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Order Picornavirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Martin BD et al. (2006) published an excellent study that described a new aspect of the use of CPMV as a scaffold and bright tag for the cargo capture and transport by Drosophila kinesin-driven microtubules in standard gliding assays. The capture occurred through both NeutrAvidin (NA)-biotin and antibody (IgG)-antigen interactions. The microtubules were derivatized with rabbit antichicken IgG or biotin, and the virus was conjugated with chicken IgG or NA. Since CPMV was a very effective scaffold for fluorescent dye molecules and was able to accommodate up to 60–120 dyes per virus depending on the mutant used, the fluorescent CPMV appeared to be superior to fluorescent polystyrene spheres of the same size, as both a reporter tag and a scaffold for the microtubule-transported cargo proteins, because of its negligible nonspecific adsorption and superior brightness (Martin BD et al. 2006).
Chimeric VLPs
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
In 2011, the phage MS2 was chosen to meet the growing requirements for the quality of bio-artificial tracers for the inline measurement of virus retention in membrane processes (for more detail, see Chapter 6). With this aim, the surface of the phage MS2 was modified by the grafting of enzymes, namely, horseradish peroxidase (HRP) (Soussan et al. 2011a,b). The HRP catalyzed a highly specific and rapid reaction, permitting its detection, and could be operated in drinking water without inhibition by the molecules present in solution. This tracer was thus built to enable direct detection of its induced enzymatic activity, notably by an amperometric method. The generation of the MS2 tracer was started with covalent binding of activated biotin to the lysine residues of the phage. This was not the case for bigger and less hydrophilic activated molecules, such as the activated HRP enzymes that were tested at the very beginning for the direct grafting method. For this reason, the neutravidin–HRP conjugates were chosen as enzymatic probes for the second labeling on biotin molecules, notably because of the highly specific, strong interaction between neutravidin and biotin. Moreover, a spacer present before the terminal reactive group of the chosen activated biotin allowed the steric hindrance between enzymatic probes and capsid proteins to be reduced (Soussan et al. 2011a). The novel MS2-based tracer was found as representative as possible of native pathogenic viruses (Soussan et al. 2011b).
Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2
Published in mAbs, 2021
Shion A. Lim, Josef A. Gramespacher, Katarina Pance, Nicholas J. Rettko, Paige Solomon, Jing Jin, Irene Lui, Susanna K. Elledge, Jia Liu, Colton J. Bracken, Graham Simmons, Xin X. Zhou, Kevin K. Leung, James A. Wells
For each phage clone, four different conditions were tested – Direct: Spike-RBD-Fc, Competition: Spike-RBD-Fc with an equal concentration of Spike-RBD-Fc in solution, Negative selection: ACE2-Fc/Spike-RBD-Fc complex, and Control: Fc. 384-well Nunc Maxisorp flat-bottom clear plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL of NeutrAvidin in PBS overnight at 4°C and subsequently blocked with PBSTB for 1 h at room temperature. Plates were washed 3X with PBS containing 0.05% Tween-20 (PBST) and were washed similarly between each of the steps. 20 nM of biotinylated Spike-RBD-Fc, ACE2-Fc/Spike-RBD-Fc complex, or Fc diluted in PBSTB was captured on the NeutrAvidin-coated wells for 30 min, then blocked with PBSTB + 10 μM biotin for 30 min. Phage supernatant diluted 1:5 in PBSTB was added for 20 min. For the competition samples, the phage supernatant was diluted into PBSTB with 20 nM Spike-RBD-Fc. Bound phage was detected by incubation with anti-M13-horseradish peroxidase conjugate (Sino Biologics catalog number 11973-MM05-H, 1:5000) for 30 min, followed by the addition of TMB substrate (VWR International). The reaction was quenched with the addition of 1 M phosphoric acid and the absorbance at 450 nm was measured using a Tecan M200 Pro spectrophotometer.
Commercialization challenges for drug eluting contact lenses
Published in Expert Opinion on Drug Delivery, 2020
Olivia L. Lanier, Keith G. Christopher, Russell M. Macoon, Yifan Yu, Poorvajan Sekar, Anuj Chauhan
Multiple studies in the literature have incorporated liposomes into contact lens formulations. A study by Gulsen et al. [85] synthesized HEMA hydrogels with dimyristoyl phosphatidylcholine (DMPC) liposomes and showed that ophthalmic drugs were released for up to 8 days, which is significantly greater than control lenses. Another study by Danion et al. [92] immobilized PEG-biotinylated lipid liposomes to the surface of a commercial contact lens (Hioxifilcon B). First, polyethylenimine was covalently bounded onto the hydroxyl groups; then, NHS‐PEG‐biotin molecules were bound to the surface amine groups by carbodiimide chemistry. NeutrAvidin was bound to the PEG-biotin layer and the liposomes were bound to the NeutrAvidin. Consecutive layers of NeutrAvidin and liposomes were created. The lenses showed release of carboxyfluorescein for up to 12 days [92].
Purity and yield of melanoma exosomes are dependent on isolation method
Published in Journal of Extracellular Vesicles, 2020
Shin La Shu, Yunchen Yang, Cheryl L. Allen, Edward Hurley, Kaity H. Tung, Hans Minderman, Yun Wu, Marc S. Ernstoff
The setup of the compact SPR biosensor system and the SPR biochip fabrication methods were described previously, with minor revisions [19]. Briefly, sapphire glass slides (600 ± 10 μm) were cleaned subsequently by acetone, methanol and deionized water (DI water). Then 2 nm Ti adhesion layers followed by 49 nm Au thin layers were deposited on the cleaned glass slides via the e-beam evaporator (Indel system). For biochip surface modification of antibodies, the biochip was first incubated with the mixture of methyl-polyethylene glycol-thiol (PEG) (PEG200, MW = 200 g/mol, Catalogue number: 26132, Thermofisher Scientific, USA) and biotinylated PEG thiol (PEG1000, MW = 1000 g/mol, Catalogue number: PG2-BNTH-1k, Nanocs, USA) at the molar ratio of 3:1 with a final concentration of 10mM in PBS at rtp for 1 h. Unbound PEG was washed off with PBS and 0.05 mg/mL of NeutrAvidin (Catalogue number: 31000, Thermofisher Scientific, USA) was added to react with biotin at rtp for 1 h. Thereafter unreacted NeutrAvidin was washed away with DI-water. Biotinylated anti-PD-L1 antibodies (13-5983-82, Thermofisher Scientific, USA) or biotinylated anti-IgG antibodies (13-4714-85, Thermofisher Scientific, USA) were added at the concentration of 0.05 mg/mL and incubated at rtp for 1 h. After washing away unbound antibodies with DI-water, the biochip was ready to use.