy-chain antibodies (HcAbs). Unlike antibodies from other species, these HcAbs contain a single variable domain and two constant domains (CH2 and CH3). HcAbs are a novel type of immunoglobulin-like, antigen binding protein with beneficial pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. Since the antigen-binding site of dromedary HcAb is comprised in one single domain, it was referred to as nanobody. In the present work, the different IgG subclasses from immunized camel (Camelus dromedairus) were purified employing their different affinity for protein A column (PA) and protein G column (PG). Characterization of IgG subclasses was done by using 12% SDS-PAGE under reducing conditions. Protein bands were visualized after staining with Coomassie Brilliant Blue, showing two bands at 50 kDa and 30 kDa in case of IgG1 while IgG2 and IgG3 produce only one band at 46 kDa and 43 kDa respectively. The induction of apoptosis by either conventional or nanobodies was evaluated on two different cell lines, Colon and Hepatic cancer cell (HCT116 and HepG2), using the comet assay. Induced apoptosis were confirmed by visualizing DNA fragmentation bands on 2% agarose gel, and the gel was photographed under UV light. This study demonstrates the successful targeting of human cancer colon cell lines by nanobodies in vitro. It may open perspectives for their future use as tumor target vehicle, due to their small size, soluble behavior and they interact with epitopes that are less antigenic for conventional antibodies.

Restrained management of copper level enhances the antineoplastic activity of imatinib in vitro and in vivo

“…Finally, the comet assay of the treated cells further confirmed the pattern of the previously mentioned results. The assay is believed to distinguish the cells if they undergo apoptosis and necrosis after any treatment 31 , 32 . Finally, the results of flow cytometry and confocal microscopy were found in agreement with the previously mentioned findings under present study.…”

“…In the present study, this experiment was performed with the method developed by Singh et al . 52 with few adjustments 31 , 32 , 53 . They full grown HepG2 cells were subjected to the test chemicals for three hours in different Petri-dishes (60 × 15 mm, Greiner) in the suitable media and conditions as mentioned in Methods 2.2.…”

A conformation-based phage-display panning to screen neutralizing anti-VEGF VHHs with VEGFR2 mimicry behavior

“…Due to their excellent potential for cancer therapy, mAbs currently get attention. As a matter of fact, most of them are directed against a growth factor or a growth factor receptor, the key drivers of tumor growth and survival [44,45]. Commonly used anti-VEGF antibodies are bevacizumab and ranibizumab, both neutralize the biological activity of VEGF through steric hindrance of receptor binding [46][47][48].…”

“…Bevacizumab, a humanized monoclonal IgG, has been developed to treat various cancers such as colorectal, non-small cell lung and renal cell cancer, while ranibizumab, a Fab fragment derived from bevacizumab, is approved for agerelated macular degeneration [47,49]. Despite of beneficial role in cancer therapy, monoclonal antibodies are still amenable for improvement in the terms of stability, affinity, specificity, size, tissue penetration and pharmacokinetic properties [45]. Nowadays, a great deal of effort has been made to develop the mAb fragments that can overcome the obstacles of conventional mAbs, Fabs and scFVs.…”

Antioxidant, antiproliferative, genotoxic and cytoprotective effects of the methanolic extract ofPadina tetrastromaticaon human breast adenocarcinoma and embryonic fibroblast cell lines

“…Conventional cancer therapies have not changed dramatically for the past 25 years, despite significant advances in the understanding of the molecular aspects and pathogenesis of cancer. The toxicity and adverse side-effects brought about by conventional therapies have resulted in a remarkable increase in the clinical testing of natural products as alternative or complementary therapies in combination with existing therapies (Shaker & Melake 2012).…”