Lateral Flow Immunoassay Assembly Protocol

Chicken anti-protein A polyclonal antibody (1.0 mg/mL; Arista Biological, Allentown, PA, USA; the purity of protein A > 98% at 214 nm by HPLC) and goat anti-human IgG antibody (1.0 mg/mL; Lampire Biological Laboratories, Pipersville, PA, USA) were dispensed to the nitrocellulose membrane (UniSart CN140; Sartorius Stedim Biotech SA) to be the test and the control lines, respectively, by using a lateral flow dispenser (XYZ3000 Dispensing Platform; BioDot Inc., Irvine, CA, USA; 1 µL/cm) and dried at 37°C for 1 h. The four parts of LFIA including sample pad (cytosep 1660; Pall Gelman Sciences), nitrocellulose membrane [UniSart CN140 NCM (Sartorius Stedim Biotech SA)], conjugate pad (GF33; Whatman Schleicher & Schuell), and absorbent pad (Whatman ABS. PAD #470; Whatman Schleicher & Schuell) were assembled as described previously (8 (link)). The treated glass fiber conjugate pad, sample pad, and absorbent pad were assembled to the backing card with a 0.2 cm overlap between each component (Fig. 1). Then the LFIA was cut into 4 mm × 6 cm strips using a CM5000 Guillotine Cutter (BioDot Inc., Irvine, CA, USA). The strips were kept at 4°C in a desiccator.