Xyz3210 dispense platform

To prepare an analytical nitrocellulose membrane, each antibody was immobilized onto a nitrocellulose membrane (AE99; Whatman Schleicher & Schuell, Dassel, Germany) using XYZ3210 dispense platform (BioDot) at rate of 1 µL/cm. Purified mAb 18B7 (0.5 µg/mL) and goat anti-mouse IgG antibody (1 mg/mL; Jackson, West Grove, PA, USA) were immobilized at the test line and the control line, respectively. The immobilized membranes were incubated at 37 °C for 60 min. After incubation, the immobilized membranes were blocked with membrane blocking buffer [1% (w/v) BSA, 1% (v/v) PVP–40, 0.25% (v/v) Triton X-100, dissolved in 2 mM Na₂B₇O₄ pH 8.0] to prevent nonspecific binding and minimized background signals. The blocked membrane was incubated at 37 °C for 90 min and was ready for assemble into ICT strips.

The rSsIR-based IgG ICT kit was optimized as follows: the test line (T) was coated with 2.0 mg/mL of recombinant SsIR and the control line (C) with 1.0 mg/mL of goat anti-mouse IgG (Lampire Biological Laboratories; 0.1 µL/mm). These were sprayed on a nitrocellulose membrane (Sartorius Stedim Biotech SA, Goettingen, Germany) using an XYZ3210 Dispense Platform (BioDot, Irvine, CA, USA). The colloidal gold-conjugated mouse monoclonal anti-human IgG (Kestrel BioSciences Co., Pathumthani, Thailand) was sprayed onto a glass microfiber filter (GF33; Whatman Schleicher & Schuell, Dassel, Germany) to form the conjugate pad. The serum samples were diluted with sample buffer at a ratio of 1:100, and 5 µL of diluted serum and 100 µL of chromatography buffer were added into the buffer holes marked in Figure 1 as “S” and “B”, respectively. The results were visually interpreted (unaided) at 15 min according to the interpretation card (Figure 1). Red bands appearing at the C line and T line within 15 min indicated a positive result, whereas a red band at only the C line indicated a negative result (Figure 1). The rSsIR-based IgG4 ICT kit was optimized using a method similar to that described above, except that the colloidal gold-conjugated mouse monoclonal anti-Human IgG4 (Invitrogen) was sprayed to form the conjugate pad and the serum samples were diluted with sample buffer at a ratio of 1:5.

The components of the ICT (Figure 1A) were laminated in five layers: (1) modified backing card (paper lower cassette), (2) nitrocellulose membrane (Sartorius Stedim Biotech SA, Göttingen, Germany) of the antigen line (T-line) and anti-mouse IgG antibody (Lampire Biological Laboratories, Pipersville, PA, USA) line (C-line), (3) conjugated pad of antibody-labeled gold nanoparticle, (4) sample pad (Millipore, Massachusetts, MA, USA) and (5) absorbent pad (Whatman Schleicher & Schuell, Dassel, Germany). The laminate was cut into strips of 0.5 cm length using a guillotine (BioDot, Irvine, CA, USA). Finally, the test strip laminate was inserted into a plastic cassette cartridge, and the cassette was closed with a cover (Adtec Inc., Oita, Japan) (Figure 1). The nitrocellulose membrane was coated with the O. viverrini antigen at the T-line and the anti-mouse IgG antibody at the C-line, using the XYZ3210 Dispense Platform (BioDot), at a flow rate of 0.1 µL/mm. The monoclonal anti-human IgG antibody-labeled gold nanoparticles (Kestrel BioSciences Co., Pathumthani, Thailand) were sprayed onto a glass microfiber filter (Whatman Schleicher and Schuell) at a flow rate of 1 µL/mm to produce the conjugated pad.