Cn140

Half strips, consisting of a nitrocellulose membrane and a wick adhered to a backing card, were constructed to determine the ideal antigen and nitrocellulose combination. Two different antigens (PERI-AMA-BSA and LB-AMA-BSA conjugates) were dispensed as test lines onto six different nitrocellulose membranes. PERI-AMA-BSA was coated at 11 mg/mL and LB-AMA-BSA was coated at 1 mg/mL in PBS. Control lines were coated with goat-anti-mouse polyclonal antibodies at 1 mg/mL in PBS. The different nitrocellulose membranes were: MDI 150, GE FF120, GE FF80, MDI 90, Sartorius CN95, and Sartorius CN140. The membranes were dried for 1 hour at 40 °C and when assembled, the wicking pad (21 mm) overlapped the nitrocellulose membranes (25mm) by ~2mm. To visualize, equal aliquots of antibody-gold nanoparticles were placed into the bottom of test tubes and each membrane type was dropped into the solution and run for approximately 10 minutes.

The immunochromatographic strip included four components: A sample pad (GL-b01, GL-b02), a conjugate pad (GL0194, Ahlstrom 8964, Ahlstrom 6613), a nitrocellulose membrane (Sartorius CN 140, PALL Vivid 170), and an adsorption pad (JY-X117). Goat anti-mouse IgG antibodies and four mouse anti-DENV specific antibodies (mAb51-1.1, 33-7.1, 43-1.3, 22-1.5) were separately applied to the nitrocellulose membrane for use as control and test lines, respectively. The nitrocellulose membrane was then dried at 40°C for 10 min to fix the antibodies. The mAb82-1.1-colloidal gold conjugate was sprayed onto the conjugate pad and then lyophilized with Freeze Dryer (FD12-5S; KINGMECH SCIENTIFIC CO., LTD., Taiwan). The condenser temperature was maintained at -60°C with the drying chamber maintained under vacuum of less than 10 Pa throughout the lyophilization process. The mAb82-1.1-colloidal gold conjugate pad was lyophilized for 8 h. The sample pad, pretreated conjugate pad, nitrocellulose membrane, and adsorption pad were pasted onto a backing card (300 mm×60 mm). Using a strip cutter, the resulting sheet was cut into 4 mm-wide strips, which were then assembled to form cassettes and stored under dry conditions until use.

A glass fiber (Kinbio, RB65, China) was used as the sample pad. AMA1C and goat anti-mouse IgG (H&L) (Solarbio, China) were diluted to a concentration of 1 mg/ml in 0.01 M PBS with 3.0% trehalose. Using the Biodot XYZ3050TM Dispense System (BioDot, XYZ3050TM, United States), the diluted proteins (1 mg/ml) were deposited onto a nitrocellulose (NC) membrane (Sartorius, CN140, Germany) at 1 μl/cm to form the test line (TL) and control line (CL), respectively. Colloidal gold probe was sprayed onto the polyester fiber (Kinbio, DL98, China), which was used as the conjugate pad. The GICA strip, which includes the sample pad, conjugate pad, NC membrane, and absorbent pad, was described and assembled as shown in Fig. 1. A programmable slitter (AUTOKUN, HGS201, China) was used for constructed PVC plate, which was divided into 4-mm-wide test strips and placed in a sealed bag for later use.Fig. 1

Schematic representation of the design of gold immunochromatographic assay strip, negative, positive, and invalid results. CL represents the control line; TL represents the test line