The MSU Department of Virology headed by Professor Joseph Atabekov is studying the in vitro assembly of compositions, consisting of artificial plant virus particles and antigens, potentially attractive for vaccines development. Artificial plant virus particles are spherical particles (SPs) generated by thermal denaturation and structural remodelling of helical plant tobacco mosaic virus, a rod-shaped virus with a diameter of 18 nm and a modal length of 300 nm. It has been found that upon thermal denaturation of TMV, viral RNA is released and becomes degraded whereas viral coated protein is assembled into spherical particles. The size of SPs depends on the initial TMV concentration and particles from 50 to 800 nm may be obtained. The group of Professor Olga Karpova has shown that SPs based on TMV are stable and may adsorb a diversity of proteins. Thus, SPs represent a new type of biogenic nanoplatform attractive for binding antigens and antigenic determinants of different pathogens.
Describing the choice of NTA for this work, Dr Nikitin says "It permits us to analyze and control the size, state of aggregation and concentration of artificial plant virus particles and small spherical plant and animal viruses. Furthermore, NTA allows us to see the formation of immunogenic complexes (candidate vaccines) by using the indirect immunofluorescence or immunogold staining methods. The technique provides us with the opportunity to obtain simultaneous information concerning nanoparticle size, state of aggregation, concentration and antigenic specificity in liquid. This is particularly important for vaccine characterization and standardization."
Continuing on the benefits of NTA over other characterization methods, Dr Nikitin says "Previously, we had used transmission electron microscopy (TEM) and dynamic light scattering (DLS) for sizing SPs, isometric viruses and virus-like particles. To detect the formation of immunogenic complexes (candidate vaccines) we use immunogold TEM and immunofluorescence microscopy. For us, the main advantage of NTA over these microscopic methods is that there is no need to fix and dry the object on a supporting film which could lead to morphological deformations and aggregation of nanoparticles. NTA provides the means for analysis of samples in liquid in real-time. DLS is also available for measuring the size of nanoparticles in liquids. However, particle aggregation and any contamination of samples will lead to incorrect results. The correct size of the particles can be obtained by DLS only in the absence of aggregation and polydispersity of sample. In addition, DLS cannot estimate the number of particles per unit volume and cannot detect the retention of particles antigenic properties. NTA does not have these problems as it makes measurements particle by particle."
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NanoSight's "Nanoparticle Tracking Analysis" (NTA) detects and visualizes populations of nanoparticles in liquids down to 10 nm, dependent on material, and measures the size of each particle from direct observations of diffusion. Additionally, NanoSight measures concentration and a fluorescence mode differentiates suitably-labelled particles within complex background suspensions. Zeta potential measurements are similarly particle-specific. It is this particle-by-particle methodology that takes NTA beyond traditional light scattering and other ensemble techniques in providing high-resolution particle size distributions and validates data with information-rich video files of the particles moving under Brownian motion.
This simultaneous multiparameter characterization matches the demands of complex biological systems, hence its wide application in development of drug delivery systems, of viral vaccines, and in nanotoxicology. This real-time data gives insight into the kinetics of protein aggregation and other time-dependent phenomena in a qualitative and quantitative manner. NanoSight has a growing role in biodiagnostics, being proven in detection and speciation of nanovesicles (exosomes) and microvesicles.
NanoSight has installed more than 500 systems worldwide with users including BASF, GlaxoSmithKline, Merck, Novartis, Pfizer, Proctor and Gamble, Roche and Unilever together with the most eminent universities and research institutes. NanoSight's technology is validated by 600+ third party papers citing NanoSight results. NanoSight's leadership position in nanoparticle characterization is consolidated further with publication of an ASTM International standard, ASTM E2834, which describes the NTA methodology for detection and analysis of nanoparticles.
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