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.