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Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
A recently developed variant of the labeled antigen detection techniques, clono-GLAD, offers a way of using even such monoclonal antibodies.308 The principle of this technique is illustrated in Figure 34. Two antisera, one polyclonal and one monoclonal, against the same antigen are used. The polyclonal antiserum stains tissue, but its specificity (presence of contaminating antibodies) is doubted. The monoclonal antibody does not react with tissue, but reacts with the native, undenatured antigen. Consequently, the polyclonal and monoclonal antibodies must recognize different epitopes; otherwise both or none would react with tissue. The polyclonal antiserum is applied first. As in the original GLAD method, it will react with only one of its two antigen-combining sites. Subsequently, an antigen preparation, which may be very crude, is applied. The free antigen-combining site on the polyclonal antibodies will bind to the corresponding epitope on the applied antigen. As this antigen is unfixed and unperturbed, it will have available the epitope detected by the monoclonal antibody. Accordingly, the correct antigen will be “sandwiched” between the primary polyclonal antibody and the monoclonal antibody. The binding of the monoclonal antibody can then be detected either by directly labeling of it with gold or by application of a second gold- or peroxidase-labeled antimouse (antirat) IgG antibody.308
Serodiagnosis: Antibody and Antigen Detection
Published in Johan A. Maertens, Kieren A. Marr, Diagnosis of Fungal Infections, 2007
Christine J. Morrison, David W. Warnock
In general, antigen detection tests that use polyclonal antibodies raised against unpurified or semipurified fungal antigens have significant cross-reactions with other pathogenic fungi (5). Furthermore, assays that use polyclonal antibodies may be subject to variability among different lots of antisera. Monoclonal antibody-based antigen detection tests offer several advantages over polyclonal antibody-based procedures, including reduced batch-to-batch variability, and the ability to generate standardized reagents in almost unlimited quantities. However, monoclonal antibody-based antigen detection tests are sometimes less sensitive than otherwise identical methods that use polyclonal antibodies.
MiR-5622-3p inhibits ZCWPW1 to induce apoptosis in silica-exposed mice and spermatocyte cells
Published in Nanotoxicology, 2023
Moxuan Zhao, Guiqing Zhou, Jingjing Wang, Yue Zhang, Jinglong Xue, Jianhui Liu, Junhong Xie, Lihua Ren, Xianqing Zhou
4% paraformaldehyde solution was applied to fix testes tissue and cells. For testes, they were embedded into paraffin blocks and cut into sections. After being heated for 2 h at 60 °C, these sections were dewaxed by xylene 3 times for 10 minutes each and hydrated in gradients of ethanol (100%, 95%, 85%, 75%) and in phosphate buffer saline (PBS) thrice for 5 minutes each. Both tissues and cells were treated with 0.05% (v/v) Triton X-100 for 40 min and then blocked with fetal bovine serum (5%). After that, they were covered by γ-H2AX rabbit polyclonal antibody (1:200, CST, USA) at 4 °C for the whole night. These samples were covered with anti-rabbit IgG secondary antibody (1:300, CST, USA) for 1.5h at 25 °C the next day. DAPI was used to stain nuclei. Laser scanning confocal microscopy was applied for the image.
A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies
Published in mAbs, 2022
Sebastiaan C. de Graaf, Max Hoek, Sem Tamara, Albert J. R. Heck
Although antibody sequencing at the protein level is still not trivial, it is being applied on a steadily increasing scale in academia and industry. Efforts to extend the sequencing of antibodies to polyclonal mixtures have, however, proven extremely challenging. The first obstacle is sample availability. While recombinant mAb samples are typically available in milligram quantities, polyclonal antibody samples are often derived from clinical samples and thus only available in limited quantities. Because the median concentration of individual clones in plasma is ~1 µg/mL the available protein per individual clone is generally orders of magnitude less compared to mAbs.31 Furthermore, isolation of individual clones is extremely challenging, further complicating the sequencing process, as most software tools are exclusively designed for assembling a single antibody, and therefore fail when data represent several similar Ig sequences. Additionally, in complex endogenous polyclonal antibody mixtures, key sequence evidence on the hypervariable regions is often not detected due to a dilution effect, whereby sequence information from the conserved regions becomes amplified (as the latter is present in every clone) and thus suppresses the signal of the CDRs, which are unique for all clones. Even though the algorithms developed for mAb sequencing are not directly applicable for polyclonal antibody sequencing, they provide a great starting point for developing new tools.
The effect of chronic neuropeptide-S treatment on non-motor parameters in experimental model of Parkinson’s disease
Published in International Journal of Neuroscience, 2021
Osman Sinen, Mehmet Bülbül, Narin Derin, Ayse Ozkan, Guven Akcay, Mutay Aydın Aslan, Aysel Agar
In a separate group of rats (n = 4) underwent the same experimental procedures, briefly, the brains were fixed by 4% paraformaldehyde. Coronal frozen sections (40 μm) from SN were incubated for 30 min in 3% H2O2 and blocked with 10% normal horse serum and 0.1% Triton in PBS for two hours. The sections were then incubated with rabbit anti-TH polyclonal antibody (1:1000 dilution; AB112, Abcam, Cambridge, MA, USA) at 4 °C overnight. The following day, sections were treated with biotinylated anti-rabbit IgG ((Vector Lab. Inc., Burlingame, CA, USA) and then processed with streptavidin peroxidase. The peroxidase activity was visualized with 3,3′-Diaminobenzidine (DAB) (Sigma-Aldrich Co. LLC, Steinheim, Germany). Finally, the sections were examined with Axioplan light microscope and photographed. The number of TH-positive cells was counted by two examiners who was blind to the animal’s identity.