| MUC1, a glycoprotein with high molecular weight, is expressed slightly in the epithlial cells of normal tissues and abberantly overexpressed in various tumors, such as breast cancer, ovarian cancer and pancreatic cancer. The extracellular domain of MUC1 is constructed mainly with variable number of tandem repeats (VNTR) which can induce cytotoxic T lymphocytes (CTLs) produce immune response effectively. Accordingly, MUC1 VNTR, known as tumor associated antigen, has been demonstrated to be a good candidate against tumors. Heat shock protein (HSP) is a molecular chaperone that plays an important role in the folding and translocating of proteins. HSP can also help the foreign antigen crosspresent in dendritic cells (DC) which can induce antigen specific CTLs. It has been demonstrated that immunization of HSP-MUC1 generated MUC1 specific CTLs and provided protection against tumor expressing MUC1 in mice. Here our laboratory expressed the recombinant HSP65-MUC1 fusion protein in E.coli which is expected as a novel vaccine against tumors overexpressing MUC1. To investigate HSP65-MUC1 fusion protein in Pharmcokinetics and toxicology, we established the method of the double antibody sandwich ELISA and indirect ELISA for monitoring the quality of the HSP65-MUC1, the distribution of HSP65-MUC1 in the immunized mice and for the detecting antibodies against HSP65-MUC1. 1. The preparation of HSP65 polyclonal antibody Recombinant HSP65 was expressed in E.coli. and purified.To prepare HSP65 polyclonal antibody, we immunized the rabbit with HSP65 in complete fred adjuvant for three times. On day 3 after the last immunization, the serum was collected when the titer of anti-serum reached to 1:100000. The immunoglobulin in anti-serum was extracted by Saturation (NH4)2SO4, then stored at -20℃. HSP65 specific antibodies in the serum verified by western blot analysis and used.as binding antibodies for recognizing HSP65 of HSP65-MUC1 in double antibody sandwich ELSIA. 2. Establishment of double antibody sandwich ELISA for detection HSP65-MUC1 fusion protein To supervise the quality of HSP65-MUC1 and observe the distribution of HSP65-MUC1 in mice, we established the double antibody sandwich ELISA for detecting HSP65-MUC1. In the ELISA system, MUC1 monoclonal antibody was used as capture antibody which can combine with MUC1 in HSP65-MUC1, HSP65 polyclonal antibody was added as detecting antibody which can recongnize HSP65 in HSP65-MUC1and goat-anti-rabbit-HRP-IgG was used for binding HSP65 polyclonal antibody .The optimal condition of the ELISA was set up as follows:The concentration of the HSP65 polyclonal antibody is 1:2000 and the concentration of MUC1 monoclonal antibody is 1:1000. the coating buffer is 0.8% glutaraldehyde/0.5MTris-HCLTheconcentration of defatted milk powder for blocking is 5%. The sample diluting buffer is 0.1% Tween 20 / Tris-NaCL (PH7.2).The wash buffer is 0.5M NaCl/0.5% Tween 20 / PBS(PH7.2). We established the standard curve for detecting HSP65-MUC1.The sensitivity of the double antibody sandwich ELISA can reach to 10ng/ml. 3.Establishment of indirect ELISA for detection HSP65-MUC1 fusion protein antibody To detect antibody produce by stimulation of HSP65-MUC1 in the study of toxicology, we establish the indirect ELISA for detecting the HSP65-MUC1 antibody in mice. In this test, condition was optimized.The concentration of HSP65-MUC1 for coating is 5μg/ml. Horse-anti-mouse -HRP-IgG was diluted 50 times. 4.Detection of tissue homogenate of mouse by double antibody sandwich ELISA To observe the distrubition of HSP65-MUC1 in the tissues,the C57BL/6 mice was injected with HSP65-MUC1 subcutaneously, and then various tissues were obtained at 10 min,30 min,50 min,70 min quickly, The HSP65-MUC1 in tissue homogenate was detected by double antibody sandwich ELISA. The results showed that HSP65-MUC1 could be detected in injected skin tissue within 70min after injection. This demonstrates that the double antibody sandwich ELISA is useful tool to determine the exisitance of HSP65-MUC1 in the tissues. |
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