| Tumor is one of the most crucial disease threatening human health. The common established chemotherapeutic drugs kill tumor cells directly, and as well have poisonous side effect to normal cells. Ribonuclease inhibitor(RI) is able to break the rapid growth of tumor tissues because RI tightly binds and inhibits angiongenion(Ang) which promotes new endothelia proliferation. RI can inhibit the growth of the tumor cell by decreasing its oxygen and nutrition's supplying. Whereas RI has no poisonous side effect to normal cell. Our laboratory has proceeded a quantity works in the field of RI anti-tumor research. Experiments showed that RI, extracted from human placenta and porcine liver, could inhibit the growth of mastocarcinoma cell Ca-761, sarcoma cell S-180, and liver cancer cell H-22. It is believed that the battle of human with tumor will undoubtedly be marked by the exploration of RI as an anticarcinogen.Domestic and international researches of RI were mainly focused on its structure, and seldom reported the function of RI to neutralize tumor growth. Our laboratory has reported that RI has anti-tumor effect. In this work, basing on the former research, we extracted and purified the antibody of RI, and also studied the pharmacokinetics of RI by the method of ELISA.RI is an acid mamalian cytoplasmic glycoprotein with the molecular weight of 50kD. We immunized rabbit with the mixture of 200 u g RI and the same volume incompleted Freund's adjuvant, and collected the blood when the titre of the anti-sera reached to 5000. SDS-PAGE identified the clear band of 55 kD, which showed the antibody of RI.Recombinant Protein(rProtein) A Sepharose Fast Flow is an affinity medium. The capacity of specific binding for Fc region of IgG is enhanced by coupling protein A with Sepharose 4 Fast Flow. The IgG is perfectly purified by the covalent combination.To pursue better purifying efficiency, the method is screened and optimized. It was confirmed that 2 ml of loading-sample is optimum in the 5ml colum bed, and the highest concentration of eluting buffer was 30%. The elutes from tube 9-16(1 ml/tube) was collected. The purified IgG was loaded on SDS-PAGE, the single band of 55kD was showed. And the special band was assayed by western-blot , which proved the result of the IgG is purified. The effect value of IgG exceeded 64,000 by ELISA, and it kept stable more than one year.With the rapid development of biotechnology, the protein medicine is more valued. Being the high activity and small dose of protein medicine make its blood concentration in a low level. So the method for detection should be sensitive. ELISA was chosen because its limit detection reaches to pg/ml~ng/ml level. The standard curve was established by indirect ELISA, and the condition was optimized. At last the primary and secondary antibody were diluted 4000 times and 2000 times respectively, and the concentration of BSA for blocking was 2% in this experiment. At the same time the method of indirect ELISA was also investigated. According to the result, the limit detection of this assay was estimated to be 0.05 u g/ml. And the coefficients of variation(CV) in intra-assay and inter-assay were 9.28% and 13.56% respectively. Diluted 4000 times, the stability of the primary antibody exceeded 6 months, proving that the biology activity was still kept.Up to now, the antibody of RI cannot be purchased from domestic and international market. So, we prepared and purified the antibody of RI which could be used for not only the detection of Western-blot and ELISA, but also for the early diagnosis of tumor. There is not reported about pharmacokinetics of RI. We explore it by ELISA, which will provide a basis for the further study. |
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