ADNP Assay for CHIKV Antibody Detection

The ADNP assay was adapted from a previous publication (47 (link)). Briefly, recombinant, biotinylated CHIKV p62-E1 protein was conjugated to streptavidin-coated Alexa Fluor 488 beads (Invitrogen) for 2 h at 37°C. After washing two times in 0.1% BSA in PBS, CHIKV antigen–coated beads were incubated with hyperimmune IgG (5-fold dilutions) in cell culture medium for 2 h at 37°C to form immune complexes. Bone marrow cells were harvested from C57BL/6 mice (mADNP) or primary neutrophils were isolated from fresh ACD blood (hADNP). Cells were washed with PBS, and 5.0 × 10⁴ cells per well were added to the immune complexes and incubated for 1 h at 37°C. The following antibodies were used to stain the cells: mADNP: CD11c APC/Cy7 (clone N418; BioLegend), CD11b APC (clone M1/70; BioLegend), Ly-6C BV605 (clone HK1.4; BioLegend), Ly6G Pacific Blue (clone 1A8; BioLegend), and CD3 PE/Cy7 (clone 17A2; BioLegend) or hADNP: CD66b Pacific Blue (clone G10F5; BioLegend). Cells were fixed with 4% PFA and analyzed on a BD LSRII flow cytometer. Neutrophils were defined as CD3⁻ and CD11c⁻ cells that were Ly6C⁻, CD11b⁺, and Ly6G⁺ for mouse cells or CD66b⁺ for human cells. The phagocytic score was calculated as follows: (percentage of Alexa Fluor 488⁺ cells) × (Alexa Fluor 488 geometric mean fluorescent intensity of Alexa Fluor 488⁺ cells)/10,000.