Figure 2

Effect of the co-attachment of lectins to the microbead surfaces on the infectivity of AAV2-microbead conjugates. AAV2.CMV-LacZ was biotinylated with sulfo-NHS-LC-biotin at 50 μg/ml, followed by the removal of non-virion-associated biotinylation reagent by dialysis. AAV2-microbead conjugates were prepared by the attachment of biotinylated AAV2 particles to the surfaces of avidin-coated fluorescent microbeads (480 nm in diameter) (9.2 AAV2 particles per microbead). To these AAV2-microbead conjugates, a biotinylated form of each lectin was added in excess (0.2 μg biotinylated lectin per 10⁷ avidin-coated microbeads), followed by the removal of unbound lectin molecules by centrifugation. The infectivity of these AAV2-microbead conjugates with and without lectin was analyzed on HeLa, COLO 205, and MIP-101 cell lines. Cells were cultured in 24-well plates at 37°C for 24 hr (initial cell number per well: HeLa, 5 × 10⁴; COLO 205, 1 × 10⁵; MIP-101, 7.5 × 10⁴). AAV2-microbead conjugates bearing each lectin, along with free unmodified AAV2.CMV-LacZ, free biotinylated AAV2.CMV-LacZ, and AAV2-microbead conjugates without lectin, were applied to target cells (a total of 1 × 10⁷ AAV2 particles per well) and incubated at 37°C for 48 hr. Cells were fixed with glutaraldehyde and stained for β-galactosidase activity using X-gal as the substrate. Then, the number of infected cells in each well was counted under a light microscope. Each datum shown is the average number of infected cells per well with a standard deviation (n = 16). A, free, unmodified AAV2.CMV-LacZ; B, free, biotinylated AAV2.CMV-LacZ; C, AAV2-microbead conjugates without lectin; D – J, AAV2-microbead conjugates bearing lectin (D, Con A; E, horse gram agglutinin; F, peanut agglutinin; G, castor bean agglutinin I; H, soybean agglutinin; I, furze gorse agglutinin I; and J, wheat germ agglutinin).