| Tumor is one of the most common diseases threatening human health, which ranks the second place in the mortality of population. Many studies demonstrated that the growth of new vessculature of cancer plays an important role in tumor proliferation, invasion and metastasis. Blocking angiogenesis of the tumor can efficiently inhibit the tumor growth and sometimes lead to a complete remission. Angiogenin (Ang) has a robust capacity to induce blood vessel formation in vivo and in vitro, and it plays a key role in the process of occurrence, development of malignant tumor. Ribonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein with an estimated 50 kD molecular weight. Ang can form more stable complex by high affinity with RI (Ki=7.1×10-16M). RI can efficiently inhibit the activity of Ang after its combination with Ang.Presently, the most commonly used method in detection of RI is Western-blot analysis, which is complicated and time-consuming. Enzyme linked immunosorbent assay (ELISA) not only can keep the immunologic competence of the antigen or antibody binding with polystyrene plates, but also can keep both immunologic competence and enzyme activity of the enzyme labelling antigen or antibody. In recent years, many diagnostic methods, such as radioimmunoassay, immunofluorescence and so on, are used in the field of detecting antigen and antibody. All of these tests are specific, sensitive, but cannot be widely used because of high expense and wasting time. Indirect ELISA has more advantage comparing with other methods. So it is easy being used on a larger scale if can be made into test kit.The purpose of this study is to establish a sensitive and specific method, indirect ELISA, to analyze the content and the expression of RI antigen in serum. The main work is as followings:1. To establish indirect ELISA technique for detection of the content of RI antigen in serum and test the specificity, sensitivity and reliability of the method. 2. To analyze the dynamic level of RI in the blood of mice transinfected with RI gene by indirect ELISA3. To detect the level of RI in serum of normal people and cancer patients ( breast cancer, gastric cancer, liver cancer and lung cancer) by indirect ELISA, and analyze the difference of RI level between normal people and cancer patients.Results:1. The indirect ELISA is stable, highly sensitive and specific.2. On analyzing the dynamic change of RI in the blood of mice transinfected with RI gene, we found that the expression of RI started from the forth day, then decreased, and reached the lowest level at the 3rd week. From the 20th day, the content of RI became increase, and the result showed the concentration of RI peaked at the first month, and then descent again.3. RI level has significant difference in malignant tumor patients compared with that in normal people (P<0.05). There is no significant difference in the RI level between the benign mammary tumor patients and normal female, although the level of RI in benign mammary tumor patients is lower than that in normal female (P>0.05). There is significant difference between the benign breast tumor and breast cancer (P<0.05).Conclusions:1. Indirect ELISA could be used for detecting the RI content in serum. The results of experiment supply the practical protocols for the production of commercial test kit in the future.2. The expression level of RI can be checked if the RI gene transinfection successful or not. Our experiment provided the basis for clinical application.3. In the patients with malignant tumor, the level of RI is low. The result showed us the level of RI can be a useful marker in malignant tumor identification. |
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