2.4. Preparation and measurement of the GICA strips

The GICA strips were developed by Zoonbio Biotechnology (Nanjing, China) (49). Briefly, colloidal gold particles with a mean particle diameter of 70 nm were used to coat recombinant protein rSjSAP4. The gold-rSjSAP4 conjugate was suspended in the buffer containing 20 mM Tris, 5% (w/v) sucrose, and 2.5% (w/v) trehalose at a final concentration of 10 μg/mL, and dispensed onto conjugate pad (glass fibre membrane) at a volume of 35 μL/cm. The pad was dried in a biochemical incubator for 12 h at 37°C. By using an XYZ Biostrip Dispenser (HM3030, Shanghai Kinbio Tech. Co., Ltd., Shanghai, China), protein G (1 mg/mL) (Zoonbio Biotechnology, Nanjing, China) and mouse anti-His tag mAb (0.7 mg/mL) (Zoonbio Biotechnology, Nanjing, China) were dispensed onto the nitrocellulose (NC) membrane (CN140, Sartorius, Goettingen, Germany) at a volume of 1 μL/cm to form the test and control lines, respectively. The NC membrane was then dried at room temperature in a biochemical incubator for 6 h. The absorbent pad (filter paper), coated NC membrane, conjugate pad, and sample pad, were laminated and pasted onto a plastic-backed support card with a 1–2 mm overlap. The assembled scale board was cut lengthwise into strips measuring 3 × 60 mm using a guillotine cutter (ZQ2002, Shanghai Kinbio Tech. Co., Ltd., Shanghai, China). The resulted strips were placed in a plastic cassette, which was further packaged into a silica gel desiccant-containing aluminium foil bag, and stored at room temperature.

The GICA strips were initially tested on 40 KK-positive [KK (+)] subjects, 20 KK-negative [KK (−)] and 20 control individuals in a previous pilot study (49). For the current study, the GICA cassettes were further tested for comparison with the other diagnostics following our previous pilot study protocol (49). Briefly, for each test, 50 μL diluted serum sample (1:20) was added to each cassette which was scanned at 10 min after sample loading. All images were further analysed by a Java-based image processing program, ImageJ to determine an R value, which was defined as the intensity of the test (T) line divided by that of the corresponding control (C) line, converting the results into fully quantitative data. The tests were determined as invalid when the control band did not appear or when the tests were left to develop for more than 15 min. GICA cassettes from the same batch were used for testing all the serum samples.