Aliphatic amine latex beads

For covalent conjugation of Ab with latex and coumarin-derived dendrimer, the conjugation procedures were based on the previously developed FICT assay 27 (link), but further optimized to improve the sensitivity and specificity with higher sample volumes directly from the throat swab samples. Briefly, 10 μL of aliphatic amine latex beads (20 nm diameter) (2% w/v) (Life Technologies) were washed with phosphate-buffered saline (PBS) (pH 7.5) and 100 μL of coumarin-derived dendrimer (1 mg/mL in dimethyl sulfoxide) was mixed with latex in the presence of 1 mL conjugation buffer (0.1 M sodium bicarbonate, pH 8.5) at room temperature with shaking. After 1 hour, 8% (v/v) glutaraldehyde (0.5 mL) was added to the mixture 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 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 and resuspended in 1 mL of storage buffer (0.1% [w/v] bovine serum albumin [BSA]) and kept at 4 °C. To suppress non-specific reactivity of bioconjugate in throat swab samples, 0.1% of blocker (sucrose, fish gelatin, and BSA) were used as blockers.

Aliphatic Amine Latex Beads (2% w/v, 20 nm) were purchased from Life Technology and two monoclonal antibodies, anti-influenza A-7307 and -7304, were purchased from Medix Biochemica. Aqueous glutaraldehyde (8% in distilled water [DW]) solution and goat anti-mouse IgG were purchased from Sigma-Aldrich.