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Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Another important aspect of the avidin-biotin techniques is the properties of the avidin preparation. Natural avidin has a very high isoelectric point (above 10) and will bind nonspecifically to many tissue constituents, notably nucleic acids. The isoelectric point of avidin can be reduced by derivatizing some of its primary amino groups, and several such derivatized preparations showing less unspecific background labeling are available now. In addition, Streptomyces avidinii produce a protein, referred to as streptavidin. It possesses the strong binding avidity of egg-white avidin for biotin, yet has an isoelectric point near the neutral range and is not glycosylated.36 Streptavidin does not stick unspecifically to tissues and is the reagent of choice in the author’s laboratory. It is available from several commercial sources. A good illustration of the early problems with the stickiness of egg white avidin is that antibodies were raised to biotin and used in place of avidin. Although the use of streptavidin largely circumvents these problems, such antibodies are also commercially available, as are derivatized and less sticky avidin preparations.
Laboratory evaluation of thyroid function
Published in Pallavi Iyer, Herbert Chen, Thyroid and Parathyroid Disorders in Children, 2020
The recommended daily dietary intake of biotin for children is 5 to 25 mcg and for adults is 30 mcg. Many supplements contain significantly more biotin with doses as high as 5 to 20 mg per dose (4). Supplementation with biotin doses as low as 1.5 mg per day have been found to affect laboratory assays (2). Biotin interferes with immunoassays by irreversibly binding to streptavidin (2,4,11).
X-Ray, MRI, and Ultrasound Agents Basic Principles
Published in George C. Kagadis, Nancy L. Ford, Dimitrios N. Karnabatidis, George K. Loudos, Handbook of Small Animal Imaging, 2018
Michael F. Tweedle, Krishan Kumar, Michael V. Knopp
However, the only biological receptors that the microbubble can access are on the endothelium of the blood vessels or blood cells, and this severely limits the range of targets available to those expressed on endothelial cells, mainly. A few dozen 2 µm diameter bubbles could fit on one side of an endothelial cell, so receptor targeted imaging is nevertheless a recognized and vibrant field of research in USCA. For attachment of the targeting ligands (e.g., antibodies, peptides) to the microbubble surface, well-known techniques, such as carbodiimide, maleimide, and biotin–streptavidin coupling are used (see Figure 15.5). Biotin–streptavidin is a popular coupling strategy because biotin’s binding affinity for streptavidin is very high (>1015 M−1). Several commercial research companies supply these microbubbles ready to label with biotinylated targeting vectors (see Table 15.6).
Biomolecular assembly on interdigitated electrode nanosensor for selective detection of insulin-like growth factor-1
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2021
Yan Gu, Lijie Liu, Jian Guo, Shun Xiao, Fang Fang, Xiaoyun Yu, Subash C. B. Gopinath, Jianlie Wu, Xunqiang Liu
Figure 4(a) explains biotinylated aptamer immobilization process on IDE sensor. After each step the current changes level were noted to get the conclusion of the biomolecule immobilization on IDE sensor. CDI treated IDE sensor current level was shown as 1.64E-08 A, after drop streptavidin, it was increased to 2.28E-07 A. This current increment is clearly stated the interacting of streptavidin on CDI-modified sensor. Further, the surface was covered by ethanolamine, and the current was increased to 3.47E-07 A. Finally, the biotinylated aptamer was added, the current change was drastically dropped to 1.8E-07 A, due to the interaction of biotin with streptavidin. It has been widely accepted that single streptavidin molecule is able to capture four biotin molecules. Considering this concept, this study titrated five different ratios (1:1, 1:2, 1:3, 1:4 and 1:5) of streptavidin and biotinylated aptamer. As displayed in the Figure 4(a) (inset) could observe gradual increments until 1:4 and at 1:5 it reaches the saturation. Based on these observations, 200 nM of streptavidin and 1 uM of biotinylated aptamer were used in this study. This overall immobilization process confirms the modification of IDE surface with aptamer.
Update on Proteomic approaches to uncovering virus-induced protein alterations and virus –host protein interactions during the progression of viral infection
Published in Expert Review of Proteomics, 2020
In one example, Coyaud and colleagues analyzed which human host proteins interact with each of the Zika virus proteins [101]. Zika virus proteins were individually expressed with FLAG tags in 293 T-Rex cells. They were affinity purified with Flag-M2 agarose beads. Other cell preparations were affinity purified with biotin-streptavidin. Washed samples were trypsin digested and analyzed in a Q-Exactive HF quadrupole-Orbitrap mass spectrometer to identify host proteins that co-precipitated with each of their viral bait proteins. Coyaud et al. identified 1224 human proteins with high confidence that participated in 3033 interactions with the various expressed Zika proteins. Duplicate biological replicates were each examined by two technical replicates. Many Zika proteins commonly interacted with cellular proteins involved in nuclear, endoplasmic reticulum, Golgi and other membrane functions. Individual Zika proteins also tended to interact with specific types of cellular proteins. The Zika virus capsid protein interacted with multiple nucleolar proteins, the non-structural protein NS5 interacted with STAT2 signaling, and NS3 interacted with centrosomes, vesicular transport and the ubiquitin system [101]. Many of these interactions explain known, and previously unknown, aspects of the Zika virus life cycle and how this virus interacts with host cells.
Combining the best of two worlds: highly flexible chimeric antigen receptor adaptor molecules (CAR-adaptors) for the recruitment of chimeric antigen receptor T cells
Published in mAbs, 2019
Diana Darowski, Sebastian Kobold, Christian Jost, Christian Klein
Avidin and streptavidin are both naturally occurring biotin-binding proteins with high affinities, but most of the monomeric or dimeric constructs reported so far are unstable, prone to aggregation, and reveal low biotin affinity. Several approaches have been undertaken to design monomeric or dimeric variants of avidin or streptavidin, respectively.69,70 Making use of modified avidin as ECD incorporated into a CAR, Urbanska et al. designed biotin-binding modCAR-T cells. The group showed that only modCAR-T cells expressing dimeric avidin were able to redirect biotinylated mAb- or scFv-CAR-adaptors. ModCAR-T cells expressing monomeric avidin did not show specific retargeting or activation of T cells. Since biotin is present in human plasma, the group demonstrated that, even at supra-physiological levels, soluble biotin did not cause antigen-independent activation of dimeric avidin engineered modCAR-T cells and did not inhibit the modCAR activity by acting as a competitor molecule.23