Preparation and Purification of Gold Standard Probe
An appropriate volume of 0.1 mg/mL K2CO3was added to a 50.0 mL gold solution to adjust its pH, reacted for 30 min, and then reacted with an appropriate volume of 0.5 mg/mL p72 protein solution (or 0.1 mg/mL p30) for 45 min. Next, 10% BSA was added to a final concentration of 1%, then the reaction was continued for 45 min and the solution allowed to stand for 2 h at 4 °C. The solution was then centrifuged at 1,500 rpm for 15 min at 4 °C; the supernatant was carefully aspirated, and the precipitate was discarded. The supernatant was centrifuged at 12,000 rpm for 45 min at 4 °C, the resulting supernatant was discarded, and added the gold-labeled protein protectant to the precipitate to reach the original volume of the solution before centrifugation. The mixture was centrifuged again at 12,000 rpm for 45 min at 4 °C and the supernatant was discarded. The precipitate was resuspended in 1/20 of the original volume of the gold-labeled probe protector. Finally, 2.5 mL of p72 gold-labeled probe or 2.5 mL of p30 gold-labeled probe solution was obtained. The two gold-labeled probe solutions were mixed in a 1:1 ratio and placed in a refrigerator at 4 °C as the gold-labeled antigen.