Application of CRISP (cysteine-rich secretory protein)1 kit for diagnosing carcinoma of tongue
A technology for test strips and secreted proteins, which is used in biological tests, material inspection products, etc., to achieve the effects of simple operation, good stability, and intuitive and reliable result judgment.
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Embodiment 1
[0016] Example 1 Immunohistochemistry
[0017] Experimental materials: normal tongue tissue, tongue cancer tissue; the primary antibody is goat anti-human CRISP1 antibody, and the second antibody is donkey anti-goat Dylight594 antibody.
[0018] experimental method:
[0019] Tissue chip is to combine a large number of tissue samples orderly on the surface of a tiny substrate, and use immunohistochemistry for detection.
[0020] 1. Fix the tissue with PFA fixative for 5 minutes and wash with PBS for 10 minutes
[0021] 2. Antigen retrieval: Expose the epitope with 0.01M citrate solution. Heat the repair solution in a microwave on high heat for 3 minutes until it boils (4 minutes), and then continue to microwave for 1 minute or twice on low heat. Cool to room temperature and wash with PBS for 10 minutes. Break the membrane with 0.5% Triton for 10 minutes.
[0022] 3. Block non-specific proteins
[0023] 1) Soak in PBS for 3 minutes × 3 times
[0024] 2) 3% H 2 O 2 -Methanol (30% H 2 O 2 1...
Embodiment 2
[0036] Example 2 Enzyme-linked immunosorbent assay (Elisa)
[0037] A total of 90 cases, including 30 cases of tongue cancer (squamous cell carcinoma) group; 30 cases of other oral diseases (periodontal disease, pulp disease, oral candidiasis) group; 30 cases of healthy control group. Use the cysteine-rich secreted protein detection kit for detection, and the detection steps refer to the instruction manual of the cysteine-rich secreted protein quantitative enzyme-linked detection kit.
[0038] ELISA test results of CRISP1:
[0039] 1. The tongue cancer group is higher than the other two groups, while the other disease group has no difference with the healthy group (P=0.802).
[0040] Table 1 The diagnostic value of saliva CRISP1 in the diagnosis of tongue cancer
[0041] Diagnostic indicators AUC Sensitivity Specificity False positive rate False negative rate Youden Index Urine CRISP1 10 ng / ml0.809 88.1% 39.3% 60.7% 11.9%0.274 Urine CRISP1 40 ng / ml0.809 71.4% 87.1% 12.9% 29.6% 0...
Embodiment 3
[0044] Example 3 Preparation of gold-labeled antibody complex of test paper
[0045] 1. Preparation of colloidal gold
[0046] Add 100ml 0.005%-0.02% chloroauric acid solution into a round bottom flask and heat to boiling. After boiling, add freshly prepared 0.4%-2% trisodium citrate (Na 3 C 6 H 5 O 7 ·2H 2 O) 1-2.2ml aqueous solution, boil for 10-25 minutes, continue to stir and cool to room temperature. During this process, the color change of the solution can be seen: golden yellow→black→purple→dark blue→bright red. When the color of the solution is completely changed to transparent bright red, the required colloidal gold is obtained. After cooling, put it into a dialysis bag and dialyze the ultrapure water (1:5000) three times. Finally, transfer the dialyzed colloidal gold to a clean glass bottle with a screw cap, and store it at 4°C in a dark environment.
[0047] 2. The connection between gold colloid and protein
[0048] 1. The adsorption of colloidal gold to protein mainly de...
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