A quick look at the A-TEEM contours in Figure 2A-2C supports the idea that the A-TEEM method may be sensitive to differences between biotinylated AAV capsids and AAVs complexed with a protein-dye conjugate. However, the presence of the Alexa Fluor 568 dye might be expected to obscure native fluorescence that could be useful in quantifying these samples. We show in this note that it is possible to differentiate biotinylated AAVs from AAV-dye complexes for undialyzed samples, with significant free dye and protein-dye conjugate present. The PARAFAC analysis separates the signal for the complex into two main components which seem to be attributable to AAV and treptavidin. With standard reference samples of each of these, we anticipate that A-TEEM could be used to create a quantitative model against which unknown samples could be quantified for complexation efficiency. PCA was used to easily distinguish AAVs from proteindye conjugates, and from AAV-protein-dye complexes. We were also able to quantify the removal of free dye and unbound streptavidin from the AAV Complex solution during dialysis.
Thus we conclude that the Aqualog and A-TEEM, when coupled with standard multivariate analysis techniques such as PARAFAC, PCA and HCA, can be used to effectively monitor binding and purification of AAV materials important to research and industrial applications.
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