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Magnetic Nanosensors
Published in Vinod Kumar Khanna, Nanosensors, 2021
In a different study (Kim et al. 2007), a slightly dissimilar scheme was implemented to detect the beta subunit of human chorionic gonadotrophin (HCG-β), a glycoprotein hormone composed of 244 amino acids with a molecular mass of 36.7 kDa, and a biomarker concerned with prostate and ovarian cancers. In this case, two different monoclonal antibodies that bind different, nonoverlapping epitopes (localized regions on the surface of an antigen that is capable of eliciting an immune response and of combining with a specific antibody to counter that response) on the HCG-β protein were attached to separate CLIO nanoparticle populations. Monoclonal antibodies are the highly specific antibodies, compared with polyclonal antibodies, which can be produced in large quantity by the clones of a single hybrid cell formed in the laboratory.
GMR Spin-Valve Biosensors
Published in Evgeny Y. Tsymbal, Igor Žutić, Spintronics Handbook: Spin Transport and Magnetism, Second Edition, 2019
Jung-Rok Lee, Richard S. Gaster, Drew A. Hall, Shan X. Wang
The second antibody, known as the detection antibody, is delivered in solution and binds to a second epitope on the captured protein of interest. The detection antibody is typically polyclonal and pre-modified with a reactive chemistry, enabling facile attachment of the detection antibody to the tag of interest. A polyclonal antibody solution is one in which all the antibodies react with the same protein; however, they may bind to different epitopes on that protein. Therefore, the Fab region is not identical across all the antibodies in a polyclonal solution. Typically, the antibody is modified with biotin and the tag is modified with streptavidin, since the biotin–streptavidin interaction is one of the strongest non-covalent receptor–ligand interactions in biochemistry (dissociation constant KD ≈ 10 fM). In the ELISA, the tag of interest is typically fluorescent or enzymatic. However, when using GMR biosensors, the tag of interest is magnetic. Therefore, the more protein that is present in the system, the more detection antibody binds, and the more magnetic tag binds. As the number of magnetic tags increases over the sensor, the magnetoresistance in the underlying GMR sensor changes proportionally, producing larger signals [16]. In this way, quantitative protein detection is possible with this assay.
Biologic Drug Substance and Drug Product Manufacture
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Ajit S. Narang, Mary E. Krause, Shelly Pizarro, Joon Chong Yee
Structurally, Ig is commonly represented in a typical Y-arm structure (Figure 8.1) consisting of two large/heavy and two small/light polypeptide chains joined by disulfide bridges. Antibody fragments consist of a (mostly) constant region (designated, Fc) and an antigen-binding region (designated, Fab). Antibodies that recognize multiple sites of an antigen are termed polyclonal, whereas antibodies that target only a specific site are monoclonal. Identical immune cells make monoclonal antibodies, whereas polyclonal antibodies are produced by a mass of immune cells that may produce antibodies against different regions of the antigen. In industrial applications, monoclonal antibodies are prepared using recombinant DNA technology in cultured cells. For human clinical applications, monoclonal antibodies are generally preferred. Polyclonal antibodies are utilized for diagnostic and lab use such as immunohistochemistry.
Production of polyclonal antibody against human Neuritin and its application of immunodetection
Published in Preparative Biochemistry and Biotechnology, 2019
Na Wang, Yu Wei, Wen Zhang, Xingyi Li, Jingling Zhu, Liya Shan, Chunyan Liu, Wumei Yuan, Jin Huang
Antiserum was obtained by direct injection of protein[13,14] and the rhNeuritin was used to immunize Sprague-Dawley rats to prepare the polyclonal antibody. A total of three immunizations were administered within 35 days. The antiserum was collected and purified by immunoaffinity chromatography and showed high sensitivity, with titers as high as 1:16,000 as measured by indirect ELISA. The antibody recognized both exogenous and endogenous Neuritin as assessed by immunoblotting. However, affinity of the antibody to endogenous Neuritin was higher than exogenous Neuritin. Therefore, we investigated whether the antibody could also be used in other immunoassays. The antibody was used in immunofluorescence and successfully bound to Neuritin localized within lipid rafts in the cell membrane.[10]
Involvement of nerve growth factor in mouse hippocampal neuronal cell line (HT22) differentiation and underlying role of DNA methyltransferases
Published in Journal of Toxicology and Environmental Health, Part A, 2018
Ming Zhang, Hongxia Zheng, Xiaolu Zhang, Xiaoli Tian, Shengdi Xu, You Liu, Shuyuan Jiang, Xiaolei Liu, Rui Shi, Kerui Gong, Shaochun Yan, He Wang, Guo Shao, Zhanjun Yang
Following 24 hr incubation cells were washed with phosphate-buffered saline (PBS) prior to addition of radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Shanghai, China) and kept on ice for 10 min. Protein was extracted and concentrations determined using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Ill., USA). Subsequently, equal amount of proteins were separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Roche Diagnostics, Indianapolis, IN, USA). The membranes were blocked with 5% nonfat milk and incubated with rabbit anti-DNMT1, 3A and 3B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), mouse anti-β-actin monoclonal antibody (Sigma Chemical Company, St.Louis, MO, USA) (Liu et al. 2017). Blots were detected by enhanced chemiluminescence detection system (ECL) (Beyotime, Haimen, Jiangsu, China).Primary antibodies were used at a dilution of 1:1000.The optical densities of the bands were quantified by using an image analysis system with ImageJ (Scion Corporation, Torrance, CA, USA). Data of western blots were normalized to β-actin.
Serum from differently exercised subjects induces myogenic differentiation in LHCN-M2 human myoblasts
Published in Journal of Sports Sciences, 2018
D. Vitucci, E. Imperlini, R. Arcone, A. Alfieri, A. Canciello, L. Russomando, D. Martone, A. Cola, G. Labruna, S. Orrù, D. Tafuri, A. Mancini, P. Buono
To obtain extracts of total proteins, cells were washed twice with PBS to remove residual media, and lysed using cold lysis buffer as reported elsewhere (Mancini et al., 2017). Cells were scraped off the plate and the extract was centrifuged at 14,000 x g for 10 min at 4°C. The supernatant was recovered and protein concentration was measured using the Bradford method. Protein extracts (each 40 µg) were resolved with 8‒12% polyacrylamide gels. The membranes were immunoblotted with the mouse anti-SQSTM1, also known as “ubiquitin-binding protein p62 monoclonal antibody” (Abcam, Cambridge, UK), the rabbit anti-Bcl-2 polyclonal antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA), the rabbit anti-poly-ADP-ribose polymerase-1 (PARP-1) polyclonal antibody (Santa Cruz Biotechnology Inc.), and the mouse anti-GAPDH monoclonal antibody (Abcam). Blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibody and target proteins were visualised by enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA). Densitometric measurements were carried out using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) as reported elsewhere (Spaziani et al., 2014). The GAPDH protein was used to estimate the total amount of loaded proteins. Results were normalised as a percentage of control in each membrane.