Streptavidin

The content of PrP^C in saliva and serum samples was detected by ELISA. PrP^C WH2 antibody (Affiliated Cancer Hospital, Institute of Guangzhou Medical University) was added to the appropriate wells of ELISA plates, incubated overnight at 4 ℃ and blocked with 10% fetal bovine serum. After removal of the liquid from each well, 200 μl aliquot of each saliva or serum sample or 200 μl of PrP^C standard solution was added into each well for 2 hours, then coated with biotin labeled secondary antibody (Affiliated Cancer Hospital, Institute of Guangzhou Medical University). Next, washed and added streptavidin (solarbio, Beijing, China) into wells for 30 minutes at RT, then washed and added 100 μl of TMB substrate solution to each well. After that, the plates were kept at RT for 15 minutes. In the end, 100 μl stop solution was added to each well to terminate the enzyme reaction, and the absorbance was read at 450 nm by using a spectrophotometer.

The PCR Master Mix Assay kit and the DL500 DNA marker were purchased from Takara Bio Inc. (Dalian, China). GoldView-I nucleic acid stain, streptavidin, PEG-20000, and sodium chloride were obtained from Solarbio Science & Technology Co. Ltd. (Beijing, China). Tris-(hydroxymethyl) aminomethane (Tris), HAuCl₄·3H₂O, and Tween-20 were bought from Sangon Biotechnology Inc. (Shanghai, China). A MagIso DNA/RNA isolation kit was obtained from Xi'an Tianlong Science and Technology Co. Ltd. (Xian, China). A sample pad, a conjugate pad, absorbent paper, and a PVC baseboard were obtained from Jie Yi Biotechnology Co. Ltd. (Shanghai, China). UniSart CN 140 nitrocellulose membranes were obtained from Millipore (Billerica, MA, USA). Synthetic oligonucleotides were obtained from Tsingke Biotechnology Co. Ltd. (Beijing, China).

The final reaction solution above (KRAS-1U or 134–2U) was used as the template for further PCR assays. First, the labeled DNA was further labeled and amplified via PCR according to the method reported(2,27), with some modifications: templates (the final reaction solution, 1 μl), dNTPs (400 μM), dTPT3TP^biotin (20 μM), dNaMTP (50 μM), MgSO₄ (2.2 mM), primers (400 nM each) OneTaq DNA Polymerases (0.018 U/μl), and DeepVent DNA polymerase (0.007 U/μl) in 1 × reaction buffer (a total of 25 μl), under the thermocycling conditions: 20 × (96°C, 30 s; 50°C, 10 s; 68°C, 4 min) with a final extension for 68°C, 5 min. The products (5 μl) were incubated with streptavidin (1 μg, Solarbio) for 30 min at 37°C. Then samples were mixed with loading dye and separated by a 6% (134-2U) or 10% (KRAS-1U) non-denaturant polyacrylamide gel electrophoresis. The shift strips were eluted by shaking and soaking at 37°C for 2 h, quantified by NanoDrop™ One^C, and used as templates for bridge base PCR (0.5–2 ng per sample). Bridge PCR was performed as described above, and inherent dU’s preference PCR as control was also performed with the same procedure as that of bridge PCR.