Application of Cystatin S and CYFRA21-1 (Cytokeratin Fragement 21-1) in preparation of markers for diagnosing and predicting esophagus cancer
A marker, esophageal cancer technology, applied in the field of medical detection, can solve problems such as differential survival rate, and achieve the effect of improving sensitivity and specificity, easy to carry, and convenient to use
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Embodiment 1
[0025] Example 1 Establishment of Cystatin S Serum Detection Reaction System and Its Optimization
[0026] Coat the ELISA plate with a mouse anti-human Cystatin S monoclonal antibody at a concentration of 5 μg / mL. The coating condition is to coat overnight at 4°C, wash the plate, and then block it in 2% BSA at room temperature for 2 hours. After washing the plate, the Cystatin S protein standard substance (the amino acid sequence of encoding Cystatin S such as SEQ ID NO.1) and samples were added to the closed plate, reacted at 37°C for 1 hour, and washed the plate; then reacted with rabbit anti-human Cystatin S polyclonal antibody labeled with HRP at a concentration of 0.5 μg / mL at 37°C for 1 hour, Wash the plate; react with tetramethylbenzidine (TMB) for 2-3 minutes, and finally stop the reaction with 2M sulfuric acid, and detect the OD value at 450nm ( figure 1 ). Depend on figure 1 It can be seen that the linear range of the Cystatin S enzyme-linked immunoassay kit is 50...
Embodiment 2
[0033] Embodiment 2, Cystatin S ELISA kit
[0034] According to the Cystatin S serum detection system established in Example 3, the Cystatin S enzyme-linked immunoassay kit was constructed, and the specific components are as shown in Table 1:
[0035] Table 1. Cystatin S ELISA Kit Components
[0036]
[0037]
[0038] Evaluate the Cystatin S ELISA kit: Use the Cystatin S ELISA kit to detect the Cystatin S positive quality control product, and repeat the detection 10 times at the Cystatin S protein concentration of 160pg / mL and 80pg / mL respectively. It shows that the coefficient of variation CV≤10%; the same sample is tested with 3 batches of kits, and the inter-assay coefficient of variation CV of 3 batches of kits is ≤15%. The research on the stability of the kit shows that it can be kept stable after being 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 3
[0039] Example 3 Establishment of CYFRA21-1 Serum Detection Reaction System
[0040] The CYFRA21-1 enzyme-linked immunosorbent assay (ELISA) detection kit used in this example was a commercial CYFRA21-1 ELISA kit purchased from Beijing Huamaike Biotechnology Co., Ltd.
[0041] The CYFRA21-1 ELISA kit and the Cystatin S ELISA kit constructed in Example 2 were used to form a Cystatin S-CYFRA21-1 combined detection kit for the detection of esophageal cancer markers CYFRA21-1 and Cystatin S , wherein the amino acid sequence of the tested marker CYFRA21-1 is shown in SEQ ID NO.2.
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