This DLS assay offers exceptional convenience, ease-of-use and reproducibility and should be useful for monitoring Adenovector preparations for the formation of aggregates. Ultimately, the entire population can aggregate to such an extent that monomeric particles can no longer be detected in the sample. These changes are also observed in the visual histogram generated by the DLS instrument. During vector particle aggregation, both the Z ave and PDI values significantly increase, leading to the formation of a heterogeneous population of aggregates. This includes both antibody- (polyclonal anti-Adenovirus) and temperature-(37☌) induced viral particle aggregation events. Experimental conditions have also been investigated in order to provide insight into the assay's capability of detecting aggregates. The polydispersity index (PDI), which is an expression of sample homogeneity, is typically observed to be <0.2 in these preparations. Under these optimized conditions, purified Adenovector particles are normally observed to be a monodisperse suspension, having a mean particle size (Z ave) in the range of 94 to 104 nm. Latex microsphere standards are utilized to establish system suitability criteria, thereby assuring assay reproducibility and accuracy. The viscosity and refractive index of the sample matrix are incorporated into the analysis in order to ensure data consistency. This instrument accommodates a wide range of sample concentrations and enables analysis of samples in a temperature-controlled environment. 2.2 Dynamic (or quasi-elastic) light scattering in dilute solution Fluctuations in light scattering intensity on the microsecond time scale are collected by the instrument, and the correlation between the intensity measured at any given time t and the intensity at an arbitrary elapsed time t + is expressed as an autocorrelation function. A single cuvette method has been established using a helium-neon laser-based High Performance Particle Sizer (Malvern Instruments HPPS 5001). This microrheology te chnique only requires a small sample volume of 12 mLto measure up to six decades in time of rheological behavi or. We have developed a method for determining vector particle aggregation that employs dynamic light scattering and enables particle size analysis in the “native” state. We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. Conventional biochemical methods for analysis of the viral proteome (SDS-PAGE, Reversed-phase HPLC, MALDI-TOF) are disruptive to the structure and organization of the viral particles and therefore do not provide sufficient insight into the aggregation phenomenon. Sinck L, Richer D, Howard J, Alexander M, Purcell DF, Marquet R, Paillart J-C (2007) In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs.Modifications that can possibly lead to vector particle aggregation.Sinck L, Richer D, Howard J, Alexander M, Purcell DF, Marquet R, Paillart J-C (2007) In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs.Abd El-Wahab EW, Smyth RP, Mailler E, Bernacchi S, Vivet-Boudou V, Hijnen M, Jossinet F, Mak J, Paillart J-C, Marquet R (2014) Specific recognition of the HIV-1 genomic RNA by the gag precursor.Figure 5: Sensing techniques and protocols, showing (top) pulse-timing. Kuzembayeva M, Dilley K, Sardo L, Hu WS (2014) Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles. Dynamic Light Scattering Studies of Biopolymers: Effects of Charge, Shape.Mailler E, Bernacchi S, Marquet R, Paillart J-C, Vivet-Boudou V, Smyth RP (2016) The life cycle of the HIV-1 gag-RNA complex.Paillart J-C, Shehu-Xhilaga M, Marquet R, Mak J (2004) Dimerization of retroviral RNA genomes: an inseparable pair.Maguire CM, Rösslein M, Wickc P, Prina-Mello A (2018) Characterisation of particles in solution – a perspective on light scattering and comparative technologies.Patel TR, Chojnowski G, Koul A, McKenna SA, Bujnicki JM (2017) Structural studies of RNA-protein complexes: a hybrid approach involving hydrodynamics, scattering, and computational methods.Friksen B (2001) Revisiting the method of cumulants for the analysis of dynamic light scattering data.A new tool for the dynamic study of biological macromolecules. Fujime S (1972) Quasi-elastic scattering laser light.Sutherland W (1905) A dynamical theory of diffusion for non-electrolytes and the molecular mass of albumin.Wyatt PJ (1993) Light scattering and the absolute characterization of macromolecules.Mie G (1908) Beitrage zur Optik trüber Medien, speziell kolloidaler Metallosungen.Einstein A (1905) über einen die Erzeugung und Verwandlung des Lichtes betreffenden heuristischen Gesichtspunkt.Strutt JW (1871) On the scattering of the light by small particles.
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