Application of cystatin SN and AFP in preparing markers for diagnosis and prediction of liver cancer
A marker and inhibitor technology, applied in the field of medical detection, can solve the problem that the specificity and sensitivity of liver cancer cannot meet the monitoring of liver cancer, etc., and achieve the effects of portability, convenience and good repeatability.
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Embodiment 1
[0024] Example 1 Establishment of CystatinSN Serum Detection Reaction System and Its Optimization
[0025] Coat the ELISA plate with a mouse anti-human CystatinSN monoclonal antibody at a concentration of 5 μg / mL, coat it overnight at 4°C, and wash the plate; then block it in 2% BSA at room temperature for 2 hours, and wash the plate; then The CystatinSN protein standard (encoded CystatinSN protein amino acid sequence such as SEQ ID NO.1) and serum samples Add them to the blocking plate respectively, react at 37°C for 1 hour, and wash the plate; then use the rabbit anti-human CystatinSN polyclonal antibody labeled with HRP at a concentration of 0.5 μg / mL, react at 37°C for 1 hour, and wash the plate; Base benzidine (TMB) for 2-3 minutes, and finally stop the reaction with 2M sulfuric acid, and detect the OD value under the condition of 450nm ( figure 1 ). Depend on figure 1 It can be seen that the linear range of the CystatinSN enzyme-linked immunoassay kit is 50pg / mL-1600p...
Embodiment 2
[0032] Embodiment 2 CystatinSN enzyme-linked immunoassay kit
[0033]According to the establishment of the CystatinSN serum detection system in Example 1, the CystatinSN enzyme-linked immunoassay kit was constructed, and the specific components are as shown in Table 1:
[0034] Table 1. CystatinSN ELISA Kit Components
[0035]
[0036]
[0037] Evaluation of CystatinSN ELISA kit: Use CystatinSN ELISA kit to detect CystatinSN positive quality control products, and repeat the detection 10 times at two levels of CystatinSN protein concentration of 160pg / mL and 80pg / mL respectively, the results show the coefficient of variation CV ≤10%; if 3 batches of kits are used to detect the same sample, the inter-assay coefficient of variation (CV) of the 3 batches of kits is ≤15%. The research on the stability of the kit shows that it can be stored at 4°C for 8 months, stored at 4°C for 2 months after opening, and transported at 0-4°C for 7 days.
Embodiment 3A
[0038] Example 3 AFP enzyme-linked immunosorbent assay (ELISA) detection kit
[0039] The AFP-ELISA detection kit uses the product of Beijing Rejing Biotechnology Co., Ltd. (Cat. No. 1003). The amino acid sequence of the detected AFP marker is shown in SEQ ID NO.2.
[0040] The CystatinSN ELISA detection kit constructed in Example 2 was combined with the AFP-ELISA detection kit in this example to form a CystatinSN-AFP combined detection kit.
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