Optimizing Influenza Nucleoprotein Bioconjugates

Bioconjugation were performed as described previously [9 (link)]. To optimize the biocojugate for cloacal samples, different blocking agents were added during the blocking step. Briefly, 10 μL of aliphatic amine latex beads (20 nm diameter; 2 % w/v) (Life Technologies, Carlsbad, USA) were washed with phosphate-buffered saline (PBS; pH 7.5) and 100 μL of coumarin-derived dendrimer (1 mg/mL in dimethyl sulfoxide) was dispersed with the amine latex in 1 mL sodium bicarbonate buffer (0.1 M; pH 8.5). After 1 h, 0.5 mL of glutaraldehyde (8 % v/v) was additionally mixed with the complex of latex beads, and incubated for 30 min. After washing the latex beads twice with PBS, coumarin-derived dendrimer-conjugated latex beads were resuspended in 50 µL of 1 mg/mL anti-influenza nucleoprotein (NP). After vortexing, the conjugate mixture was incubated at 4 °C for 2 h. After centrifugation at 27,237 × g for 5 min, the collected bioconjugates were blocked for 30 min in different blocking buffers (0.1 % bovine serum albumin [BSA], 0.1 % gelatin, 0.1 % sucrose, 0.1 % casein, and mixture of 0.1 % casein and 0.1 % sucrose) and resuspended in 1 mL of storage buffer (0.1 % w/v BSA in PBS, pH 7.6) and kept at 4 °C.