2.5. Lateral Immune Detection Methods for the Quantification of CHIKV E1/E2

The E1/E2 lateral flow test was validated using 29 fever serum samples, of which 19 samples were from Honduras and 10 were from Colombia. Dipstick and lateral flow assays were constructed in the lab using an adapted protocol. Briefly, forty-nanometer gold nanoparticles (Innova Biosciences, Cambridge, UK) were conjugated to the CHIKV antibodies according to the manufacturer’s instructions. The antibody was first diluted to 0.1 mg/mL in the supplied dilution buffer. Next, 12 µL of diluted antibody were mixed with 42 µL of reaction buffer. Forty-five microliters of the mix were then used to suspend the lyophilized gold nanoparticles (OD₂₀). The antibody-nanoparticle mix was incubated for 10 min at room temperature, followed by the addition of 5 mL of quencher solution to stop the coupling reaction. After adding the quencher solution, 100 mL of 1% Tween 20 in PBS and 50 mL of 50% sucrose in water were added to the conjugates before use in immunochromatography. Dipsticks were used to screen antibodies and collect limit of detection values. Lateral flows were used to collect limit of detection values and test patient samples in the field.